Wolczuketal.2018

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Structure of the alimentary tract in the Atlantic mudskipper Periophthalmus
barbarus (Gobiidae: Oxudercinae): anatomical, histological and ultrastructural
studies
Article in Zoology · April 2018
DOI: 10.1016/j.zool.2018.04.002
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Accepted Manuscript
Title: Structure of the alimentary tract in the Atlantic
mudskipper Periophthalmus barbarus (Gobiidae:
Oxudercinae): anatomical, histological and ultrastructural
studies
Authors: Katarzyna Wołczuk, Maciej Ostrowski, Agnieszka
Ostrowska, Teresa Napiórkowska
PII:
DOI:
Reference:
S0944-2006(18)30006-0
https://doi.org/10.1016/j.zool.2018.04.002
ZOOL 25639
To appear in:
Received date:
Revised date:
Accepted date:
18-1-2018
23-4-2018
24-4-2018
Please cite this article as: Wołczuk, Katarzyna, Ostrowski, Maciej,
Ostrowska, Agnieszka, Napiórkowska, Teresa, Structure of the alimentary
tract in the Atlantic mudskipper Periophthalmus barbarus (Gobiidae:
Oxudercinae): anatomical, histological and ultrastructural studies.Zoology
https://doi.org/10.1016/j.zool.2018.04.002
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Structure of the alimentary tract in the Atlantic mudskipper Periophthalmus barbarus (Gobiidae:
Oxudercinae): anatomical, histological and ultrastructural studies
Katarzyna Wołczuka, Maciej Ostrowskib, Agnieszka Ostrowskac, Teresa Napiórkowskad
a
Nicolaus Copernicus University, Department of Vertebrate Zoology, Lwowska 1, 87-100 Torun,
b
c
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Poland
Nicolaus Copernicus University, Department of Biochemistry, Lwowska 1, 87-100 Torun, Poland
Warsaw University of Life Science SGGW, Analytical Centre, Ciszewskiego 8, 02-786 Warsaw,
d
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Poland
Nicolaus Copernicus University, Department of Invertebrate Zoology, Lwowska 1, 87-100 Torun,
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Poland
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Corresponding author: Katarzyna Wołczuk, Department of Vertebrate Zoology, Nicolaus Copernicus
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University, Lwowska 1, 87-100 Torun, Poland. Tel.: +48566114996. E-mail address:
k.wolczuk@gmail.com
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Highlights
 The alimentary tract of mudskipper does not have an anatomically distinct stomach.
 However, the stomach is well-distinguished histologically.
 Features of gastric glands and surface gastric epithelium indicate that the stomach is a
functional organ.
Abstract
The alimentary tract of oxudercine gobies is characterized by a lack of an anatomically distinct
stomach, owing to which they are classified as stomachless. Since the environment, food
requirements, and feeding habits have a significant impact on the anatomy of the alimentary tract of
fish, it was assumed that predominantly carnivorous, semi-terrestrial mudskippers would have a
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stomach. In order to verify this hypothesis, anatomical, histological, histochemical and
ultrastructural analysis of the alimentary tract of the Atlantic mudskipper Periophthalmus barbarus
was performed. The results revealed that despite a lack of clear anatomical distinction within the
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alimentary tract, there were four well-distinguished sections visible at the histological level:
oesophagus, stomach, intestine, and rectum. The division was enhanced by the presence of a pyloric
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sphincter and an ileorectal valve. The stomach contained tubular glands composed of
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oxynticopeptic cells. Gland cells had pepsinogen granules and a well-developed tubulovesicular
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network of smooth membranes, which indicates the secretion of gastric juice. The presence of
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neutral mucus in the apical region of surface epithelial cells as protection against hydrochloric acid
as well as the presence of active pepsin also confirm gastric function. However, low pepsin activity
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seems to implies low protein digestion. The results of this study indicate that the Atlantic
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mudskipper P. barbarus has a functional stomach.
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Keywords: gut sections; functional stomach; pepsin; mucus; oxudercine goby.
1. Introduction
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The alimentary tract in fishes shows structural and functional diversity (Diaz et al., 2003; Dos
Santos et al., 2015), connected with different food requirements, feeding habits, phylogeny, body
mass and shape, and even sex (Al-Hussaini, 1949; Kapoor et al., 1976; Banan Khojasteh et al.,
2009; Day et al., 2014). Major differences, observed at macroscopic and microscopic levels, have
been observed in the length of the gut, degree of its anatomical diversification, and histological
features of its particular sections. Compared to other sections, the stomach shows the greatest
variation in shape, size, and structure (Smith et al., 2000; Day et al., 2011). The true stomach is the
widening of the lumen of the anterior portion of the alimentary canal, separated from the intestine
by the pyloric sphincter. It is characterized by the presence of gastric glands, which produce
hydrochloric acid and pepsinogen. Its main functions include food storage as well as protein
denaturation and degradation in an acidic environment (Smith et al., 2000). Compared to other
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segments of the alimentary tract, the true stomach in fish appeared quite late in evolution and its
origins have never been fully explained. Its formation may be associated with the appearance of
jaws and a gradual transition from microphagy to macrophagy. It may as well be connected with
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active foraging and predation (Barrington, 1942; Ugolev, 1985). Although most fish species have a
stomach, there are cases of secondary loss of this organ among Teleostei i.e. Cyprinidae, Labridae,
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Scaridae, Cobitidae, Blennidae (Kapoor et al., 1976; Koelz, 1992; Barton, 2007; Wilson and Castro,
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2010). According to some researchers, this phenomenon may result from specific food (especially a
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particulate diet) and the formation of structures used for grinding food, i.e. pharyngeal teeth (Koelz,
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1992; Walker and Liem, 1994; Wilson and Castro, 2010).
The relationship between feeding habits and stomach development can be easily observed in the
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ontogenesis of amphibians. All carnivorous salamanders and cecilians have a well-developed
stomach in both larval and adult life. In anurans the stomach is present only in adult specimens
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(except for some rare cases), which feed on meat. Anuran larvae, which eat algae and detritus, do
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not have the true stomach. The exceptions include the egg-eating Philatus carinensis larvae and the
obligatory carnivorous Lepidobrachus laevis larvae (Wassersug et al., 1981; Ruibal and Thomas,
1988; Smith et al., 2000). In terrestrial vertebrates the absence of the functional stomach is
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extremely rare. There are only two species lacking the stomach: the platypus and short-beaked
echidna, both belonging to Monotremes feeding mainly on small invertebrates (Koelz, 1992).
Although their alimentary tract includes a gastric section, it does not contain gastric glands to
produce gastric acid.
Many studies have investigated the mechanisms of ingestion, digestion and absorption of food in
fish. The adaptations of the alimentary tract to different feeding habits and environments have also
been studied (Al-Hussaini, 1949; Kappor et al., 1976; Wilson and Castro, 2010; Dos Santos et al.,
2015). Despite extensive literature on the alimentary tract in teleost fish, the research on the
alimentary tract in gobioid fish, has been very limited (Wilson and Castro, 2010). This seems
puzzling in view of the fact that gobies, owing to their diverse food requirements and habitats, may
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serve as perfect models for investigating this issue.
Gobiidae is one of the largest fish families, with representatives living in seas, estuaries and fresh
waters throughout the world. Species belonging to the subfamily Oxudercinae are also well adapted
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to amphibian lifestyle (Polgar et al., 2010; Thacker and Roje, 2011). Gobies include carnivores,
herbivores, omnivores and detritivores (Geevarghese, 1983; Clayton, 1993; Pogoreutz and Ahnelt,
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2014). Their diverse feeding habits and habitats imply that their alimentary tract is also varied in
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terms of structure and that it has several morphological and physiological adaptations that enable
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them to survive under various conditions. So far, however, the histological structure of the
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alimentary tract has been studied only in several species, mainly from the subfamilies Gobiinae and
Gobionellinae (Kobegenova and Dzhumaliev, 1991; Hur et al., 2005; Jaroszewska et al., 2008;
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Wołczuk et al., 2015). Relatively little is known about the alimentary tract of species from the
subfamily Oxudercinae. Apart from the description of the histological structure of the alimentary
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tract in Periopthalmus sp. delivered by Kobegenova and Dzhumaliev (1991), there are no more data
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available. Limited knowledge of the structure and functioning of the alimentary tract in
Oxudercinae contributed to the variety of views on the presence/absence of the stomach in these
fishes. Takahashi et al. (2006) did not report the presence of a stomach in carnivorous
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Periophthalmus modestus, concluding that mudskippers do not have this organ. Before, Dhage and
Mohamed (1977) failed to find a stomach in Periophthalmus koelreuteri (=P. barbarus). Suyehiro
(1942) described a simple stomach in herbivorous Boleophthalmus pectinirostris and carnivorous
Periophthalmus cantonensis (=P. modestus), but his observations were based only on gross
morphology. Kobegenova and Dzhumaliev (1991) provided histological evidence for the presence
of gastric glands (as a form of a stomach) in Periophthalmus sp., but these were recognized as nonfunctional. In the light of the foregoing, it seemed interesting to undertake research into whether the
functional stomach is present in mudskippers.
Semi-terrestrial mudskippers inhabit the intertidal mudflats in tropical river estuaries
(Stebbins and Kalka, 1961). Some species have been reported to spend over 90% of their time on
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land (Gordon, 1998), where they can feed efficiently (Stebbins and Kalka, 1961; Michel et al.,
2016). The Atlantic mudskipper Periophthalmus barbarus, selected for this study, is dominantly
carnivorous (Clayton, 1993; Bob-Manuel, 2011; Chukwu and Deekae, 2013).
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With the Atlantic mudskipper as a model, the hypothesis was tested that carnivorous feeding
habits is a good predictor of functional segmentation of the alimentary tract and the presence of a
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stomach. A more specific objective of this study was to determine whether mudskippers have a
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functional stomach and whether it plays a role in protein digestion. Considering the amphibious
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lifestyle and feeding habits of this species, it was assumed that their alimentary tract would be more
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diversified than that of previously investigated fully aquatic Gobiinae and Gobionellinae species
2015).
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2. Material and methods
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(Kobegenova and Dzhumaliev, 1991; Hur et al., 2005; Jaroszewska et al., 2008; Wołczuk et al.,
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2.1. Sample collection
Seventeen adult mudskippers P. barbarus ranging 7.1 to 12.4 g body mass and 64.1 to 98.0 mm
standard body length (Ls) were used in the study. The specimens were obtained from a pet shop
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immediately after importation from Nigeria, West Africa. The alimentary tract was dissected after
fish were killed with tricaine solution (0.1% MS 222, Sigma Aldrich).
2.2. Anatomy of the alimentary tract
The shape and length of the alimentary tract was drawn on the paper from a stereoscopic
microscope equipped with a specialized eyepiece and measured with an electronic curvaturer
(Planwheel SA2, Scale Corporation) to the nearest of 0.01 mm. The length of the alimentary tract
was measured for determining relative length of the gut (RLG), which was calculated as ratio of the
alimentary canal length to Ls.
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2.3. Light microscopy
Alimentary tracts of nine individuals were fixed in 10% neutralized formalin. The fixed material
was rinsed under running water, dehydrated in series of alcohol solutions and embedded in paraffin.
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Transverse and longitudinal sections (5 µm thick) of alimentary tracts were stained using four
methods: Delafield’s haematoxylin and eosin (H-E) for general histological examination, Alcian
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Blue – Periodic Acid Schiff technique (AB-PAS) for detection of acid (AB positive), neutral (PAS
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positive) and mixed (AB-PAS positive) mucus.
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The prepared slides were used for histological and histochemical analyses, as well as measurement
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of the thickness of tissue layers forming the wall of oesophagus, stomach, intestine and rectum. The
thickness of the mucosa (epithelium and lamina propria), muscularis (outer and inner layer) and
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serosa were measured in all sections of the alimentary tract. In the stomach, the height and width of
the tubular gland, as well as the length of glandular zone were measured. For each of these
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parameters, 20 measurements were taken and their mean values were calculated. The results were
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presented as mean ± SD values. The measurements were performed using a light microscope
Olympus CX21 equipped with a calibrated eyepiece.
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2.4. Transmission electron microscopy
Small fragments of two stomachs were fixed for 24h with 2.5% glutaraldehyde in 0.1 M phosphate
buffer (pH 7.2) containing 5% sucrose. Next, the samples were postfixed in 1% osmium tetraoxide
in ddH2O for 1h, washed three times in ddH2O, dehydrated in graded alcohol solutions and
embedded in Epon resin. Thin section were stained with uranyl acetate and lead citrate (Reynollds,
1983) and examined using a Joel JEM 1220 electron microscope equipped with SIS Morada 11
Megapixel camera.
2.5. Pepsin assay
Pepsin activity was analyzed using the method detailed by Anson (1958) modified by Newton et al.
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(2015) using hemoglobin as substrate. The stomachs of six individuals were removed and
immediately frozen at −20°C for the biochemical analysis of pepsin.0.5 g of tissue were
homogenized in 5 ml of 50 mM Tris-HCl buffer (pH 7.8) using UltraTurax T25 homogenizer (IKA,
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Germany). The homogenate was centrifuged at 4°C for 10 min at 12000 g (Sigma Sartorius 3K30
Centrifuge, Germany). The incubation mixture contained 100 µl of a supernatant fluid and 100 µl of
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2% (w/v) horse hemoglobin (Sigma-Aldrich) (a final concentration of 0.155 M) in 60 mM HCl.
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This mixture was incubated for 10 min at 25°C. Next, the enzymatic reaction was stopped by
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adding 1000 µl of cold 5% (w/v) trichloroacetic acid (TCA). After that, the mixture was centrifuged
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(6000 g, 6 min, 4°C) and the supernatant fluid was used for spectrophotometric assay. The blank
sample contained 50 mM Tris-HCl buffer pH 7.8 against the tissue supernatant. The tyrosine
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released upon enzymatic reaction was determined spectrophotometrically (UVmini-1240 UV-Vis
Spectrophotometer, Shimadzu, Japan) by measurement of absorbance at 280 nm. The pepsin
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activity was calculated using molar absorption coefficient for tyrosine (εTyr) of 1.25 µmol -1 cm-1.
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One unit (U) of pepsin activity = one µmol of tyrosine released during 1 min of the reaction.
Enzyme activity was expressed as U per g of wet tissue (i.e., U g tissue-1).
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3. Results
3.1. Anatomy of the alimentary tract
RLG of P. barbarus was 0.66 ± 0.08. Particular sections of the alimentary tract were not well
defined anatomically (Fig. 1), and their identification was possible only at the microscopic level.
The oesophagus was short and smoothly transitioned into the I-shaped stomach. The short intestine
consisted of two loops and was separated from the rectum by a small narrowing.
3.2. Histological structure of the alimentary tract
The following parts were distinguished within the alimentary tract: oesophagus, stomach (fundic
and pyloric region), intestine, and rectum.
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The walls of all gastrointestinal sections of P. barbarus consisted of mucosa, and muscularis and
serosa. The mucosa was divided into epithelium and lamina propria. The muscularis consisted of
the inner and outer layer. The serosa was built of a thin layer of connective tissue covered with a
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simple squamous epithelium.
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3.2.1. Oesophagus
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The mucosa formed longitudinal folds. Epithelium of the oesophagus was a stratified squamous
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epithelium containing numerous sac-like mucous cells (Fig. 2b). The mucous cells contained a
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mixture of acid and neutral mucus. The distal part of the oesophagus contained mainly cells with
neutral mucus. Numerous taste buds were present in the epithelium of the proximal part of the
presented in Table 1.
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oesophagus. The thickness of the epithelium and its contribution to the total wall thickness is
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Lamina propria of the mucosa was composed of the connective tissue, the structure of which was
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dense directly beneath the epithelium and slightly less dense within the deeper layers (Fig. 2b). It
consisted mainly of collagen fibres.
Muscularis consisted of two layers of striated muscles. The inner layer consisted of longitudinal
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bundles of muscle fibres, located only in the side walls of the oesophagus. Bundles of muscle fibres
were separated by abundant connective tissue and some of the fibres penetrated into the lamina
propria of the mucous membrane (Fig. 2b). The outer layer consisted of bundles of circular muscle
fibres located over the entire circumference of the oesophagus (Fig. 2b). The thickness of both
layers and their contribution to the formation of the wall are presented in Table 1.
The outer wall of the oesophagus was covered with a very thin serosa (Table 1).
3.2.2. Stomach
There was a distinctive transition zone between the oesophagus and the stomach. The stratified
squamous epithelium lining the oesophagus was replaced by a simple columnar epithelium (Fig. 2a-
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c). The epithelium of transition zone was made by columnar cells secreting neutral mucus (Fig. 2c).
These cells were distinguished by the presence of microvilli, dark (electron dense) mucous
granules, an abundance of subapical mitochondria, and prominent lateral interdigitations of plasma
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membrane (Fig. 3a). Furthermore, the arrangement of muscle layers forming the muscularis
changed. The inner longitudinal and outer circular layers of striated muscles forming the muscularis
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of the oesophagus developed into the inner circular and outer longitudinal layers of smooth muscles
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forming the muscularis of the stomach.
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The gastric mucosa was distinguished by longitudinal folds. The surface epithelium was
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composed of columnar cells secreting neutral mucus (Fig. 2c-e). Columnar epithelial cells lining the
fundic and pyloric lumen closely adhered to each other and had microvilli on the luminal surface. In
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the apical part of these cells, the cytoplasm contained dark mucus granules, although more
numerous than epithelial cells in the transition zone (Fig. 3a, b). Within the epithelium there were
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no goblet cells but ovoid rodlet cells with large rod-shaped cytoplasmic granules (Fig. 2d).
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Lamina propria of the mucosa contained multicellular glands of tubular type, which were found
only in to the fundic region (Fig. 2c-e). The length of the gastric glandular region was 3.8 ± 0.5 mm.
The height of tubules forming the glands was 63.59 ± 9.88 m, and their width, 23.52 ± 1.67 m.
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Cells building the wall of the glands, i.e. the so-called oxynticopeptic cells, had microvilli on the
luminal surface, numerous mitochondria, a well-developed tubulovesicular network of smooth
membranes, and a few pepsinogen granules (Fig. 3c, d). The activity of pepsin in the gastric region
was 0.23 ± 0.03 U g of tissue-1. A layer of well-vascularised connective tissue (contained in lamina
propria of the mucosa) was found below the glands. Due to the presence of the glands, the lamina
propria formed a relatively thick layer (Table 1).
Muscularis of the stomach was mostly composed of smooth muscles, while bundles of striated
muscles forming the outer layer of oesophageal muscles were found in the gastric fundus. Smooth
muscles were arranged in two layers: the inner layer with a circular arrangement of myocytes and
the outer layer with a longitudinal arrangement. The inner layer was better developed and its
the pyloric region of the stomach, where it formed a pyloric sphincter (Fig. 2e).
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The serosa of the stomach wall was also thin (Table 1).
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contribution to the wall structure was higher (Table 1). The inner circular muscle layer was thick in
3.2.3. Intestine
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The intestinal mucosa formed high slender folds which were arranged in a longitudinally directed
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zigzag pattern.. In the transverse section of the intestine the folds were seen as villi-like projections
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(Fig. 2f). The epithelium covering the mucosa consisted of a single layer of columnar cells, the oval
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nuclei of which were located in the centre or at the base of the cell. Goblet cells, evenly scattered
between the columnar cells, contained mixed mucus (Fig. 2f).
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Lamina propria of the mucosa consisted mainly of collagen fibres, few cellular elements, and blood
vessels (Fig. 2f). As in the stomach, the muscularis consisted of two smooth muscle layers; the inner
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circular layer of muscles was thicker compared to the outer longitudinal layer (Fig. 2f, Table 1).
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The serosa had a small thickness (Table 1).
An ileorectal valve was located on the border between the intestine and the rectum. It consisted of
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prominent mucosal folds.
3.2.4. Rectum
The rectal mucosa formed high, longitudinal folds (Fig. 2g). The epithelium lining the rectum
lumen consisted of a single layer of columnar cells, which contained small supranuclear vacuoles
(Fig. 2h). Numerous goblet cells with mixed mucus were found within the epithelium (Fig. 2g).
Lamina propria of the mucosa was composed of well-vascularised connective tissue, consisting
mainly of collagen fibres. However, these fibres were more loosely arranged compared to the
intestine (Fig. 2f, g).
Muscularis formed two layers of smooth muscles, the pattern of which was identical as in the
intestine (Fig. 2f, g).
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Thin serosa served as an outer layer of the wall (Table 1).
4. Discussion
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This study confirms the hypothesis that mudskippers have a stomach and provides structural
and functional evidence of its presence. Although the stomach, contrary to our expectations, was not
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well distinguished in gross morphology, it was histologically well defined. Low pepsin activity
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indicated that protein digestion in an acidic environment was less significant than expected of
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predominantly carnivorous fish. Compared to the alimentary tract of previously studied Gobiinae or
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Gobionellinae fish (Kobegenova and Dzhumaliev, 1991; Hur et al., 2005; Jaroszewska et al., 2008;
Wołczuk et al., 2015), the alimentary tract of mudskippers seems anatomically more diversified.
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These results challenge the view that oxudercine gobies do not have a functional stomach
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(Kobegenova and Dzumaliev, 1991).
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4.1. Anatomy of the alimentary tract
The alimentary tract of P. barbarus is relatively short, which reflects the feeding habits of
this species. According to the Geevarghese classification (1983) regarding the relationship between
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the relative length of the alimentary tract of gobioids fish and their trophic level, the low RGL < 1
(Relative Gut Length) in P. barbarus (i.e. 0.66), indicates the dominantly carnivorous nature of this
species. This is in line with the observations on the Atlantic mudskipper’s food, which consists
mainly of crustacean appendages (36%), insects (20-30%), polychaetes(21%), and fish (23%) (BobManuel, 2011; Chukwu and Deekae, 2013). A similar coefficient was recorded in other Gobiidae,
particularly in species feeding on fishes and cruastaceans (Geevarghese, 1983). Simple structure
and the lack of a distinct anatomical differentiation found within the alimentary tract of P. barbarus
have also been observed in other gobies (Geevarghese, 1983; Kobegenova and Dzhumaliev, 1991;
Jaroszewska et al., 2008; Wołczuk et al., 2015). This characteristic of the Gobiidae was frequently a
sufficient basis for classifying them as stomachless (Wołczuk et al., 2015). Therefore it can be
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assumed that the morphology of their alimentary tract reflects their phylogeny. On the other hand, it
is known that in fish the size and capacity of the stomach depends on their feeding behaviour,
particularly the nature of the food, and the size of the prey (Ghosh and Chakrabarti, 2015b).
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Similarly to other gobiids, Atlantic mudskipper is benthivorous and eats small portions of food at
short intervals (Pezel, 1950; Bob-Manuel, 2011). According to researchers a large, well-developed
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stomach, whose maintenance requires a large amount of energy, is not necessary with this feeding
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behaviour (Ugolev, 1985; Koelz, 1992). However, it is found in fish which eat large portions of
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food at long intervals (Barrington, 1942; Ugolev, 1985; Koelz, 1992; Smith et al., 2001). The
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absence of a well-defined stomach may also be associated with the presence of pharyngeal teeth,
found in many benthic-feeding species including P. barbarus and other gobiids (Sponder and
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Lauder, 1981; Clayton, 1993; Parenti and Thomas, 1998; Fugi et al., 2001; Kramer et al., 2009;
Kramer et al., 2012). Numerous studies have indicated that species with well-developed pharyngeal
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teeth do not have a well-distinguished stomach because its mechanical function was partially taken
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over by these structures (Gerking, 1994; Fugi et al., 2001; Wilson and Castro, 2010). The
development of pharyngeal teeth is often considered as one of the causes of the stomach loss
(Wilson and Castro, 2010).
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4.2. Histological structure of the alimentary tract
Histological analysis revealed that the alimentary tract in P. barbarus consisted of the
oesophagus, stomach, intestine, and rectum. The sections were clearly separated by sphincters: the
pyloric sphincter between the stomach and the intestine, and the ileorectal valve, referred to as
prerectal (Oliveira-Ribeira and Fanta, 2000) or intestino-rectal (Ezeasor and Stokoe, 1980) valve,
between the intestine and the rectum. Both structures are also found in other species from the
subfamily Gobiinae, i.e. Mesogobius batrachocephalus, Neogobius ophiocephalus (Kobegenova
and Dzhumaliev, 1991), and Neogobius (=Babka) gymnotrachelus (Jaroszewska et al., 2008). Welldeveloped sphincters control food transportation between segments of the alimentary canal, prevent
food regurgitation, and divide the alimentary tract into functionally different compartments.
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The straight, I-shaped stomach was one of the histologically distinguished sections, with
several features indicative of its functionality: a) the presence of well-developed gastric glands, b)
the presence of active pepsin, c) the presence of neutral mucus in the apical region of the surface
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epithelial cells.
Gastric glands in fishes take the form of tubules and alveoles (Wilson and Castro, 2010) and their
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degree of development and distribution depend on food-intake (Kapoor et al., 1976; Diaz et al.,
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2008; Gosh and Chakrabarti, 2015a). Gastric glands in P. barbarus were tubular and densely
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distributed in the gastric mucosa and they were absent in the pyloric stomach. Among Gobiidae,
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well-developed gastric glands are also present in carnivorous representatives of the Gobiinae and
Gobionellinae subfamilies: Rhinogobius giurinus, P. semilunaris and Mesogobius batrachocephalus
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(Kobegenova and Dzumaliev, 1991; Hur et al., 2005; Wołczuk et al., 2015). However, gastric
glands in these fish take the form of alveoles, are less densely packed, and are distributed over a
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smaller area (Kobegenova and Dzumaliev, 1991; Hur et al., 2005; Jaroszewska et al., 2008;
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Wołczuk et al., 2015). There are almost no relevant data on the subfamily Oxudercinae, except for
the information provided by Kobegenova and Dzumaliev (1991). The authors reported tubular
glands in a representative of the genus Periophthalmus, but they did not provide the name of the
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species. Gastric glands in P. barbarus were composed of oxynticopeptic cells, similarly to those
observed in other fishes, amphibians, reptiles, and birds (Wołczuk et al. 2015). The main
characteristics of these cells include: the presence of a large number of mitochondria, the presence
of the well-developed tubulovesicular network of smooth membranes associated with the secretion
of hydrochloric acid, and the presence of pepsinogen in the form of secretory granules. However, in
P. barbarus pepsinogen granules were observed only occasionally and were very small. A low
number of pepsinogen granules does not determine non-functionality of gastric glands, especially in
view of the fact that our research indicates the production of hydrochloric acid and pepsin in the
stomach of this species. The presence of active pepsin was confirmed directly in biochemical
analysis, whereas the presence of neutral mucus in the apical region of epithelial cells lining the
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stomach lumen provides indirect evidence for the production of active pepsin and hydrochloric acid.
The role of neutral mucus is to protect the mucosa against aggressive gastric juices (Wilson and
Castro, 2010; Naguib et al., 2011), whose presence is one of the indicators of stomach functionality
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(Wilson and Castro, 2010; Naguib et al., 2011). In the majority of examined gobies, including a
representative of Periophthalmus genera, there was no protective mucus-secreting epithelium lining
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the stomach. This led to the conclusion that these fishes do not produce gastric acid (Kobegenova
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and Dzumaliev, 1991; Jaroszewska et al., 2008). However, the examination of the oesophagogastric
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section in carnivorous P. semilunaris demonstrated that in this species neutral mucus is produced by
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epithelial cells, hence the conclusion that gastric glands produce hydrochloric acid (Wołczuk et al.,
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2015).
We provide evidence for the functionality of gastric glands in P. barbarus, but low pepsin activity
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and a small number of pepsinogen granules in oxynticopeptic cells suggest that the acid-peptic digestion of proteins is less significant than that of carnivorous fish with a well-developed stomach
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i.a. yellowtail (Kofuji et al., 2005) or tuna (Nalinanon et al., 2008). The explanation may lie in the
fact that a large amount of pepsin is not necessary for digesting small portions of food consumed at
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short intervals. In addition, the incurred metabolic cost of producing a large amount of digestive
enzymes would be wasted, especially when low levels of substrates for these enzymes are ingested
(German et al., 2004). Although mudskipper diet may contain a significant amount of protein (protein content in invertebrates ranges from 20 to 76%) (Lira et al., 2007; Maleknejad et al., 2014; Kurimska and Adamkova, 2016), the access to that protein is limited due to the exoskeleton covering
the body of the prey. Owing to the presence of sharp, canine-like pharyngeal teeth (Sponder and
Lauder, 1981) of P. barbarus can puncture the shells of their prey and inject enzymes to their soft
inner parts (Kramer et al., 2009). However, shells occupy the space in the stomach which could
otherwise be used for receiving additional portions of protein. On the other hand, easy availability
of food consumed by Atlantic mudskippers implies that its storage in the stomach is not necessary.
Numerous studies indicate that the food strategy used by this species (frequent feeding), is very
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common in fish feeding on small invertebrates, and has evolved in many phylogenetically unrelated
species (Pezel, 1950). Living both on land and in water enables mudskippers to use food resources
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available in each of these environments (Michel et al., 2016). The provided explanation of low pepsin activity in mudskippers seems convincing, the more so because similarly low activity of this
enzyme was also found in other fish feeding on invertebrates (Yufera and Darias, 2007; Chaudhuri
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et al., 2012). However, the activity of digestive enzymes can be modified by a number of factors
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(not discussed in this study), and therefore this issue remains open.
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The oesophagus, which, together with the oral cavity and the pharynx, forms a functional
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unit responsible for food movement towards the stomach or directly into the intestine (in
stomachless fishes) has a well-developed muscularis. In P. barbarus, muscularis accounted for ca.
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48% of the total thickness of the oesophageal wall. Its inner layer consisted of longitudinal bundles
of muscle fibres separated by the connective tissue. A similar structure of muscularis was observed
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in other gobies (Kobegenova and Dzhumaliev, 1991; Jaroszewska et al., 2008; Wołczuk et al.,
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2015). According to Murray et al. (1994), this arrangement of oesophageal muscles enables the fish
to reject tasteless food, which would explain the presence of taste buds in the initial part of the
oesophagus in P. barbarus. In addition it provides reinforcement to the oesophagus, which is
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subjected to violent contracting and stretching when it propels food. Moreover, the oesophagus in
P. barbarus has a large number of goblet cells, densely distributed between epithelial cells lining the
oesophagus lumen. As in other fish species from the family Gobiidae (Kobegenova and
Dzhumaliev, 1991; Hur et al. 2005; Wołczuk et al., 2015; Hur et al., 2016), in P. barbarus goblet
cells produce a mixture of acid and neutral mucus. As can be assumed, numerous goblet cells
provide a large amount of mucus, which coats the surface of the mucosa and facilitates the
swallowing of solid material prevailing in the food of a mudskipper. The mucus also protects the
mucosa against microorganisms and mechanical injuries. The ability of goblet cells to produce both
acid and neutral mucus may constitute a mechanism facilitating adaptations to changing
environmental conditions. In P. barbarus and other fish species from the family Gobiidae, this may
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indicate an adaptation to diversified food as well as varying water salinity. The latter would involve
the osmoregulatory function of the oesophagus. The oesophagus of euryhaline and marine fishes
plays an important role in maintaining the ionic balance (Marshall and Grosell, 2006; Grosell,
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2010). According to Abaurrea-Equisoain and Ostos-Garrido (1996), mucus produced by
oesophageal goblet cells allows the passive diffusion of ions. The osmoregulatory function is
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attributed to columnar epithelial cells in the distal part of the oesophagus and oesogastric regions
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(transition zone) in euryhaline and marine fishes (Wilson and Castro, 2010). The epithelium in this
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region is devoid of goblet cells. Epithelial cells are characterised by the presence of microvilli, an
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abundance of subapical mitochondria, and prominent lateral connection with neighbouring cells
through plasma membrane interdigitations, desmosomes, and intercellular dilation (Yamamoto and
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Hirano, 1978; Arellano et al., 2001; Wilson and Castro, 2010). Cells of the transition zone in the
euryhaline P. barbarus have the qualities which may indicate the osmoregulatory function of the
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epithelium in this region.
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The intestine in P. barbarus has a uniform histological structure along its entire length. The
mucosa is strongly folded, which significantly increases the absorptive surface in the short intestine
of P. barbarus. Given the size of epithelial cells, it may be concluded that digestion and absorption
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occur mainly in the intestine, which contains the highest cells. The presence of vacuoles in the
rectal epithelium of P. barbarus shows that the absorption of nutrients takes place also in the rectal
segment of the alimentary tract. Rectal absorption of protein by endocytosis has been reported in
many fish species, including stomachless goby Neogobius (=Babka) gymnotrachelus (Jaroszewska
et al., 2008). According to researchers, it may prevent excessive loss of protein (HernandezBlazquez and Silva, 1998).
There are numerous goblet cells in the intestinal epithelium, which, similarly to those in other
species from the family Gobiidae (Kobegenova and Dzhumaliev, 1991; Hur et al., 2005) – produce
both acid and neutral mucus. The production of mixed mucus may facilitate digestion in different
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environments.
5. Conclusions
The present study indicates that the structure of the alimentary tract in P. barbarus reflects the type
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and size of consumed food, although the relationship between this structure and phylogeny of the
species is also clear. The alimentary tract of Atlantic mudskipper contains a poorly defined
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anatomically stomach, whose small capacity is probably related to the species’ food, particularly
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small portions of consumed food. As evidenced by histological, histochemical and biochemical
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analysis, the stomach of P. barbarus is a functional organ, secreting hydrochloric acid and pepsin.
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Low pepsin activity in the stomach suggests that its participation in protein digestion may be less
significant than in carnivorous fish with w well-developed stomach. Further research into digestive
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enzymes, particularly proteolytic enzyme secretion in relation to the varied diet, may clarify this
issue.
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Declarations of interest: none.
Acknowledgements
This study was supported by funds for statutory research from the Nicolaus Copernicus University
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in Toruń, Poland. We would like to thank J. Niedojadło and M. Świdziński for technical assistance,
and M. Wojciechowski for critical comments on the manuscript.
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Figure captions
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Fig. 1 –Anatomy of the uncoiled alimentary tract of Periophthalmus barbarus.
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Fig. 2 –Histology of the alimentary tract of Periophthalmus barbarus. –(a). Longitudinal section
through the oesophagus (OE), transitional zone (TZ), fundic (FR) and pyloric (PR) regions of the
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stomach (S), pyloric sphincter (PS) and intestine (I); AB-PAS staining. –(b). Transverse section
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through the oesophagus showing numerous sac-like mucous cells (arrows) in the epithelium (E,
epithelium; LP, lamina propria of the mucosa; CM, circular layer of the muscles; LM, longitudinal
layer of the muscles); AB-PAS staining. –(c). Longitudinal section through the transitional zone
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showing simple columnar epithelium (E) with neutral mucus (arrow) and lamina propria (LP)
without gastric glands (CM, circular layer of the muscles; LM, longitudinal layer of the muscles);
AB-PAS staining. –(d). Longitudinal section trough the fundic stomach showing tubular gastric
glands (GG) in the lamina propria of the mucosa, (E, surface epithelium; arrow, neutral mucus; CM,
circular layer of the muscles; LM, longitudinal layer of muscles; LP, lamina propria of the mucosa;
R, rodlet cell); AB-PAS staining. –(e). Longitudinal section of pyloric stomach showing lamina
propria (LP) of the mucosa without gastric glands (E, epithelium; arrow, neutral mucus; CM,
circular layer of the muscles; LM, longitudinal layer of the muscles); AB-PAS staining. –(f).
Transverse section trough the intestine showing high, slender, villi-like mucosal folds (MF) (E,
epithelium; LP, lamina propria of the mucosa, goblet cells (arrows); M, muscularis); AB-PAS
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staining. –(g). Transverse section trough the rectum (E, epithelium; LP, lamina propria of the
mucosa, goblet cells (arrows); M, muscularis); AB-PAS staining. –(h). Transverse section of rectal
folds showing vacuolated epithelial cells (E, epithelium; arrows, vacuoles); Methylene blue
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staining.
Fig. 3 –Electron micrographs of the transitional zone and stomach epithelium of Periophthalmus
barbarus. –(a). Surface epithelium of the transitional zone showing cylindrical cells with microvilli
(M) in the luminal surface, mucus granules (arrows), interdigitations of the lateral cell membrane
(I), and nucleus (N) at the base of the cell. –(b). Surface epithelium of the fundic stomach showing
cylindrical cells with microvilli (M) in the luminal surface, mucus granules (arrows) in the
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cytoplasm and nucleus (N) at the base of the cell. –(c-d). The gastric gland cells with microvilli
projecting into the gland lumen (LG), well-developed tubulovesicular network of smooth
membranes (TN) in the supranuclear region, numerous mitochondria (Mi), spherical nucleus (N)
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and few pepsinogen granules (arrows).
Table 1 Thickness (µm) of tissue layers forming the wall of the alimentary tract sections and the
percentage (%) of the layers in the wall thickness of the individuals of Periophthalmus barbarus
(values are means ± S.D., n = 7)
Intestine
Inner layer
Outer layer
µm
62.97 ± 8.92
71.27 ± 8.88
3.58 ± 0.80
%
10.72 ± 1.52
39.85 ± 2.02
22.57 ± 1.86
25.56 ± 1.57
1.29 ± 0.27
µm
25.64 ± 2.68
84.71 ± 8.10
12.12 ± 3.75
10.58 ± 3.99
2.59 ± 0.10
%
19.02 ± 2.23
62.53 ± 2.86
8.84 ± 2.24
7.69 ± 2.48
1.92 ± 0.17
µm
30.18±3.10
16.54 ± 2.92
17.04 ± 1.73
10.82 ± 1.67
2.53 ± 0.08
%
39.07 ± 2.47
21.33 ± 2.26
22.23 ± 2.68
14.07 ± 2.02
3.31 ± 0.27
µm
21.14 ± 3.64
24.26 ± 16.46
21.37 ± 7.90
43.15 ± 17.86
2.55 ± 0.09
%
20.09 ± 4.04
20.57 ± 6.02
19.09 ± 4.40
37.70 ± 5.85
2.56 ± 0.86
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N
Rectum
View publication stats
Serosa
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Stomach
Muscularis
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Oesophagus
Mucosa
Lamina
Epithelium
propria
29.72 ± 4.43 110.63 ± 7.55
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Region