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Morphological characteristics of the blackspot seabream (Pagellus bogaraveo) tongue: A structural and immunohistochemical study

Abstract The blackspot seabream (Pagellus bogaraveo, Brünnich, 1768) is an omnivorous, predominantly carnivorous fish. In aquaculture, it is fed with pellets rich in proteins and fat. The morphological and functional aspects of the fish tongue, the feeding modality and the tasting capacity are strictly related. Therefore, the aim of this study was to describe by scanning electron, light and confocal laser microscopy, the morphological characteristics of the tongue in this species. It showed an apex, a body and a root. There were rows of teeth on the edges of the mouth and taste pores on all the tongue dorsal surface with folds and furrows. In addition, body and root showed several fungiform‐like papillae in the mucosa of the folds, covered by a weakly keratinized stratified squamous epithelium, can be observed. The papillae were innervated by S100 positive fibres. In the apex, a mesenchymal tissue with vimentin positive star‐shaped stem cells was evident. The results could give a support for a wider use of the blackspot seabream as a farmed species, considering the morphological data as correlated with the potentiality of food discrimination. This provides a basis for possible applications in feeding strategies. The presence, localization and characteristics of the mesenchymal stem cells, as seen also in previous studies, could represent a further basis for future applications in clinical trials.

the European seabass (Dicentrarchus labrax, Linnaeus) and the rainbow trout (Onchorinchus mykiss). In the last years, there is an increasing interest for new aquaculture species, through diversifying the aquaculture industry. The blackspot seabream is one of these possible alternatives to the commonly used fish for the high quality of its flesh and the high nutritive and commercial importance. The breeding of this species is already widely accepted in Spain, especially in the Azores. The blackspot seabream is also used to produce fish meal and oil. These are alternative and equivalent protein and lipid sources easily available, cheaper and with less environmental impact comparable to those of vegetable origin (Iaconisi et al., 2017;Micale et al., 2011). Therefore, this study aims to evaluate the morphology of the oral cavity, especially of the tongue, in relation to function. It has been shown that the capacity and modality of feeding are strictly related to the morphofunctional adaptations of fish to the environment, thus explaining the several differences observed in the presence, morphology, abundance and distribution of taste buds, papillae with mechanic or sensitive properties and teeth of different shapes in several species of fish (Abbate et al., 2006(Abbate et al., , 2020b(Abbate et al., , 2020a(Abbate et al., , 2012b(Abbate et al., , 2012aGermanà et al., 2009;Amato et al., 2012;Dos Santos et al., 2015;Sadeghinezhad et al., 2015;Guerrera et al., 2015 ;Mahmoud et al., 2016;Kettratad et al., 2017;Levanti et al., 2017;Ik pegbu et al., 2019;Kasumyan, 2019). Also, a strict comparative correlations with numerous studies carried out in other vertebrates like birds and reptiles are significative (Abbate et al., 2020c(Abbate et al., , 2010(Abbate et al., , 2008Erdoğan and Alan, 2012;Erdoğan and Iwasaki, 2014;Herrel et al., 2014;Cizek et al., 2019;Bels et al., 2020). In addition, recent data demonstrate that the role of some hormones is strictly related to the anatomical aspects of the digestive system and particularly of the teleosts oral cavity (Montalbano et al., 2016(Montalbano et al., , 2018aMania et al., 2017;Audira et al., 2018;Carnovali et al., 2018). The relevance of this study is also demonstrated in previously investigated farmed fish (Abbate et al., 2020b(Abbate et al., , 2020a(Abbate et al., , 2012b(Abbate et al., , 2012a, showing that is strictly related to the correlations between the tongue morphology and the feeding habits. The adaptive changes in the morphology of fish oral cavity are in close relationship with the feeding function, with a significant influence on the processes of food intake and taste. This could help to identify new and better methods of farmed fish breeding, with regard to nutrition. It will help to improve the quality of the fish product. There are scant data on the anatomy of the oral cavity and tongue in Sparidae, and none to our knowledge exist for blackspot seabream. The aim of this investigation was to describe by scanning electron, light and confocal laser microscopy the characteristics of the tongue in blackspot seabream.

| MATERIAL S AND ME THODS
The heads of 20 specimens (12 male and 8 female) of fresh blackspot seabreams of commercial size, (between 800 and 900 g) obtained from the fish markets of Messina, were used. The commercial size is typical of adult fish of this species that came from the aquaculture companies as certified by the seller. All these fish were intended for human consumption. After decapitation, better exposure of the tongue, the temporo-mandibular joints were also disarticulated and cut. The tongues were dissected out and washed in 5% neutral Extran (Merck), which is a cleansing solution commonly used to remove the mucus.

| Scanning electron microscopy
The samples of 10 fresh specimens (6 male and 4 female) were fixed in 2.5% glutaraldehyde in Sorensen phosphate buffer 0.1 M. After several rinsing in the same phosphate buffer, they were dehydrated in a graded alcohols series (50°, 70°, 80°, 95° and 100°, 1 hr for each step), critical point dried in a Balzers CPD 030 and then sputter-

Sections were mounted on microscope slides and stained with
Masson's trichrome and with aniline blue 04-010802 (Bio-Optica).
Weigert's iron haematoxylin for nuclei staining and aniline blue for connective tissue stain were used. After rinsing in distilled water, the sections were submitted to the reagents, as mentioned in the product datasheet, washed in distilled water and rapidly dehydrated through ascending alcohols, clarified in xylene and mounted using

| Laser confocal microscopy
The samples of 5 fresh specimens (3 male and 2 female) were fixed in Bouin's fixative for 24 hr and processed for routine paraffin embedding. The blocks were cut in 10 µm thick sections, as carried out for light microscopy. Serial transverse, horizontal and sagittal sections were mounted on gelatin-coated microscope slides and processed for immunofluorescence. Following dehydration, rehydrated sections were washed with Tris-HCL (0.05 M, pH 7.5) containing 0.1% bovine serum albumin and 0.2% Triton X-100. The endogenous peroxidase activity and non-specific binding were blocked using 3% hydrogen peroxide and 50% foetal bovine serum. Thereafter, sections were incubated in a humid chamber overnight at 4°C with mouse anti-vimentin (Serotec) No: 201100, dil. 1:100) and FLEX Polyclonal Rabbit Anti-S100 Ready-to-Use (Dako Omnis) at dilution of 1:200.
Subsequently, the sections were rinsed in PBS buffer. Then, they were incubated for 90 min at room temperature, respectively, with Alexa fluor 488-donkey anti-mouse IgG (H+L) (Invitrogen, A 21202) at dilution of 1:300 and Alexa fluor 594 Goat anti-rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody Catalog # A 11012 (Thermo Fisher).
For negative controls, representative sections were incubated with non-immune rabbit sera instead of the primary antibodies, or omitting the primary antibodies, following the same procedure previously described (Viña et al., 2015). Under these conditions, no positive immunostaining was observed (data not shown).
The study was carried out on fresh fish obtained from common markets and intended for human consumption; therefore, no approval was necessary from local and/or national institutional animal use committee. However, all experimental processes followed the EU Directive 2010/63/EU in the use of animals for experimental purposes.

| Gross results
The snout was distinct with a shorter diameter than that of the eye

| Scanning electron microscopy
An external row of pointed conical teeth was present in the oral cavity floor, with a slightly curved apex, an oro-aboral orientation and an innermost series of slightly smaller cardiform teeth. Cardiform teeth are pointed, thin and short teeth, often numerous, present in many fishes that have multiple rowed teeth as American catfish (Ictaluridae), perches (Percidae) and many sea basses (Serranidae).
They are followed, posteriorly, by two-three rows of molariform teeth (Figure 3a

| Light and confocal laser microscopy
The dorsal surface of the tongue was covered by a weakly keratinized stratified squamous epithelium. In the apex, body and root, profiles of fungiform-like papillae projected among the epithelial laminae (Figure 5a). In the tongue, the weakly keratinized epithelium was followed by layers of dense fibrillar connective tissue that evolved into loose connective tissue with interspersed amorphous substance. This layer was formed by unilocular adipose tissue formed by groups of polyhedric fat cells (Figure 5b). At the apex, below the adipose tissue, the connective tissue deepens to delineate lodges within an amorphous extracellular matrix, containing vimentin positive star-shaped cells immersed in abundant extracellular matrix, thus forming a mesenchymal tissue (Figures 5c, 6a, b). In the body and root, there were folds of mucosa with abundant fungiform-like papillae ( Figure 7a) and presence of taste pores (Figure 7b). Within the papillae, S100 positive nervous fibres were observed (Figure 7c).
In the deeper layers of the body, the dense connective tissue There were no remarkable differences between the male and female specimens used in this study.

| DISCUSS ION
The aim of our study was to analyse the morphological characteristic of the tongue to demonstrate the possible correlations to the mechanisms of prehension and ingestion of food, by scanning electron, light and confocal laser microscopy. So far, the numerous data available regarding the morphology of the oral cavity of fishes (for a review see Kapoor & Khanna, 1994) and the close interrelationships between the oral cavity and feeding habits, and different modes of deglutition have been clearly demonstrated. In addition, the taste system is affected by environmental changes caused by pollution and contaminant (Kasumyan, 2019). The blackspot seabream was chosen in this study because it may become a more widely bred species soon. Prehension and swallowing, in this species, is achieved by the presence of cardiform teeth whose orientation gives important support to ingestion, and their activity is strongly supported by rows of molariform teeth. Several fungiformlike papillae, through the tongue dorsal surface, whereas no taste buds, were observed. The absence of taste buds has been previously demonstrated in the swordfish and Atlantic salmon tongue, but unlike these two species, in the blackspot seabream the presence of several taste pores was observed, with, under the papillae, S100 positive nervous fibres, thus demonstrating, a taste capacity that should be considered, in aquaculture, in the possible food choices (Benetti et al., 2009;Igbokwe et al., 2021). The presence of folds, several papillae, teeth with different appearance and taste pores could also be compared with previous data obtained, using the same techniques, in gilthead seabass (Dicentrarchus labrax) and seabream  (Musaelyan et al., 2018). The mesenchymal/ stem stromal cells can be found in adult mesenchymal tissues other than bone marrow (Bernardo et al., 2009;Campagnoli et al., 2001) and mesenchyme, as a matrix with morphogenetic properties for

| CON CLUS ION
Considering that there are limited data so far available on the morphological characteristics of the tongue in the blackspot seabream, the current results could contribute to the anatomical knowledge of the digestive system in fish, giving a support for the future wider use of this fish as a farmed species in aquaculture. The morphological data regarding the tongue structure could be utilized as basis for further physiological and nutritional studies aimed at optimization of the blackspot seabream farming, using well-targeted nutritional protocols. It is known that a better nutrition reflects in better fish welfare and therefore optimum production for human nutrition.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.