Received: 17 March 2021 Accepted: 29 July 2021

DOI: 10.1111/jfb.14868

REGULAR PAPER FISH

Morphological and histochemical features of the digestive tract

of Leiarius marmoratus (Gill, 1870)

Valquiria Aparecida Matheus1 | Claudemir Kuhn Faccioli2 |

3 4
Renata Alari Chedid | José Augusto Senhorini |
Irene Bastos Franceschini-Vicentini3,5 | Carlos Alberto Vicentini3,5

1
Department of Biochemistry and Biology
Tissue, Institute of Biology, University of Abstract
Campinas (UNICAMP), Campinas, Brazil Leiarius marmoratus, a freshwater catfish from Pimelodidae family, shows great bio-
2
Department of Human Anatomy, Institute of
logical and commercial relevance because of its geographic distribution and adapta-
Biomedical Sciences, Federal University of
Uberlândia (UFU), Uberlândia, Brazil tion to fish-farm. The knowledge of the morphological characteristics of the digestive
3
Faculty of Sciences, Department of Biological tract is fundamental to the understanding of fish physiology and nutrition, which
Sciences, S~ao Paulo State University (UNESP),
Bauru, Brazil helps in the planning of diets to provide better management and success in fish farm-
4
National Center for Research and ing. Thus, this work described the morphology and histochemistry of the digestive
Conservation of Continental Aquatic
tract of L. marmoratus adults. After euthanasia, the animals were dissected for analy-
Biodiversity ICMBio/CEPTA, Pirassununga,
Brazil sis of the digestive tract. The oesophagus is a short and distensive organ with longitu-
5
Aquaculture Center of UNESP, S~ao Paulo dinal folds that allow the passage of large food, e.g., other fishes. Oesophageal
State University (UNESP), Jaboticabal, Brazil
mucosa layer shows a stratified epithelium with goblet cells and club cells. The secre-
Correspondence tion of goblet cells is composed of neutral and acidic mucins that are anchored in the
Valquiria Aparecida Matheus, Department of
Biochemistry and Biology Tissue, Institute of
epithelium luminal face by epithelial cells fingerprint-like microridges, lubricating the
Biology, University of Campinas (UNICAMP), surface to facilitate the food sliding. Club cells have protein secretion that can be
Cidade Universitária Zeferino Vaz – Bar~ao
Geraldo, Campinas – SP, 13083-970, Brazil.
involved in alarm signals when epithelium is damaged and in immunological defence.
Email: valmath@unicamp.br The saccular stomach is highly distensible to store large food. Gastric mucosa layer is

Funding information
Fundaç~ao de Amparo à Pesquisa do Estado de
S~ao Paulo, Grant/Award Number: 2011/
23055-0
composed of epithelial cells with intense secretion of neutral mucin to protect
against self-digestion of gastric juice. Cardiac and fundic regions of stomach show
well-developed gastric glands composed of oxynticopeptic cells. These cells have
numerous mitochondria, highlighting their intense activity in the synthesis of acid
and enzymes. The intestine is divided into three regions: anterior, middle and poste-
rior. Although it is a short tube, intestine shows longitudinal folds and microvilli of
enterocytes to increase the contact surface. These folds are higher in the anterior
region of the intestine, highlighting their function in digestion and absorption. Intesti-
nal goblet cells have acidic and neutral mucins that lubricate the epithelium and aid in
digestive processes. These cells increase in number towards aboral, and they are
related to the protection and lubrication to expulsion of faecal bolus.

KEYWORDS
anatomy, histochemistry, histology, mucin, mucous cells, ultrastructure

J Fish Biol. 2021;1–10. wileyonlinelibrary.com/journal/jfb © 2021 Fisheries Society of the British Isles. 1
2
FISH
1 | I N T RO DU CT I O N
The digestive tract is anatomically formed by a long hollow tube with
associated glands. Functionally, this tube has distinct regions special-
ized in the processes of digestion, absorption and transport of ions
(Ray & Ringø, 2014). Histologically, the digestive tract has four distinct
layers: mucosa, submucosa, muscularis and serosa (Pawlina &
Ross, 2021). The mucosa layer is in constant contact with the luminal
content. For this reason, in addition to participating in the processes
of digestion, absorption and ionic regulation, the mucosa layer plays
an important role as a selective barrier defining the luminal content of
the individual's internal environment (Cao & Wang, 2009;
Shephard, 1984; Sundh et al., 2010).
The mucosa layer also acts as an immune barrier (Grove et al., 2006;
McGuckin et al., 2011; Ringø et al., 2003). Specialized mucosal cells syn-
thesize and secrete mucins related to the individual's defence against
pathogens. Mucins also perform other essential functions for homeosta-
sis such as lubrication, protection against digestive enzymes and osmo-
regulation (Cao & Wang, 2009; Leknes, 2015; Petrinec et al., 2005). In
this context the morphological analysis of the digestive tract is relevant
to the understanding of fish nutrition, in the identification of pathological
or physiological changes, contributing to better fish management and
 n et al., 2006; Wilson & Castro, 2010).
conservation (Carrasso
Leiarius marmoratus (Gill, 1870) is a freshwater catfish from
Pimelodidae family distributed in the basins of the Amazon, Essequibo
and Orinoco rivers, which shows omnivorous habit but with adaptations
for each basin (Ramírez-Gil & Ajíaco-Martínez, 2011). This is reflected in
the ease of handling it in captivity, and the species ability to assimilate
pelleted food (Cruz-Casallas et al., 2010). According to Sánchez et
al. (2009), L. marmoratus has great gastronomic value because of the
quality of its meat (light colour, mild flavour and the presence of few
intramuscular bones), in addition to great commercial relevance, as it is
a species widely used to obtain hybrids (Bernardes et al., 2016; Fortes-
Silva et al., 2017; Oliveira-Almeida et al., 2015; Paulino et al., 2020). In
the search for a better understanding of the digestive and nutritional
characteristics of L. marmoratus, this work aims to describe the morpho-
logical and histochemical characteristics of the different segments of
the digestive tract of L. marmoratus, because it is the mucosa layer that
is in constant contact with the luminal contents.
2 | MATERIALS AND METHODS
2.1 | Fish samples
Ten adult male specimens of L. marmoratus were obtained from the
National Center for Research and Conservation of Continental
Aquatic Biodiversity ICMBio/CEPTA, Pirassununga – SP. The fish
were raised in earthen pond. Two days before euthanasia, they were
acclimated in plastic tanks and deprived of food deprivation. The ani-
1
mals were euthanized with a saturated benzocaine solution (10 g L ).
Then, a longitudinal incision was made in the ventral region of the ani-
mals for analysis and removal of the digestive tract organs
MATHEUS ET AL.
(oesophagus, stomach and intestine). Fragments of oesophagus, stom-
ach (cardiac, fundic and pyloric regions) and intestine (anterior, middle
and posterior) were fixed in appropriate solutions for each study as
described below: gross anatomy, histology, histochemistry and ultra-
structure. This study is in agreement with the ethics principles in ani-
mal experimentation, adopted by the Animal Experimentation Ethics
Committee of the National Center for Research and Conservation of
Continental Aquatic Biodiversity ICMBio/CEPTA, Pirassununga – SP,
Brazil.
2.2 | Gross anatomy
The digestive tract was dissected and photographed. Then, fragments
of oesophagus, stomach and intestine were removed and fixed in 10%
formalin to describe the macroscopic features. The photo-
documentation was performed with a Leica IC80HD camera attached
to a Leica M50 stereo microscope.
2.3 | Histology
Tissue fragments of digestive tract were fixed in Karnovsky's solution
(2.5% glutaraldehyde, pH 7.2, 0.1 M phosphate buffer and 8% para-
formoldehyde). After 24 h, the samples were washed in 70% ethanol,
dehydrated in graded ethanol series and embedded in historesin (His-
toresin embedding kit, Leica). Histological sections of 3 μm thick were
obtained using an automated microtome (Leica RM 2265, Germany).
Subsequently, the sections were stained with 1% toluidine blue,
haematoxylin and eosin, and Masson's trichrome (Suvarna et
al., 2018). The images were taken using the Olympus BX40f4 photo-
microscope attached with an Olympus DP12 camera.
2.4 | Histochemistry
For histochemical analysis, the samples were fixed in Bouin's solution.
After fixation, fragments were washed in 70% ethanol, dehydrated in
graded ethanol series and embedded in Paraplast Plus (McCormick Scien-
tific; 39503002, Leica, Germany). Histological sections 7 μm thick were
cut using the American Optical microtome. The sections were prepared
for the detection of mucin by submerging them in histochemical reagents.
The periodic acid-Schiff (PAS) technique was performed for the detection
of neutral mucins and the Alcian blue (AB) technique pH 1.0 and 2.5 for
acidic mucins (Cao & Wang, 2009; Suvarna et al., 2018; Vidal et al., 2020).
The photomicrographs were obtained using an Olympus BX40f4 micro-
scope attached with an Olympus DP12 camera.
2.5 | Ultrastructure
Fragments of the digestive tract were fixed in Karnovsky's solution, at
4 C for 24 h. Then, the samples were post-fixed for 2 h in 1% osmium
MATHEUS ET AL.
tetroxide (pH 7.4), dehydrated in a graded acetone series and embed-
ded in Araldite resin. Resin polymerization was completed in an oven

at 60 C for 48 h. Ultra-thin sections (60 and 80 nm) were mounted on
copper grids and contrasted with uranyl acetate and lead citrate. The
analysis and photodocumentation were performed using a Philips
CM100 transmission electron microscope (TEM).
3 | RESULTS
The digestive tract of L. marmoratus extends ventrally along the body
cavity (Figure 1a,b). The oesophagus is a short tubular organ with lon-
gitudinal high folds (Figure 1c). The stomach has a saccular shape, is
richly vascularized and is divided macroscopically into cardiac, fundic
and pyloric regions (Figure 1c). The cardiac and fundic regions con-
tained thick longitudinal folds. The pyloric region has a thicker wall
and a developed musculature forming a pyloric sphincter. The intes-
tine shows three regions: anterior, middle and posterior. Along its
length, loops or curvatures are observed, mainly in the middle region
(Figure 1a,b). Intestinal folds are longitudinal. In anterior intestine, the
folds are numerous, thin and tall, becoming less numerous and thicker
in posterior direction (Figure 1d-h).
The oesophageal mucosa layer of L. marmoratus consists of a
stratified epithelium, composed of three distinct cell types: epithelial
cells, goblet cells and club cells (Figure 2a). Epithelial cells occur in all
strata of epithelium showing a columnar shape in the basal portion
and became squamous in the apical portion. The plasma membrane of
all cells of oesophageal epithelium showed many interdigitations and
desmosomes (Figure 2b). In squamous epithelial cells, plasma mem-
brane presents fingerprint-like microridges on the luminal face (Figure
2b). These squamous cells contain heterochromatic nucleus, and the
cytoplasm contains many small mitochondrial organelles and electron-
lucent vacuoles (Figure 2b).
Goblet cells are located in the apical region of the oesophageal
epithelium, distributed among the squamous epithelial cells, which
open into the oesophageal lumen (Figure 2a,c-g). Basal nucleus pre-
sents a predominance of euchromatin (Figure 2d). Around the nucleus,
well-developed rough endoplasmic reticulum and golgi complex were
observed, but the cytoplasm is occupied almost entirely by secretion
granules (Figure 2c,d). Secretory granules show different electron
TABLE 1 Histochemical reactions of mucin in the mucosa layer of the digestive tract of Leiarius marmoratus
Staining Stomach Stomach
technique used Oesophagus Oesophagus cardic fundic
PAS +++ +++ +++
AB pH 1.0 + +
AB pH 2.5 +++
AB pH 2.5 + PAS +++ +++ +++
Cell types “club” cell Goblet cell Columnar Columnar
cell cell
Note. Staining intensity: ( ) negative, (+) weak; (++) moderate; (+++), strong.
AB: Alcian blue; PAS: periodic acid-Schiff.
FISH 3
density in TEM and PAS-positive and AB pH 2.5-positive in histo-
chemical technique (Table 1) (Figure 2e-g).
Club cells are located in the basal region of the oesophageal epi-
thelium (Figure 2a,c). These are the largest cells of epithelium, show-
ing a homogeneous cytoplasm and a central euchromatic nucleus
(Figure 2c). Few organelles, the mitochondria mainly, were observed
near the nucleus. As observed in epithelial cells, club cells membrane
shows many interdigitations. These cells show no opening to the
oesophageal lumen and no reaction to histochemical technique (Fig-
ure 2e-g). Oesophageal mucosa layer also shows connective tissue of
lamina propria that is continuous with the submucosa because of the
absence of muscularis mucosae (Figure 2a). Muscularis layer consists
of striated muscle and is divided into inner longitudinal and outer cir-
cular layers. A serosa layer coats the oesophagus externally.
The gastric mucosa layer is lined by simple columnar epithelium
(Figure 3a-c). The columnar cells have a centro-basal nucleus that shows
euchromatin predominance and evident nucleoli (Figure 3c,d). In the
cytoplasm around the nucleus, well-developed rough endoplasmic retic-
ulum was observed (Figure 3d). In the apical portion, columnar cells
have elongated electron-dense granules (Figure 3c,f). In histochemical
testing, these granules show a strongly positive reaction (Figure 3e).
In the cardiac and fundic regions, the mucosa layer contains gastric
glands, which open into the gastric crypts (Figure 3b). These tubular
glands are composed of oxynticopeptic cells with a pyramid shape with
a rounded euchromatic nucleus and evident nucleolus (Figure 3g,h).
Several mitochondria of different shapes, well-developed rough endo-
plasmic reticulum and golgi complex are observed in the cytoplasm (Fig-
ure 3g,h). Oxynticopeptic cells have many spherical and electron-lucent
granules and a tubulovesicular system in apical region (Figure 3g,h).
Stomach shows a lamina propria of connective tissue and a
muscularis mucosae of longitudinal smooth fibres separating from con-
nective tissue of submucosa (Figure 3a). Muscularis layer consists of
smooth muscle divided into inner circular and outer longitudinal layers.
Inner circular layer is well developed in pyloric, forming the sphincter.
The serosa layer constitutes both connective tissue and mesothelium.
The intestinal mucosa layer is composed of simple columnar epi-
thelium that has two predominant cell types: absorptive cells or
enterocytes and goblet cells (Figure 4a,b). Enterocytes present numer-
ous microvilli on their apical surface (Figure 4d-f). The oval basal
nucleus shows predominance of euchromatin and evident nucleolus
Stomach Anterior Middle Posterior
piloric intestine intestine intestine
+++ +++ +++ ++
+ +
+++ +++ ++
+++ +++ +++ ++
Columnar Goblet cell Goblet cell Goblet cell
cell
4
FISH MATHEUS ET AL.

F I G U R E 1 Anatomical characteristics of the digestive tract of Leiarius marmoratus. (a) Fresh digestive tract; (b) digestive tube fixed in bouin;
(c) cut of the stomach showing the cardiac (ca), fundic (fd), pyloric (py) and muscle layers (arrow); cross section of the anterior intestine (d) and the
posterior intestine (e), showing the muscular layer (arrow with two heads) and intestinal folds (*). (f, g, h) Anterior, medium and posterior intestinal
folds (*), respectively. AI: anterior intestine; ES: oesophagus; MI: medium intestine; PI: posterior intestine; RT: direct; ST: stomach. Scale bars: (a),
(b) and (c) 1 cm; (d) and (e) 0.125 cm; (f) 2 mm; (g) 1 mm; (h) 500 μm
MATHEUS ET AL.
FISH 5

F I G U R E 2 Histological, histochemical and ultrastructural characteristics of the oesophagus of Leiarius marmoratus. (a) Photomicrograph of
stratified glandular epithelium composed of epithelial cells (ec), goblet cells (gc) and club cells (cc), and the underlying lamina propria and the
submucosa (lp/sm); (b) electron micrograph of epithelial cell with emphasis on desmosomes (arrowhead), interdigitations (i) and microcrystals
(arrows); (c) electron micrograph of the basal portion of the oesophageal epithelium with emphasis on club cells (cc) and goblet cells (gc); (d)
electron micrograph of the endoplasmic reticulum (rer) close to the nucleus of goblet cells; (e, f, g) photomicrograph of goblet cells (gc). Stains and
reactions: (a) haematoxylin–eosin, (e) periodic acid-Schiff (PAS), (f) Alcian blue (AB), (g) combined PAS + AB reaction. Scale bars: (a) 30 μm; (b)
5 μm;(c) 5 μm; (d) 500 nm; (e) 30 μm; (f) 20 μm; (g) 30 μm

(Figure 4d). Plasma membrane presents junctional complexes, formed
by the tight junctions, communicating junctions and desmosomes
observed by TEM (Figure 4e). Several elongated or rounded mito-
chondria are evident in the cytoplasm of enterocytes (Figure 4e).
Intestinal goblet cells increase in number towards aboral direction.
Goblet cells show a basal nucleus with predominance of euchromatin.
Cytoplasm is filled by numerous granules (Figure 4f). Granules show
content of homogeneous electron density in TEM and positive reac-
tion to PAS and AB in the histochemical technique (Figure 4g-i).
Intestinalmucosa layer also shows connective tissue of lamina propria
continuous with the submucosa (Figure 4a-c). Muscularis layer
consists of smooth muscle divided into innercircular and outer longi-
tudinal layers (Figure 4c). The serosa layer constitutes both connective
tissue and mesothelium (Figure 4c).
4 | DI SCU SSION
L. marmoratus is an omnivorous species (Cruz-Casallas et al., 2010),
but it shows a classical digestive tract as described for carnivorous
6
FISH MATHEUS ET AL.

F I G U R E 3 Histological, histochemical and ultrastructural characteristics of the stomach of Leiarius marmoratus. (a) Photomicrograph of the
stomach of L. marmoratus with emphasis on the muscularis mucosa (mm) between the lamina propria (lp) and the submucosa (sb); (b)
photomicrograph of the stomach showing the epithelium e composed of mucous cells and the tubular gastric glands (gg) immersed in the lamina
propria (lp); (c) electron micrograph of the apical portion of the stomach with an emphasis on granules of secretion (arrowhead); (d) electron
micrograph of the endoplasmic reticulum (rer) close to the nucleus (n) of the mucous epithelial cells; (e) photomicrograph of the lining epithelium
of the stomach with strongly positive periodic acid-Schiff (PAS) cells (arrowhead); (f) electromicrograph of mitochondria; (g, h) electron
micrography with emphasis on oxyntic cells of pyramid shape (ox) that have a nucleus with de-densified chromatin with mitochondria dispersed in
the cytoplasm (*) and spherical and electron-lucent granules (★). Stains and reactions: (a) Masson's trichrome; (b) haematoxylin–eosin; (e) PAS.
Scale bars: (a) 200 μm; (b) 60 μm; (c) 5 μm; (d) 500 nm; (e) 30 μm; (f) 500 nm; (g) 5 μm; (h) 1 μm

and piscivorous teleosts, consisting of: a short distensible oesophagus, contrary, this species does not have pyloric caeca, structures that
a saccular distensible and richly vascularized stomach, and a short have been associated with carnivorous fish (Layman et al., 2005),
intestine (Faccioli et al., 2014; Wilson & Castro, 2010). On the although they are related to phylogeny and not eating habits.
MATHEUS ET AL.
FISH 7

F I G U R E 4 Histological, histochemical and ultrastructural characteristics of the intestine of Leiarius marmoratus. (a, b) Photomicrograph of the
intestinal lining epithelium composed of columnar epithelial cells (enterocytes – e) with elongated nucleus (n) located in the basal region, goblet
cells (*) and the lamina pro pria (lp); (c) Longitudinal section of the intestine with emphasis on the muscular layer; (d, e, f) Electron micrograph of
enterocytes showing the brush border (arrows) formed by microvilli, mitochondria (m) and tight junctions (arrowhead). (g, h, i) Goblet cells (*)
dispersed throughout the intestinal epithelium (e). Stains and reactions: (a, b) Toluidine blue, (c) Masson's trichrome, (g) periodic acid-Schiff (PAS),
(h) Alcian blue (AB), (i) PAS + AB. Scale bars: (a) 60 μm; (b) 20 μm; (c) 200 μm; (d) 5 μm; (e) 2 μm; (f) 5 μm; (g) 20 μm; (h) 30 μm; (i) 30 μm
8
FISH
The short oesophagus shows longitudinal folds that allow great
distensibility to facilitate the passage of large food, like fish (Faccioli et
al., 2014; Mello et al., 2019; Rodrigues & Cargnin-Ferreira, 2017; Vidal
et al., 2020). Oesophageal epithelium of studied species is composed
of epithelial cells, goblet cells and club cells. All these cells have many
interdigitations and desmosomes, as observed in fish oesophagus that
eat large food and so are in constant distension and abrasion (Faccioli
et al., 2014).
The oesophageal epithelium of L. marmoratus shows large amount
of goblet cells with neutral and acidic mucins secretion for the organ
lubrication. Mucins are anchored in the luminal face of epithelium by
fingerprint-like microridges of squamous epithelial cells, forming a gel
on the oesophageal surface (Faccioli et al., 2016a). Functionally, neu-
tral mucins lubricate the luminal surface (Kalhoro et al., 2019), may
indicate pre-gastric digestion (Cao & Wang, 2009) and are related to
emulsification of food in chyme (Hopperdietzel et al., 2014; Humbert
et al., 1984; Leknes, 2015). Acidic mucins increase the viscosity of
secretions that are associated with protecting the tissue against path-
ogens and trapping water-soluble particles (Schauer, 2004;
Tibbetts, 1997). As observed in Hemisorubim platyrhynchos (Faccioli et
al., 2014), there is a predominance of carboxylated acidic mucins over
sulphated ones, in the oesophagus of L. marmoratus. Carboxylated
acidic mucins are involved in lubrication and sulphated acidic mucins
to protect against microorganisms and increase secretion viscosity
(Cao & Wang, 2009; Tibbetts, 1997).
L. marmoratus oesophageal epithelium also shows club cells. It is
possible that these cells are typical of the oesophagus of Neotropical
Siluriformes, because these cells have been described in the oesopha-
gus of Trachelyopterus striatulus (Santos et al., 2015), H. platyrhynchos
(Faccioli et al., 2014), Pimelodus maculatus (Santos et al., 2007),
Pseudoplatystoma coruscans (Cal, 2006) and Trichomycterus brasiliensis
(Ribeiro & Fanta, 2000). In turn, they are absent in the oesophagus of
Siluriformes from other regions of the world, such as Pelteobagrus
fulvidraco of Asia (Cao & Wang, 2009). Club cells are found in the skin
of other teleost species, as Cyprinus carpio (Iger et al., 1994), Liobagrus
mediadiposalis (Park et al., 2003) and Arius tenuispinis (Al-Banaw et
al., 2010). Club epidermal cells may be related to the release of sub-
stances involved in alarm signals when they are damaged by a preda-
tor attack (Reverter et al., 2018). Another possible function of these
cells may be immunological, because they increase in number when
exposed to pathogens (James et al., 2009; Nakamura et al., 2001).
According to Lauriano et al. (2020), club cells may be involved with
photoprotective function. Although some functions of epidermal club
cells have been described, the presence of these cells in the
oesophageal epithelium is still poorly understood. Nonetheless, club
cells may be associated with protection against epithelial damage cau-
sed by the intake of large food, as seen in carnivorous fish (Faccioli et
al., 2014). In this sense, L. marmoratus oesophagus also shows a thick
connective tissue constituting the lamina propria and submucosa, and
a thick muscularis composed of striated fibres, enabling great distensi-
bility and the apprehension of live prey, respectively.
The stomach of L. marmoratus is lined by a simple columnar epi-
thelium composed of epithelial cells with neutral mucins secretion,
MATHEUS ET AL.
similar to other species (Cao & Wang, 2009; Gosavi et al., 2019;
Suvarna et al., 2018; Vidal et al., 2020). Neutral mucins have an impor-
tant role in protecting the gastric epithelium against proteolytic degra-
dation of enzymes secreted by the gastric glands, as well as a
buffering effect on high stomach acidity (Domeneghini et al., 2005). In
addition to the protective function, the presence of neutral mucins is
involved in the absorption and transport of macromolecules across
the membrane (Pedini et al., 2005). The secretion granules of colum-
nar epithelial cells are strongly electron-dense in TEM, which may
indicate the presence of lipids that get electron-dense after fixation in
osmium tetraoxide. According to Kao and Lichtenberger (1987), sur-
face-active phospholipids are essential component of the protective
hydrophobic lining stomach, synthesized by the gastric epithelial cells.
Gastric mucosa layer of studied specie has numerous tubular
glands in the cardiac and fundic regions. These glands are composed
of oxynticopeptic cells, which secrete hydrochloric acid and pepsino-
gen (Ostos Garrido et al., 1993; Wilson & Castro, 2010). These cells
show electron-lucent euchromatic nucleus and large amount of mito-
chondria. According to Faccioli et al. (2018), oxynticopeptic cells are
highly active and require high energy demand. Moreover, the stomach
of L. marmoratus was the only organ of digestive tract with an evident
muscularis mucosae. This structure composed of longitudinal smooth
fibres can help the movement of the mucosa layer to promote the
secretion of gastric juice. The muscularis layer promotes bolus move-
ment for mixing with enzymes and the pyloric sphincter keeps food in
the stomach until it is digested (Pawlina & Ross, 2021).
The intestine of L. marmoratus is short, but it presents longitudinal
folds to increase the contact surface (Kalhoro et al., 2019; Wilson &
Castro, 2010). Anterior intestine has many tall folds, highlighting its
function as the main area of digestion and absorption of the intestine
(Løkka et al., 2014; Faccioli et al., 2016b). Along the intestine,
enterocytes have microvilli that react to PAS, indicating neutral
glycoconjugates that promote emulsification of food into chyme and
absorption of nutrients (Clarke & Witcomb, 1980; Stroband et
al., 1979).
The secretion of intestinal goblet cells of L. marmoratus has sev-
eral functions and histochemistry allowed its characterization into
acidic and neutral mucins. Acidic mucins protect the intestinal epithe-
n et al., 2006). Sulphated acidic
lium against degradation (Carrasso
mucins increase secretion viscosity (Cao & Wang, 2009), although the
mucus produced in the intestine is less viscous than the other regions
of the digestive tract, which maintains intestinal transport with
greater fluidity (Tibbetts, 1997). Such observations corroborate the
weak reaction of goblet cells of L. marmoratus with AB (pH 1.0). In
turn, neutral mucins combined with alkaline phosphatase contribute
to the digestion providing cofactors for enzymatic breakdown and
assisting in the absorption of macromolecules (Murray et al., 1996).
Cao and Wang (2009) relate the large amount of neutral mucins in the
anterior intestine of P. fulvidraco to a large activity of digestion and
absorption functions. L. marmoratus shows a strong reaction of neutral
mucins in the anterior and middle regions of intestine, confirming that
these regions are the main sites of digestion and absorption. On the
contrary, the increase in the number of goblet cells towards aboral
MATHEUS ET AL.
side observed in L. marmoratus is related to the protection and lubrica-
tion of the digestive tract for expulsion of faecal bolus (Murray et
al., 1996; Petrinec et al., 2005).
In conclusion, although some authors classify the species L. mar-
moratus as omnivorous, the results obtained in the current study indi-
cate that the species has a digestive tract with remarkable
morphological characteristics of carnivorous fish. These features include
oesophagus with tall longitudinal folds and well-developed musculature
to swallow large prey and many goblet cells and club cells for epithelial
lubrication and protection, a distensible saccular and glandular stomach
to stock and digest large food and short and convoluted intestine with
longitudinal folds to digest and absorb easily digestible food and many
goblet cells in posterior region for expulsion of faecal bolus.
ACKNOWLEDGEMEN TS
The authors thank the Laboratory of Morphology of Aquatic Organ-
isms, School of Sciences, Bauru, SP, the Center for Electronic Micros-
copy of the School of Biosciences, Botucatu, SP, and the National
Center for Research and Conservation of Continental Aquatic Biodi-
versity ICMBio/CEPTA, Pirassununga – SP for technical assistance
and obtaining samples. This work was funded by grants from FAPESP
(Grant# 2011/23055-0, Brazil).
AUTHOR CONTRIBUTIONS
V.A.M., C.K.F. and R.A.C. worked on the morphological procedures,
analysis of the data and writing of the manuscript. J.A.S. contributed
to the collection, macroscopic analysis and the biology of the animals.
I.B.F.V. and C.A.V. contributed to the analysis of the data and final
writing. In the end, all the authors have read and agreed with the final
version of this work.
CONF LICT S OF INTE R ES T
The authors declare that they have no conflicts of interest.
E TH I CS S T A TE M E N T
All procedures performed in studies involving animals were in accor-
dance with the ethical standards of the institution and conformed to
the animal welfare laws, guidelines and policies.
ORCID
Valquiria Aparecida Matheus https://orcid.org/0000-0001-8528-
8004
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Carlos Alberto Vicentini https://orcid.org/0000-0002-6816-033X
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How to cite this article: Matheus, V. A., Faccioli, C. K., Chedid,
R. A., Senhorini, J. A., Franceschini-Vicentini, I. B., & Vicentini,
C. A. (2021). Morphological and histochemical features of the
digestive tract of Leiarius marmoratus (Gill, 1870). Journal of
Fish Biology, 1–10. https://doi.org/10.1111/jfb.14868