Digestion
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Digestion is the process by which food molecules are broken down into smaller molecules that are able to be absorbed by the gut tissue. Most food molecules requiring digestion are polymers such as proteins and starch, and are sequentially digested through three phases.
Primary digestion is the dispersion and reduction in molecular size of the polymers and results in oligomers. During intermediate digestion, these undergo a further reduction in molecular size to dimers, which in final digestion form monomers.
Digestion usually occurs under the action of digestive enzymes from the midgut, with minor or no participation of salivary enzymes. In most insects, midgut pH is either mildly acidic or neutral.
Lepidopteran and trichopteran larvae, scarabaeid beetles, and nematoceran flies have alkaline midguts, whereas cyclorrhaphous flies have a very acidic section in the middle of the midgut. The midgut is, as a rule, an oxidizing site, although in some wool-digesting insects it is a reducing site, a condition necessary to break disulfide bonds in keratin, thus facilitating enzymatic hydrolysis.
Digestion of Proteins
Initial digestion of proteins is carried out by proteinases (endopeptidases), which are enzymes able to cleave the internal peptide bonds of proteins (Fig. 1). Different endopeptidases are necessary to do this because the amino acid residues vary along the peptide chain (R is a variable group in Fig. 1).
Proteinases may differ in specificity toward the reactant protein (substrate) and are grouped according to their reaction mechanism into the subclasses: serine, cysteine, and aspartic proteinases. Trypsin, chymotrypsin, and elastase are serine proteinases that are widely distributed in insects and have molecular masses in the range 20 to 35 kDa and alkaline pH optima.
Trypsin preferentially hydrolyzes (its primary specificity) peptide bonds in the carboxyl end of amino acids with basic R groups (Arg, Lys); chymotrypsin is preferential toward large hydrophobic R groups (e.g., Phe, Tyr) and elastase, toward small hydrophobic R groups (e.g., Ala).
The activity of the enzymes also depends on the amino acid residues neighboring the bond to be cleaved. This may explain the differences in susceptibilities of insects to strains of Bacillus thuringiensis, because the deleterious effects depend on the previous proteolysis of the bacterial endotoxin.
Related to this is the growing evidence that insects fed on trypsin inhibitor-containing food express new trypsin molecules insensitive to the inhibitors. These inhibitors are proteins and their binding to the enzyme has molecular requirements similar to those of the substrate.
Cysteine and aspartic proteinases are the only midgut proteinases in hemipterans and they occur in addition to serine proteinases in cucujiformia beetles. Their occurrence in Hemiptera is interpreted as a consequence of the loss of the usual digestive serine proteinases associated with the adaptation of hemipteran ancestors to a diet lacking proteins (plant sap), followed by the use of lysosome-like enzymes in adapting to a new predatory habit.
The presence of cysteine and aspartic proteinases in cucujiformia beetles is likely an ancestral adaptation to circumvent proteolytic inhibition caused by trypsin inhibitors in ingested seeds.
Cysteine and aspartic proteinases have pH optima of 5.5 to 6.0 and 3.2 to 3.5 and molecular masses of 20 to 40 kDa and 60 to 80 kDa, respectively. Because of their pH optima, aspartic proteinases are not very active in the mildly acidic midguts of Hemiptera and cucujiformia beetles, but are very important in the middle midguts (pH 3.5) of cyclorrhaphous flies.
Intermediate digestion of proteins is accomplished by exopeptidases, enzymes that remove amino acids from the N-terminal (aminopeptidases) or C-terminal (carboxypeptidases) ends of oligopeptides (fragments of proteins) (Fig. 1).
Insect aminopeptidases have molecular masses in the range 90 to 130 kDa, have pH optima of 7.2 to 9.0, have no marked specificity toward the N-terminal amino acid, and are usually associated with the microvillar membranes of midgut cells. Therefore, the action of aminopeptidase is restricted to the surface of midgut cells. Because aminopeptidases are frequently active on dipeptides, they are also involved in protein-terminal digestion together with dipeptidases.
Aminopeptidases may account for as much as 55% of the midgut microvillar proteins in larvae of the yellow mealworm, Tenebrio molitor. Probably because of this, in many insects aminopeptidases are the preferred targets of B. thuringiensis endotoxins. These toxins, after binding to aminopeptidase (or other receptors), form channels through which cell contents leak, leading to insect death.
The most important insect carboxypeptidases have alkaline pH optima, have molecular masses in the range 20 to 50 kDa, and require a divalent metal for activity. They are classified as carboxypeptidase A or B depending on their activity upon neutral/acid or basic C-terminal amino acids, respectively.
Digestion of Carbohydrates
Initial and intermediate digestion of starch (or glycogen) is accomplished by α-amylase. This enzyme cleaves internal bonds of the polysaccharide until it is reduced to small oligosaccharides or disaccharides (Fig. 2). The amylases are not very active in digesting intact starch granules, making mastication prior to ingestion important.
Insect amylases depend on calcium ions for activity or stability, they are activated by chloride ions (amylases in Lepidoptera are exceptions), their molecular masses are found in the range 48 to 68 kDa, and their pH optima vary widely (4.8-9.8) depending on the insect taxon. As described for trypsin, insects feeding on amylase inhibitor-containing food express new amylase molecules insensitive to the inhibitors.
The final digestion of starch chains occurs under α-glucosidases, enzymes that sequentially remove glucosyl residues from the nonreducing ends of short oligomaltosac-charides. If the saccharide is a disaccharide, it is named maltose (Fig. 2). Because of that, α-glucosidase is also called maltase.
As a rule, sucrose (glucose α1,β2-fructose) is hydrolyzed by α-glucosidase. If an enzyme is able to hydrolyze sucrose, but not maltose, it is likely a β-fructosidase, an enzyme attacking sucrose by the fructosyl residue. Sucrose is found in large amounts in nectar and phloem sap and in lesser amounts in some fruits and leaves.
The important insect hemolymph and fungal sugar trehalose (glucose α1,α1-glucose) is hydrolyzed only by the specific enzyme trehalase. This digestive enzyme occurs in luminal contents or immobilized at the surface of midgut cells and also as an enzyme at the midgut basal cell membrane, making available glucose from hemolymph trehalose.
Although cellulose is abundant in plants, most plant-feeding insects, such as caterpillars and grasshoppers, do not use it. Cellulose is a nonramified chain of glucose units linked by β-1,4 bonds (Fig. 3) arranged in a crystalline structure that is difficult to disrupt. Thus, cellulose digestion is unlikely to be advantageous to an insect that can meet its dietary requirements using more easily digested food constituents.
The cellulase activity found in some plant feeders facilitates the access of digestive enzymes to the plant cells ingested by the insects. True cellulose digestion is restricted to insects that have, as a rule, nutritionally poor diets, as exemplified by termites, woodroaches, and cerambycid and scarabaeid beetles.
There is growing evidence that insects secrete enzymes able to hydrolyze crystalline cellulose, challenging the long-standing belief that microbial symbionts are necessary for cellulose digestion. The end products of cellulase action are glucose and cellobiose (Fig. 3); the latter is hydrolyzed by a β-glucosidase.
Hemicellulose is a mixture of polysaccharides associated with cellulose in plant cell walls. They are β-1,4- and/or β-1,3-linked glycan chains made up mainly of glucose (glucans), xylose (xylans), and other monosaccharides.
The polysaccharides are hydrolyzed by a variety of enzymes from which xylanases, laminarinases, and lichenases are the best known. The end products of the actions of these enzymes are monosaccharides and β-linked oligosaccharides.
The final digestion of those chains occurs under the actions of β-glycosidases that sequentially remove glycosyl (usually glucosyl, galactosyl, or xylosyl) residues from the nonreducing end of the β-linked oligosaccharides. As these may be cellobiose, β-glycosidase is frequently also named cellobiase. Thus, β-glycosidases complete the digestion of cellulose and hemicelluloses.
A special β-glycosidase (aryl β-glycosidase) acts on glycolipids and in vivo probably removes a galactose from monogalactosyldiacylglycerol that together with digalactosyl-diacylglycerol is a major lipid of photosynthetic tissues. Digalactosyldiacylglycerol is converted into monogalactosyl-diacylglycerol by the action of an α-galactosidase.
The aryl β-glycosidase also acts on plant glycosides that are noxious after hydrolysis. Insects circumvent these problems by detoxifying the products of hydrolysis or by repressing the synthesis and secretion of this enzyme while maintaining constant the synthesis and secretion of the other β-glycosidases.
Digestion of Lipids and Phosphates
Oils and fats are triacylglycerols and are hydrolyzed by a triacylglycerol lipase that preferentially removes the outer ester links of the substrate (Fig. 4) and acts only on the water-lipid interface. This interface is increased by surfactants that, in contrast to the bile salts of vertebrates, are mainly lysophosphatides. The resulting 2-monoacylglycerol may be absorbed or further hydrolyzed before absorption.
Membrane lipids include glycolipids, such as galactosyl-diacylglycerol and phosphatides. After the removal of galactose residues from mono- and digalactosyldiacylglycerol, which leaves diacylglycerol, it is hydrolyzed as described for triacylglycerols.
Phospholipase A removes one fatty acid from the phosphatide, resulting in a lysophosphatide (Fig. 4) that forms micellar aggregates, causing the solubilization of cell membranes. Lysophosphatide seems to be absorbed intact by insects.
Nonspecific phosphatases remove phosphate moieties from phosphorylated compounds to make their absorption easier. Phosphatases are active in an alkaline or acid medium.
Digestion is the process by which food molecules are broken down into smaller molecules that are able to be absorbed by the gut tissue. Most food molecules requiring digestion are polymers such as proteins and starch, and are sequentially digested through three phases.
Primary digestion is the dispersion and reduction in molecular size of the polymers and results in oligomers. During intermediate digestion, these undergo a further reduction in molecular size to dimers, which in final digestion form monomers.
Digestion usually occurs under the action of digestive enzymes from the midgut, with minor or no participation of salivary enzymes. In most insects, midgut pH is either mildly acidic or neutral.
Lepidopteran and trichopteran larvae, scarabaeid beetles, and nematoceran flies have alkaline midguts, whereas cyclorrhaphous flies have a very acidic section in the middle of the midgut. The midgut is, as a rule, an oxidizing site, although in some wool-digesting insects it is a reducing site, a condition necessary to break disulfide bonds in keratin, thus facilitating enzymatic hydrolysis.
Digestion of Proteins
Initial digestion of proteins is carried out by proteinases (endopeptidases), which are enzymes able to cleave the internal peptide bonds of proteins (Fig. 1). Different endopeptidases are necessary to do this because the amino acid residues vary along the peptide chain (R is a variable group in Fig. 1).
Proteinases may differ in specificity toward the reactant protein (substrate) and are grouped according to their reaction mechanism into the subclasses: serine, cysteine, and aspartic proteinases. Trypsin, chymotrypsin, and elastase are serine proteinases that are widely distributed in insects and have molecular masses in the range 20 to 35 kDa and alkaline pH optima.
Trypsin preferentially hydrolyzes (its primary specificity) peptide bonds in the carboxyl end of amino acids with basic R groups (Arg, Lys); chymotrypsin is preferential toward large hydrophobic R groups (e.g., Phe, Tyr) and elastase, toward small hydrophobic R groups (e.g., Ala).
The activity of the enzymes also depends on the amino acid residues neighboring the bond to be cleaved. This may explain the differences in susceptibilities of insects to strains of Bacillus thuringiensis, because the deleterious effects depend on the previous proteolysis of the bacterial endotoxin.
Related to this is the growing evidence that insects fed on trypsin inhibitor-containing food express new trypsin molecules insensitive to the inhibitors. These inhibitors are proteins and their binding to the enzyme has molecular requirements similar to those of the substrate.
Cysteine and aspartic proteinases are the only midgut proteinases in hemipterans and they occur in addition to serine proteinases in cucujiformia beetles. Their occurrence in Hemiptera is interpreted as a consequence of the loss of the usual digestive serine proteinases associated with the adaptation of hemipteran ancestors to a diet lacking proteins (plant sap), followed by the use of lysosome-like enzymes in adapting to a new predatory habit.
The presence of cysteine and aspartic proteinases in cucujiformia beetles is likely an ancestral adaptation to circumvent proteolytic inhibition caused by trypsin inhibitors in ingested seeds.
Cysteine and aspartic proteinases have pH optima of 5.5 to 6.0 and 3.2 to 3.5 and molecular masses of 20 to 40 kDa and 60 to 80 kDa, respectively. Because of their pH optima, aspartic proteinases are not very active in the mildly acidic midguts of Hemiptera and cucujiformia beetles, but are very important in the middle midguts (pH 3.5) of cyclorrhaphous flies.
Intermediate digestion of proteins is accomplished by exopeptidases, enzymes that remove amino acids from the N-terminal (aminopeptidases) or C-terminal (carboxypeptidases) ends of oligopeptides (fragments of proteins) (Fig. 1).
Insect aminopeptidases have molecular masses in the range 90 to 130 kDa, have pH optima of 7.2 to 9.0, have no marked specificity toward the N-terminal amino acid, and are usually associated with the microvillar membranes of midgut cells. Therefore, the action of aminopeptidase is restricted to the surface of midgut cells. Because aminopeptidases are frequently active on dipeptides, they are also involved in protein-terminal digestion together with dipeptidases.
Aminopeptidases may account for as much as 55% of the midgut microvillar proteins in larvae of the yellow mealworm, Tenebrio molitor. Probably because of this, in many insects aminopeptidases are the preferred targets of B. thuringiensis endotoxins. These toxins, after binding to aminopeptidase (or other receptors), form channels through which cell contents leak, leading to insect death.
The most important insect carboxypeptidases have alkaline pH optima, have molecular masses in the range 20 to 50 kDa, and require a divalent metal for activity. They are classified as carboxypeptidase A or B depending on their activity upon neutral/acid or basic C-terminal amino acids, respectively.
Digestion of Carbohydrates
Initial and intermediate digestion of starch (or glycogen) is accomplished by α-amylase. This enzyme cleaves internal bonds of the polysaccharide until it is reduced to small oligosaccharides or disaccharides (Fig. 2). The amylases are not very active in digesting intact starch granules, making mastication prior to ingestion important.
Insect amylases depend on calcium ions for activity or stability, they are activated by chloride ions (amylases in Lepidoptera are exceptions), their molecular masses are found in the range 48 to 68 kDa, and their pH optima vary widely (4.8-9.8) depending on the insect taxon. As described for trypsin, insects feeding on amylase inhibitor-containing food express new amylase molecules insensitive to the inhibitors.
The final digestion of starch chains occurs under α-glucosidases, enzymes that sequentially remove glucosyl residues from the nonreducing ends of short oligomaltosac-charides. If the saccharide is a disaccharide, it is named maltose (Fig. 2). Because of that, α-glucosidase is also called maltase.
As a rule, sucrose (glucose α1,β2-fructose) is hydrolyzed by α-glucosidase. If an enzyme is able to hydrolyze sucrose, but not maltose, it is likely a β-fructosidase, an enzyme attacking sucrose by the fructosyl residue. Sucrose is found in large amounts in nectar and phloem sap and in lesser amounts in some fruits and leaves.
The important insect hemolymph and fungal sugar trehalose (glucose α1,α1-glucose) is hydrolyzed only by the specific enzyme trehalase. This digestive enzyme occurs in luminal contents or immobilized at the surface of midgut cells and also as an enzyme at the midgut basal cell membrane, making available glucose from hemolymph trehalose.
Although cellulose is abundant in plants, most plant-feeding insects, such as caterpillars and grasshoppers, do not use it. Cellulose is a nonramified chain of glucose units linked by β-1,4 bonds (Fig. 3) arranged in a crystalline structure that is difficult to disrupt. Thus, cellulose digestion is unlikely to be advantageous to an insect that can meet its dietary requirements using more easily digested food constituents.
The cellulase activity found in some plant feeders facilitates the access of digestive enzymes to the plant cells ingested by the insects. True cellulose digestion is restricted to insects that have, as a rule, nutritionally poor diets, as exemplified by termites, woodroaches, and cerambycid and scarabaeid beetles.
There is growing evidence that insects secrete enzymes able to hydrolyze crystalline cellulose, challenging the long-standing belief that microbial symbionts are necessary for cellulose digestion. The end products of cellulase action are glucose and cellobiose (Fig. 3); the latter is hydrolyzed by a β-glucosidase.
Hemicellulose is a mixture of polysaccharides associated with cellulose in plant cell walls. They are β-1,4- and/or β-1,3-linked glycan chains made up mainly of glucose (glucans), xylose (xylans), and other monosaccharides.
The polysaccharides are hydrolyzed by a variety of enzymes from which xylanases, laminarinases, and lichenases are the best known. The end products of the actions of these enzymes are monosaccharides and β-linked oligosaccharides.
The final digestion of those chains occurs under the actions of β-glycosidases that sequentially remove glycosyl (usually glucosyl, galactosyl, or xylosyl) residues from the nonreducing end of the β-linked oligosaccharides. As these may be cellobiose, β-glycosidase is frequently also named cellobiase. Thus, β-glycosidases complete the digestion of cellulose and hemicelluloses.
A special β-glycosidase (aryl β-glycosidase) acts on glycolipids and in vivo probably removes a galactose from monogalactosyldiacylglycerol that together with digalactosyl-diacylglycerol is a major lipid of photosynthetic tissues. Digalactosyldiacylglycerol is converted into monogalactosyl-diacylglycerol by the action of an α-galactosidase.
The aryl β-glycosidase also acts on plant glycosides that are noxious after hydrolysis. Insects circumvent these problems by detoxifying the products of hydrolysis or by repressing the synthesis and secretion of this enzyme while maintaining constant the synthesis and secretion of the other β-glycosidases.
Digestion of Lipids and Phosphates
Oils and fats are triacylglycerols and are hydrolyzed by a triacylglycerol lipase that preferentially removes the outer ester links of the substrate (Fig. 4) and acts only on the water-lipid interface. This interface is increased by surfactants that, in contrast to the bile salts of vertebrates, are mainly lysophosphatides. The resulting 2-monoacylglycerol may be absorbed or further hydrolyzed before absorption.
Membrane lipids include glycolipids, such as galactosyl-diacylglycerol and phosphatides. After the removal of galactose residues from mono- and digalactosyldiacylglycerol, which leaves diacylglycerol, it is hydrolyzed as described for triacylglycerols.
Phospholipase A removes one fatty acid from the phosphatide, resulting in a lysophosphatide (Fig. 4) that forms micellar aggregates, causing the solubilization of cell membranes. Lysophosphatide seems to be absorbed intact by insects.
Nonspecific phosphatases remove phosphate moieties from phosphorylated compounds to make their absorption easier. Phosphatases are active in an alkaline or acid medium.