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 (Fig. 1). 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 mid-gut 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. Microbes may provide insects with nutrients or help detoxifying noxious substances with a negligible role in digestion.
DIGESTION OF PROTEINS
Initial digestion of proteins is carried out by proteinases (endopepti-dases), which are enzymes able to cleave the internal peptide bonds of proteins (Fig. 1A). For this different endopeptidases are necessary, because the amino acid residues vary along the peptide chain (R is a variable group in Fig. 1A). 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 protein-ases that are widely distributed in insects and have molecular masses in the range 20-35 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. Also, insects fed on trypsin inhibitor-containing food may express new trypsin molecules insensitive to the inhibitors, due to changes in their primary specificities or binding properties of their subsites.
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 protein-ases associated with the adaptation of hemipteran ancestors to a diet usually 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 protein-ases have pH optima of 5.5-6.0 and 3.2-3.5 and molecular masses of 20-40 kDa and 60-80 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 exopepti-dases, enzymes that remove amino acids from the N-terminal (aminopeptidases) or C-terminal (carboxypeptidases) ends of oli-gopeptides (fragments of proteins) (Fig. 1A). Insect aminopeptidases have molecular masses in the range 90-130 kDa, have pH optima of 7.2-9.0, have no marked specificity toward the N-terminal amino acid, and are usually associated with the microvillar membranes 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 20-50 kDa, and require a divalent metal for activity. They are classified as car-boxypeptidase 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 a-amylase. This enzyme cleaves internal bonds of the polysac-charide until it is reduced to small oligosaccharides or disaccharides (Fig. 1B). 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 48-68 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 a-glucosi-dases, enzymes that sequentially remove glucosyl residues from the nonreducing ends of short oligomaltosaccharides. If the saccharide is a disaccharide, it is named maltose (Fig. 1B) . Because of that, a-glucosidase is also called maltase. As a rule, sucrose (glucose a 1,( 2-fructose) is hydrolyzed by a -glucosidase.
The important insect hemolymph and fungal sugar trehalose (glucose a1,a1-glucose) is hydrolyzed only by the specific enzyme trehalase. This digestive enzyme occurs in luminal contents or is immobilized at the surface of midgut cells and also as an enzyme
FIGURE 1 Digestion of important nutrient classes. Arrows point to bonds cleaved by enzymes. (A) Protein digestion; R, different amino acid moieties. (B) Starch digestion. (C) (3-linked glucoside. (D) Lipid digestion; PL, phospholipase; R, fatty acyl moieties.
at the midgut basal cell membrane, making available glucose from hemolymph trehalose. Trehalase is inhibited by plant glucosides like amygdalin and esculin with deleterious consequences.
Although cellulose is abundant in plants, most plant-feeding insects, such as caterpillars and grasshoppers, do not use it. Cellulose is a chain of glucose units linked by (3-1,4 bonds (Fig. 1C) 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. The end products of cellulase action are glucose and cellobiose (Fig. 1C); the latter is hydrolyzed by a ( -glucosidase.
Hemicellulose is a mixture of polysaccharides associated with cellulose in plant cell walls. They are 3-1,4-, and/or (3-1,3-linked glycan chains made up mainly of glucose (glucans), xylose (xylans), and other monosaccharides. The polysaccharides are hydrolyzed by a variety of enzymes, of which xylanases, laminarinases, and lichenases are the best known. The end products of the actions of these enzymes are monosaccharides and (3-linked oligosaccharides. The final digestion of those chains occurs under the actions of ( -glycosidases that sequentially remove glycosyl residues from the nonreducing end of the p-linked oligosaccharides. As these may be cellobiose, p-glycosi-dase is frequently also named cellobiase.
A special p-glycosidase (aryl p-glycosidase) acts on glycolipids and in vivo probably removes a galactose from monogalactosyldiacylglyc-erol that together with digalactosyldiacylglycerol is a major lipid of photosynthetic tissues. Digalactosyldiacylglycerol is converted into monogalactosyldiacylglycerol by the action of an a -galactosidase. The aryl p-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 p-glycosidases.
The rupture of the bacterial cell wall catalyzed by lysozyme is important in insects like the house fly larvae. Fungi are nutrients especially for detritivorous insects. The fungal wall is broken by digestive chitinases and laminarinases.
DIGESTION OF LIPIDS AND PHOSPHATES
Oils and fats are triacylglycerols and are hydrolyzed by a triacylglyc-erol lipase that preferentially removes the outer ester links of the substrate (Fig. 1D) 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 galactosyldiacylglyc-erol 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. 1D) that forms micellar aggregates, causing the solubilization of cell membranes. Lysophosphatide seems to be absorbed intact by insects.
Nonspecific phosphatases remove phosphate moieties from phos-phorylated compounds to make their absorption easier. Phosphatases are active in an alkaline or acid medium.
