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Aquaculture, 61(1987) 241-257 Elsevier Science Publishers B.V., Amsterdam




in The Netherlands

Development of the Digestive Tract of Milkfish,Chanos chanos (Forsskal) : Histology and Histochemistry R. P. FERRARIS*,

J. D. TAN and M. C. DE LA CRUZ’

Aquaculture Department, Southeast Asian Fisheries Development Center, Tigbauan, Iloilo (Philippines) ‘Faculty of Applied Science, Hiroshima University, Fukuyama 720 (Japan) SEAFDEC




No. 192

20 August 1986)


ABSTRACT Ferraris, R. P., Tan, J. D. and De la milkfish, Chanos chanos (Forsskal):

Cruz,M. C., 1987. Development histology and histochemistry.

of the digestive tract of Aquaculture, 61: 241-257.

The digestive tract of the newly hatched milkfish larva is a simple undifferentiated tube. Three days after hatching, differentiation of the esophagus begins with development of mucous-secreting cells. At this time, the intestine can be distinguished from the anterior portion of the digestive tract by its tall columnar cells with centrally located nuclei and brush border with cytoplasmic projections. After 14 days, mucosal folds develop in the esophagus. In 21-day-oldlarvae, the stomach differentiates into the cardiac and pyloric regions while goblet cells start to develop in the intestine. In fish undergoing metamorphosis ( 2 42 days old), the mucosal cells of the cardiac stomach develop into two distinct cell types: the columnar cells which make up the folds nearest the lumen, and the cuboidal cells which constitute the gastric glands. The cardiac stomach is the only region in the digestive tract where mucus secretion is not acidic. From 3-day-old larvae up to the older stages, alkaline phosphatase is localized only at the brush border of the intestinal epithelial cells. Aminopeptidase is also found only in the brush border of enterocytes, but only in 21day and older milkfish. Intestinal e&erases are present not only in the brush border but are also diffusely distributed in the cytoplasm of enterocytes of 3-day or older fish. E&erase is also found in both the columnar and gland cells of the cardiac stomach, but only in postmetamorphic (60day or older) fish. These morphological and hi&chemical changes of the gut seem to parallel dietary and habitat shifts throughout development, which encompasses life stages spent in pelagic, coastal or inland waters.


The milkfish ( Chanos chanos Forsskal) is a euryhaline, eurythermal species that inhabits a variety of habitats throughout its life history. The larvae are *Present (U.S.A.)



of Physiology,

UCLA Medical

0 1987 Elsevier Science Publishers



Los Angeles, CA 90024


generally found in shallow marine coastal waters, the juveniles from estuarine, lacustrine or riverine areas, and the subadults and adults from pelagic waters. These various stages may subsist on organisms that inhabit these areas so that feeding habits may change from fry through juvenile to adult (Tampy, 1958; Chandy and George, 1960). Similar to most species, larval milkfish tend to be carnivorous, feeding mainly on zooplankton, while juveniles and adults are omnivorous and select from a wide range of plant, animal and detrital material (Chacko, 1945; Juliano and Rabanal, 1963; Vicencio, 1977; Liao et al., 1979; Senta et al., 1979; Ferraris et al., 1986). Morphological changes of the digestive tract that accompany growth were therefore studied, because such changes may be related to diet, habitat as well as body form. Because 0.75 million hectares are being utilized to culture milkfish in several Asian and Pacific countries, and because efforts to breed milkfish in captivity have intensified (Juario et al., 1985)) information gathered from this study may be utilized in diet formulation for the different life stages. Several authors have described the digestive tract of milkfish, but earlier studies were limited to macroscopic, histological or histochemical observations of a single anatomical region or life stage (Chacko, 1945,1949; Kapoor, 1954; Chandy, 1956; Chandy and George, 1960). To obtain information on the structure of and enzyme distribution in the major organs of the digestive tract, the esophagus, cardiac stomach, pyloric stomach and intestine from milkfish of various ages were therefore studied using histological and histochemical techniques. MATERIALS AND METHODS

Fish were obtained from hatchery (for larvae up to metamorphosis; temperature range: 26-29’ C; salinity range: 27-33 ppt ) , pond ( for juveniles; 24-36’ C; 15-24 ppt) and cage-reared (for subadults; 25-33°C; 25-34 ppt) stock. Larvae were reared according to Juario et al. (1984) ; juveniles were fed natural food (lablab) and rice bran; subadults were maintained on commercial pellets (20% protein) given twice daily at 1.5% body weight. The following stages were sampled: larvae at hatch, and at days 3,5,7,14, and 21, during metamorphosis (42 days), juveniles (2 and 7 months) and subadults (2 and 4 years). At least three individuals from each stage were examined. Regions studied were the esophagus, cardiac and pyloric stomach, and the anterior, middle and posterior intestine. Tissues for histology were fixed in Bouin’s fluid, embedded in paraplast, and 6-pm sections from each region were stained with hematoxylin and eosin, Mallory trichrome, and alcian blue-periodic acid-Schiff (Humason, 1979). Tissues for histochemistry were fixed in 80% isopropyl alcohol, absolute acetone or calcium formalin. These were then embedded in paraffin (45’ C ) . The methods of Gomori (1941)) Gomori (1955)) and McManus and


Fig. 1. General morphology of milkfish digestive tract. E=Esophagus, CS=cardiac stomach, PS =pyloric stomach, PC =pyloric caeca, I= intestine, R=rectum. In vivo the entire digestive tract is highly coiled, and forms a compact mass in the abdominal cavity.

Mowry (1960) were used to localize alkaline phosphatase, ylic esterase, and leucine aminopeptidase, respectively.




At hatch, the alimentary canal is a simple, undifferentiated tube throughout its length. Three days later, the digestive tract becomes differentiated into the esophagus, stomach and intestine. After 21 days, it attains the four-tissuelayer arrangement characteristic of adult vertebrates. Fig. 1 shows the fully differentiated digestive tract of a 110-g juvenile milkfish. The intestinal to fork length ratio increases significantly as a function of size, so that in 2-g fish, it is 2.10t0.07 (50) [mean+s.e. (n)], in 10 g, 2.63t0.10 (42), and in 110 g, 3.06-+0.14 (26).The regions shown can be distinguished macroscopically after metamorphosis of the larvae to juveniles.


Fig. 2. Cross-section of the esophagus from a 42-day-old milkfish. The longitudinal muscle layer (LM) is well defined. In the esophagus, this layer is more interior to the mucosal than to the circular muscle layer (CM). The mucosal cells (C) as well as the mucoid secretory products (MSP) form the mucosal epithelium. x 132.

Esophagus The esophagus can be distinguished from other regions by the appearance of secretory products from the epithelial cells 3 days after hatching. These structures stain blue in all developmental stages with alcian blue-PAS, indicating the presence of acid mucopolysaccharides. The development of the musocal folds is first observed in the 14-day old larvae. In the various stages of development, the mucosa is not directly separated from the submucosa because the lamina propia cannot be differentiated from the submucosa. Both lamina propia and submucosa are made up of fibrous connective tissue. When the larvae become 21 days old (fry stage), the striated circular muscle layer and adventitia become distinct, and the esophagus acquires the four-layer arrangement characteristic of adults. During metamorphosis, the striated muscular layer subdivides into inner longitudinal and outer circular layers, an arrangement found only in the esophagus (Fig. 2). In the 2-month-old juvenile stage, the mucosal folds first show secondary branching. Fat cells and a third muscle layer (oblique) are first observed in the 7-month-old fish. Epithelial cells in the esophagus of 2-year-olds become organized into lobules (Fig. 3). Throughout the various stages, the adventitia remains thin and uniform.

Fig. 3. Cross-section of the esophagus from 2-year-old fish. The mucosal cells (C) are organized into distinct lobules (L) which sometimes display a common channel (CH) leadingto the mucoid secretory products (MSP) which line the lumen of the esophagus. x 33.

Stomach Three days after hatching, the stomach is distinguished from the intestine by the presence of cuboidal cells with central nuclei. Cytoplasmic projections into the lumen are not present. Differentiation into the cardiac and pyloric regions does not occur until 21 days after hatching. Cardiac region. In larvae 21 days old, a thin layer of circular striated muscle develops in the cardiac region (Fig. 4). The epithelial cells become columnar and are organized into folds 42 days after hatching (Fig. 5). The secretions which form a distinct non-cellular layer above the columnar cells stain red with alcian blue-PAS, indicating that they are neutral or basic mucopolysaccharides. Apparently, this is the only region of the milkfish digestive tract where mucus secretion is not acidic. In addition, glands also develop next to the epithelial or gastric cells (Fig. 5 ) . The transition between gland cells and epithelial cells is abrupt and distinctive. Cuboidal cells which constitute these glands seem to be of one kind only. The glands are simple and tubular. A striated, circular muscle layer also appears in the region below the glands. In the 7month-old milkfish, the cardiac glands proliferate while the acellular matrix becomes thicker relative to the columnar cell folds. Fat cells become evident in the submucosa and/or muscle layers. An oblique muscle layer is inserted throughout the circular layer in the 4-year-old milk&h.


Fig. 4. Cardiac stomach of milkfish larvae. After 21 days, the circular muscle layer ( CM 1 appears between the serosa (S) and the mucosa (M) . The mucosal cells (C) become columr rar mrhile their nuclei (N) occupy a position more basal than that in 3-day-old larvae. x 132

Fig. 5. Cardiac stomach during metamorphosis. After 42 days, the mucosal cells de7relol ) into two distinct cell types: the columnar cells (C ) which are lightly stained and which mak :e up I the folds nearest the lumen, and the dark-staining cuboidal cells (Cu) which compose the c:ardi iac glands (CG) found below the columnar cells. An acellular matrix (AM) lines the lumen of stomach. x 132.


Pyloric region. The pyloric region is distinguished

by the development of thick, smooth circular muscle in the 21-day-old larvae (Fig. 6). During metamorphosis, the tall, columnar, mucosal cells are arranged into folds, and an acellular matrix begins to line the lumen (Fig. 7). In the older stages, the pyloric region shows the same features, with mucosal cells growing taller. The mucosal folds become deeper relative to the cardiac region, and the smooth muscle layer and acellular matrix become thicker. Although the circular muscle layer hypertrophies, the longitudinal muscle layer cannot be found even in the 4-year-old specimen. The epithelial cells stain blue with alcian blue-PAS indicating their acid mucopolysaccharide content.

Three days after hatch, the intestinal region becomes differentiated from the rest of the digestive tract by the presence of tall columnar cells with (a) centrally located nuclei, and (b) brush border with cytoplasmic projections (Fig. 8). The length of these projections (sometimes called cilia) is about 0.125 x the average length of the cell. No qualitative histological differences were observed among the various regions of the intestine as described by Chandy and George (1960). The smooth circular muscle layer develops and goblet cells

Fig. 6. Pyloric stomach of milkfish larvae. In the younger stages, the pylorus can be distinguished from the cardiac region because of the thick circular muscle layer (CM) and tall columnar cells (C) with basal nuclei (NJ. X 132.


Fig. 7. Pyloric stomach during metamorphosis. A thick acellular matrix (AM) begins to line the lumen while the mucosal cells (C) are organized into folds. SM = Submucosa, C:M = circular muscle layer. x 132.

Fig. 8. Cross-section of intestine from milkfish larvae. Three days after hatct ting, the intestine may be distinguished from the other regions by the presence of cytoplasmic prc ejections (CP) on the brush border (BB) of the columnar cells (C) with centrally located nuclei (N). x330.


start to proliferate only during the metamorphosis stage. Goblet cells of all developmental stages stain blue with alcian blue-PAS, indicating their acid mucopolysaccharide content. In the 7-month-old milkfish, the mucosal epithelium shows secondary branching and the outer longitudinal muscle layer becomes distinct. Enzyme histochemistry Both alkaline phosphatase and aminopeptidase are localized only at the brush border of the intestinal epithelial cells (Table 1) . Alkaline phosphatase appears in the intestine of milkfish 3 days or older. Aminopeptidase, however, is not present in the early larval stages, and may be found only in larvae and postlarvae 21 days or older. Esterase is present in the brush border as well as being diffusely distributed in the cytoplasm of enterocytes. Intestinal esterases (both brush border and cytoplasmic) are found in 3-day-old larvae or older fish. Esterase is also intensely localized in the cytoplasm of both the columnar and gland cells of the cardiac stomach but only after metamorphosis (60-day-old and older fish ) .

TABLE 1 Histochemical Enzymes

distribution of enzymes in the digestive tract of milkfish Esophagus




Alkaline phosphatase Aminopeptidase E&erase


Brush border
















+++ +++



- = Absent; + =diffuse;

+ + =moderate;


+ + + =intense.


The development of the digestive system of milkfish during the yolk sac stage is similar to most other larval teleosts examined (Tanaka, 1973; Lopez, 1982)) probably because larval fish require only the most rudimentary system for yolk resorption. During metamorphosis and further growth, this system can be developed to adapt to the feeding habits of the fish. Differentiation of the digestive tract seems asynchronous, proceeding from distal and proximal


to central (presumptive stomach) regions. Differentiation soon after hatching would allow early feeding. By the time yolk is resorbed after 2-3 days, differentiation is distinct, so that it coincides not only with development of the ability to produce certain enzymes (intestinal esterase and alkaline phosphatase) , but also with eye pigmentation and onset of first feeding. The first of two “critical periods” observed in larval culture of milkfish occurs at this developmental stage (Juario et al., 1984) when larvae shift from endogenous to exogenous nutrition.

Esophagus Because its cells multiply to form a pseudostratified layer of cells and secretory products in the mucosa, a relatively complex tissue organization may be attained by the esophagus 2 or 3 days after hatching. The turgid appearance of the mucus cells suggests active secretion. Early development of this region may be important at the onset of first feeding and/or essential in other functions such as osmoregulation. If the function of these structures is primarily digestive, then the mucus may facilitate the movement of food and the mucosal folds would allow for distention during food consumption, or increase the area for digestive activities. Some authors have reported the presence of enzymes in the esophagus of tilapia and perch (Fish, 1960; Nagase, 1964). Milkfish, however, does not ingest bulky food. Moreover, the folds develop very early in its life stage when the main food items are zooplankton. Because habitat changes also occur at this time (Table 2)) it is possible that these developmental events in the esophagus of milkfish larvae are preparatory and connote an osmoregulatory preadaptation leading to the development of euryhalinity (Hoar, 1976). The esophagus is an important osmoregulatory organ in the euryhaline fish Anguillu anguillu (Hirano and Mayer-Gostan, 1973). During changes in environmental salinity, the esophagus of A. anguilla undergoes morphological changes (Yamamoto and Hirano, 1978) so that during freshwater transformation, there is an increase in the luminal area of the esophagus due to development of complex folds, and mucus is substantially extruded (Meister et al., 1983). The second critical period observed during mass culture of milkfish occurs on the 7th day after hatching, when milk&h are markedly stenohaline (P. S. Young, personal communication, 1986; Duenas and Young, 1983). At this time, the only major event observed in the digestive tract is the start of the development of mucosal folds in the esophagus (Table 2). Thus, the changes in the esophagus of milkfish larvae in the process of entering less saline habitats seem to exhibit some similarity to those described for the eel (A. anguilla) undergoing freshwater transformation, and coincides with the second critical period of milkfish culture.

251 TABLE 2 Summary

of developmental

events occurring

in milkfish digestive tract (see text for details


Age 3-5



42 days

2-7 months

2-4 years


Secretions from epithelial cells

Mucosal folds develop

Presence of four tissue layers

Inner longitudinal and outer circular muscles

Mucosal folds show secondary branching

Lobular organization of cells


Cuboidal cells with central nuclei

Cardiac and pyloric regions develop

Glands develop in cardiac: acellular matrix in pyloric

Cardiac glands proliferate

Circular muscle hypertrophies in pyloric


Brush border with cytoplasmic projections

Goblet cells proliferate

Mucosal epithelium shows secondary branching


Enzymes Alkaline phosphatase

Present in brush border of enterocytes



Present in brush border of enterocytes Intestinal esterases

Esterases cardiac stomach


Assumed food Zooplankton

and some phytoplankton



Habitat Pelagic (?)




Estuarine and freshwater


When milkfish return to oceanic environments as subadults, the esophagus again undergoes distinct transformation ( Fig. 3 ) . The elaborate development of the mucosa in adults suggests that one of the chief functions of the esophagus is still mucus production. The mucus layer alters permeability to ions,


and its composition seems to differ between freshwater and seawater eels (Olivereau and Lemoine, 1971a, b) . Taste buds have not been detected in milkfish esophagus in this or in a previous study (Chandy, 1956). This suggests that milkfish may not be as selective of its food as other species (Reifel and Travill, 1977; Sis et al., 1979; Hirji, 1983). The acid mucopolysaccharides found in the esophagus in all developmental stages are characteristic of most teleosts (Tanaka, 1973). The arrangement of inner longitudinal and outer circular layers of the muscularis in the milkfish esophagus differs from the rest of the digestive tract as in other fishes ( Al-Hussaini, 1947; Liem, 1967). In milkfish, the inner longitudinal muscle seems to be embedded in the submucosa as in perch (Hirji, 1983 ) . Stomach The development of the stomach occurs later than the other regions of the digestive tract. Although the oesophageal and intestinal regions can already be distinguished 3 days after hatching, the presumptive stomach region between the esophagus and intestine remains simple and undifferentiated until the development of muscle layers at 21 days. The age (21 days) at differentiation of both the cardiac and pyloric regions is similar to most teleost larvae (Tanaka, 1973; Lopez, 1982)) and coincides with the time when milkfish shifts from oceanic to coastal and estuarine waters. The development of the gastric glands, however, occurs later in milkfish (42 days) than some species (20-25 days for Pagrus major and Acanthopagrus schlegeli) , and earlier than others (about 100 days in Plecoglossus altiuelis and Salmo gairdneri; Tanaka, 1973). Prior to the development of these glands, milkfish may rely on mechanical digestion, proper food selection and intestinal enzymes to compensate for the absence of gastric enzymes. The absence of .these glands should be considered when formulating diets for milkfish larvae. Cardiac. The mucosa of the corpus or cardiac stomach is composed primarily of two functional layers: the simple columnar cells lining the luminal surface and secreting neutral or basic mucopolysaccharides, and the gastric glands which are simple, tubular and apparently comprising only one cell type which probably secretes both hydrochloric acid and enzymes (Kapoor et al., 1975). The gastric histology of milkfish corpus is therefore similar to other teleostean species (Bucke, 1971; Reifel and Travill, 1978). Unlike some carnivorous species possessing a cardiac stomach (Esox spp., Reifel and Travill, 1978) the surface epithelial cells of milkfish contain neutral mucosubstances, the only region in the digestive tract where non-acidic mucopolysaccharides are secreted. These mucopolysaccharides may serve to protect these cells from auto-digestion by the HCl and enzymes produced by the gastric glands. In the other fishes


examined by Tanaka (1973)) gastric glands develop simultaneously with the elongation of the stomach, appearance or elaboration of some muscle layers, and an increase in the number of teeth and of intestinal goblet cells. Pyloric. The development of the pyloric stomach coincides with cessation of pelagic life at 18-21 days, suggesting that the onset of mechanical digestion may be related to a change in dietary habits. The mucosal cells lining the lumen of the pylorus are much taller than those of the cardiac stomach. These cells may be responsible for the secretion of acidic mucopolysaccharides and the acellular matrix. This matrix and the thick muscle layer become very prominent at 42 days, although Chandy and George (1960) did not observe this even with older specimen (fingerlings). The microscopic structure and function of the pylorus may be comparable to that of an avian gizzard (Chandy and George, 1960). Unlike the pylorus of carnivorous esocids (Reifel and Travill, 1978)) absence of glands in the pylorus of milkfish suggests that the pylorus may not contribute enzymes or hydrochloric acid to further chemical digestion, but may only serve to facilitate such digestion by mechanical means. Intestine Except for changes in cell dimension, thickness of muscle layers and the degree of mucosal folding, the histological structure of the milkfish intestine, unlike those of other regions, is similar across all developmental stages except the earliest ones. The histological characteristics of intestinal epithelial cells of various teleostean species are also very similar (Ozaki, 1965; Yamamoto, 1966; Kapoor et al., 1975). This similarity in intestinal structure across developmental stages and across species is probably related to the relative homogeneity of partially or fully digested food items (e.g. mono- and disaccharides, free amino acids, short peptides) that this hindmost region of the digestive tract digests and/or absorbs. In contrast, the more anterior regions of the digestive tract are in closer contact with the environment, and encounter food in a greater variety of forms (e.g., intact proteins and complex sugars in the stomach; tissues in the esophagus). The structural similarity of milkfish intestine across intestinal regions is like that of many herbivorous fish(Ferraris and Ahearn, 1984). Prior to the development of gastric glands and a muscular pylorus, the milkfish relies primarily on the intestine for digestion and absorption. Thus, 3 days after hatching, the intestinal mucosal cells become similar to those of adults as these become fully differentiated from the rest of the digestive tract. While some teleost larvae also have intestinal mucosal cells which display cilia projecting into the lumen interior, these cilia disappear in the postlarval stage (Tanaka, 1973). However, milkfish and some other teleosts (Kapoor et al., 1975) retain this apparently primitive characteristic, which may function to


aid in food movement, even in adults when the development of smooth muscle layers can already assume this function. This is not surprising because Chunos chanos is a relatively primitive species (Greenwood et al., 1966). Secondary mucosal folds develop in 7-month-old milkfish, thereby increasing absorptive area without necessitating any increase in length of the gut. This may be advantageous because at this stage the ratio of the intestinal to body length no longer increases as rapidly as in the younger stages. The change is apparently correlated with return to pelagic existence and subsistence on marine life (Table 2 ) . Hktochemistry

Absence or low concentrations of enzymes during the yolk sac interval (O-2 days) of milkfish is similar to that found for acipenserids (O-10 days) when their embryos are dependent on endogenous nutritional reserves for metabolism (Buddington and Christofferson, 1985). From onset of active feeding to metamorphosis, Buddington and Christofferson then describe a rapid increase in enzyme diversity as well as in amylase and lipase concentrations. The latter is an adaptation to a zooplanktivorous diet whereby the high lipolytic activity would enhance the digestion of large amounts of lipids (up to 50% of body weight; Tessier and Goulden, 1982) and wax esters in this type of diet. Along with alkaline phosphatase, we observe esterase activity in milkfish enterocytes at the onset of feeding (3 days old), but not aminopeptidase activity. Thus, esterases may be more essential than aminopeptidases to milkfish larvae, which subsist on a diet of rotifers, copepods and brine shrimp (Juario et al., 1984) at this stage of their development. Aminopeptidase is localized only in the brush border of the milkfish intestinal cells (Table 1) as in many other teleosts examined so far ( Al-Hussaini, 1949; Chao, 1973; Hirji and Courtney, 1982). This enzyme system appears late in the larval stage (Table 2). Alkaline phosphatase has been demonstrated only in the brush border of milkfish intestine (Table 1) , a finding similar to other teleosts (Prakash, 1961; Chao, 1973; Goel and Sastry, 1973; Tanaka, 1973)) and its development may be coincident with the appearance of the brush border. Its function appears to be associated with nutrient transport in fish intestine (Stroband et al., 1979)) hence the presence of this enzyme may be essential before the intestine can become fully functional at the onset of feeding (Prakash, 1961; Tanaka, 1973). Some studies, however, reported that this enzyme has been detected in other parts of the digestive tract, namely, the stomach, pyloric caeca, liver and pancreas (Prakash, 1961; Goel and Sastry, 1973). Localization of non-specific carboxylic esterases in the cardiac stomach soon after metamorphosis when the gland cells develop coincides with the time when milk&h begin to inhabit brackish and freshwater areas and become opportun-


istic and omnivorous feeders. This suggests that development of the cardiac stomach and many of its enzymes may be related to a change in feeding habits from larvae (fry) to fingerling ( > 42 days old) stages, just as development of the intestine and its esterases may be related to a change from yolk (endogenous) to zooplankton nutrition. ACKNOWLEDGEMENTS

This study was partially supported by the International Development Research Centre (IDRC) of Canada under grant no. 3-P-74-0146 to the milkfish project. We thank R. Rudio, S. Torrento, and S. Gentuya for their technical assistance, and R. Buddington (UCLA), G. Enriquez (UP) and P. Young for valuable discussion and for criticizing an earlier version of the manuscript.

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