Ultrastructural Changes in the Epithelium of the Stomach of Aphanius dispar ( Cyprinodontidae ) , Due to Stress from Starvation

ةـصلاخ : ةيلاتلا تارتفلل عيوجتلا ةجيتن يللآا رهجملا مادختساب دصلا كمس ةدعمل يئلاطلا ءاشغلل تثدح يتلا تاريّغتلا فصو مت 120,96,72,48,24 ةعاـس . دعب يئلاطلا ءاشغلا يف ريّغتلا ةيلمع تأدب 24 تلصو ىتح عيوجتلا ةرتف ةدايز عم تدادزاو ةعاس لا ىلإ ةورذ يف 96 ةعاس . لكآتو ةيطاخملا ايلاخلاو ايردنكوتيملا لكش ريغتو ةينهدلا تاريطقلا ءافتخا ةيادب تاريّغتلا هذه لمشتو ةدعملا دئاوز فارـطأ . أ ةساردلا هذه دكؤتو كمس ةدعمل ةيكيجولوفرملا تارييغتلا يف اريبك ارود بعلي ةليوط تارتفل عيوجتلا ن دصلا .   ABSTRACT: Ultrastructural changes in the epithelium of the stomach of Aphanius dispar, a cyprinodont fish, due to starvation have been described. The changes in the epithelium after 24, 48, 72, 96, and 120 hours have been discussed. The degeneration of the epithelial cells commenced after 24 hours and steadily progressed till 96 hours at which the maximum change was observed. Changes in response to starvation include the disappearance of lipid droplets, mitochondrial damage, goblet cells degeneration, morphological aberration of the nuclei and overall abnormalities in the structural integrity of the rugae. This study confirms that stress due to starvation causes significant pathomorphological changes in the stomach in four days.

istological studies describing the structure and ultrastructure of fish digestive tracts are many (Osman and Caceci, 1991;Grau et al., 1992;Gargiulo et al., 1997Gargiulo et al., , 1998)).Most of these have also attempted to relate structures to different types of feeding, nutritional requirements, digestive functions and adaptations (Kuperman and Kuz'mina, 1994;Murray et al., 1996).However, investigations exploring the histological changes imposed by physiological stress are very few.The food supply available to fish in its natural environment is not always optimum and periods of food shortage cause stress due to starvation.
Aphanius dispar (Rüppell 1828), a widely distributed cyprinodont fish in the fresh and brackish waters of the Middle East, is physiologically capable of coping with a variety of adverse environmental conditions.Recently, we described the histology of the stomach of A. dispar using light microscopy and specifically discussed the changes in the structure and number of goblet cells in response to starvation (Ba-Omar et al., 1998).This study describes the ultrastructure of the epithelium of the stomach and its changes in response to stress caused by starvation.

Materials and Methods
Specimens of A. dispar with a total length (TL) range of 30.4-46.3 mm, weighing from 0.43-0.70gm were collected from Wadi Al-Khod near the Sultan Qaboos University, Sultanate of Oman (Lat.23 ο 30' N; Long.58 ο 40'E).Fish were acclimatized for two weeks in an aerated holding tank and were fed with Tetramin flakes, ad libitum, twice daily.An experimental tank was prepared with dechlorinated water and held a large perspex visijar in its center containing the same water, with the same water level as that of the outside tank.After a stabilization period of seven days, 36 fish, irrespective of sex were randomly transferred from the holding tank to the outer area of the experimental tank.The feeding regimen for these fish was the same as that for those in the holding tank.The experiment was conducted after another acclimatization period of seven days.At the start of the experiment, 30 fish were randomly transferred to the central visijar and were deprived of food.The six remaining control fish in the outer area of the experimental tank were fed ad libitum, twice daily as usual.The water in the central visijar was replaced with dechlorinated, prestabilized water at short intervals to prevent coprophagy.Six starving fish were randomly sacrificed at each interval of 24, 48, 72, 96 and 120 hr for histological studies.At the end of the experiment, the six control fish, fed throughout the experimental duration were also sacrificed for histological investigations. .The fish were placed in ice bath and were immediately decapitated.The entire stomach from each fish was then removed and immediately fixed at room temperature.The methods for the preparation of specimens examined under light microscopy are given elsewhere (Ba-Omar et al., 1998).
For transmission electron microscopy, the stomach was fixed in Karnovsky fixative buffered with sodium cacodylate to a pH of 7.4 for four hours and then cut into small pieces.These were washed in cacodylate buffer and then post-fixed in 1% aqueous solution of osmium tetroxide for 1 hour and dehydrated in a series of alcohol before embedding in Agar 100 resin.Semi-thin and ultrathin sections were cut using Reichert ultramicrotome.The semi-thin sections were stained with toluidine blue and the ultra-thin sections were stained with uranyl acetate and post-stained in lead citrate.The sections were examined using a Philip109 transmission electron microscope.

Results
The stomach of A. dispar is a hollow organ with four different layers: mucosa, submucosa, muscularis and serosa.The mucosa, thrown into folds of variable lengths (rugae) is composed of columnar epithelium with striated border (     After 24 hours of starvation, the epithelial cells of the mucosa are columnar in shape with most of the nuclei elongated in shape (Figure . 5).Most of the mitochondria were seen toward the apical side.The damage caused to the mucosa was similar in fish starved for 96 and 120 hours (Figures. 10,11).The damage to the apical part of the rugae was severe characterised by extensive cell degeneration and the disappearance of microvilli (Figures. 10,11).Most of the goblet cells were round and seen away from the surface and close to the basal part of the rugae (Figure .11).

Discussion
The general histology of the stomach of A. dispar and its similarities to that of other teleosts was earlier discussed by Ba-Omar et al. (1998).What is referred to as stomach in this work is the morphologically distinct enlarged, sac-like portion of the gut separated from intestine by a constriction.The ultrastructure of the stomach as revealed by electron microscopy here, also confirms striking similarities between A. dispar and other freshwater teleosts (Gargiulo et al. 1997(Gargiulo et al. , 1998)).The stomachs of A. dispar with large number of well defined goblet cells in the apical region of the mucosa seem typical of omnivorous and herbivorous feeders.Osman and Caceci (1991) suggested that the gastric epithelial cells specialized for the secretion of neutral mucin and the increase in surface area provided by the dense striated (= brush) border were adaptations for the surface absorption of nutrients in Oreochromis niloticus.Although A. dispar has similar morphology of the mucosa, its role in nutrient absorption needs to be verified by histochemical techniques.The large number of lipid droplets seen in well fed A. dispar, are probably absorbed from the commercial food with at least 5% lipid content.The turnover time for lipid in the intestinal epithelium is only a few hours.Therefore, it is not surprising that the epithelia of fish starved for 24 hours were devoid of lipid droplets.
The ability to cope with stress is an important parameter determining the survival of fish.Stress is known to induce physiological and pathomorphological changes (Boddingius, 1976;1993;Pottinger and Pickering, 1992, Pottinger et al. 1994, Szakolczai, 1997).In the European eel, stress caused the atrophy of the glandular tissue layer of the stomach (Peters, 1982).Stress during harvest and transport of common carp resulted in the reduction of the number of goblet cells and the detachment of columnar epithelial cells from the basement membrane (Szakolczai, 1997).
Studies on pathological changes caused by stress due to starvation are only a few.In several teleosts including A. dispar, the responses to starvation include the reduction in mucosal mass and the decline in the number of goblet cells (Boge et al., 1981;McLeese and Moon, 1981;Ba-Omar et al., 1998).It is well known that goblet cells secrete mucuous which is used for lubrication and for protection of the mucosa against chemical and physical damage.Since there is a reduction in the number of goblet cells in the stomach then this will result in the damage of the epithelial cells and other tissues (Ba-Omar et al 1998).This study documents, at the ultrastructural level, the process of gastric erosion caused by starvation.The significant sequence of events from 24 -120 hours of starvation are, the disappearance of lipid droplets, withdrawal of goblet cells from the apical surface, damage to the striated border, a steady degeneration of mitochondria, degeneration of goblet cells and the disappearance of microvilli.

Figure 1 .
Figure 1.Light micrograph (LM) of a transverse section of the control fish showing rugae of the stomach (large open arrowheads) and the striated border (Sb) (x100).

Figure 5 .
Figure 5. Transmission electron micrograph (TEM) of fish after 24 hours of starvation showing the the columnar cells with their nuclei (N), Goblet cells (Gc) and the striated borders (Sb) (x980).

Figure 8 .
Figure 8. Transmission electron micrograph (TEM) of fish after 72 hours of starvation showing darkly stained cells and their degeneration characteristics such as the mitochondria (small arrowheads).It shows also a round Goblet cell (Gc) and the striated borders (Sb) (x2800).

Figure 9 .
Figure 9. Transmission electron micrograph (TEM) of fish after 72 hours of starvation showing a basal part of a rugae with darkly stained cells showing degeneration characteristics such as empty spaces (arrowheads) and irregularly shaped nuclei (N) (x7500).

Figure 10 .Figure 11 .
Figure 10.Transmission electron micrograph (TEM) of fish after 96 hours of starvation showing a damaged apical part of a rugae (large open arrowhead), round Goblet cells (Gc) and degenerated part of the rugae (Dc) (X1300).