Research Article |
Corresponding author: Grace E. Okuthe ( ageokuthe@gmail.com ) Academic editor: Carolina Arruda Freire
© 2020 Grace E. Okuthe, Bongile Bhomela.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Okuthe GE, Bhomela B (2020) Morphology, histology and histochemistry of the digestive tract of the Banded tilapia, Tilapia sparrmanii (Perciformes: Cichlidae). Zoologia 37: 1-14. https://doi.org/10.3897/zoologia.37.e51043
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This study described anatomical, histological and histochemical features of the mucosal layer of the digestive tract of Tilapia sparrmanii Smith, 1840, an omnivorous freshwater fish endemic to Southern Africa. This species exhibited a short thick oesophagus with long deep longitudinal folds (466.68 ± 16.91 µm), and a thick (173.50 ± 10.92 µm) muscular layer that allow the passage of large food items. The mucosa was lined with stratified secretory epithelium rich in goblet cells that secreted neutral and acid mucins. The stomach was a sac-like structure with simple tubular glands surrounded by connective tissue. The mucosa was lined with simple columnar epithelium and the lamina propria exhibited a well-developed layer of gastric glands that occupied the entire length of the cardio-fundic region. The stomach mucosa consisted of epithelial cells with intense neutral mucin secretion which protects against gastric juice. Neck cells of gastric glands synthesized neutral and acid mucins. The intestine was highly coiled and presented a complex pattern of transversal folds internally (villi). Villi length decreased progressively from the anterior to the posterior intestine (p < 0.0001). Tunica muscularis of the mid-intestine had the thinnest thickness among all parts of the intestine (p < 0.0001). Goblet cells whose numbers increased towards the rectum secreted both acid and neutral mucins. The results indicate structural similarities of T. sparrmanii GIT with other tilapia species and will be useful for understanding the physiology of the digestive systems as well as functional components of the GIT.
Fish, gastrointestinal tract, histo-architecture
The basic plan of the gastrointestinal tract (GIT) of fish is similar to that of other vertebrates (
The GIT in fish plays a critical role in growth, nutrition and survival of the fish under various environmental conditions. The mouth serves to capture and pre-process food before it enters the oesophagus. The latter is a short and muscular, mucous-secreting connection leading to the stomach. The stomach, when present, is the portion of the GIT that displays most variations (
The ability of fish to utilize ingested nutrients depends on the presence of appropriate enzymes in appropriate locations in the wall and along the lumen of the GIT. Mucus helps to protect epithelial surfaces against mechanical damage while enabling rapid removal of various types of aggressive pathogens as well as irritants (
The GIT is one of the major organ systems of fishes that interacts with the environment and plays a critical role in growth, nutrition, as well as survival under stressful environments. Further, several functions of various segments of the GIT are controlled by endocrine cells, which can vary in frequency and distribution depending on the fish and prevailing environmental conditions (
Tilapia sparrmanii Smith, 1840, commonly known as Banded tilapia (
Fish were collected from a freshwater dam in Mthatha in the Eastern Cape Province, South Africa (31°32’S; 28°43’E) during routine sampling. Twenty-one adult specimens of T. sparrmanii, body mass about 0.32–0.40 kg, 110–120 mm total length (TL) were used in the current study. Fish were sacrificed by immersing in an overdose of tricaine methane sulphonate (MS-222, Sigma Chemicals, MO, USA). The whole GIT; oesophagus, stomach (glandular and non-glandular), and three intestinal segments (anterior, middle and posterior) was explanted from each specimen (Figs
(1) Image of an adult of Tilapia sparrmanii in the lateral view; (2) gross morphology of the gastrointestinal tract (GIT) of T. sparrmanii in ventral view of fish showing the relationship of the gastrointestinal tract with other organs in the abdominal cavity. Oesophagus (O) connected to pharynx (Ph) and stomach (S), which overlapped by the liver (L) and heart (H). Notice the highly coiled intestine (I). (3) Gross morphology of the gastrointestinal tract (GIT) of T. sparrmanii, showing the stomach (S). The intestine is divided into anterior intestine (AI), middle intestine (MI), posterior intestine (PI) and rectum (R).
Histochemical procedures comprised periodic acid Schiff (PAS). PAS staining was done according the packet insert and according to the manufacturer’s instructions (Sigma-Aldrich, St. Louis, MO, USA) with or without heametoxylin.
PAS and Alcian blue (AB) pH 2.5 staining was done to reveal neutral and acid mucins, respectively. Here tissue sections of the GIT were deparaffinized and hydrated to distilled water, incubated in 3% acetic acid. Thereafter, sections were stained in alcian blue for 30 minutes and washed in running tap water. Tissue sections were counterstained in nuclear fast red for three minutes, rinsed in tap water and dehydrated. Sections were cleared in HistoChoice® clearing agent and mounted with Leica CV mount (Leica Microsystems Nussloch, GmbH).
Prepared slides were used for the measurement of individual thickness of the layers making up the wall of the GIT. For each of these parameters, 20 measurements were taken of which an average was calculated. Results were presented as means ± SD values. Intensity of histochemical labelling with PAS and AB pH 2.5 was evaluated semi quantitatively according to observed intensity of color reaction, i.e., (−) no staining, (+) low, (++) medium, and (+++) strong staining (Table
Intensity of histochemical labeling of mucins in the GIT of T. sparrmanii. (AB) Alcian blue, (ES) Epithelium surface, (PAS) periodic acid Schiff.
Procedure | Oesophagus | Stomach | Anterior intestine | Mid intestine | Posterior intestine | ||||||||||||||
ES | Goblet cells | ES / Neck cells | Goblet cells | ES | Goblet cells | ES | Goblet cells | ES | Goblet cells | ||||||||||
Reaction | Distribution | Reaction | Distribution | Reaction | Distribution | Reaction | Distribution | Reaction | Distribution | ||||||||||
Pas | + | ++ | +++ | +++ / ++ | ++ | ++ | – | +++ | +++ | + | +++ | ++ | + | +++ | +++ | ||||
Ab ph 2.5 | + | +++ | +++ | – / ++ | – | ++ | – | +++ | +++ | + | ++ | ++ | + | ++ | +++ |
All measurements were performed using Leica LAS imaging software version 4.5, (magnification 10x). Means of various parameters of intestinal segments was compared using one-way ANOVA. Scheffe’s test was used to compare values between the segments using Statistical Packet of Social Science (SPSS), version 13. The significance level was set at 0.0001.
Experimental protocols for the study were approved by the Animal Ethics Screening Committee, Faculty of Natural Sciences, Walter Sisulu University.
The Gross morphology of gastrointestinal tract (GIT) in T. sparrmanii (Fig.
Histologically the GIT of T. sparrmanii follows the general plan as in other vertebrates, consisting of four layers: the mucosa, sub-mucosa, muscularis and serosa. The three segments of the intestine had the same basic organization of the muscular layer as in the stomach, consisting of an external longitudinal and internal circular muscle layers.
Microscopically the mucosa of the esophagus exhibited deep longitudinal folds which extended the entire length of the oesophageal tube (Fig.
The distinct feature of mucosal epithelium was that it was made up of stratified epithelium consisting of basal cuboidal cells, intermediate columnar mucous cells and superficial layer of flattened cells (Figs
(4) Photomicrograph of the oesophagus of T. sparrmanii showing distinct layers; mucosa (M), submucosa (SM), muscularis which consisted of inner circular (IC) and outer linear and a serosa. H&E stain. (5) An enlarged transverse section of the area marked by box in Fig.
The stomach in T. sparrmanii is morphologically differentiated into two regions: the glandular (cardiac and fundic) and non-glandular (pyloric) regions (Figs
The lumen of the pyloric stomach was narrow due to the extensive development of rugae; mean rugae width and depth of 304.29 ± 35.27 µm and 686.87 ± 76.54 µm respectively (Fig.
The sub-mucosa of the stomach was thin and highly vascularized, composed of a network of connective tissue, collagen, including blood vessels, which projected into the mucosal folds forming the lamina propria (Figs
(8) Photomicrograph of the cardiac stomach of T. sparrmanii showing mucosal fold consisting of lamina propria with numerous gastric glands (GG), and muscularis, which consisted of inner circular (IC) and outer longitudinal (OL), the serosa (S) and the epithelial layer with gastric pits (arrow). H&E stain. (9) An enlarged photomicrograph of the cardiac stomach of T. sparrmanii showing the different layers; the sub mucosa (SM), muscularis consisting of inner circular (IC) and outer longitudinal (OL) muscle layers, the serosa (S) and gastric glands (GG). H&E stain. (10) Photomicrograph of the pyloric stomach of T. sparrmanii showing villi like projections into the gastric lumen. Note the absence of gastric glands. (11) Photomicrograph showing the transition between the oesophagus and the stomach (arrows). Note the absence of AB (pH 2.5) positive cell in the pyloric region of the stomach (*). Scale bars: 8, 10, 11 = 200 µm, 9 = 50 µm.
(12, 13) Photomicrographs of the cardiac stomach of T. sparrmanii showing numerous gastric glands (GG), and AB (pH 2.5) positive neck cells (arrows). (14, 15) Photomicrographs of the stomach of T. sparrmanii showing numerous gastric glands (GG), and PAS positive epithelial and mucous cells (arrows). Lamina propria (LP); Mucosa (M); submucosa (SM). Scale bars: 12–14 = 50 µm, 15 = 200 µm.
Typically, the mucosa of the anterior intestine was folded into long and relatively narrow branched villi (Fig.
Photomicrographs of the anterior intestine (AI): (16) An overview of the anterior intestine with emphasis for zig-zag shaped villi, with columnar epithelial cells, which are well endowed with goblet cells. H&E stain. (17) Shows an enlarged image of a segment of Fig.
The mucosa of the mid-intestine was made of short longitudinal unbranched folds similar in structure to those seen in the anterior intestine except that the mid-intestine had relatively few but larger goblet cells (Figs
The posterior intestine was characterized by shorter intestinal transverse villi (Table
Thickness (µm) of tissue layers forming the wall of the intestine in T. sparrmanii. p < 0.001.
Intestinal segment | Muscle tissue layer | Villi width | Villi length |
Anterior | 105.3379 ± 9.884823a | 108.5855 ± 5.959744a | 625.9519 ± 42.06772a |
Mid-intestine | 38.20750 ± 2.045053b | 118.1115 ± 6.403478a | 368.8625 ± 11.84932b |
Posterior | 58.26950 ± 3.524109b | 214.1985 ± 13.67820b | 155.7320 ± 9.017996c |
p | * | * | * |
Photomicrographs of the middle and posterior intestine of T. sparrmanii. (22, 23) Photomicrographs of transverse sections of the middle intestine, showing mucosa, (M); lamina propria, (LP); submucosa, (SM); internal circular muscular layer, (IC); external longitudinal muscle layer, (OC) H&E stain. (24) Photomicrographs of transverse sections of the middle intestine, showing AB (Ph 2.5 positive cells (arrows). (25) Photomicrographs of transverse sections of the middle intestine, showing PAS positive cells (arrows). (26, 27) Photomicrographs of transverse sections of the posterior intestine, showing mucosa, (M); lamina propria, (LP); submucosa, (SM); internal circular muscular layer, (IC); external longitudinal muscle layer, (OC); serosa (S) and the epithelium (EP) H&E stain. Scale bars: 50 µm.
In all intestinal segments, the muscular layer had same basic organization, with an internal circular and external longitudinal muscle layers. Unlike the anterior and mid-intestine, the posterior intestine had thick muscle fibres. As in the anterior region, the distal intestine had layers of nervous tissue between the muscular layers. The serosa of the intestine was very thin with squamous epithelium. The cells were flattened with scant cytoplasm and a compressed oval nucleus. Goblet cells of the distal intestine revealed moderate AB (pH 2.5) labelling (Table
Photomicrographs of the middle and posterior intestine of T. sparrmanii. (28, 29) Photomicrographs of transverse sections of the posterior intestine, showing AB (Ph 2.5 positive cells (arrows). (LP), lamina propria; (M), mucosa; (EP), epithelium. (30, 31) Photomicrographs of transverse sections of the posterior intestine, showing PAS positive cells. PAS/haematoxylin stain. (LP), lamina propria; (M), mucosa; (EP), epithelium.
Biodiversity in tropical freshwaters ecosystems is very high however; many tropical fish species are yet to be described. For most of these tropical fish, basic baseline morphological information is very scant in the literature. This necessitated among others histological and histochemical characterization of GIT of T. sparrmanii.
Morphological characterization of the GIT in fish is essential to understanding the biology of species under various physiological and pathological conditions. The GIT of fish display remarkable difference in their morphology and functions however, as is in the current study, the stomach and intestinal walls in many teleost fish exhibit four distinct layers; the mucosa, sub-mucosa, muscular and serosa.
A thick layer of connective tissue was a characteristic feature of oesophagus of T. sparrmanii. Connective tissues aid in maintaining oesophageal wall integrity as reported in other fish species (
The presence of PAS positive cells in tissues often indicates the presence of neutral mucins, whereas AB (pH 2.5) positivity indicates presence of mucins with carboxyl groups. The presence of these two types of mucous producing cells in the oesophagus as seen in the current study has been reported in other fish such as the Pike (
The presence of neutral mucins in the oesophagus of T. sparrmanii as in other fish species may facilitate food transport into the stomach and protect the oesophageal wall from mechanical damage including participation in enzymatic food digestion and its transformation into chime (
When present, the main function of fish stomach is that of food storage and production of hydrochloric acid (HCl) to aid digestion. Morphologically the stomach in T. sparrmanii has the same basic organization in terms of cell types as in most teleost fish (
The strong PAS reaction by columnar epithelial cells in the stomach in T. sparrmanii suggests the predominance of neutral mucins in epithelial cells along the mucosal border of the stomach. Neutral mucins serve to protect mucosal surface against microorganisms and high acidity of the stomach contents. This is in agreement with the findings of
Structurally, the intestine in T. sparrmanii is similar to those described for other fish. However, the intestine is highly coiled, a characteristic which is usually seen in fish with herbivory diet. The highly coiled intestine has been described in other fish species and is believed to aid in absorption processes (
In the current study absorptive cells decreased in size towards the distal intestine, whereas goblet cells increased in size and number from the mid-intestine towards the posterior. The gradual increase in the number goblet cells from the anterior to the posterior region of the intestine has been reported in a number of fish species with different feeding habits including cichlids (
In conclusion, the functional structure of the GIT in T. sparrmanii was investigated and considered as an adaptation to its omnivore feeding behavior (having a small stomach and a long intestine). The histo-architecture and the morphology of the different regions of the GIT, including the histochemical features of the various cells lining the alimentary canal has been highlighted. These results will provide background information on gross morphology as well as some functional components of the GIT in T. sparrmanii, which are essential for the generation of data for the fish under investigation. However, further detailed studies are required at the ultrastructural level to confirm these findings.
This research work was supported by a grant from the National Research Foundation (NRF, South Africa) under TTK1207112657 grant #IUD: 84357.