Integrated Sensory and Immune Cell Organization in the Lip Skin of the Goldfish (Carassius auratus, Linnaeus, 1758)

TL;DR

This study explores the specialized lip skin of goldfish, revealing a unique combination of sensory and immune cells that work closely together.

Contribution

The study is the first to simultaneously identify multiple immune and sensory cell types in goldfish lip skin using histological, ultrastructural, and immunohistochemical methods.

Findings

Goldfish lip skin contains a high density of sensory and immune cells, including neuromasts, Merkel cells, and macrophages.

Telocytes and synapse-like structures were observed, suggesting roles in cellular communication and sensory processing.

Immunohistochemical markers confirmed the presence of specific cell types and their spatial organization.

Abstract

Fish lips are specialized sensory regions involved in foraging, substrate exploration, and environmental detection. In goldfish, the lip skin contains a high concentration of sensory and immune cells, yet its detailed structure has not been fully described. Histological and ultrastructural observations revealed diverse cell types, including Merkel cells, rodlet cells, eosinophilic granular cells, and intraepidermal macrophages, as well as specialized sensory organs such as neuromasts and tuberous-like sensory units. Immunohistochemical labeling patterns for CK20, S100, CD68, CD64, CD117, and E-cadherin were consistent with the corresponding histological and ultrastructural observations. Telocytes were also identified in the dermis, indicating roles in cellular communication. Our results highlight the structural specialization of the lips as a region where sensory and immune-related elements are closely associated.

This study examines the histological, ultrastructural, and immunohistochemical features of the lip skin of the goldfish (Carassius auratus, Linnaeus, 1758), a sensory-rich region that plays an essential role in feeding and environmental perception. Our findings highlight the coexistence and close association of immune, epithelial, and sensory cells within the epidermis and dermis. For the first time in goldfish, intraepidermal macrophages, eosinophilic granular cells, rodlet cells, Merkel cells, and several specialized sensory structures—neuromasts, taste buds, and tuberous-like sensory units—were simultaneously identified within the same integumentary field. Quantitative morphometry demonstrated a high density of eosinophilic granular cells, rodlet cells, and neuromasts per unit epithelial area, reinforcing the functional specialization of the goldfish lip as a sensory–immune interface. Immunohistochemical markers (CK20, S100, CD68, CD64, CD117, and E-cadherin) were applied as complementary tools to describe phenotypic labeling patterns. These findings are interpreted cautiously as supportive evidence consistent with epithelial, neural-associated, stromal, and immune cell distributions observed morphologically. Transmission electron microscopy further uncovered fine structural details such as synapse-like contacts in taste buds and Merkel cells, dense-core granules in eosinophilic granular cells, and telocyte–nerve fiber associations in the dermis. By integrating cellular, structural, and immunohistochemical perspectives, this study provides a novel descriptive reference for the goldfish lip skin as a region characterized by the close spatial association of sensory and immune-related elements, underscoring its value as a model for vertebrate cutaneous biology and neuroimmunology.

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1. Introduction

The lips of many teleost fish represent specialized sensory fields that contribute significantly to feeding behavior, substrate exploration, and environmental monitoring [1,2]. In cyprinids, the lip epithelium contains a high density of mechanosensory and chemosensory structures, making it distinct from the more generalized body skin [3]. Previous studies have shown that fish lips may house neuromasts, taste buds, Merkel cells, and other sensory units involved in detecting hydrodynamic changes, chemical cues, and fine tactile stimuli [4,5]. In addition to their sensory roles, the lips are in constant contact with the surrounding environment and may therefore also function as an active barrier containing diverse immune cell populations [6].

Unlike the general body surface, teleost lip skin exhibits a thickened epidermis [5] enriched with sensory structures [7], mucous cells [8], and immune-related elements that support direct interaction with the aquatic environment [9]. The lip epidermis commonly contains superficial neuromasts, taste buds, and Merkel-like cells, which contribute to mechanosensory, chemosensory, and tactile functions, respectively [10,11,12]. Comparative studies have demonstrated pronounced interspecific variation in lip skin organization related to feeding strategy and habitat [13], with silver carp showing a highly specialized upper lip rich in taste buds, club cells, and mucous cells adapted to turbid environments [3], as well as similar regional specialization reported in ornamental fishes [5].

This study focused specifically on the lip region because it contains a high density of cutaneous sensory structures, which are less abundant or absent in other body regions. The lip epidermis of cyprinids, including Carassius auratus (Linnaeus, 1758), is considered a functionally enriched sensory field, making it an appropriate model for integrated histological and immunohistochemical analysis [5,14]. Despite their functional importance, the cellular composition and integrated organization of the goldfish lip skin remain insufficiently documented. Most published descriptions of goldfish integument focus on the trunk or general epidermis, with little attention given to the specialized lip region. Understanding the sensory and immune landscape of this area is essential, as it is a primary site of environmental interaction and food acquisition. Therefore, the present study focuses exclusively on the lip skin of the goldfish. Through combined histological, ultrastructural, and immunohistochemical approaches, we aim to characterize the sensory structures and immune cell populations in this region and evaluate their spatial relationships. Our findings establish a detailed reference for the sensory–neural–immune organization of goldfish skin and its significance in vertebrate cutaneous biology.

2. Materials and Methods

2.1. Sample Collection

The Ethics Committee of Assiut University, Egypt, approved the study (ethical number: 06/2024/0191). Twenty-four mature, healthy goldfish (C. auratus) were used. Goldfish were kept in the laboratory for a 2-week acclimatization period before the study to guarantee ideal health conditions. Water temperature was kept at 22 ± 1 °C, with pH maintained at 7.0–7.5 and dissolved oxygen at 6–7 mg/L using continuous aeration. Photoperiod was controlled at 12 h light:12 h dark. Fish were fed a commercial pelleted diet twice daily at a rate of 2% of body weight. Stocking density was maintained at 1 fish per 5 L to prevent crowding stress. All environmental parameters were monitored daily, and only fish kept under consistent, stable conditions were included in the study. After this, healthy fish with an average weight of 34.50 ± 3.70 g and a standard length of 9.80 ± 1.2 cm were chosen at random from the aquariums. Sex was not determined because goldfish outside the breeding season lack reliable external sexual dimorphism, and no gonadal examination was performed since this study did not involve internal organs. Individuals showed no external signs of reproductive activity, injury, or disease. Fish exhibiting skin lesions, discoloration, or abnormalities were excluded. Only specimens with intact lip epidermis and dermis were included to ensure consistency across samples. Fish were euthanized via their exposure to an overdose (250 mg/L) of tricaine methanesulfonate (E10521, Sigma-Aldrich, St. Louis, MO, USA) in water that was 4 °C, buffered to a pH of 7.0–7.2 using sodium bicarbonate to prevent acidification [15]. In addition to the fish being unable to resume active motions in the recovery tank, fish death was verified two minutes after the last opercular movements. The exposure time and death confirmation were controlled according to ARRIVE guidelines.

2.2. Histological and Histochemical Analysis

Skin of the lip region was fixed in Bouin’s fluid for 22 h. The fixed samples were dehydrated with ethanol and cleared by methyl benzoate 99%, then embedded in paraffin wax. Serial transverse (5 μm thick) paraffin sections were taken and stained with Harris hematoxylin and eosin, PAS, and Crossmon’s trichrome [16].

2.3. Immunohistochemistry

For immunohistochemical analysis, sections were processed using the Pierce Peroxidase Detection Kit (36000, Thermo Fisher Scientific, Whaltam, MA, USA). After deparaffinization in xylene and rehydration in graded ethanols, antigen retrieval was performed by heating sections in citrate buffer (pH 6.0) at 95–98 °C for 10 min, followed by cooling at room temperature for 30 min. Sections were rinsed in TBS with 0.05% Tween-20 (TBST, pH 7.4) and treated with peroxidase suppressor for 30 min to block endogenous peroxidase. A biotin-blocking system (Avidin/Biotin Blocking Kit, Invitrogen, Carlsbad, CA, USA) was applied prior to secondary antibody incubation. Nonspecific binding was reduced using Universal Blocker™ (Thermo Fisher Scientific, Waltham, MA, USA) in TBS for 30 min at room temperature.

The sections were incubated overnight at 4 °C with diluted primary antibodies against rabbit polyclonal S100 protein (Z0311, Dako, Glostrup, Denmark, 1:100), rabbit polyclonal anti-CD117 (c-kit) (1:100, Dako, Glostrup, Denmark), mouse monoclonal anti-CD68 (sc-17832, Santa Cruz Biotechnology, Heidelberg, Germany), mouse monoclonal anti-E-cadherin (1:100, 13-1900, Thermo Fisher Scientific, Waltham, MA, USA), rabbit polyclonal anti-CD64 (PA5-102382, 1:100, Thermo Fisher Scientific, Waltham, MA, USA), and monoclonal mouse anti-cytokeratin 20 (sc-271183, Santa Cruz Biotechnology, Dallas, TX, USA, 1:200). In parallel, tissue specimens, in which the S100 protein or CK20 primary antibody was omitted and replaced with buffer, served as negative controls (Figure S1). All primary antibodies were selected based on reported cross-reactivity in teleost studies and their use in previous morphological analyses in fish species [17,18]. The slides were washed two times for 3 min with wash buffer and incubated with secondary antibodies for 30 min at room temperature: diluted (1:1000) goat anti-rabbit IgG (65-6140, Invitrogen, Carlsbad, CA, USA) and diluted (1:100) goat anti-mouse IgG (31800, Invitrogen, Carlsbad, CA, USA) secondary antibodies for 30 min at room temperature. Following this, the slides were washed three times for 3 min each with a wash buffer, and the tissues were incubated with the diluted (1:500) Avidin-HRP (43-4423, Invitrogen, Carlsbad, CA, USA) in universal blocking buffer for 30 min. The slides were then washed three times for 3 min each with a wash buffer. The tissues were incubated with 1 × metal-enhanced DAB substrate working solution (by adding stable peroxide buffer to the 10 × DAB/metal concentrate) for 5–15 min until the desired staining was achieved. Finally, the sections were washed twice for 3 min each with a wash buffer, counterstained with Harris modified hematoxylin, and mounted with mounting media. Positivity criteria included (1) chromogenic deposition confined to cytoplasm or processes of target cells, (2) intensity greater than background and negative controls, and (3) consistency across biological replicates.

2.4. Semithin Sections and Transmission Electron Microscopy (TEM) Preparations

For electron microscopy, tissues were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) [19]. After rinsing in the same buffer, samples were post-fixed in 1% osmium tetroxide for 1 h, dehydrated in a graded ethanol series, and embedded in epoxy resin. Semithin sections (0.5–1 µm) were cut using a Reichert Ultracut ultramicrotome (Reichert-Jung, Munich, Germany), stained with toluidine blue, and screened to localize sensory and immune cell clusters. Ultrathin sections (~70 nm) were prepared on the same instrument, mounted on copper grids, stained with uranyl acetate and lead citrate [20], and examined using a JEOL transmission electron microscope (JEOL Ltd., Akishima, Tokyo, Japan).

From each fish, two resin blocks were prepared from anatomically matched lip regions. For each block, 3–4 ultrathin sections were collected, and 10–12 fields per section were systematically sampled using a systematic random sampling approach to avoid selection bias. Across all specimens, this yielded a minimum of 60–80 ultrastructural fields per structure type (neuromasts, taste buds, rodlet cells, eosinophilic granular cells, macrophages, and Merkel-like cells).

2.5. Histomorphometry and Quantitative Analysis

Quantitative cell counts were performed to assess the density of eosinophilic granular cells (EGCs), rodlet cells (RCs), and neuromasts within the lip epidermis. For each fish, five non-overlapping microscopic fields were captured at 1000× magnification using a calibrated digital microscope camera. EGCs and rodlet cells were counted within a standardized 10,000 µm^2^ area of epidermis, measured using ImageJ software v.1.54p (NIH, Bethesda, MD, USA). Counts were performed on toluidine-blue-stained semithin sections. Neuromast density was calculated as the number of neuromasts per millimeter of lip length, measured along the epithelial surface. All measurements were performed by two independent observers blinded to sample identity. Data are presented as mean ± standard deviation (SD). Inter-observer variability was <5%. Statistical analyses were descriptive in nature, as the study did not include treatment groups

3. Results

3.1. Histological Analysis

Light microscopic examination of the goldfish lip skin revealed a multilayered epidermis overlying a loose connective tissue dermis. In H&E-stained sections, the epidermis contained club cells that appeared as acidophilic polygonal cells. The surface epithelium contained scattered mucous cells (Figure 1A). Superficial neuromasts appeared as oval-to-round sensory units embedded within the epidermal layer. These structures were composed of a central cluster of pale-staining, columnar sensory cells surrounded by smaller, more basophilic supporting cells. The sensory epithelium was arranged in a compact, mound-like profile that projected slightly above the surrounding epidermis (Figure 1B).

Several rounded taste buds were observed within the superficial layers of the epidermis. These units appeared as well-circumscribed, circular-to-ovoid profiles composed of centrally located sensory epithelial cells surrounded by smaller supporting cells. The apical cytoplasm of the sensory cells showed a distinct PAS-positive reaction, producing a magenta coloration that sharply contrasted with the more lightly stained surrounding epidermal cells. This PAS reactivity was most prominent at the apical membrane and within fine cytoplasmic granules, suggesting enrichment in glycoconjugates typical of sensory epithelial specializations. Numerous rounded PAS-positive mucous cells were frequently located adjacent to these structures (Figure 1C). Crossmon’s trichrome staining (Figure 1D) demonstrated a well-defined separation between the epidermis and the collagen-rich dermis. Trichrome staining further delineated the architecture of superficial neuromasts and tuberous-like sensory units.

The semithin sections of goldfish lip skin show that it consists of two main layers:

A—The epidermis is a non-keratinized stratified epithelium composed of multiple cell types covered with microridges (Figure 2A). Rodlet cells with characterized thick capsules and cytoplasm with rodlet granules are present in different stages among superficial epidermal cells (Figure 2A). Mucous cells are scattered among pavement cells and contain large mucin-filled vacuoles (Figure 2B and Figure 3B). Eosinophilic granular cells (EGCs) are immune-related cells distributed within the middle and basal epidermis. They are rounded or oval with a central nucleus and their cytoplasm is filled with large, metachromatic granules with toluidine blue (Figure 2C,D). The epidermis also contains specialized mechanoreceptor cells, Merkel cells, in association with tuberous-like sensory units. These cells are distributed singly and characterized by pale vacuolated cytoplasm with small metachromatic granules (Figure 2D).

Many specialized sensory structures are distributed in the epidermis of goldfish, including tuberous-like sensory units and neuromasts. The tuberous-like sensory unit comprises sensory receptor cells arranged in a compact epithelial cluster, surrounded by supporting cells and basement membrane. It is covered by a thin layer of epithelial cells (Figure 2A–D).

The taste buds appear as a flask-shaped chamber lined with modified sensory receptor cells interspersed with supporting cells. Afferent nerve fibers innervate the basal side of the sensory cells (Figure 3A,B). The basal cells of the epidermis are small cells found at the base of the epidermis adjacent to the basement membrane (Figure 3A).

Neuromasts are a component of the lateral line system and are composed of sensory hair cells, supporting cells, and basal cells. Sensory hair cells are centrally located, each with stereocilia and a single kinocilium projecting into a gelatinous cupula. Nerve terminals contact the bases of hair cells (Figure 4A–D).

Quantitative evaluation of the epithelial cell populations revealed a consistent distribution pattern across all examined specimens. The mean number of eosinophilic granular cells (EGCs) was 12.4 ± 2.1 cells per 10,000 µm^2^ of epidermis. Rodlet cells (RCs) were less frequent, with 4.7 ± 1.3 cells per 10,000 µm^2^. EGCs were widely distributed through the mid and basal layers, whereas rodlet cells were often clustered near sensory structures. The density of neuromasts along the lip margin averaged 3.2 ± 0.4 neuromasts per mm of epithelial length.

B—The dermis is a dense connective tissue layer subdivided into the stratum spongiosum (an upper loose collagen network containing pigment cells, lymphocytes, macrophages, and blood vessels) (Figure 5A). The stratum compactum is composed of dense collagen bundles with fibroblasts, granular leucocytes, and many nerve fibers (Figure 5B,C). The hypodermis and underlying musculature lie beneath the dermis (Figure 5D).

3.2. Immunohistochemical Analysis

CK20 immunoreactivity was detected in scattered epidermal cells morphologically consistent with Merkel-like cells. These cells were frequently observed in close spatial proximity to intraepidermal nerve profiles or adjacent to neuromast structures. The labeling pattern appeared cytoplasmic with a fibrillar distribution (Figure 6A,B). CK20-positive staining was also observed within neuromast structures (Figure 6B,C). In deep canal neuromasts, sensory hair cells exhibited CK20 immunoreactivity (Figure 6D). These observations are reported descriptively as immunolabeling patterns without implying definitive molecular identity.

S100 immunoreactivity was detected in club cells within the epidermis (Figure 7A). Positive labeling was also observed in cells associated with deep canal neuromasts, including sensory receptor cells (Figure 7B), as well as within neuromast structures (Figure 7C). In the dermis, elongated interstitial cells with telocyte-like morphology formed a network and showed S100 immunoreactivity (Figure 7D).

CD68 immunoreactivity was observed in scattered epidermal cells with morphological features consistent with macrophages (Figure 8A). CD64-positive labeling was detected in epidermal macrophage-like cells and in dermal interstitial cells with telocyte morphology (Figure 8B). E-cadherin immunoreactivity was present in club cells (Figure 8C). CD117 labeling was observed in dermal telocyte-like cells (Figure 8D). All immunoreactivity patterns are described as phenotypic observations consistent with the corresponding histological and ultrastructural features.

Samples were collected from multiple individuals (n = 24), and no significant inter-animal variation was observed in the morphology, distribution, or immunohistochemical profiles of the examined structures. The consistent appearance of neuromasts, taste buds, Merkel cells, rodlet cells, and immune components across all specimens supports the reproducibility of the findings.

3.3. Electron Microscopy

The epidermis is multilayered with superficial pavement cells that are covered by microridges and the cytoplasm contains glycogen and mitochondria (Figure 9A,B). Intraepidermal macrophages are demonstrated in the superficial epidermis as they are migrating immune cells with pseudopodia. They are irregularly shaped with an eccentrically placed oval nucleus. The cytoplasm contained lysosomes, phagosomes, residual bodies, mitochondria, and rER (Figure 9A,C). Mucous cells are observed in the superficial epidermis and contain electron-lucent granules (Figure 9D). Cells with mitotic divisions are also seen in the epidermis (Figure 9D).

Merkel cells are closely opposed to intraepidermal nerve endings, forming Merkel cell–neurite complexes (tactile disks). The lateral plasma membrane forms desmosomal junctions with adjacent keratinocytes. The cytoplasm contains dense-core neurosecretory granules (~80–150 nm diameter). Synapse-like contacts are formed, specialized areas with thickened pre- and post-synaptic membranes and vesicle accumulation in the nerve terminal (Figure 10A–C). Some Merkel cells are located immediately above the basement membrane, and adjacent nerve fibers are observed (Figure 10D).

Many immune cells are distributed in the epidermis, including EGCs characterized by rounded electron-dense granules and eccentric nucleus placement (Figure 11A–C). Many lymphocytes with a high nuclear-to-cytoplasmic ratio can be detected (Figure 11A–C). Under the basement membrane in the dermis, many lymphocytes and macrophages can be observed (Figure 11D).

Taste buds appear as flask-shaped epithelial invaginations extending from the surface toward the basal lamina. The apical ends of sensory cells converge at a taste pore, a small opening through which microvilli project into the mucous layer (Figure 12A). Sensory cells are slender, elongated cells extending from the base of the bud to the taste pore, possessing bundles of microvilli projecting into the pore (Figure 12A). The cytoplasm contains mitochondria and dense vesicles (Figure 12B–D). The basal membrane forms synapse-like contacts with afferent nerve terminals containing clear synaptic vesicles (Figure 12B). The supporting cells have broader electron-dense cytoplasm. The basal cells are small, undifferentiated cells at the base of the taste bud (Figure 12A,B).

The dermis contains granular leukocytes with electron-dense granules, lymphocytes, and fibroblasts (Figure 13A). Bundles with myelinated nerve fibers are observed in the dermis (Figure 13B–D). Telocytes with long cytoplasmic processes or telopodes (Tps) are detected in association with nerve fibers (Figure 13C). Macrophages with phagocytosed materials are observed in the dermis (Figure 13D).

Telocytes are arranged in a network in the dermis. TCs and their Tps establish heterocellular contact with muscles, nerve fibers, and lymphocytes. Numerous vesicles were seen in the podoms of Tps (Figure 14A). TCs and their Tps extended around the muscles and their cell bodies showed many mitochondria, with some vesicles shed to the dermis (Figure 14B–D). TCs and their Tps were also wrapped around the nerve fibers (Figure 14E).

4. Discussion

The present study provides a detailed characterization of goldfish (C. auratus) lip skin, demonstrating that the epidermis is a structurally complex interface where sensory, epithelial, and immune elements coexist in close association. The concentration of specialized sensory organs within the lip region, including neuromasts, taste buds, Merkel cells, and tuberous receptors, supports the functional specialization of this area as a primary site for chemosensory and mechanosensory input. This configuration agrees with observations in other cyprinids, where the lips and oral margins serve as major sensory fields important for feeding, substrate exploration, and environmental assessment [1,2].

The epidermis of the goldfish was typically composed of stratified epithelium. The superficial microridges are in contact with the cuticle and offer mechanical strength to prevent abrasion [21]. Numerous cell types in the skin’s epidermis create surface mucus; mucous goblet cells in particular are known to contain a range of physiologically active macromolecules, mainly glycoproteins and mucin granules. Glycoproteins are essential for immunological responses as well as other biological functions [22,23]. Mucins are the principal molecules in mucus and are responsible for cell surface communication, pathogen entrapment, mucus viscosity, and physical protection of the skin surface [24]. Skin mucus has been found to include a few metabolites with antibacterial properties, such as hydroxyisocaproic acid, azelaic acid, and N-acetylneuraminic acid [25]. The anterior body region’s mucous cell condensation is crucial for lubricating and shielding fish from abrasive injuries sustained while looking for food on the bottom [26,27].

Our study indicates that Merkel cells (MCs) can be best identified by electron microscopy based on the presence of dense-core granules in their cytoplasm and their close proximity to intraepidermal nerve terminals. Merkel cells are neuroendocrine cutaneous cells that are located at the epidermal/dermal border and innervated by afferent somatosensory fibers, which act as mechanoreceptors [28,29]. Merkel cells were classified as members of the APUD system family because their osmiophilic granules contained biogenic amines and peptides [30].

Although the exact role of Merkel cells is uncertain, it is hypothesized that MCs act as mechanoreceptors that are activated by pressure, motion, contact, and stretching [29,31]. Since MCs may have originated from neural crest cells, they express a variety of neuropeptides and neuroendocrine markers [32,33,34,35,36]. These include substance P [37], leu-enkephalin in striped dolphins [38], PGP9.5 [36], and serotonin-like immunohistochemical reactions identified in conger eels [32] and striped dolphins [38]. However, MCs may be of epidermal origin because they express a basic epithelial cytokeratin [29]. The current study suggests the presence of MCs by immunohistochemical marker cytokeratin-20 (CK-20), which is the most specific marker for MCs [39,40]. The identification of Merkel cells, and their association with nerve endings, indicates that the goldfish lip epidermis integrates fine touch sensations with the broader lateral line system. Some authors argue that the Merkel cell population is highly diverse and that it carries out many functions, including endocrine ones [41].

Neuromasts are mechanoreceptive organs that can be found on the body’s surface (superficial neuromast or pit organs) or in a network of canals that run along the head, trunk, and tail (deep neuromasts) [42,43]. The structural features of neuromasts, including the polarity of hair cells, the presence of supporting cells, and afferent innervation, are consistent with previous descriptions in zebrafish [11], blind cavefish [7], and goby [44]. However, superficial and canal neuromasts have been previously described in the lips of cyprinids [10,27]. The detection of CK20 immunoreactivity in deep canal neuromasts represents a finding that has not been previously reported in cyprinids. CK20 is classically associated with differentiated epithelial cells and Merkel cells; therefore, its localization within neuromast sensory and supporting cells is described here as a phenotypic immunoreactivity pattern rather than as evidence of a specific functional role. This observation may reflect shared epithelial characteristics or conserved cytoskeletal elements among specialized epidermal cell types. Superficial neuromasts and hair cells of canal neuromasts in goldfish both showed immunoreactivity to the S-100 protein, a specific lateral line system marker. Furthermore, nerve fibers showed S100 immunoreactivity, which is likely a marker for Schwann cells [45]. Previous observations have shown that teleost and zebrafish display the same expression [46]. Since the neuromast in saccules of the rainbow trout (Salmo gairdnerii R.) resembles the sensory cells of the inner ear of a cichlid fish in both structure and function, as well as sharing certain immunohistochemical characteristics such as the expression of S100 protein immunoreactivity, research on these cells is quite intriguing [47].

The taste buds exhibited their characteristic arrangement of modified epithelial sensory cells, supporting cells, and non-specialized basal cells resembling Merkel cells. The TEM identification of synapse-like contacts between sensory cells and afferent terminals underscores their rapid transmission of chemical information [48]. Comparable structures have been described in the lips and olfactory epithelium of zebrafish [14] and fins of damselfish [49], suggesting conserved chemosensory architecture across teleosts. The presence of mitochondria-rich cytoplasm and numerous dense-core vesicles reflect the high metabolic activity associated with their continuous role in chemical detection and neurotransmission. These vesicles are known to contain and release various neurotransmitters, including serotonin, γ-aminobutyric acid (GABA), acetylcholine, and norepinephrine, which mediate signal transfer from receptor cells to gustatory neurons [50].

The tuberous-like sensory unit is made up of sensory cells encased in a cellular capsule, and was also investigated in this study [27]. These units perform several important functions, including the detection of active objects, contributing to orientation and spatial localization, and facilitating the remote detection of other electric fish. They also play a key role in transmitting electric organ discharges, which are essential for perceiving weak electrical stimuli [51]. Similar structures have been described in cheeks of weakly electric fish [51] and, to a lesser extent, in catfish [52], lips of koi fish [27], and lips of silver carp [4]. Their presence in goldfish suggests that cyprinids may retain rudimentary detection of low-frequency electric cues.

In addition to sensory cells, the epidermis contained many immunological cells such as eosinophilic granular cells, intraepidermal macrophages, rodlet cells, and lymphocytes. TEM analysis of eosinophilic granular cells revealed numerous electron-dense cytoplasmic granules, supporting their defensive function. These granules likely contain and release a diverse spectrum of bioactive substances, including heparin, neuropeptides, proteases, and antimicrobial peptides (AMPs), as reported in various teleost species [53,54]. Intraepidermal macrophages exhibited characteristic features of active phagocytes, including the presence of lysosomes, phagosomes, and residual bodies, confirming their role within the skin-associated lymphoid tissue (SALT) [55]. CD68 positivity within the epidermis and dermis corresponded to cells with ultrastructural features typical of macrophages. This staining pattern agrees with earlier descriptions in gills of goldfish [56], where CD68 or other macrophage-associated markers selectively label phagocytic cells involved in immune surveillance and SALT activity. Rodlet cells, distinguished by their thick capsule and cytoplasm filled with rod-shaped granules, were also observed. These cells have been attributed multiple functions, such as defensive secretory activity, regulation of pH, antimicrobial responses, and facilitation of water or electrolyte transfer and lubrication [57,58]. Moreover, rodlet cells are considered nonspecific immune effectors whose numbers increase during parasitic infections, indicating their involvement in innate immune defense [59]. The coexistence of immunological and sensory elements within the same epithelial domain highlights the multifunctional nature of the fish integument and suggests an evolutionary linkage between environmental sensing and immune protection.

Epidermal club cells (ECCs) exhibited S100 immunoreactivity in the present study. Similar S100 labeling has been reported in catfish club cells [60]. E-cadherin immunoreactivity was also observed in club cells, consistent with reports from other teleosts in which club cells display epithelial-type junctional molecules due to their incorporation within the stratified epidermis. Comparable E-cadherin labeling has been described in the trunk epidermis of zebrafish [61], supporting the interpretation of these cells as integrated epithelial elements, where these cells participate in epithelial cohesion and barrier maintenance despite their specialized secretory function. The ECCs have been given a variety of roles. The release of warning chemicals during any predator attack is the primary function of the ECCs [62,63]. Behaving as innate immune cells and taking part in various immunological processes is another suggested role for ECCs [63]. This was confirmed by Alesci and his colleagues [64] who found that zebrafish club cells expressed mitogen-activated protein kinase (MAPK) p38, Toll-like receptor (TLR)2, Piscidin1, and inducible nitric oxide synthase (iNOS) peptides.

In the dermis of the lip region, telocytes (TCs) characterized by their long, slender telopodes were observed. These interstitial cells establish close associations with collagen fibers, blood vessels, nerve fibers, and immune cells [65]. Telocytes are multifunctional, contributing to angiogenesis, immune regulation, cell differentiation, organ morphogenesis, and mechanoreception, and they possess receptors for both excitatory and inhibitory neurotransmitters [66]. Dermal telocyte-like cells (TCs) showed S100 immunoreactivity, in agreement with observations reported in tilapia [65,67]. Telocyte-like cells also exhibited CD117 (c-kit) immunoreactivity. Although CD117 is widely recognized as a marker associated with stem and certain interstitial cell populations [68], its detection here is interpreted cautiously as a phenotypic stromal labeling pattern. Morphologically, TCs were frequently distributed around dermal nerve fibers, and this spatial association may reflect structural proximity within the connective tissue microenvironment.

Interestingly, a subset of dermal telocyte-like cells exhibited CD64 (FcγRI) immunoreactivity. CD64 is classically associated with monocyte/macrophage lineages [69]; however, its expression has also been described in certain stromal populations involved in immune–stromal interactions. In the present study, CD64-positive telocyte-like cells were commonly located near nerve fibers and blood vessels. This distribution is reported descriptively and may indicate spatial association with vascular and neural elements, without implying a definitive immunological function.

Although the study focused on the lip region, this area is recognized as one of the most sensory-rich fields in cyprinids, providing an optimal site for examining the coexistence of epithelial, neural, and immune components. The consistency of the findings across all sampled fish further supports that the observed features represent typical characteristics of the species’ integument rather than individual variation.

A general methodological limitation of the present immunohistochemical analysis is the absence of orthogonal validation methods, such as species-specific antibodies, Western blotting, or RT-qPCR confirmation. This limitation applies to all markers used in this study (CK20, S100, CD68, CD64, CD117, and E-cadherin). Although mammalian antibodies are widely employed in zebrafish and other teleost models and often demonstrate cross-reactivity due to conserved epitopes, such cross-reactivity requires empirical validation for each species, tissue, and experimental condition. For goldfish specifically, direct antibody validation data remain limited or unavailable for the markers applied here. Therefore, the immunoreactivity patterns described in this study are interpreted strictly as phenotypic labeling consistent with the observed histological and ultrastructural features, rather than as definitive molecular identification. Furthermore, the use of similar markers in other teleost studies does not automatically ensure identical antibody performance, particularly if different clones, manufacturers, or antigen retrieval conditions were employed. Accordingly, all immunohistochemical findings in the present work should be regarded as descriptive and supportive observations that complement, but do not replace, the structural evidence provided by light and transmission electron microscopy.

5. Conclusions

This study provides a detailed histological and ultrastructural characterization of the lip skin of the goldfish (C. auratus), demonstrating a structurally specialized epidermis in which sensory, epithelial, and immune-related elements coexist within the same anatomical framework. The identification of diverse cell populations, including Merkel-like cells, eosinophilic granular cells, rodlet cells, and intraepidermal macrophage-like cells, together with specialized sensory structures such as neuromasts and tuberous-like sensory units, underscores the architectural complexity of this region. Immunohistochemical labeling patterns for CK20, CD64, CD68, CD117, E-cadherin, and S100 were observed in cell populations consistent with their morphological features and are interpreted here as complementary phenotypic findings rather than definitive molecular identification. Ultrastructural analysis further confirmed the cellular organization and spatial proximity of sensory and immune-related components. Collectively, these observations establish a comprehensive morphological reference for the goldfish lip skin and document the close structural association of sensory and immune elements in this specialized epidermal region.