- Email: [email protected]
Digestive System
CHAPTER
Digestive System Randal K Buddington Department of Biological Sciences, Mississippi State University, Mississippi, USA
m
I#l -4 m
Victoria Kuz'mina Institute for the Biology of Inland Waters, Borok, YaroslavI, Russia
Introduction Even though the gross anatomy of the digestive tract, particularly the alimentary canal, is highly variable among fish, the types of cells and tissues (i.e. the 'building blocks') are conserved. An appropriate analogy is how houses built for different environments may vary in structure, but the same basic materials are used for their construction. Similar to all other vertebrates the different regions of the alimentary canal consist of the same four basic tissue layers (Figure 23.1). The internal mucosal layer consists of two components. The first is the epithelium which serves as a selectively permeable membrane that separates the body from the outside (lumen). The epithelium lining the two ends of the digestive system develops from two invaginations: the stomodeum forms the mouth and the proctodeum forms the anus. The epithelium lining the remainder of the alimentary canal is derived from endoderm, with evaginations of this tissue forming the accessory organs, including the liver and gall bladder, pancreas, and air bladder. Directly under the
Copyright 9 2000 Academic Press
-4 HI
epithelium is the lamina propria, which is largely connective tissue. Capillaries are present in the lamina propria and they allow for the exchange of materials between the host and the outside environment, using the epithelium as the site of exchange. Specifically, nutrients absorbed from food are transferred to the blood in the capillaries, and conversely, the blood can deliver other materials or by-products of metabolism
i N
O O "ID "R C Z i
O Z
Dorsa
r
Z
,al O -<
Lamina p Epithelium
Figure 23.1 The organization of the tissue layers in the fish alimentary canal.
111 I--
>. i11
ILl
@ >-
O
F< Z
<
...-I
for secretion into the intestine. In the intestine lymph vessels can be seen and these are involved in the absorption of fat, other nutrients and fluids. A thin layer of smooth muscle known as the muscularis mucosa can sometimes be seen between the lamina propria and the deeper tissue layers, but it is less developed in fish compared to that of mammals. Extensions of the muscularis mucosa, when present, penetrate up into the villi and provide the villi with the ability to move, which effectively 'stirs' the layer of fluid overlying the epithelium. This reduces the thickness of the boundary layer that covers the epithelium and limits diffusion of nutrients down to the absorptive cells, and by so doing enhances the absorption of the luminal contents. The second tissue layer is called the submucosa. This layer supports the mucosa and consists mainly of connective tissue. The submucosa is penetrated by nerves and blood vessels that extend up to the mucosa. The third tissue layer is known as the muscularis. This layer is actually composed of two coats of smooth muscle. The inner circular layer is arranged such that the fibers loop around the lumen. Fibers of the second, or longitudinal, layer are oriented in a proximal to distal direction. Contractions of the muscle layers are important for the segmental contractions that mix food items and the peristaltic contractions that propel the chyme in a distal direction. The fourth, and outermost layer, is known as the serosa. In reality, the serosa is the visceral peritoneum that covers the alimentary canal and other viscera lying within the peritoneal cavity.
Z
0 F-U Z Ii
Mouth and Pharynx
U
9 U
0 U
The microscopic anatomy of the fish mouth and pharynx is basically the same as that seen in other vertebrates. Only the macroscopic structure varies widely, corresponding with the wide diversity of food habits. Mucous cells are interspersed among the epithelial cells lining the oral cavity and pharynx. Salivary gland cells are present, but unlike other vertebrates they exist as solitary cells, not multicellular glands. The secretions from the mucous and salivary gland cells serve to lubricate the food, and in some filter feeders help to 'trap' small particles. Taste buds, which consist of elongate sensory cells with accessory cells, can be found on the lips, tongue and throughout the oral cavity, but can also be located outside of the mouth, such as in the sawfish. They can
also be found on the body surface and fins of some species, with the catfish as an excellent example. The external taste buds allow for recognition of food items without ingestion and are particularly well developed in species that live in low light, poor visibility environments.
Esophagus The epithelium of the esophagus is stratified and consists mainly of cuboidal cells with numerous mucussecreting goblet cells and occasional lymphocytes. The underlying submucosa is thick because of abundant connective tissue. Striated muscle fibers can be seen in the two muscle layers. The only other region of the alimentary canal where striated muscle is seen is the anal sphincter. In some species the striated muscle is gradually replaced by smooth muscle as the esophagus extends caudally. However, in other species striated muscle can be seen throughout the esophagus, and can be even more developed in the caudal end of the esophagus. Exemplary of this is the expanded terminal region of the esophagus in carp.
Stomach The stomach is commonly separated into three regions (cardiac, fundic and pyloric) based on location. However, the three regions can also be distinguished by histological features. The cardiac region, which receives and stores food entering from the esophagus, is usually nonsecretory, or has only a few gastric glands. This region is lined by a stratified epithelium. Rugae, which are ridges with extensive and well developed muscle fibers in the mucosa, allow the cardiac region to distend and accommodate large meals. The fundic and pyloric regions have secretory functions in fish with true stomachs. Both are lined by a simple epithelium with a mixture of columnar cells that lack microvilli and numerous goblet cells that secrete a mucus with a basic pH which protects the gastric epithelium from autodigestion. In fish with true stomachs, simple and branched tubular gastric glands penetrate deep into the lamina propria of the mucosa, with the abundance and depth of the glands greater in the fundic region. Goblet cells are present along the neck of the gastric glands and in the
pyloric region are even more numerous in the necks of the glands. At the base of the glands is a population of oxyntopeptic cells that secrete both acid (HC1) and pepsin, which initiate chemical digestion of dietary inputs. The ultrastructural features of the oxyntopeptic cells is consistent with the dual functions of acid and enzyme secretion. The apical domain of the secretory cells has an extensive network of tubules that is involved with the secretion of acid. Zymogen granules that contain the digestive enzymes are located more at the base of the oxyntopeptic cells where they are associated with a well developed rough endoplasmic reticulum that is needed to support an intense level of protein synthesis. The granules are transported to the apical membrane and released by the process of exocytosis. The submucosa under the gastric epithelium consists of connective tissue with interspersed smooth muscle fibers. The next layer, the muscularis, consists of the inner circular and outer longitudinal smooth muscle layers. In some species striated muscle can be seen in the cardiac region of the stomach, and this is thought to represent an extension of the striated muscle present in the esophagus. In some species the two muscle layers of the pyloric region are highly developed forming a 'gizzard', which is used for grinding food items (e.g. the sturgeons and gizzard shad). A third muscle layer can be seen in the gizzard of some species. At the terminus of the stomach the smooth muscle layers are both thicker and constricted, forming a structure known as the pyloric sphincter. This structure regulates the passage of food items from the stomach into the intestine. The sphincter is either absent or poorly developed in stomachless fishes.
Intestine The partly digested food entering the intestine from the stomach is referred to as chyme. Enzymes, electrolytes, water and other solutes (e.g. bile) secreted by the intestine itself and associated accessory organs (pancreas and gall bladder) are added to the chyme and continue the chemical digestion started in the stomach. To a large extent, the capacity of the intestine to hydrolyze and absorb food items is directly related to the amount of surface area. Similar to other vertebrates, the mucosa of the intestine has a complex architecture that greatly increases the amount of surface area compared to that of a smooth bore cylinder. In
most species the mucosa is arranged into complex folds called villi that can give the intestinal lining the appearance of velvet. The finger-like villi seen in mammals effectively increase the surface area by about 10fold over that of a smooth-walled tube. Although the specific size and shape of the villi vary among the different species of fish, they effectively increase digestive surface area by several-fold. The depth and complexity of the mucosa, hence villus architecture, also varies among regions of the intestine, being greatest in the proximal intestine and decreasing distally. The different strategies used to increase the gross digestive surface area (longer intestines, more complex mucosal architecture, presence of pyloric ceca, or development of a spiral valve) are simply modifications of the existing intestine and do not involve the development of new tissues. For example, the ceca are simply evaginations of the adjacent intestine and they only differ from the proximal intestine in being thinner. The spiral valve present in the distal intestine of some species is formed from an extension of the mucosa and submucosa. The intestine is perhaps the organ best characterized by histology. A simple epithelium is dominated by columnar absorptive cells (enterocytes). The enterocytes are usually elongated, but can be cuboidal. In some primitive species a portion of the enterocytes are ciliated. Enterocytes are considered to be 'polarized' cells. The outer, or apical, membrane domain that is exposed to the luminal contents of the intestine is invested with enzymes that complete the final stages of hydrolyzing water-soluble nutrients and another set of proteins known as transporters that are responsible for absorbing the constituent building blocks. The other domain, known as the basolateral membrane, has another set of functional proteins, including a different group of carriers that transport absorbed nutrients into the blood. Nutrients that are water soluble are dependent on the transporters in both sets of membranes to reach the blood. Lipid-soluble nutrients are able to traverse the enterocytes passively by diffusing through both sets of membranes down concentration gradients and specialized transporters are not needed. The individual enterocytes are linked together by tight junctions forming an epithelium that effectively forms a barrier that prevents large molecules and organisms from gaining entrance to the body. However, the tight junctions are not so well developed that they prevent the movement of water and monovalent ions. This has led to the intestinal epithelium being considered as 'leaky'.
m
-,t
r: m
Ill i11 -,t I'll
@ O r
O "1"1 C Z r
6 Z r-Z -4
O ..<
tu I--
i11
O >-
O <
Z ...-I
< Z
0
F-U Z ii U
0
The enterocytes that constitute the intestinal epithelium are constantly being replaced. This is accomplished in an orderly fashion. New enterocytes are produced at the base of the villi by proliferating stem cells. As the new enterocytes migrate up the villi they differentiate and acquire functional capabilities. The enterocytes eventually migrate to the tips of the villi where they are shed. Villi grow in length when rates of enterocyte proliferation, hence replacement, exceed the rate of shedding at the villus tip, and will shorten when the rate of shedding is greater than that for replacement. In mammals the lifespan of an enterocyte is about 2-3 days, coinciding with complete replacement of the villus epithelium. The replacement process in fish is slower compared to mammals. The lifespan is even longer at colder temperatures, and in some species complete replacement of the enterocyte population can take several weeks. Interspersed among the enterocytes are goblet cells which are filled with secretory granules. The mucus secreted by the goblet cells and other digestive secretions form a boundary layer that covers the epithelium and is referred to as the glycocalyx. Other cell types that can be seen in the epithelium include immune associated cells (e.g. macrophages) and cells that appear to have endocrine secretory functions based on the contents of cytoplasmic granules. Much less is known about these other cell types despite the obvious importance they have in protecting against invasion and regulating digestive and metabolic processes. The underlying submucosa is thinner than that seen in the stomach and contains connective tissue, blood and lymph vessels, smooth muscle fibers and nerves. The muscularis has both the circular and longitudinal layers seen in all regions of the alimentary canal. The serosa can be highly pigmented in some species.
U
0 U
Functional anatomy of the intestine The concept of membrane digestion has been used to describe the functional activities of the enterocytes that dominate the epithelium in conjunction with enzymes and features of the overlying mucous layer. Therefore, the greater the surface area, the greater the digestive capacities. In addition to the gross anatomical strategies that increase the amount of surface area available for digestion, the apical membrane of each
enterocyte is lined by microvilli. These are extensions of the cell membrane that surround an inner framework composed of actin filaments that are organized by 'linker' proteins. The microvilli are estimated to increase the surface area available for hydrolysis and absorption by about 200-fold more. The length and number of microvilli vary among intestinal regions, species of fish, feeding state and environmental conditions. In contrast the diameter of the microvilli is much more consistent and ranges from about 10 to 14 pm. Although other ultrastructure characteristics are shared by enterocytes, there can be differences, such as variation of the number, degree of development, and distribution of enterocyte organelles. The enzymes associated with the apical membrane of enterocytes include disaccharidases (e.g. maltase and trehalase), and a diversity of peptidases that release smaller peptides and free amino acids. There are several classes of transporters for absorbing the different types of solutes (sugars, amino acids, vitamins, bile acids, etc.). The specific proportions of the different functional proteins vary among regions of the intestine as well as among species. Absorption of proteins and carbohydrates is largely dependent on the luminal and brush border enzymes that release the constituent amino acids, peptides and sugars, which are subsequently absorbed. The process by which fats are absorbed and transported to the blood of fish differs from that for the other nutrients, but appears to be similar to that known for mammals and other vertebrates. Briefly, fatty acids and monoglycerides released from dietary fats by the action of luminal lipases are absorbed, reesterified in the enterocyte, and then transferred to the blood as a component of particles synthesized by the endoplasmic reticulum. The enterocytes of many, if not most, fish are also capable of macromolecular absorption. This ability has been demonstrated numerous times using intraluminal injections of horseradish peroxidase or other macromolecules and then being able to detect them inside the enterocytes using cytochemical methods. The capacity to take in macromolecules is particularly well developed in larval fish that do not yet have a 'mature' digestive system and in adults of other species that lack true secretory stomachs. The proportions of the different enzymes and transporters present in the brush border membrane are matched to the composition of the natural diet. For example, the activities of carbohydrases and rates of glucose transport are higher in omnivorous and herbivorous species compared to strict carnivores.
Despite the diversity of feeding habits, fish must eat sufficient quantities of food to meet requirements for protein and essential amino acids. Because the requirements do not differ widely among fish, the activities of the peptidases and amino acid transporters are not as variable among fish as those seen for the carbohydrases and glucose transport. The epithelium lining the intestine can be damaged by some components that are used to formulate some production diets. For example, feeding salmonids diets that contain soybean meal causes an inflammation (enteritis) in the distal intestine that is associated with a reduction in the amount of mucosa and a decrease in the activities of certain digestive functions. Although the causes for the enteritis are not yet known, they are suggestive of an allergic reaction to a component of soybeans. Although the majority of research has been directed at understanding the responses of the brush border functions to the composition of the diet, it is now recognized that the densities, types, and functional characteristics of the different enzymes and transporters present in the membranes of enterocytes are also responsive to changes in environmental conditions such as temperature and salinity. Multicellular glands are not present in the mucosa, but throughout the stomach and intestine there are large individual cells that are granulated and have been shown to have secretory functions. A number of different types of endocrine cells have been identified in the intestine with most characterized as being or similar to enterochromaffin cells. Although many of the secretory cells are located in the mucosa, endocrine and other secretory cell types can be detected throughout other tissues of the digestive system. Most of the secretory cells synthesize polypeptides that can act locally (paracrine) or systemically (endocrine). Exemplary are the cells in the proximal intestine that synthesize cholecystokinin and secretin to regulate exocrine pancreatic functions in response to the chemical composition of the chyme in the intestine. The best known examples of digestive system endocrine cells are those of the pancreas. The endocrine pancreas produces several hormones, and the four best known are insulin, glucagon, somatostatin and pancreatic polypeptide. Each hormone is synthesized by a specific type of cell. When the numerous different secretory cell types are considered collectively, the digestive system represents the largest endocrine organ in the body and the associated secretions are critical for regulating physiological and metabolic processes throughout the body.
Although endocrine cells of the intestine are known to be important for regulating digestion and metabolism in mammals, the specific roles they play in fish have not been adequately studied and defined. In addition to the influence of endocrine secretions, intestinal functions are also subject to regulation by the multitude of neurons that comprise the 'enteric nervous system'. The intestinal epithelium is also an important tissue for osmoregulation. This is particularly true for marine species that must drink sea water to compensate for the osmotic loss of body fluids to the environment. This is accomplished by intestinal absorption of monovalent ions present in sea water (e.g. Na and C1), which drives the movement of water out of the gut into the blood. The excess ions are then disposed of by the gills. Corresponding with the changes in functional demands placed on the intestine during adaptation to different salinities, there are changes in hydrolytic and transport functions. Another key function of the intestine is the defense against invasion. This is accomplished by both non-specific and specific defense mechanisms. The non-specific mechanisms include the tight junctions that link the enterocytes together and effectively form a barrier. The mucus secreted by the goblet cells also provides a barrier that reduces the ability of pathogens to invade. Additionally, phagocytic cells in the epithelium and underlying tissue layers are able to respond to organisms and particles that manage to pass the epithelial barrier. The lymphocytes present in the epithelium are known as intraepithelial lymphocytes (IEL), with additional lymphocytes found in the underlying tissue layers. The lymphocytes are able to recognize and react to specific antigens present in the intestines of fish. The collection of lymphocytes and associated tissues present in the tissues of the alimentary canal is often referred to as the mucosal associated lymphoid tissue (MALT).
m
m
lgl 4.4 m
O O -o
-H C Z -4
5 Z
i-Z -4
O
Accessory organs The pancreas develops as an extension of the alimentary canal epithelium just distal to the future stomach and the ducts that link the pancreas to the intestine (or ceca) are the remnants of the embryological origin. The pancreas has both endocrine and exocrine functions. In many, if not most species offish, the pancreas is a diffuse organ, but is a compact organ in some fish as it is in other vertebrates. In most species the
-<
U,I
I-u'l >.
111
I--
ul
O >-
O F-
pancreatic tissue is hard to detect macroscopically. Patches of exocrine and endocrine pancreatic tissue can be seen in several sites, including the intestine, the visceral mesenteries, and alongside blood vessels near the intestine. Pancreatic tissue has also been reported in the liver and spleen. The exocrine pancreatic cells are more abundant than the endocrine cells and are detected by staining for the presence of zymogen granules, which represent the digestive enzymes, or by using antibodies that are selective for specific enzymes. The digestive secretions enter the proximal intestine through a single duct or multiple ducts. In addition to secreting a multitude of digestive enzymes (e.g. several proteases, amylase, lipase, deoxy and ribonucleases), the acinar cells of the pancreas produce bicarbonate to neutralize gastric acid and other electrolytes. The liver also develops as an outgrowth of the epithelium of the developing intestine and at about the same level as the presumptive pancreas. The bile duct, much like the pancreatic duct, is a remnant of the embryologic origin of the liver and it provides a connection with the intestine. Most hepatic cells, like the enterocytes, are 'polarized' and have two distinct membrane domains. One domain is exposed to the blood whereas the other membrane domain faces the caniculi that collect secretions that eventually constitute the bile and are secreted into the intestine.
Z
<
_.1
< z 0 utz u D_
0u 0 u
References B~everfjord, G. and Krogdahl, A. (1996). Development and regression of soybean meal induced enteritis in
Atlantic salmon, Salmo salar L., distal intestine: a comparison with the intestines of fasted fish. J. Fish Dis. 19,375-387. Buddington, R.K., Krogdahl, A. and Bakke-McKellep, A.M. (1997). The intestines of carnivorous fish: structure and functions and the relations with diet. Acta Physiol. Scand. 161 (Suppl. 638), 67-80. F~inge, R. and Grove, D. (1979). Digestion. In Fish Physiology, vol. 8 (eds W.S. Hoar, D.J. Randall and J.R. Brett), pp. 161-260. Academic Press, New York. Kuperman, B.I. and Kuz'mina, V.V. (1994). The ultrastructure of the intestinal epithelium in fishes with different types of feeding.J. Fish Biol. 44, 181-193. Kuz'mina, V.V. and Gelman, A.G. (1997). Membranelinked digestion in fish.Rev. Fish. Sci. 5, 99-129. Iwama, G. and Nakamishi, T. (eds) (1996). The Fish Immune System: Organism, Pathogen, and Environment. Academic Press, New York. Reinecke, M., M/iller, C. and Segner, H. (1997). An immunohistochemical analysis of the ontogeny, distribution, and coexistence of 12 regulatory peptides and serotonin in endocrine cells and nerve fibers of the digestive tract of the turbot, Scophthalmus maximus (Teleostie).Anat. Embryol. 195, 87-102. Romer, A.S. (1970). The Vertebrate Body. W.B. Saunders, Philadelphia. Suyehiro, Y. (1941). A study on the digestive system and feeding habits of fish.Jap. J. Zool. 10,1-303. Yasutake, W.T. and Wales, F.H. (1983). Microscopic Anatomy of Salmonids: An Atlas. United States Department of the Interior, Resource Publication 150, Washington, DC. Zapata, A.G. and Cooper, E.L. (eds) (1990). The Immune System: Comparative Histopathology. John Wiley, New York.
Digestive System Randal K Buddington Department of Biological Sciences, Mississippi State University, Mississippi, USA
m
I#l -4 m
Victoria Kuz'mina Institute for the Biology of Inland Waters, Borok, YaroslavI, Russia
Introduction Even though the gross anatomy of the digestive tract, particularly the alimentary canal, is highly variable among fish, the types of cells and tissues (i.e. the 'building blocks') are conserved. An appropriate analogy is how houses built for different environments may vary in structure, but the same basic materials are used for their construction. Similar to all other vertebrates the different regions of the alimentary canal consist of the same four basic tissue layers (Figure 23.1). The internal mucosal layer consists of two components. The first is the epithelium which serves as a selectively permeable membrane that separates the body from the outside (lumen). The epithelium lining the two ends of the digestive system develops from two invaginations: the stomodeum forms the mouth and the proctodeum forms the anus. The epithelium lining the remainder of the alimentary canal is derived from endoderm, with evaginations of this tissue forming the accessory organs, including the liver and gall bladder, pancreas, and air bladder. Directly under the
Copyright 9 2000 Academic Press
-4 HI
epithelium is the lamina propria, which is largely connective tissue. Capillaries are present in the lamina propria and they allow for the exchange of materials between the host and the outside environment, using the epithelium as the site of exchange. Specifically, nutrients absorbed from food are transferred to the blood in the capillaries, and conversely, the blood can deliver other materials or by-products of metabolism
i N
O O "ID "R C Z i
O Z
Dorsa
r
Z
,al O -<
Lamina p Epithelium
Figure 23.1 The organization of the tissue layers in the fish alimentary canal.
111 I--
>. i11
ILl
@ >-
O
F< Z
<
...-I
for secretion into the intestine. In the intestine lymph vessels can be seen and these are involved in the absorption of fat, other nutrients and fluids. A thin layer of smooth muscle known as the muscularis mucosa can sometimes be seen between the lamina propria and the deeper tissue layers, but it is less developed in fish compared to that of mammals. Extensions of the muscularis mucosa, when present, penetrate up into the villi and provide the villi with the ability to move, which effectively 'stirs' the layer of fluid overlying the epithelium. This reduces the thickness of the boundary layer that covers the epithelium and limits diffusion of nutrients down to the absorptive cells, and by so doing enhances the absorption of the luminal contents. The second tissue layer is called the submucosa. This layer supports the mucosa and consists mainly of connective tissue. The submucosa is penetrated by nerves and blood vessels that extend up to the mucosa. The third tissue layer is known as the muscularis. This layer is actually composed of two coats of smooth muscle. The inner circular layer is arranged such that the fibers loop around the lumen. Fibers of the second, or longitudinal, layer are oriented in a proximal to distal direction. Contractions of the muscle layers are important for the segmental contractions that mix food items and the peristaltic contractions that propel the chyme in a distal direction. The fourth, and outermost layer, is known as the serosa. In reality, the serosa is the visceral peritoneum that covers the alimentary canal and other viscera lying within the peritoneal cavity.
Z
0 F-U Z Ii
Mouth and Pharynx
U
9 U
0 U
The microscopic anatomy of the fish mouth and pharynx is basically the same as that seen in other vertebrates. Only the macroscopic structure varies widely, corresponding with the wide diversity of food habits. Mucous cells are interspersed among the epithelial cells lining the oral cavity and pharynx. Salivary gland cells are present, but unlike other vertebrates they exist as solitary cells, not multicellular glands. The secretions from the mucous and salivary gland cells serve to lubricate the food, and in some filter feeders help to 'trap' small particles. Taste buds, which consist of elongate sensory cells with accessory cells, can be found on the lips, tongue and throughout the oral cavity, but can also be located outside of the mouth, such as in the sawfish. They can
also be found on the body surface and fins of some species, with the catfish as an excellent example. The external taste buds allow for recognition of food items without ingestion and are particularly well developed in species that live in low light, poor visibility environments.
Esophagus The epithelium of the esophagus is stratified and consists mainly of cuboidal cells with numerous mucussecreting goblet cells and occasional lymphocytes. The underlying submucosa is thick because of abundant connective tissue. Striated muscle fibers can be seen in the two muscle layers. The only other region of the alimentary canal where striated muscle is seen is the anal sphincter. In some species the striated muscle is gradually replaced by smooth muscle as the esophagus extends caudally. However, in other species striated muscle can be seen throughout the esophagus, and can be even more developed in the caudal end of the esophagus. Exemplary of this is the expanded terminal region of the esophagus in carp.
Stomach The stomach is commonly separated into three regions (cardiac, fundic and pyloric) based on location. However, the three regions can also be distinguished by histological features. The cardiac region, which receives and stores food entering from the esophagus, is usually nonsecretory, or has only a few gastric glands. This region is lined by a stratified epithelium. Rugae, which are ridges with extensive and well developed muscle fibers in the mucosa, allow the cardiac region to distend and accommodate large meals. The fundic and pyloric regions have secretory functions in fish with true stomachs. Both are lined by a simple epithelium with a mixture of columnar cells that lack microvilli and numerous goblet cells that secrete a mucus with a basic pH which protects the gastric epithelium from autodigestion. In fish with true stomachs, simple and branched tubular gastric glands penetrate deep into the lamina propria of the mucosa, with the abundance and depth of the glands greater in the fundic region. Goblet cells are present along the neck of the gastric glands and in the
pyloric region are even more numerous in the necks of the glands. At the base of the glands is a population of oxyntopeptic cells that secrete both acid (HC1) and pepsin, which initiate chemical digestion of dietary inputs. The ultrastructural features of the oxyntopeptic cells is consistent with the dual functions of acid and enzyme secretion. The apical domain of the secretory cells has an extensive network of tubules that is involved with the secretion of acid. Zymogen granules that contain the digestive enzymes are located more at the base of the oxyntopeptic cells where they are associated with a well developed rough endoplasmic reticulum that is needed to support an intense level of protein synthesis. The granules are transported to the apical membrane and released by the process of exocytosis. The submucosa under the gastric epithelium consists of connective tissue with interspersed smooth muscle fibers. The next layer, the muscularis, consists of the inner circular and outer longitudinal smooth muscle layers. In some species striated muscle can be seen in the cardiac region of the stomach, and this is thought to represent an extension of the striated muscle present in the esophagus. In some species the two muscle layers of the pyloric region are highly developed forming a 'gizzard', which is used for grinding food items (e.g. the sturgeons and gizzard shad). A third muscle layer can be seen in the gizzard of some species. At the terminus of the stomach the smooth muscle layers are both thicker and constricted, forming a structure known as the pyloric sphincter. This structure regulates the passage of food items from the stomach into the intestine. The sphincter is either absent or poorly developed in stomachless fishes.
Intestine The partly digested food entering the intestine from the stomach is referred to as chyme. Enzymes, electrolytes, water and other solutes (e.g. bile) secreted by the intestine itself and associated accessory organs (pancreas and gall bladder) are added to the chyme and continue the chemical digestion started in the stomach. To a large extent, the capacity of the intestine to hydrolyze and absorb food items is directly related to the amount of surface area. Similar to other vertebrates, the mucosa of the intestine has a complex architecture that greatly increases the amount of surface area compared to that of a smooth bore cylinder. In
most species the mucosa is arranged into complex folds called villi that can give the intestinal lining the appearance of velvet. The finger-like villi seen in mammals effectively increase the surface area by about 10fold over that of a smooth-walled tube. Although the specific size and shape of the villi vary among the different species of fish, they effectively increase digestive surface area by several-fold. The depth and complexity of the mucosa, hence villus architecture, also varies among regions of the intestine, being greatest in the proximal intestine and decreasing distally. The different strategies used to increase the gross digestive surface area (longer intestines, more complex mucosal architecture, presence of pyloric ceca, or development of a spiral valve) are simply modifications of the existing intestine and do not involve the development of new tissues. For example, the ceca are simply evaginations of the adjacent intestine and they only differ from the proximal intestine in being thinner. The spiral valve present in the distal intestine of some species is formed from an extension of the mucosa and submucosa. The intestine is perhaps the organ best characterized by histology. A simple epithelium is dominated by columnar absorptive cells (enterocytes). The enterocytes are usually elongated, but can be cuboidal. In some primitive species a portion of the enterocytes are ciliated. Enterocytes are considered to be 'polarized' cells. The outer, or apical, membrane domain that is exposed to the luminal contents of the intestine is invested with enzymes that complete the final stages of hydrolyzing water-soluble nutrients and another set of proteins known as transporters that are responsible for absorbing the constituent building blocks. The other domain, known as the basolateral membrane, has another set of functional proteins, including a different group of carriers that transport absorbed nutrients into the blood. Nutrients that are water soluble are dependent on the transporters in both sets of membranes to reach the blood. Lipid-soluble nutrients are able to traverse the enterocytes passively by diffusing through both sets of membranes down concentration gradients and specialized transporters are not needed. The individual enterocytes are linked together by tight junctions forming an epithelium that effectively forms a barrier that prevents large molecules and organisms from gaining entrance to the body. However, the tight junctions are not so well developed that they prevent the movement of water and monovalent ions. This has led to the intestinal epithelium being considered as 'leaky'.
m
-,t
r: m
Ill i11 -,t I'll
@ O r
O "1"1 C Z r
6 Z r-Z -4
O ..<
tu I--
i11
O >-
O <
Z ...-I
< Z
0
F-U Z ii U
0
The enterocytes that constitute the intestinal epithelium are constantly being replaced. This is accomplished in an orderly fashion. New enterocytes are produced at the base of the villi by proliferating stem cells. As the new enterocytes migrate up the villi they differentiate and acquire functional capabilities. The enterocytes eventually migrate to the tips of the villi where they are shed. Villi grow in length when rates of enterocyte proliferation, hence replacement, exceed the rate of shedding at the villus tip, and will shorten when the rate of shedding is greater than that for replacement. In mammals the lifespan of an enterocyte is about 2-3 days, coinciding with complete replacement of the villus epithelium. The replacement process in fish is slower compared to mammals. The lifespan is even longer at colder temperatures, and in some species complete replacement of the enterocyte population can take several weeks. Interspersed among the enterocytes are goblet cells which are filled with secretory granules. The mucus secreted by the goblet cells and other digestive secretions form a boundary layer that covers the epithelium and is referred to as the glycocalyx. Other cell types that can be seen in the epithelium include immune associated cells (e.g. macrophages) and cells that appear to have endocrine secretory functions based on the contents of cytoplasmic granules. Much less is known about these other cell types despite the obvious importance they have in protecting against invasion and regulating digestive and metabolic processes. The underlying submucosa is thinner than that seen in the stomach and contains connective tissue, blood and lymph vessels, smooth muscle fibers and nerves. The muscularis has both the circular and longitudinal layers seen in all regions of the alimentary canal. The serosa can be highly pigmented in some species.
U
0 U
Functional anatomy of the intestine The concept of membrane digestion has been used to describe the functional activities of the enterocytes that dominate the epithelium in conjunction with enzymes and features of the overlying mucous layer. Therefore, the greater the surface area, the greater the digestive capacities. In addition to the gross anatomical strategies that increase the amount of surface area available for digestion, the apical membrane of each
enterocyte is lined by microvilli. These are extensions of the cell membrane that surround an inner framework composed of actin filaments that are organized by 'linker' proteins. The microvilli are estimated to increase the surface area available for hydrolysis and absorption by about 200-fold more. The length and number of microvilli vary among intestinal regions, species of fish, feeding state and environmental conditions. In contrast the diameter of the microvilli is much more consistent and ranges from about 10 to 14 pm. Although other ultrastructure characteristics are shared by enterocytes, there can be differences, such as variation of the number, degree of development, and distribution of enterocyte organelles. The enzymes associated with the apical membrane of enterocytes include disaccharidases (e.g. maltase and trehalase), and a diversity of peptidases that release smaller peptides and free amino acids. There are several classes of transporters for absorbing the different types of solutes (sugars, amino acids, vitamins, bile acids, etc.). The specific proportions of the different functional proteins vary among regions of the intestine as well as among species. Absorption of proteins and carbohydrates is largely dependent on the luminal and brush border enzymes that release the constituent amino acids, peptides and sugars, which are subsequently absorbed. The process by which fats are absorbed and transported to the blood of fish differs from that for the other nutrients, but appears to be similar to that known for mammals and other vertebrates. Briefly, fatty acids and monoglycerides released from dietary fats by the action of luminal lipases are absorbed, reesterified in the enterocyte, and then transferred to the blood as a component of particles synthesized by the endoplasmic reticulum. The enterocytes of many, if not most, fish are also capable of macromolecular absorption. This ability has been demonstrated numerous times using intraluminal injections of horseradish peroxidase or other macromolecules and then being able to detect them inside the enterocytes using cytochemical methods. The capacity to take in macromolecules is particularly well developed in larval fish that do not yet have a 'mature' digestive system and in adults of other species that lack true secretory stomachs. The proportions of the different enzymes and transporters present in the brush border membrane are matched to the composition of the natural diet. For example, the activities of carbohydrases and rates of glucose transport are higher in omnivorous and herbivorous species compared to strict carnivores.
Despite the diversity of feeding habits, fish must eat sufficient quantities of food to meet requirements for protein and essential amino acids. Because the requirements do not differ widely among fish, the activities of the peptidases and amino acid transporters are not as variable among fish as those seen for the carbohydrases and glucose transport. The epithelium lining the intestine can be damaged by some components that are used to formulate some production diets. For example, feeding salmonids diets that contain soybean meal causes an inflammation (enteritis) in the distal intestine that is associated with a reduction in the amount of mucosa and a decrease in the activities of certain digestive functions. Although the causes for the enteritis are not yet known, they are suggestive of an allergic reaction to a component of soybeans. Although the majority of research has been directed at understanding the responses of the brush border functions to the composition of the diet, it is now recognized that the densities, types, and functional characteristics of the different enzymes and transporters present in the membranes of enterocytes are also responsive to changes in environmental conditions such as temperature and salinity. Multicellular glands are not present in the mucosa, but throughout the stomach and intestine there are large individual cells that are granulated and have been shown to have secretory functions. A number of different types of endocrine cells have been identified in the intestine with most characterized as being or similar to enterochromaffin cells. Although many of the secretory cells are located in the mucosa, endocrine and other secretory cell types can be detected throughout other tissues of the digestive system. Most of the secretory cells synthesize polypeptides that can act locally (paracrine) or systemically (endocrine). Exemplary are the cells in the proximal intestine that synthesize cholecystokinin and secretin to regulate exocrine pancreatic functions in response to the chemical composition of the chyme in the intestine. The best known examples of digestive system endocrine cells are those of the pancreas. The endocrine pancreas produces several hormones, and the four best known are insulin, glucagon, somatostatin and pancreatic polypeptide. Each hormone is synthesized by a specific type of cell. When the numerous different secretory cell types are considered collectively, the digestive system represents the largest endocrine organ in the body and the associated secretions are critical for regulating physiological and metabolic processes throughout the body.
Although endocrine cells of the intestine are known to be important for regulating digestion and metabolism in mammals, the specific roles they play in fish have not been adequately studied and defined. In addition to the influence of endocrine secretions, intestinal functions are also subject to regulation by the multitude of neurons that comprise the 'enteric nervous system'. The intestinal epithelium is also an important tissue for osmoregulation. This is particularly true for marine species that must drink sea water to compensate for the osmotic loss of body fluids to the environment. This is accomplished by intestinal absorption of monovalent ions present in sea water (e.g. Na and C1), which drives the movement of water out of the gut into the blood. The excess ions are then disposed of by the gills. Corresponding with the changes in functional demands placed on the intestine during adaptation to different salinities, there are changes in hydrolytic and transport functions. Another key function of the intestine is the defense against invasion. This is accomplished by both non-specific and specific defense mechanisms. The non-specific mechanisms include the tight junctions that link the enterocytes together and effectively form a barrier. The mucus secreted by the goblet cells also provides a barrier that reduces the ability of pathogens to invade. Additionally, phagocytic cells in the epithelium and underlying tissue layers are able to respond to organisms and particles that manage to pass the epithelial barrier. The lymphocytes present in the epithelium are known as intraepithelial lymphocytes (IEL), with additional lymphocytes found in the underlying tissue layers. The lymphocytes are able to recognize and react to specific antigens present in the intestines of fish. The collection of lymphocytes and associated tissues present in the tissues of the alimentary canal is often referred to as the mucosal associated lymphoid tissue (MALT).
m
m
lgl 4.4 m
O O -o
-H C Z -4
5 Z
i-Z -4
O
Accessory organs The pancreas develops as an extension of the alimentary canal epithelium just distal to the future stomach and the ducts that link the pancreas to the intestine (or ceca) are the remnants of the embryological origin. The pancreas has both endocrine and exocrine functions. In many, if not most species offish, the pancreas is a diffuse organ, but is a compact organ in some fish as it is in other vertebrates. In most species the
-<
U,I
I-u'l >.
111
I--
ul
O >-
O F-
pancreatic tissue is hard to detect macroscopically. Patches of exocrine and endocrine pancreatic tissue can be seen in several sites, including the intestine, the visceral mesenteries, and alongside blood vessels near the intestine. Pancreatic tissue has also been reported in the liver and spleen. The exocrine pancreatic cells are more abundant than the endocrine cells and are detected by staining for the presence of zymogen granules, which represent the digestive enzymes, or by using antibodies that are selective for specific enzymes. The digestive secretions enter the proximal intestine through a single duct or multiple ducts. In addition to secreting a multitude of digestive enzymes (e.g. several proteases, amylase, lipase, deoxy and ribonucleases), the acinar cells of the pancreas produce bicarbonate to neutralize gastric acid and other electrolytes. The liver also develops as an outgrowth of the epithelium of the developing intestine and at about the same level as the presumptive pancreas. The bile duct, much like the pancreatic duct, is a remnant of the embryologic origin of the liver and it provides a connection with the intestine. Most hepatic cells, like the enterocytes, are 'polarized' and have two distinct membrane domains. One domain is exposed to the blood whereas the other membrane domain faces the caniculi that collect secretions that eventually constitute the bile and are secreted into the intestine.
Z
<
_.1
< z 0 utz u D_
0u 0 u
References B~everfjord, G. and Krogdahl, A. (1996). Development and regression of soybean meal induced enteritis in
Atlantic salmon, Salmo salar L., distal intestine: a comparison with the intestines of fasted fish. J. Fish Dis. 19,375-387. Buddington, R.K., Krogdahl, A. and Bakke-McKellep, A.M. (1997). The intestines of carnivorous fish: structure and functions and the relations with diet. Acta Physiol. Scand. 161 (Suppl. 638), 67-80. F~inge, R. and Grove, D. (1979). Digestion. In Fish Physiology, vol. 8 (eds W.S. Hoar, D.J. Randall and J.R. Brett), pp. 161-260. Academic Press, New York. Kuperman, B.I. and Kuz'mina, V.V. (1994). The ultrastructure of the intestinal epithelium in fishes with different types of feeding.J. Fish Biol. 44, 181-193. Kuz'mina, V.V. and Gelman, A.G. (1997). Membranelinked digestion in fish.Rev. Fish. Sci. 5, 99-129. Iwama, G. and Nakamishi, T. (eds) (1996). The Fish Immune System: Organism, Pathogen, and Environment. Academic Press, New York. Reinecke, M., M/iller, C. and Segner, H. (1997). An immunohistochemical analysis of the ontogeny, distribution, and coexistence of 12 regulatory peptides and serotonin in endocrine cells and nerve fibers of the digestive tract of the turbot, Scophthalmus maximus (Teleostie).Anat. Embryol. 195, 87-102. Romer, A.S. (1970). The Vertebrate Body. W.B. Saunders, Philadelphia. Suyehiro, Y. (1941). A study on the digestive system and feeding habits of fish.Jap. J. Zool. 10,1-303. Yasutake, W.T. and Wales, F.H. (1983). Microscopic Anatomy of Salmonids: An Atlas. United States Department of the Interior, Resource Publication 150, Washington, DC. Zapata, A.G. and Cooper, E.L. (eds) (1990). The Immune System: Comparative Histopathology. John Wiley, New York.