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Introduction

Paiche (Arapaima gigas Schinz, 1822) is the largest scale fish of the Amazonian rivers, being found mainly in locations with low or no currents, such as marginal lakes (Dória, Catâneo, Torrente-Vilara, & Vitule, 2020). A. gigas is considered the most important and emblematic species of Amazonian ichthyofauna, not only for its large size, but also for the historical and economic context of artisanal fishing (Farias et al., 2015). Among the fish of the Order Osteoglossiformes, paiche is the best known and, such as others of this order, it is characterized by the presence of parasphenoid bone (Nelson, 1994).

However, due to the high exploitation of natural resources and lack of inspection, paiche is in a worrying situation, mainly due to the reduction of natural resources and near disappearance of its habitat (Nunes, Franco, Mársico, & Neves, 2012). On the other hand, it presents itself as an important fishing economic activity, as it is a neotropical fish, of large size, high rusticity, easy adaptation to high rates of density and high temperatures, as well as good feed conversion and excellent weight gain in low water quality environments (Azevedo, Morey, & Malta, 2017). However, knowledge of the fishing biology of these species is necessary, especially, to understand eating peculiarities, aiming to offer rational measures of exploitation (Dantas Filho et al., 2021). The architecture and the structure of the gastrointestinal tract have been described in several fish species, mainly due to the interest of wide variations in both morphology and functions related to nutritional dynamics, physiological changes in aquaculture target species and digestive pathologies (Hernández, Pérez, & Domitrovic, 2009; Lokka, Austbø, Falk, Bjerkås, & Koppang, 2013; Faccioli, Chedid, Amaral, Franceschini, & Vicentini, 2014).

According to Rodrigues and Cargnin-Ferreira (2017), the morphological aspects of the digestive tract of the A. gigas, in the juvenile phase is similar to other carnivorous teleosts, allowing the species to ingest, store and digest large foods. In particular, such studies are relevant to understand morphophysiological particularities related to seizure, digestion, and absorption of food in target aquaculture species, and thus reduce adaptive flexibility in response to changes in diet, due to the decrease in the variation in nutrient composition in natural environment, administered in captivity production (Buddington, Krogdahl, & Bakke-Mckellep, 1997).

Based on the aforementioned, the propose of this investigation is to describe the morphological and structural aspects of the esophagus and stomach of juvenile pirarucu (Arapaima gigas), raised in commercial creations in excavated tanks.

Material and methods

The research was presented and approved by the Research Ethics Committee on the use of animals of the Universidade Federal de Rondônia, with protocol No. 043/2019.

Thus, esophagus and stomach were collected from six specimens of Paiche (Arapaima gigas), in the juvenile phase, raised in an intensive system of tanks excavated and fed with a pelletized commercial diet, from an aquaculture property in the municipality of Pimenta Bueno, Rondônia, Brazil. The fish were caught for the consumption of the property, by artisanal fishing, and after removal of the viscera, the esophagus and stomach were washed in running water, removing the food content present inside and fixed in 10% buffered formaldehyde. The sample was processed at Facilite Advanced Center for Diagnostic Imaging, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo (CADI- FMVZ-USP).

Organ dissection and mucosal exposure were performed on a plank to macroscopically observe the different structures. Then these organs were later documented with a photographic camera. For microscopic analysis, tissue samples were dehydrated in a series of increasing concentrations of ethanol (70 to 100%) and diaphanized in xylol, with subsequent inclusion in histological liquid paraffin and then microtomized at 5μm thick and subsequently stained with Hematoxylin-Eosin (HE). The images were obtained with the Nikon Eclipse E-800 light microscope. The complete description of the methodology mentioned above can be found in the research developed by Santos et al. (2016).

Part of the material was processed for scanning electron microscopy (SEM). Samples fixed in 10% formaldehyde were dehydrated in increasing sets of alcohols at concentrations of 50, 70, 90, and 100%, dried in LEICA critical point apparatus in CPD300 (FMVZ-USP), fixed with carbon glue in aluminum metal bases (stub) and metallized (sputting) with gold in the EMITECH K550 metallizer (FMVZ-USP), being analyzed and photographed under a LEO 435VP scanning electron microscope (FMVZ-USP). The complete description of the methodology mentioned above can be found in the research developed by Santos et al. (2016).

Results

Anatomically, the esophagus and stomach of the A. gigas are tubular organs composed of muscle fibers that connect the pharyngeal cavity to the proximal intestine (Figure 1A). The esophagus is presented in form of a muscular and short rectilinear tube (Figure 1A and C), but at the pharyngo-esophageal junction (cranial portion), a marked dilution of the fan-shaped organ (a) is observed, and in the caudal portion a visible reduction of the lumen (b). On the other hand, the stomach is a J-shaped muscle sac (Figure 1A), located on the left side of the cavity, extending from the esophagus to the proximal intestinal portion. The esophageal mucosa is characterized by numerous longitudinal folds, most evident in the medial and caudal region (Figure 1C).

As the esophageal mucosa is replaced by gastric, such folds become thicker and more visible. However, after stomach fixation in 10% formaldehyde it was possible to differentiate it into three distinct portions, mainly by the color and shape of the distribution of the gastric folds, in: cranial or cardiac portion, with a lighter aspect and the central sulcus and the mucosa folds parallel to the central axis of the organ (c). In the medial or fundus portion, gastric folds are more evident, with the presence of transverse sulcus (d) and in the caudal or pyloric portion, gastric folds are higher with deep sulcus (e). During the opening of the paiche’s stomach (Figure 1B) the presence of small gastroliths was observed in the fundus and pyloric portions (d, e).

In the microscopic study of light, it was observed that the esophagus is lined by a stratified epithelium (Figure 2A) (line) with three different cell types: the simple epithelial cells, the mucous cells and the claviform cells. The number of layers varied from 1 to 12 layers of cells. The mucous cells presented higher numbers and they were distributed among the layers of the stratified epithelium. By histochemical methods, with hematoxylin and eosin (H/E) two cell types could be differentiated: acidophil and basophil (Figure 2A). Morphologically, the acidophilic cells presented cubiform shape, enucleated, plasma membrane well demarcated and acidophylic cytoplasm of granulous aspect. The basophilic cells are larger, presenting round-shaped and basophilic cytoplasm. The epithelial cells present varied morphology from columnar to cuboidal (Figure 2B) and they are observed in greater number in places where the epithelium is composed of up to two layers of cells. Goblet cells (Figure 2A) are observed in smaller numbers, nucleated, presenting a light halo around the nucleus, being usually located in the medial region of the epithelium. The lamina propria of the mucosa and submucosa (Figure 2B) are composed of dense, not presenting gland, well-developed connective tissue and with no separation between them. Bundles of smooth muscles infiltrating the connective tissue of the mucous tunica were also observed, being more evident in the cranial portion of the esophagus. The muscle wrap is formed by bundles of muscles in a internal circular More specifically, the muscle envelope is formed by bundles of smooth muscle arranged in two layers arranged in a circular and longitudinal manner. By scanning electron microscopy (SEM) it was observed that esophageal mucosa is composed of numerous folds and sulcus in different directions (Figure 2D and E), and it is lined by epithelial cuboid-shaped cells, with the apical portion of some cells discontinued, suggesting the release of cytoplasmic content to lumen in the form of cellular explosion, and large amount of mucus on the surface and inside the interdigitations of the mucosa. However, in the cranial portion of the esophagus, mucosal folds are not observed, and the lining epithelium is formed by cells rounded to polyhedral, with a large amount of mucus. Furthermore, numerous projections in taste bud-shapes (Figure 2C) are observed on the epithelial surface, randomly distributed by the epithelium line. Such projections are intra-epithelial structures formed by grouping of support cells of the pavement type and in the center, numerous sensory cells with microvilli immersed in the central pore (white arrowhead). However, the surface is formed by epithelial cells with digitiform micro-saliences in the caudal portion of the esophagus.

The stomach mucosa of the A. gigas is covered with the simple columnar epithelium with the presence of numerous crypts and gastric folds, of morphology, height and in different numbers between the portions. In the cardiac portion (Figure 3A), the crypts have the end of a rounded-to-round aspect, with the presence of smaller secondary crypts among them. The lamina propria of the mucosa is formed by loose connective tissue with glands, which flow into the region of the base of the crypts.

In the fundus portion (Figure 3B), the epithelial cells of wrapping are columnar, with slightly eosinophilic cytoplasm and the crypts are elevated and slender with the pointed end, besides being more frequent compared to the cardiac portion. In the mucosal lamia propria, a greater number of glands in the connective tissue is observed, which are larger than the cardiac portion.

In the pyloric portion the epithelial cells present a columnar to balloon form, with areas of epithelium hyperplasia, with up to three layers of cells. Such cells are also observed in glands located in the mucosal lamina propria. The gastric crypts of this region are thicker and shorter.

There is no separation between the mucous wrap and mucosal lamina propria, however the connective tissue of the mucous wrap is formed of thicker and denser collagen than the mucosal lamina propria. The muscular wrap is formed of bundles of smooth muscles arranged in two layers, horizontally and vertically. Or rather, the muscle wrap is formed by bundles of smooth muscles arranged in two layers arranged in a circular and longitudinal way.

The ultramicroscopic scanning study of the surface of the paiche’s stomach mucosa presented it is formed of numerous folds and interdigitations (D). These are overlaid by cuboid epithelial cells (E) with micro-orifices (arrowhead) on the surface of the plasma membrane of these cells.

At the ends of the mucosa folds, epithelial cells present dilation of the intercellular spaces, which allows the visualization of desmosome (arrows). Inside the interdigitations, cuboid epithelial cells are replaced by epithelial cells with the plasma membrane (Figure 3D) with digitiform micro-salience. These cells reduce in quantity in the fundus portion and they were not found in the pyloric part. Mucus was observed in the three regions of the stomach; however the pyloric and cardiac portion were the sites with the highest amount, also presenting mucus and more flattened villi (Figure 3F).

Discussion

The anatomical arrangement of the digestive system of the paiche (A. gigas) is similar to other teleosts. However, the stomach has varied shapes and regions; structurally adapted to food types. Anatomically, the stomach of the A. gigas is elongated in J-shape, similar to the species Rhamdia quelen (Hernández et al., 2009), with a chemical region, and another mechanical, highly muscularized, with crushing function of the ingested food, also observed in piscivore stomachs (Lopes et al., 2017).

The esophagus of the A. gigas is a muscular organ, with several folds of mucosa and a fan-shaped dilation in the cranial portion with marked reduction of lumem in the medial portion, thus enabling distension during ingestion of large foods. Generally, this anatomical aspect has been observed in other carnivorous fish such as Hoplias malabaricus (Menin & Mimura, 1993), Salminus brasiliensis (Rodrigues & Menin, 2008), and H. lacerdae (Hani et al., 2018), as well as in other teleosts with different eating habits (Fernandes, Cruz, Costa, & Perry, 2012; Schuningues, Lima, Lima, Martins, & Costa, 2013; Hani et al., 2018). According to Rodrigues and Cargnin-Ferreira (1997) and Scadeng et al. (2020) the several amount of folds of mucosa supports the integrity of the esophageal wall, especially during the sudden distension motivated by quick ingestion.

By the scanning microscopy and light microscopy, a variation in the morphology of epithelial cells of esophageal wrapping was observed, thus, in the cranial portion, the mucosa was covered by rounded to polyhedral cells with the presence of taste corpuscle and in the other portions, secretory epithelial cells. The gustatory system of teleosts is activated by water-soluble substances, and these are involved in detection, selection, and ingestion (Scadeng et al., 2020).

According to Santos, Santos, and Araújo (2011) there is a close relationship between the distribution pattern of taste corpuscles and the way fish locate and select foods, in carnivores usually present in the buccopharyngeal cavity and little observed in the esophagus. The presence of such corpuscles in the cranial portion of the paiche esophagus, allow them to eject the already ingested food and even the eversion of the stomach when they feed on something strange or not palatable, as well as observed in salmon (Gioda et al., 2017), and other teleosts (Santos et al., 2011).

In addition to the taste corpuscles, three different cell types were also observed in the stratified epithelium of the esophagus, mainly in the medial and caudal portions, the basophilic, acidophilic and claviform mucous cells. The epithelium of the teleosts is quite variable according to species, which may vary from stratified squamous to stratified columnar with three cell types: secretory cells of neutral, acidic and mixed mucopolysaccharides (Santo, Arantes, Pessali, & Santos, 2015), such cells were also observed in the paiche’s esophagus of this study. According to Rodrigues and Cargnin-Ferreira (1997) the goblet cells produce mucus that protect the esophageal wall against chemical and mechanical action during the passage of food. Nevertheless, acid mucus produced by acidophilic cells, is associated with binders capable of aggregating food particles, besides efficiently protecting the epithelium (Sundt & Björkblom, 2011; Naldoni et al., 2009).

Bertin (1958) describes on stomach of the teleosts different anatomical conformations and it can be grouped into three types: siphonal, cecum, and linear. The siphonal stomach has two branches, one descending, corresponding to the cardiac and the pyloric branch that ascends. The cecum stomach on the other hand, has two branches that interpose in the union region, the blind and rectilinear fundus and a muscular tube. However, it was possible to anatomically and histologically differentiate the stomach of the A. gigas in three distinct portions: the cardiac, fundus, and pyloric, all glandular and presenting different histological characteristics, mainly at the ends of the gastric crypts, as well as the shape and number of glands of the mucosal lamina propria. Such morphological conformations corroborate the classification suggested by Cardoso et al. (2015), that divide the stomach according to the anatomical structures and the presence or not of gastric glands in three parts, as described in the paiche.

The study of the digestive tract of several fish species, particularly teleosts, has attracted the attention of many researchers, mainly due to the wide structural variation with diversity of eating habits and behaviors, which are not found in other vertebrates. Based on anatomical and histological characteristics of the digestive tract, we can comprehend the feeding of fish (Machado et al., 2013). The observation of gastrolithic pebbles inside the paiches’s stomach of this study may be associated with an adaptation to the type of pelletized food. For Magalhães et al. (2017) the presence of pebbles in the stomach is related to the maceration process of pelletized foods, aided by stomach peristaltic waves. However, considering the low number of specimens in these studies, further research with native and captive fish are necessary to verify whether the presence of these pebbles is a food adaptation.

On the ultramicroscopic scanning study of the surface of the paiche stomach mucosa, it was observed that it is formed by folds and interdigitations, also it is overlaid with different cell types such as cuboid epithelial cells, which secrete mucus, and epithelial cells with digitiform micro-salience. Cells with digitiform micro-salience were also observed in the caudal portion of the paiche’s esophagus. Morphological studies based on Colossoma macropomum also observed the presence of such cells, overlaying the esophagus (Cavali et al., 2020). According to Carrassón, Grau, Dopazo, and Crespo (2006), these cells protect the epithelial surface and anchor the mucus secreted by goblet cells, besides being associated with fluids and ions absorption.

Conclusion

Based on anatomical and morphological aspects observed in the esophagus of this study, the anatomical fan-shaped of the cranial portion, as well as the presence of taste corpuscles, enables the storage of food before passing to the stomach and the neurostimulation of flavors by taste corpuscles stimulate the bundles of muscle present in the submucosa wrap, carrying out the regurgitation or deglutition. Then morphology found was consistent with the eating habits and management of distinct characteristics of the digestive tract. However, these studies were carried out in juvenile fish, raised in aquaculture systems, fed with pelletized feed, which may not be observed in A. gigas in natural environment.

Acknowledgements

To CAPES, through PROCAD/Amazônia, granted a postdoctoral scholarship to Jerônimo Vieira Dantas Filho. We also thank the Advanced Center for Diagnostic Imaging, Faculty of Veterinary Medicine and Animal Science, Universidade de São Paulo (CADI-FMVZ-USP).

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