307

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 70: 307-318, January-December 2022 (Published May 18. 30, 2022)

Anatomy and histology of the digestive tract

and immunolocalization of Npy in the fish Cichlasoma dimerus

(Cichliformes: Cichlidae)

Andrés Breccia1,2; https://orcid.org/0000-0003-4902-1024

Mónica Alejandra Palmieri1; https://orcid.org/0000-0002-3652-3589

María Paula Di Yorio1,2; https://orcid.org/0000-0003-3225-9327

Ariadna Gabriela Battista2; https://orcid.org/0000-0001-9374-9309

Paula Gabriela Vissio1,2; https://orcid.org/0000-0002-0240-9534

Daniela Irina Pérez Sirkin1,2*; https://orcid.org/0000-0002-5854-7995

1. Departamento de Biodiversidad y Biología Experimental, Facultad de Ciencias Exactas y Naturales, Universidad de

Buenos Aires, Ciudad Universitaria, Buenos Aires, Argentina; breccia94@gmail.com,

monica.ale.palmieri@gmail.com, mariapauladiyorio@gmail.com, pvissio@gmail.com, daniperezsirkin@gmail.com

(*Correspondence)

2. Instituto de Biodiversidad y Biología Experimental y Aplicada (IBBEA), Consejo Nacional de Investigaciones

Científicas y Técnicas- Universidad de Buenos Aires (CONICET-UBA), Buenos Aires, Argentina;

ariadnabattista@gmail.com

Received 01-XI-2021. Corrected 04-III-2022. Accepted 12-V-2022.

ABSTRACT

Introduction: The digestive tract of Neotropical cichlids has not been deeply studied, and it is a fundamental

topic for understanding fish physiology, nutrition, trophic associations, and evolution.

Objective: To describe anatomically and histologically the digestive tract of the Neotropical cichlid fish

Cichlasoma dimerus and to immunolocalize the orexigenic peptide (Npy) along the intestine.

Methods: We euthanized 14 adult individuals and fixed the organs in Bouin’s solution; we stained 7 μm thick

paraffin sections for general description and with Alcian Blue (pH = 2.5, AB) and Periodic acid-Schiff (PAS)

to identify acid or neutral glycoconjugates, respectively. Additionally, we performed immunohistochemistry for

Npy in 3 adult individuals. We manually counted PAS- and AB-positive cells, and Npy-immunoreactive cells

per fold.

Results: There is a short oesophagus, a sac-like stomach, and a tubular intestine with two loops. The oesophagus

has a stratified epithelium with a high density of PAS- and AB-positive goblet cells and striated muscle fibers

in the tunica muscularis. The stomach mucosa is formed by simple columnar epithelium. The intestine has a

simple columnar epithelium, with brush border and interspersed PAS- and AB-positive goblet cells, and Npy-

immunoreactive cells. There is an ileorectal valve in the transition between the posterior intestine and the rectum.

This last gut portion has goblet cells and a thicker tunica muscularis.

Conclusions: C. dimerus shares features with other Neotropical cichlids, but the goblet cells and gastric glands

distribution seems to be unique for the species. To our understanding, this is the first work to describe Npy-

immunoreactive cells distribution in the intestine of a Neotropical cichlid fish.

Key words: goblet cells; histomorphology; histochemistry; immunohistochemistry; neotropical cichlids.

https://doi.org/10.15517/rev.biol.trop..v70i1.48957

VERTEBRATE BIOLOGY

308 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 70: 307-318, January-December 2022 (Published May 18, 2022)

Cichlids are one of the higher diversity

families among bony fishes with more than

200 genera and 1 700 species recognized (Nel-

son et al., 2016). Their geographical distribu-

tion ranges from Africa, Middle East, and

India to Central and South America, these last

being referred to as New World or Neotropical

cichlids (Nelson et al., 2016). One factor that

contributed to the high diversification of this

family is the differential ability of each species

to consume different food types (Axelrod &

Burgess, 1979). Gross morphology examina-

tion of the digestive tract and feeding structures

in cichlid fish has allowed establishing com-

mon features and trends among species, with

respect to their diets, ontogeny, life histories,

and ecological and phylogenetic relationships

(López-Fernandez et al., 2012; Wagner et al.,

2009; Zihler, 1981).

Histological and/or histochemical char-

acterizations of the digestive tract improve

knowledge about certain topics of fish biology,

like trophic preferences and nutrition require-

ment (Rašković et al., 2011), digestive physiol-

ogy (Beveridge & Baird, 2000), parasitology

(de Oliveira et al., 2019), toxicology (Cengiz

& Unlu, 2006; Dane & Şişman, 2020), and phe-

notypic plasticity (Gaucher et al., 2012; Pervin

et al., 2020; Vidal et al., 2014). Considering the

number of cichlids species recognized, there

are few works about the histological features

of their digestive tract of adult animals. To our

knowledge, these are mainly focused on the

African cichlids species, particularly on genus

Oreochromis (Al-Hussaini, 1949; Caceci et al.,

1997; Gargiulo et al., 1997; Morrison & Wright

Jr, 1999; Osman & Caceci, 1991; Pasha, 1964;

Scocco et al., 1996; Scocco et al., 1997; Scocco

et al., 1998), and there are few works for Neo-

tropical cichilds, (Arman & Ucuncu, 2017; da

Silva et al., 2012; Hopperdietzel et al., 2014;

Ramírez Espitia et al., 2020).

Gastrointestinal endocrine cells are dis-

tributed in the mucosa of the digestive tract

(Banan Khojasteh, 2012). Their secretion con-

trol digestion, gut motility, enzyme secretion,

nutrient absorption, among others (Volkoff,

2016). An important hormone involved in

feeding behaviour is the neuropeptide Y (Npy).

This peptide was described in both the brain

and gastrointestinal tract of a vast number

of species, acting as a powerful orexigenic

neuropeptide, stimulating food intake (Mat-

suda et al., 2012). The number and distribution

of Npy immunoreactive (Npy-ir) cells in the

gastrointestinal tract vary depending on the

species and the nutritional status (Hernández

et al., 2018; Pereira et al., 2015; Vigliano et

al., 2011). In cichlids, there is only one study

analyzing the distribution of these cells in

Mozambique Tilapia Oreochromis mossambi-

cus (Pereira et al., 2017).

Cichlasoma dimerus (Heckel 1840) is a

Neotropical cichlid fish that inhabits the Parana

River Basin in South America (Kullander,

1983). Adults can reach 12 cm of standard

length (SL) and their diet mainly consists of lit-

tle aquatic insects, such as larvae of chironomi-

dae, ephemeroptera, odonata, and trichopteran

and, in lower proportion, little fishes and crus-

taceans (Almirón et al., 2015). In recent years,

it has become an excellent animal model for

different kinds of studies (Pandolfi et al., 2009),

that include more than 60 research. Given the

importance of the digestive tract in the overall

biology of fish and the scarce information

published about this topic in Neotropical cich-

lids, the work aims to describe anatomical,

histological, and histochemical features of the

digestive tract of C. dimerus, together with the

immunolocalization of Npy-ir cells.

MATERIALS AND METHODS

Animals: C. dimerus adults of both sexes

were captured in ‘‘Esteros del Riachuelo’’, Cor-

rientes, Argentina (27°12’50’’ S & 58°11’50’’

W) and transferred to the laboratory, where

they were acclimated and maintained in 130

L freshwater tanks under a 14:10 light/dark

cycle and a temperature of 25 ± 2 °C for a year.

Animals were fed ad libitum once a day with

commercial pellets (Kilomax Iniciador, #310;

Mixes del Sur, Provincia de Buenos Aires,

Argentina). Fish were handled following the

Principles of Laboratory Animal Care, which

309

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 70: 307-318, January-December 2022 (Published May 18. 30, 2022)

were approved by the Comisión Institucional

para el Cuidado y Uso de Animales de Labo-

ratorio (CICUAL), FCEyN, UBA, Argentina

(Protocol #26).

Morphometric parameters and histo-

logical characterization: Adult fish (14 indi-

viduals) were randomly selected, and SL, and

body weight (BW) were measured. Then, 4 h

after the last feeding, they were euthanized by

decapitation after anesthetized with benzocaine

0.1 %. Digestive tracts were extracted, and the

intestinal lengths (IL) were measured. With

those parameters, we calculated the Zihler

index (ZI) and the Relative Intestinal Length

(RIL) as ZI = IL/(BW^1/3) and RIL = IL/

SL, according to Al-Hussaini (1949), Zihler

(1981), and Karachle and Stergiou (2010). The

statical comparison of BW, SL, IL, ZI, and RIL

between males (N = 7) and females (N = 7)

was made by different one-way ANOVA using

the Infostat 2017 software (FCA, Universidad

Nacional de Córdoba, Argentina). Results are

expressed as mean ± SEM.

Digestive tracts were split into four rec-

ognizable regions: oesophagus, stomach,

intestine, and rectum. As in fish no marked

morphological distinction could be observed

along the intestine (Banan Khojasteh, 2012),

we divided it into equal sections for the his-

tological process. The organs were fixed in

Bouin’s solution for 24 h and they were dehy-

drated through crescent-gradient ethanol solu-

tions (70°, 90°, 96°, 100°), cleared in xylene,

and embedded in paraplast (Fisherbrand; Fisher

Scientific, Washington, DC, USA) (Pérez Sir-

kin et al., 2013). The samples were cut in 7 µm

coronal or parasagittal sections and mounted

on gelatin-coated slides in a way that the same

area of interest was available for the different

histological procedures.

For histological characterization, differ-

ent sections from all the collected organs

were stained with modified Masson’s trichrome

(mMT) and haematoxylin and eosin (H&E).

The histochemical stains Alcian blue (AB) (pH

= 2.5) and periodic acid-Schiff (PAS), both

plus haematoxylin, were performed to identify

acid or neutral glycoconjugates, respective-

ly. The slides were examined and digital-

ly photographed with a NIKON Microphot

FX microscope.

Npy-ir cells localization: For immunohis-

tochemistry, the samples (N = 3) obtained and

cut as it was described above were deparaf-

finized in xylene and rehydrated in a descend-

ing series of ethanol solutions (100°, 96°, 90°,

70°), and then, in a phosphate-buffered saline

(PBS) (pH 7.4). Later, endogenous peroxidase

activity was blocked by incubation in 0.3

% hydrogen peroxide solution. The reduc-

tion of nonspecific staining was performed

by incubation in PBS containing 5 % (w/v)

non-fat dry milk at room temperature (RT).

After that, the slides were maintained at 4 °C

overnight with the primary anti-NPY antibody

(dilution 1:2 500 in PBS, rabbit anti-porcine

NPY serum, Peninsula Laboratories Inc., CA).

The next day, they were washed in PBS and

incubated for 1 hour with biotinylated anti-

rabbit immunoglobulin (Ig) G (dilution 1:500

in PBS; Sigma-Aldrich, St Louis, MO, USA)

at RT. Subsequently, peroxidase-conjugated

streptavidin (dilution 1:500 in PBS; Invitrogen,

Carlsbad, CA, USA) was applied for 1 hour at

RT. The final reactive products were visual-

ized with DAB Substrate Kit (Cell Marque,

Rocklin, CA, USA). Then, the sections were

rehydrated, slightly counter-stained with hae-

matoxylin, and mounted with synthetic Canada

balsam (Biopack, Buenos Aires, Argentina).

The specificity of the antiserum was previously

performed in this species with primary antisera

preadsorbed with porcine NPY (Pérez Sirkin et

al., 2013). Omission of first antibody control

was performed.

Specific cell abundance: The abundance

of Npy-ir, AB-, and PAS-positive cells were

analyzed in randomly selected slides (seven

slides per region) of the different organs stud-

ied. The numbers of positive cells per fold were

counted manually under a light microscope,

with the precaution that the same cell was not

considered twice, and then, the average of each

310 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 70: 307-318, January-December 2022 (Published May 18, 2022)

region was estimated. Results are presented

qualitatively, reflecting the density of positive

cells per fold.

RESULTS

Morphometric parameters and histo-

logical characterization: The C. dimerus indi-

viduals (BW = 29.52 ± 3.18 g and SL = 8.28

± 0.24 cm) presented a digestive tract with

a short oesophagus and a blind-sac stomach.

From the left side of the junction between them

arose an intestine (IL = 10.81 ± 0.50 cm), with

two loops in its path: on the left side, the ante-

rior portion turned out to a cranial direction,

whereas, on the right side, the second loop

returned to a caudal direction concluding in

the rectum and the anus (Fig. 1A). No pyloric

ceca were observed in any of the animals. The

mean RIL and ZI were 1.22 ± 0.07 and 3.29

± 0.20, respectively. All the parameters evalu-

ated did not significantly differ between males

(RIL = 1.29 ± 0.07; ZI = 3.49 ± 0.17) and

females (RIL = 1.19 ± 0.10; ZI = 3.20 ± 0.29).

Histologically, the typical four concentric

tunicae of fish digestive tubes (Genten et al.,

2009) were recognized in the examined organs:

mucosa, submucosa, muscularis, and serosa.

The oesophagus (Fig. 1B, Fig. 1C, Fig. 1D,

Fig. 1E, Fig. 1F, Fig. 1G) consisted of a tunica

mucosa with stratified epithelium, numerous

folds (Fig. 1B, Fig. 1C), and abundant AB-

(Fig. 1D, Fig. 1F) and PAS-positive (Fig. 1E,

Fig. 1G) goblet cells the latter being more

abundant all along the oesophagus. The AB-

positive cell number increased from anterior to

posterior, while the PAS-positive cell number

decreased in the same direction (Table 1 and

Fig. 1D, Fig. 1E, Fig. 1F, Fig. 1G). It was

not possible to distinguish the lamina propria

from tunica submucosa due to the absence of

a continuous layer of muscular fibers. In both

cases, abundant connective tissue with fibro-

cytes, collagenous fibers, and blood vessels

Fig. 1. A. Drawing of the ventral view of the digestive tract. Bar = 1cm. (B. C. D. E. F. and G. Microphotographs of the

oesophagus of Cichlasoma dimerus. B. Oesophagus transversal panoramic view. mMT. Bar = 200 μm. C. Longitudinal

section of the oesophagus. mMT. Bar = 35 μm. D. AB positive goblet cells in the anterior oesophageal mucosa. AB pH = 2.5.

Bar = 20 μm. E. PAS positive goblet cells in the anterior oesophageal mucosa. PAS. Bar = 20 μm. F. AB positive goblet cells

in the posterior oesophageal mucosa. AB pH = 2.5. Bar = 20 μm. G. PAS positive goblet cells in the posterior oesophageal

mucosa. PAS. Oesophagus (Oe), stomach (St), anterior intestine (AI), posterior intestine (PI), rectum (Re), anterior (ANT),

posterior (POST), left (L), right (R), epithelium (Ep), connective tissue (CT), inner longitudinal muscle layer (ILM), outer

circular muscle layer (OCM).

311

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 70: 307-318, January-December 2022 (Published May 18. 30, 2022)

was observed. No glands projecting to the con-

nective tissue were observed. The tunica mus-

cularis consisted of two striated muscle layers:

a longitudinal inner one, with some bundles of

muscle projected to the connective tissue, and

a circular outer one.

In the junction between the oesophagus,

the stomach, and the intestine, the striated mus-

cle was gradually replaced by smooth muscle

fibers. The muscle layers orientation alternated

concerning that described above: the longitu-

dinal layer became external and the circular

internal in the stomach and the intestine. The

oesaphagus’ epithelium was abruptly replaced

with the typical epithelium of the stomach and

the intestine.

The stomach (Fig. 2) was a blind sac that

presented a tunica mucosa with simple colum-

nar epithelium (Fig. 2A, Fig. 2B) and oxynti-

copeptic (gastric) glands in the lamina propria

(Fig. 2C). The apical border of the epithelium

was PAS-positive (Fig. 2D) and not goblet cells

were detected. The underlying connective tis-

sue of the tunica submucosa presented a dense

bundle of collagen fibers under the epithelium

and a loose one near the tunica muscularis.

Two regions could be distinguished in the

stomach, according to the presence/absence

of gastric glands and their relative anatomical

position (Fig. 2E): a glandular stomach, that

is proximal, next to the oesophagus and the

intestine, with the presence of gastric glands

except in the most anterior portion, and a

non-glandular stomach, that is in the distal

region of the blind sac. The gastric glands

presented polyhedral cells with a spherical

nucleus, prominent nucleolus, and acidophilic

cytoplasm (Fig. 2C). They formed clusters sur-

rounded by connective tissue from the lamina

propria and scattered smooth muscle fibers.

Regarding the intestine (Fig. 3), modifica-

tions of the tube diameter, length and morphol-

ogy of the folds, and the abundance of some

cellular types were analysed. The intestinal

mucosa presented a simple columnar epithe-

lium, with enterocytes with brush border and

interspersed AB- (Fig. 3C) and PAS- (Fig.

3D) positive goblet cells. These goblet cells

decreased in number from anterior to posterior,

with a greater abundance of AB- positive cells

all along the intestine (Table 1). The PAS-

positive cells slightly increase in density in the

middle intestine and then decrease in the last

portion of the gut. The mucosa folds decreased

in number and height from anterior to posterior.

In the foregut, they presented thin finger-like

projections (Fig. 3A, Fig. 3B), whilst in the

hindgut, they were shorter and with a folia-

ceous appearance (Fig. 3E). Particularly, in this

region, enterocytes with large clear vacuoles

in the apical cytoplasm were found (Fig. 3F).

All along the intestine, the connective tissue

layer of the lamina propria and the tunica

submucosa was thinner than in the oesophagus

and the stomach, with isolated smooth muscle

fibers. The two layers of the tunica muscularis

present similar width of smooth muscle fibers.

The myenteric plexus between the two muscle

layers were observed all along the intestine.

TABLE 1

Relative abundance of Alcian Blue (AB), Periodic Acid Schiff (PAS) and Npy-immunoreactive (-ir) positive cells

along the different regions of the digestive tract of Cichlasoma dimerus

Technique

Region

Oesophagus Stomach Intestine Rectum

Anterior Posterior Cardiac Fundic Pyloric Anterior Posterior

AB pH 2.5 +++ ++++ - - - +++ +++ +++ ++ +

PAS ++++ +++ ∆ ∆ ∆ ++ ++ +++ + +

Npy NE NE NE NE NE °°° °° °° ° -

Note: (-) null; (NE) not evaluated; (+) low, (++) medium, (+++) high and (++++) very high density of goblet cells per fold;

(∆) reactivity in the apical border of the epithelial cells; (°) low, (°°) medium and (°°°) high density of Npy-ir cells per fold.

312 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 70: 307-318, January-December 2022 (Published May 18, 2022)

The intestinal diameter decreased towards the

posterior regions.

The transition between the hindgut and

the rectum was abruptly marked by an ileo-

rectal valve where thickening of the circular

muscle layer, a sudden change in gut diameter,

and fewer mucosal folds were observed (Fig.

3G). The rectum (Fig. 3H) presented a simple

columnar epithelium with a high density of

AB- and PAS-positive goblet cells. The inner

circular muscle layer was thicker than the

outer longitudinal.

Npy-ir cells localization: We found Npy-

ir cells scattered between the enterocytes of the

intestine (Fig. 3I), decreasing in density from

anterior to posterior (Table 1). These cells were

open-type (Barrios et al., 2020), with a narrow

and elongated shape and a round nucleus. The

cytoplasm was projected to the apical and basal

regions, with strong immunolabel in the peri-

nuclear zone. In the last portion of the intestine

and in the rectum, no Npy-ir cells were detect-

ed in the epithelium. Besides, Npy-ir fibers into

the myenteric plexus between muscle layers of

the tunica muscularis were observed all along

the intestine and the rectum.

DISCUSSION

The ability of Neotropical cichlids to diver-

sify and colonize a great variety of habitats

along Central and South America has become

this group in the subject of numerous studies

related to their behavioral, biogeographical,

ecomorphological, and phylogenetic aspects

Fig. 2. Microphotographs of the stomach of Cichlasoma dimerus. A. Stomach transversal panoramic view. mMT. Bar 260

μm. B. Enlarged image from A. mMT. Bar = 100 μm. C. Gastric glands underlying the gastric epithelium. mMT. Bar = 8

μm. D. Gastric mucosa PAS positive in the apical border. PAS Bar = 20 μm. E. Longitudinal section shows the boundaries

(dotted line) between glandular and non-glandular region of the stomach given by the absence of gastric glands in the latter.

mMT. Bar = 20 μm. Epithelium (Ep), connective tissue (CT), gastric glands (GG), inner circular muscle layer (ICM), outer

longitudinal muscle layer (OLM), glandular stomach (GS), non-glandular stomach (NGS).

313

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 70: 307-318, January-December 2022 (Published May 18. 30, 2022)

(Malabarba & Malabarba, 2020). Given that

there is limited information about the digestive

tube of new-world cichlids, C. dimerus has

arisen as an interesting study model that could

provide new perspectives on the feeding biol-

ogy of this group.

The stomach’s extendible blind pouch, the

left-hand exit of the stomach to the anterior

intestine, and the first intestinal loop on the left

side observed in C. dimerus, are considered

synapomorphies within the Cichlidae fam-

ily (Zihler, 1981). Also, we observed a short

Fig. 3. Microphotographs of the intestine of Cichlasoma dimerus. A. Anterior intestine transversal panoramic view. Mucosal

folds are long, finger-like projections. H&E. Bar = 200 μm. B. Anterior intestine mucosa with enterocytes with brush border

and interspersed goblet cells (arrowheads). mMT. Bar = 20 μm. C. AB positive goblet cells in the anterior intestinal mucosa.

AB pH = 2.5. Bar = 30 μm. D. PAS positive goblet cells in the posterior intestinal mucosa. PAS. Bar = 30 μm. E. Overview

of the posterior intestine, with shorter and fewer mucosal folds. Bar = 200 μm. F. Enterocytes of the posterior region with

supranuclear vacuoles (arrowheads) in the apical cytoplasm. H&E. Bar = 20 μm. G. Transition zone between posterior

intestine and rectum (ileorectal valve). H&E. Bar = 200 μm. H. Coronal section of the rectum. mMT. Bar = 125 μm. I.

Anterior intestinal mucosa with Npy-ir cells (arrowheads) interspersed in the epithelium. Npy IHQ + haematoxylin. Bar =

20 μm. Epithelium (Ep), connective tissue (CT), inner circular muscle layer (ICM), outer longitudinal muscle layer (OLM).

314 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 70: 307-318, January-December 2022 (Published May 18, 2022)

intestine with little coiling and low values of ZI

and RIL. These morphological characteristics

could be related to the dietary habits and/or the

evolutionary history of the Neotropical cichlids.

Regarding the first, many works have linked

the gross morphology of the gastrointestinal

tract with the dietary habits of a wide variety

of fish species (Al-Hussaini, 1949; Karachle

& Stergiou, 2010; Kramer & Bryant, 1995;

Zihler, 1981). Both indexes obtained are close

to the values described for omnivorous species

with a preference for carnivory (Karachle &

Stergiou, 2010). In this sense, C. dimerus diet

in wild environments consists of microinverte-

brates (Almiron et al., 2015). Concerning the

phylogenetic context, many Neotropical cichlid

species have lower values of ZI and RIL, with

shorter intestines with little or no coiling too

(Kramer & Bryant, 1995; Zihler, 1981). These

species tend to be substrate-sifting invertivores,

epibenthic invertebrate gleaners, and piscivores

(López-Fernández et al., 2012; Montaña &

Winemiller, 2013).

Some of the functions described for the

oesophagus in fish have been related to food

passage, digestion, and osmoregulation (Wil-

son & Castro 2010). In this sense, the stratified

epithelium with mucus cells observed in our

species could act as a mechanical and chemical

barrier. The mucopolysaccharides secreted to

the oesophageal lumen may help in lubrication,

defence against virus and bacteria, and diges-

tion (Genten et al., 2009; Wilson & Castro,

2010). The great abundance of mucus cells is

common along the oesophagus of both Neo-

tropical (da Silva et al., 2012; Hopperdietzel

et al., 2014) and African cichlids (Morrison

& Wright Jr, 1999; Okuthe & Bhomela, 2020;

Scocco et al., 1998), although some differences

were reported about their distribution pattern.

For example, in Amatitlania nigrofasciatta,

PAS-positive cells are abundant in the anterior

portion; the AB-positive cells are uniformly

distributed (Hopperdietzel et al., 2014). In

our study PAS-positive cells present a similar

distribution, but the AB-positive cells increase

from anterior to posterior.

Concerning the transition zone between

the oesophagus, the stomach, and the intestine,

the abrupt change in the epithelial morphology

and the alternate of striated and smooth muscle

fibers are shared features in most of the cich-

lids’ alimentary tracts described so far (Ansari

et al., 2020; Caceci et al., 1997; da Silva et

al., 2012; Hopperdietzel et al., 2014; Mor-

rison & Wright Jr, 1999; Okuthe & Bhomela,

2020; Pasha, 1964). Because of the presence

of a thicker lamina muscularis with a high

abundance of striated muscle fibers, this region

may function as a pyloric sphincter, prevent-

ing that food passes directly into the intestine

without being chemically digested (Ansari et

al., 2020) and allowing regurgitation of food

from the stomach into the oesophagus (Hop-

perdietzel et al., 2014).

In Neotropical cichlid species, there are

some common traits in the histology of the

stomach wall, such as the simple columnar

epithelium with PAS-positive apical border,

the absence of goblet cells, and the presence

of gastric glands in all the lamina propria (da

Silva et al., 2012; Hopperdietzel et al., 2014;

Ramírez Espitia et al., 2020). We found these

features in C. dimerus, although the blind-end

of the stomach lacks gastric glands. Despite its

phylogenetical proximity, in African cichlids,

there are different histological arrangements

of the gastric mucosa across the species. For

example, gastric glands can be present through-

out its extension or absent in the pyloric region,

and different types and distribution of mucous

glands have been reported (Ansari et al., 2020;

Gargiulo et al., 1997; Morrison & Wright Jr,

1999; Okuthe & Bhomela, 2020; Pasha, 1964;

Scocco et al., 1996).

We identified two distinct secretory cells

in the gastric mucosa: the oxyntincopeptic and

the epithelial ones with a PAS-positive api-

cal border. The oxynticopeptic cells secrete

HCl, denaturing protein and converting the

pepsinogen (secreted by the same cell) into

the active proteolytic form pepsin (Bakke et

al., 2010). The Neutral glycoconjugates on the

apical border protect the mucosa from the acid

environment (Ghosh & Chakrabarti, 2015).

315

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 70: 307-318, January-December 2022 (Published May 18. 30, 2022)

Additionally, this secretion allows the absorp-

tion of disaccharides and short-chain fatty acids;

and the mixing and emulsion of food particles

with digestive enzymes (Mokhtar et al., 2017).

Along the intestine, we observed changes

in the histology of the tunica mucosa related to

the digestive physiology of the organ. The char-

acteristic mucosal fold shape of the anterior

region may facilitate the absorption of water-

soluble molecules in the brush border and

short and medium-chain fatty acids (Bakke et

al., 2010). In the posterior region, enterocytes

exhibit supranuclear vacuoles, which are prob-

ably due to the absorption of large peptides by

endocytosis (Van den Ingh et al., 1991). Goblet

cell general distribution seems to be a common

feature in several carnivore species (Pereira et

al., 2020), and in the omnivorous Centroameri-

can cichlid A. nigrofasciata (Arman & Ucuncu,

2017). It is proposed that intestinal neutral gly-

coproteins would be involved in the absorption

and transportation of molecules through the

membranes, whereas the acidic glycoproteins

would be a lubricative and protective secretion

(Arman & Ucuncu, 2017).

In C. dimerus, the transition between the

distal portion of the intestine and the rectum

is marked by an ileorectal valve, which would

help to control faecal egestion. This valve was

also observed in cichlids such as O. niloticus

(Morrison & Wright Jr, 1999). The rectum has

abundant goblet cells and a thick muscular

layer, which may facilitate the egestion of fae-

cal materials to the anus (Mokhtar et al., 2017).

Npy is considered one of the most potent

orexigenic peptides in fish acting on the central

nervous system (Matsuda et al., 2012; Volkoff,

2016). In C. dimerus, Npy-ir neurons localiza-

tion and Npy-ir fibers abundance have been

already studied (Pérez Sirkin et al., 2013).

Despite its central function, the knowledge

about the specific functions of this neuropeptide

on the digestive system is scarce. To our knowl-

edge, this is the first report to describe Npy-ir

cells in a Neotropical cichlid species. The pat-

tern observed in C. dimerus was also described

for several species, as O. niloticus (Pereira et

al., 2017), Rhamdia quelen (Hernández et al.,

2018), Odontesthes bonariensis (Vigliano et

al., 2011), Chanos chanos (Lin et al., 2017),

Salminus brasiliensis (Pereira et al., 2015),

Ictalurus punctatus (Min et al., 2009), Prochi-

lodus lineatus (Barrios et al., 2020), Colossoma

macropomum, Pseudoplatystoma reticulatum

× Leiarius marmoratus (Pereira et al., 2020).

The anterior portion of the intestine would be

a source of peripheral signals capable of stimu-

lating food intake when the gut is empty (Vigli-

ano et al., 2011). Pereira et al. (2017) argued

that the density of Npy-ir cells observed in

the anterior intestine of several species is high

because of the connection with vagal afferents

that send nutritional information to the central

nervous system (Olsson, 2010).

To sum up, in this work, we described the

digestive tract of the South American cichlid

fish C. dimerus. The gross morphology and the

calculated intestinal indexes are close to those

reported for omnivorous Neotropical cichlids

with a preference for carnivory. Besides, the

histological and histochemical characteristics

found present some unique features, such as

the distribution of the goblet cells and gastric

glands. Considering the morphological and

histological differences in the digestive tract

across the cichlids, as well as the scarce infor-

mation about this topic in Neotropical species,

the present study provides new perspectives on

this issue, in relation to feeding habits, diges-

tive physiology, and phylogenetic context.

Ethical statement: the authors declare

that they all agree with this publication and

made significant contributions; that there is no

conflict of interest of any kind; and that we fol-

lowed all pertinent ethical and legal procedures

and requirements. All financial sources are

fully and clearly stated in the acknowledge-

ments section. A signed document has been

filed in the journal archives.

ACKNOWLEDGMENTS

We are grateful for the support of

Consejo Nacional de Investigaciones

Científicas y Técnicas (CONICET; PIP

316 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 70: 307-318, January-December 2022 (Published May 18, 2022)

2014-2016:11220130100501CO to P.G.V.),

Universidad de Buenos Aires (UBACyT

20020190100294BA to P.G.V.) and Agen-

cia Nacional de Promoción Científica y

Tecnológica (PICT-2018- 02577 to P.G.V.).

RESUMEN

Anatomía e histología del tracto digestivo e

inmunolocalización del Npy (péptido orexigénico) en

el pez Cichlasoma dimerus (Cichliformes: Cichlidae)

Introducción: El tracto digestivo de los cíclidos neotro-

picales no ha sido profundamente estudiado y es un tema

fundamental para entender la fisiología, nutrición, asocia-

ciones tróficas y evolución de los peces.

Objetivo: Describir anatómica e histológicamente el tracto

digestivo del pez cíclido neotropical Cichlasoma dimerus

e inmunolocalizar el péptido orexigénico (Npy) a lo largo

del intestino.

Métodos: Sacrificamos 14 individuos adultos y fijamos los

órganos en solución de Bouin; teñimos secciones de para-

fina de 7 μm de espesor para una descripción general y con

azul alcián (pH = 2.5, AB) y ácido periódico-Schiff (PAS)

para identificar glicoconjugados ácidos o neutros, respec-

tivamente. Además, en 3 individuos adultos se realizaron

inmunohistoquímicas contra Npy. Contamos manualmente

las células PAS y AB positivas, y las células inmunorreac-

tivas a Npy por pliegue.

Resultados: Hay un esófago corto, un estómago en forma

de saco y un intestino con dos vueltas. El esófago tiene

un epitelio estratificado con una alta densidad de células

caliciformes PAS- y AB- positivas y fibras esqueléticas

estriadas en las capas musculares. La mucosa del estómago

está revestida por epitelio simple cilíndrico. El epitelio

intestinal es simple cilíndrico con chapa estriada y células

caliciformes PAS- y AB- positivas intercaladas, y células

inmunorreactivas a Npy. Hay una válvula ileorrectal en la

transición entre el intestino posterior y el recto. Esta última

porción intestinal tiene células caliciformes y una túnica

muscular más gruesa.

Conclusiones: C. dimerus comparte características con

otros cíclidos neotropicales, pero la distribución de las

células caliciformes y las glándulas gástricas, serían ras-

gos propios de esta especie. A nuestro entender, este es

el primer trabajo que describe la distribución de célu-

las inmunorreactivas a Npy en el intestino de un pez

cíclido neotropical.

Palabras clave: células caliciformes; histomorfología;

histoquímica; inmunohistoquímica; cíclidos neotropicales.

REFERENCES

Al-Hussaini, A. H. (1949). On the functional morphology

of the alimentary tract of some fish in relation to

differences in their feeding habits: anatomy and

histology. Journal of Cell Science, 3(10), 109–139.

Almirón, A. E., Casciotta, J. R., Ciotek, L., & Giorgis,

P. (2015). Guía de los peces del Parque Nacio-

nal Pre-Delta (2nd Ed.). Administración de Parques

Nacionales.

Ansari, M. H., Ebrahimi, M., & Esmaeili, H. R. (2020).

Morphohistological characteristic of digestive tract

of an endemic cichlid fish, Iranocichla hormuzen-

sis Coad, 1982 (Teleostei: Cichlidae). International

Journal of Aquatic Biology, 8(1), 1–8.

Arman, S., & Ucuncu, S. I. (2017). Histochemical cha-

racterization of convict cichlid (Amatitlania nigro-

fasciata) intestinal goblet cells. Pakistan Journal of

Zoology, 49(2), 445–453.

Axelrod, H. R., & Burgess, W. R. (1979). African Cichlids

of Lakes Malaŵi and Tanganyika. Tropical Fish

Hobbyist Publications.

Bakke, A. M., Glover, C., & Krogdahl, Å. (2010). Feeding,

digestion and absorption of nutrients. In M. Grosell,

A. P. Farrell, & C. J. Brauner (Eds.), Fish physiology:

The multifunctional gut of fish (pp. 57–110). Acade-

mic Press.

Banan Khojasteh, S. M. (2012). The morphology of the

post-gastric alimentary canal in teleost fishes: a brief

review. International Journal of Aquatic Science,

3(2), 71–88.

Barrios, C. E., Santinón, J. J., Domitrovic, H. A., San-

chez, S., & Hernandez, D. R. (2020). Localization

and distribution of CCK-8, NPY, Leu-ENK-, and

Ghrelin-in the digestive tract of Prochilodus lineatus

(Valenciennes, 1836). Anais da Academia Brasileira

de Ciências, 92.

Beveridge, M. C. M., & Baird, D. J. (2000). Diet, feeding

and digestive physiology. In M. C. M. Beveridge, &

B. J. McAndrew (Eds.), Tilapias: Biology and Exploi-

tation (pp. 59–87). Springer.

Caceci, T., El-Habback, H. A., Smith, S. A., & Smith, B.

J. (1997). The stomach of Oreochromis niloticus

has three regions. Journal of Fish Biology, 50(5),

939–952.

Cengiz, E. I., & Unlu, E. (2006). Sublethal effects of com-

mercial deltamethrin on the structure of the gill, liver

and gut tissues of mosquitofish, Gambusia affinis:

a microscopic study. Environmental Toxicology and

Pharmacology, 21(3), 246–253.

317

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 70: 307-318, January-December 2022 (Published May 18. 30, 2022)

Dane, H., & Şişman, T. (2020). A morpho-histopathological

study in the digestive tract of three fish species

influenced with heavy metal pollution. Chemosphere,

242, 125212.

da Silva, M. R., Natali, M. R. M., & Hahn, N. S. (2012).

Histology of the digestive tract of Satanoperca pap-

paterra (Osteichthyes, Cichlidae). Acta Scientiarum.

Biological Sciences, 34(3), 319–326.

de Oliveira, M. I., de Matos, L. V., da Silva, L. A., Cha-

gas, E. C., da Silva, G. S., & Gomes, A. L. (2019).

The digestive tube of Piaractus brachypomus: gross

morphology, histology/histochemistry of the mucosal

layer and the effects of parasitism by Neoechinor-

hynchus sp. Journal of Fish Biology, 94(4), 648–659.

Gaucher, L., Vidal, N., D’Anatro, A., & Naya, D. E. (2012).

Digestive flexibility during fasting in the characid

fish Hyphessobrycon luetkenii. Journal of Morpho-

logy, 273, 49–56.

Gargiulo, A. M., Ceccarelli, P., Dall’Aglio, C., & Pedini,

V. (1997). Ultrastructural study on the stomach of

Tilapia spp. (Teleostei). Anatomia, Histologia, Embr-

yologia, 26(4), 331–336.

Genten, F. (2009). Atlas of Fish Histology. Science

Publishers.

Ghosh, S. K., & Chakrabarti, P. (2015). Histological and

histochemical characterization on stomach of Mystus

cavasius (Hamilton), Oreochromis niloticus (Lin-

naeus) and Gudusia chapra (Hamilton): Comparative

study. The Journal of Basic & Applied Zoology, 70,

16–24.

Hernández, D. R., Barrios, C. E., Santinón, J. J., Sánchez,

S., & Baldisserotto, B. (2018). Effect of fasting and

feeding on growth, intestinal morphology and ente-

roendocrine cell density in Rhamdia quelen juveniles.

Aquaculture Research, 49(4), 1512–1520.

Hopperdietzel, C., Hirschberg, R. M., Hünigen, H., Wolter,

J., Richardson, K., & Plendl, J. (2014). Gross mor-

phology and histology of the alimentary tract of the

convict cichlid Amatitlania nigrofasciata. Journal of

Fish Biology, 85(5), 1707–1725.

Karachle, P. K., & Stergiou, K. I. (2010). Intestine mor-

phometrics of fishes: a compilation and analysis of

bibliographic data. Acta Ichthyologica et Piscatoria,

40(1), 45–54.

Kramer, D. L., & Bryant, M. J. (1995). Intestine length in

the fishes of a tropical stream: 2. Relationships to

diet—the long and short of a convoluted issue. Envi-

ronmental Biology of Fishes, 42(2), 129–141.

Kullander, S. O. (1983). A revision of the South Ameri-

can cichlid genus Cichlasoma (Teleostei, Cichlidae).

Swedish Museum of Natural History.

Lin, X., Wang, P., Ou, Y., Li, J. E., & Wen, J. (2017). An

immunohistochemical study on endocrine cells in

the neuroendocrine system of the digestive tract of

milkfish Chanos chanos (Forsskal, 1775). Aquacultu-

re Research, 48(4), 1439–1449.

López-Fernández, H., Winemiller, K. O., Montaña, C., &

Honeycutt, R. L. (2012). Diet-morphology correla-

tions in the radiation of South American geophagine

cichlids (Perciformes: Cichlidae: Cichlinae). PLOS

ONE, 7(4), e33997.

Malabarba, L. R., & Malabarba, M. C. (2020). Phylogeny

and classification of neotropical fish. In B. Baldise-

rotto, E. Urbinati, & J. Cyrino (Eds.), Biology and

physiology of freshwater neotropical fish (pp. 1–19).

Academic Press.

Matsuda, K., Sakashita, A., Yokobori, E., & Azuma, M.

(2012). Neuroendocrine control of feeding behavior

and psychomotor activity by neuropeptide Y in fish.

Neuropeptides, 46(6), 275–283.

Min, H. E., Wang, K. Y., & Zhang, Y. (2009). Immunocyto-

chemical identification and localization of diffuse

neuroendocrine system (DNES) cells in gastrointes-

tinal tract of channel catfish (Ictalurus punctatus).

Agricultural Sciences in China, 8(2), 238–243.

Mokhtar, D. M. (2017). Fish histology: from cells to

organs. CRC Press.

Montaña, C. G., & Winemiller, K. O. (2013). Evolutionary

convergence in Neotropical cichlids and Nearctic

centrarchids: evidence from morphology, diet, and

stable isotope analysis. Biological Journal of the

Linnean Society, 109(1), 146–164.

Morrison, C. M., & Wright Jr, J. R. (1999). A study of the

histology of the digestive tract of the Nile tilapia.

Journal of Fish Biology, 54(3), 597–606.

Nelson, J. S., Grande, T. C., & Wilson, M. V. (2016). Fishes

of the World. John Wiley & Sons.

Okuthe, G. E., & Bhomela, B. (2020). Morphology, his-

tology and histochemistry of the digestive tract of

the Banded tilapia, Tilapia sparrmanii (Perciformes:

Cichlidae). Zoologia, 37, e51043.

Olsson, C. (2010). The enteric nervous system. In M. Gro-

sell, A. P. Farrell, & C. J. Brauner (Eds.), Fish physio-

logy: The multifunctional gut of fish (pp. 319–340).

Academic Press.

Osman, A. H. K. & Caceci, T. (1991). Histology of the

stomach of Tilapia nilotica (Linnaeus, 1758) from

the River Nile. Journal of Fish Biology, 38, 211–223.

Pandolfi, M., Cánepa, M. M., Meijide, F. J., Alonso, F.,

Vázquez, G. R., Maggese, M. C., & Vissio, P. G.

(2009). Studies on the reproductive and develop-

mental biology of Cichlasoma dimerus (Percifomes,

Cichlidae. Biocell, 33(1), 1–18.

318 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 70: 307-318, January-December 2022 (Published May 18, 2022)

Pasha, K. (1964). The anatomy and histology of the ali-

mentary canal of herbivorous fish Tilapia mossam-

bica (Peters). Proceedings of the Indian Academy of

Science B, 54, 340–349.

Pereira, R. T., Costa, L. S., Oliveira, I. R. C., Araújo, J. D.

C., Aerts, M., Vigliano, F. A., & Rosa, P. V. (2015).

Relative distribution of gastrin-, CCK-8-, NPY-and

CGRP-immunoreactive cells in the digestive tract

of dorado (Salminus brasiliensis). Tissue and Cell,

47(2), 123–131.

Pereira, R. T., de Freitas, T. R., de Oliveira, I. R. C., Costa,

L. S., Vigliano, F. A., & Rosa, P. V. (2017). Endocrine

cells producing peptide hormones in the intestine of

Nile tilapia: distribution and effects of feeding and

fasting on the cell density. Fish Physiology and Bio-

chemistry, 43(5), 1399–1412.

Pereira, R. T., Nebo, C., de Paula Naves, L., Fortes-Silva,

R., Regina Cardoso de Oliveira, I., Paulino, R. R., &

Rosa, P. V. (2020). Distribution of goblet and endo-

crine cells in the intestine: A comparative study in

Amazonian freshwater Tambaqui and hybrid catfish.

Journal of Morphology, 281(1), 55–67.

Pérez Sirkin, D. I., Suzuki, H., Cánepa, M. M., & Vissio, P.

G. (2013). Orexin and neuropeptide Y: tissue specific

expression and immunoreactivity in the hypothala-

mus and preoptic area of the cichlid fish Cichlasoma

dimerus. Tissue and Cell, 45(6), 452–459.

Pervin, M. A., Jahan, H., Akter, R., Omri, A., & Hossain, Z.

(2020). Appraisal of different levels of soybean meal

in diets on growth, digestive enzyme activity, antio-

xidation, and gut histology of tilapia (Oreochromis

niloticus). Fish Physiology and Biochemistry, 46(4),

1397–1407.

Ramírez Espitia, E. J., Hurtado Giraldo, H., & Gómez

Ramírez, E. (2020). Anatomía general, histología

y morfometría del sistema digestivo del pez Ptero-

phyllum scalare (Perciformes: Cichlidae). Revista de

Biología Tropical, 68(4), 1371–1383.

Rašković, B. S., Stanković, M. B., Marković, Z. Z., &

Poleksić, V. D. (2011). Histological methods in the

assessment of different feed effects on liver and

intestine of fish. Journal of Agricultural Sciences,

56(1), 87–100.

Scocco, P., Accili, D., Menghi, G., & Ceccarelli, P. (1998).

Unusual glycoconjugates in the oesophagus of a tila-

pine polyhybrid. Journal of Fish Biology, 53, 39–48.

Scocco, P., Ceccarelli, P., & Menghi, G. (1996). Glycohis-

tochemistry of the Tilapia spp. stomach. Journal of

Fish Biology, 49(4), 584–593.

Scocco, P, Menghi, G., & Ceccarelli, P. (1997). Histoche-

mical differentiation of glycoconjugates occurring

in the tilapine intestine. Journal of Fish Biology, 51,

848–857.

Van den Ingh, T. S. G. A. M., Krogdahl, Å., Olli, J. J., Hen-

driks, H. G. C. J. M., & Koninkx, J. G. J. F. (1991).

Effects of soybean-containing diets on the proximal

and distal intestine in Atlantic salmon (Salmo salar):

a morphological study. Aquaculture, 94(4), 297–305.

Vidal, N., Zaldúa, N., D’Anatro, A., & Naya, D. E.

(2014). Are the more plastic the more abundant? An

assessment of the interplay between physiological

flexibility and community structure for a Neotropical

fish assamblage. PLOS ONE, 9(3), e92446.

Vigliano, F. A., Muñoz, L., Hernández, D., Cerutti, P.,

Bermúdez, R., & Quiroga, M. I. (2011). An immuno-

histochemical study of the gut neuroendocrine system

in juvenile pejerrey Odontesthes bonariensis (Valen-

ciennes). Journal of Fish Biology, 78(3), 901-911.

Volkoff, H. (2016). The neuroendocrine regulation of food

intake in fish: a review of current knowledge. Fron-

tiers in Neuroscience, 10, 540.

Wagner, C. E., McIntyre, P. B., Buels, K. S., Gilbert, D. M.,

& Michel, E. (2009). Diet predicts intestine length in

Lake Tanganyika’s cichlid fishes. Functional Ecolo-

gy, 23(6), 1122–1131.

Wilson, J. M., & Castro, L. F. C. (2010). Morphological

diversity of the gastrointestinal tract in fishes. In

M. Grosell, A. P. Farrell, & C. J. Brauner (Eds.),

Fish physiology: The multifunctional gut of fish (pp.

1–55). Academic Press.

Zihler, F. (1981). Gross morphology and configuration of

digestive tracts of Cichlidae (Teleostei, Perciformes):

phylogenetic and functional, significance. Nether-

lands Journal of Zoology, 32(4), 544–571.