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Stoffolano (1995) has referred to the midgut as the least studied, but largest, endocrine tissue in insects.

2.2.3 Regulation of protein and carbohydrate intake

Waldbauer and Friedman (1991) defined self-selection of optimal diets as a continuous regulation of intake involving frequent shifts between foods. The fact that insects perceive nutritional deficiencies, and alter behaviour to correct them, has been clearly illustrated by application of the geometrical approach to protein and carbohydrate intake in Locusta migratoria. Many aspects of nutritional regulation in this species stem from interactions between these two macronutrients (Raubenheimer and Simpson 1999). Animals given a balanced diet, or two or more unbalanced but complementary diets, can satisfy their nutrient requirements (arrive at the same point in nutrient space) and achieve similar growth performances (Fig. 2.2). It must be emphasized that the nutritional needs reflected in intake targets are not static. A flight of 2 h duration moves the intake target of adult L. migratoria towards increased carbohydrate levels, and targets also vary as requirements change throughout development (Raubenheimer and Simpson 1999).

When fed unbalanced diets and prevented from reaching their intake targets, the grass-feeding species L. migratoria is less willing to eat an unwanted nutrient than the polyphagous Schistocerca gregaria, perhaps because the latter has a better chance of encountering new host plants of different composition (Raubenheimer and Simpson 1999). Put another way, the amount eaten of the unbalanced food should reflect the probability of encountering an equally and oppositely unbalanced food. This is supported by comparisons of nutritional regulation in solitary and gregarious phases of S. gregaria (Simpson et al. 2002). Solitary locusts are less mobile and encounter fewer host plants, and so experience less nutritional heterogeneity. Gregarious locusts, not subject to these constraints, ingest more of the excess nutrient in unbalanced foods. Locusts respond rapidly to nutritional deficiencies: compensatory selection for either protein or carbohydrate was evident after a single deficient meal in L. migratoria, but not in another relatively sessile herbivore, Spodoptera littoralis, in which the response may take longer to develop (Simpson et al. 1990). Interactions between nutrients and allelo-chemicals in locust feeding are considered below (Section 2.4.3). On a more detailed level, Phoetaliotes nebrascensis grasshoppers are able to select individual amino acids from a background mixture of amino acids and sucrose applied to glass fibre discs (Behmer and Joern 1993, 1994). This selection is determined by nutrient requirements: Nymphs but not adults preferred diets high in phenylalanine (needed for cuticle production), while adult females but not males preferred high proline concentrations (probably because of the protein demands of egg production).

When diet selection was investigated in Blatella germanica (Blattaria, Blatellidae) using paired foods differing in protein and carbohydrate content, the intake target was biased towards carbohydrate because symbionts contributed to nitrogen balance, and cellulose digestion compensated for inadequate levels of soluble carbohydrate in diluted diets (Jones and Raubenheimer 2001). Kells et al. (1999) investigated the nutritional status of the same cockroach species in the 'field' (low income apartments). In spite of the reputation of cockroaches as successful generalists, the apartment diet was considered suboptimal (low in protein) compared to rodent chow because the uric acid content of field cockroaches was much lower. Stored uric acid is utilized by symbiont bacteria (see Section 2.4.2).

Nutritional homeostasis involves not only long-term regulation of feeding, but also of post-ingestive utilization. The geometric approach draws attention to the fact that regulating intake of one nutrient often involves ingesting, and then removing, excesses of another. There is so far little evidence in insects for pre-absorptive removal of excess nutrients by the most likely mechanisms of increasing gut emptying rate, decreasing enzyme secretion, or reducing absorption rates (for references see Simpson et al. 1995). Instead, the major site of differential regulation appears to be post-absorptive. Nymphs of L. migratoria feeding on unbalanced foods remove excess nitrogen by increased uric acid excretion (Fig. 2.3b) and excess carbon by increased CO2 output, that is, 'wastage' respiration (Zanotto et al. 1993, 1997). Use of chemically defined diets with sucrose radiolabelled in either the glucose or fructose moiety has shown that the pea aphid, Acyrthosiphon pisum, preferentially assimilates and respires the fructose from ingested sucrose, while converting the glucose into oligosaccharides which are excreted in the honeydew (Ashford et al. 2000).

2.3 Digestion and absorption of nutrients

Some digestion may occur in the crop, as a result of salivary enzymes or midgut enzymes moving anteriorly (as in beetles, Terra 1990), but most biochemical transformation occurs in the midgut. Occasionally the midgut has a storage function, like the anterior midgut of Rhodnius prolixus (Hemiptera, Reduviidae) (exploited by researchers wishing to obtain blood non-invasively from bats, Helverson et al. 1986). Dow's (1986) review brought together information on ultrastucture, ion transport, enzymes, and detoxification for midguts from all main insect feeding types shown in Fig. 2.1. In addition to the hindgut (Section 4.1.3), the midgut is a major site of ion regulation (which is fundamental to nutrient absorption) and the best understood transport epithelium in insects.

The midgut, as a primary interface between insect and environment, is a target for insect control. Insecticides based on Bt toxin from the soil bacterium Bacillus thuringiensis are highly effective against certain pests, particularly larvae of Lepi-doptera, Coleoptera, and Diptera, and transgenic crops expressing Bt genes are now in widespread use. The toxin, which is activated by midgut proteases, is thought to bind to receptor proteins on the columnar cell microvilli; it then undergoes a conformational change and inserts into the membrane in aggregates, forming pores that result in osmotic lysis and disintegration of the epithelium (Pietrantonio and Gill 1996). Assays of osmotic swelling in membrane vesicles from the midguts of target insects can be used to measure susceptibility to the toxins produced by different strains of Bt (Escriche et al. 1998). Young, actively feeding larvae are most affected, and the success of Bt products as microbial insecticides is due to their specificity to the target insects. Possible harmful effects on nontarget species, including natural enemies, are the focus of active research (Zangerl et al. 2001; Dutton et al. 2002).

2.3.1 Digestive enzymes and the organization of digestion

Digestive enzymes

Insect digestive enzymes are all hydrolases, show general similarities to mammalian enzymes and are classified using standard nomenclature based on the reactions they catalyse (Applebaum 1985; Terra and Ferreira 1994; Terra et al. 1996a). The biochemistry and molecular biology of purified enzymes is currently an active field. Molecular biology is now used as an alternative to exhaustive protein purification, especially for the large arrays of serine proteinase genes present in disease vectors and other insects (Muharsini et al. 2001). For example, there are about 200 genes encoding serine protei-nases in the genome sequence of D. melanogaster (Rubin et al. 2000).

Serine proteinases (of which the trypsins and chymotrypsins are well characterized) hydrolyse internal peptide bonds, while carboxypeptidases and aminopeptidases remove terminal amino acids. The term protease, thus, includes both proteinases (endopeptidases) and exopeptidases. Serine proteinases of blood-sucking insects are important in vector-parasite relationships: infection requires that the parasite survive protease activity in the midgut. For phytophages, naturally occurring proteinase inhibitors are important secondary plant compounds (Section 2.4.3), and when chewing breaks up plant cell walls some of the plant enzymes released are also active in the gut lumen (Appel 1994). In bruchid beetle larvae, which specialize on a diet of legume seeds, reduced levels of proteases are complemented by additional enzymes obtained from the seeds (Applebaum 1964). The main digestive proteinases of Bruchidae are not serine but cysteine proteinases, which require a lower pH and are common in the midguts of Hemiptera and some Coleoptera. The distribution of cysteine proteinases in beetles has a phylo-genetic basis. These enzymes first appeared in an

□ Cysteine proteinases absent ■ Cysteine proteinases present

\ Equivocal

Figure 2.5 Hypothesized cladogram showing the distribution of cysteine proteinases in the major superfamilies of Coleoptera. Note: The cladogram is based on data for 52 species representing 17 families. Some secondary loss of cysteine proteinases has occurred in the more derived and phytophagous superfamilies.

Source: Reprinted from Comparative Biochemistry and Physiology B, 126, Johnson and Rabosky, 609-619. © 2000, with permission from Elsevier.

ancestor of the series Cucujiformia (Fig. 2.5), perhaps in response to a seed diet rich in trypsin inhibitors, and in total occur in five related super-families (Johnson and Rabosky 2000). Cysteine proteinases have been secondarily lost in some Cerambycidae (superfamily Chrysomeloidea), suggesting reversion to a digestive strategy utilizing serine proteinases. Although polymer digestion is considered unnecessary in aphids, Cristofoletti et al. (2003) have recently demonstrated substantial cysteine proteinase activity in the midgut of the pea aphid, A. pisum.

Carbohydrases fall into two broad categories according to whether they are active on poly-saccharides or on smaller fragments. Amylases (active on starch or glycogen) and cellulases (see Section 2.4.1) cleave internal bonds in polysaccharides. Lysozyme is involved in the digestion of bacterial cell walls in the midgut of cyclo-rrhaphan Diptera. The second category includes glucosidases and galactosidases, which hydrolyse oligosaccharides and disaccharides. Use of the term 'sucrase' does not differentiate between sucrose hydrolysis by a-glucosidases or less common b-fructosidases. Trehalase, which hydrolyses tre-halose into glucose, is widespread in insect tissues and, in the midgut, may counteract back-diffusion of trehalose into the lumen (first suggested by Wyatt 1967).

Insect lipases are less easily studied than proteases and carbohydrases because of their lower activities. Moreover, reaction between the enzymes, which are water soluble, and their substrates, which are not, requires a suitable emulsion. These are difficult to prepare experimentally (Applebaum 1985). Phospholipases act on membrane lipids and cause cell lysis. Triacylglycerols are major lipid components of the diet and are hydrolysed to fatty acids and glycerol. In general, lipid digestion is poorly understood in insects (Arrese et al. 2001).

Regulation of enzyme levels

Do the levels of digestive enzymes vary according to the quantity or quality of food? Continuous and discontinuous feeders will obviously have different requirements regarding control of digestive enzyme secretion. Ultrastructural evidence indicates that secretion usually follows soon after synthesis, even in discontinuous feeders, although there are examples of storage of enzymes in the latter group (Lehane and Billingsley 1996). Synthesis and secretion are controlled in two ways: 'secretagogue' stimulation according to the amount of relevant substrate in the gut, or hormonal regulation. These are not necessarily mutually exclusive, and may operate on different time scales (Applebaum 1985). Unfortunately, experimentally distinguishing these types of control is difficult. The best evidence for hormonal influences comes from mosquitoes (Lehane etal. 1995). Because the term 'secretagogue' can be confusing, the latter authors have proposed that direct interaction of a component of the meal with enzyme-producing cells should be termed a prandial mechanism. They also distinguish between paracrine and endocrine mechanisms: a paracrine effect is a local hormonal effect on neighbouring cells. The diffuse endocrine system of the insect midgut remains a major obstacle to differentiating between prandial and endocrine control.

Many studies have investigated the effect of proteins on protease levels in haematophagous Diptera, because of their large and infrequent blood meals, and their role as disease vectors. Diverse soluble proteins stimulate trypsin secretion into the incubation medium of midgut homogenates of the stable fly Stomoxys calcitrans (Diptera, Muscidae), and the effect is concentration-dependent (Blakemore et al. 1995). This method distinguishes between effects on synthesis and on secretion, because new synthesis over the time scale of the incubations is considered negligible. Insoluble proteins, small peptides and amino acids do not stimulate trypsin secretion. Regulation of levels can vary within an enzyme family. In female Anopheles gambiae (Diptera, Culicidae), for example, some trypsins are constitutively expressed, while others, produced in larger amounts, are induced by blood feeding (Muller et al. 1995). Rapid advances at the molecular level are being driven by interest in the regulation of serine proteinases of blood-sucking insects (Lehane et al. 1995). This interest extends to midgut immunity through the recently discovered defensin family of peptides. Hamilton et al. (2002) have shown that midgut defensins of S. calcitrans are colocalized with a serine proteinase during storage, and that the complex dissociates on secretion into the lumen, the defensins protecting the stored blood meal from bacterial attack. Insects adapt to proteinase inhibitors in their diet by hyperproduction of proteinases or by switching to novel proteinases that are insensitive to these plant defences (see Section 2.4.3).

Peritrophic matrix and the organization of digestion The midgut cells of most insects (Hemiptera excluded) secrete a multilayered peritrophic matrix (although frequently called a peritrophic membrane, it lacks cellular structure) consisting of chitin, proteins, and proteoglycans. This functions as a physical barrier to protect the epithelium from mechanical abrasion, toxic plant allelochemicals, and pathogens, and also allows compartmentaliza-tion of the gut lumen and the spatial organization of digestive processes. Large macromolecules are hydrolysed by soluble enzymes inside the peritrophic matrix, until they are small enough to diffuse into the space between the peritrophic matrix and midgut epithelium, where digestion is completed by membrane-bound enzymes which may be integral proteins of the microvillar membranes (Terra et al. 1996b). Structural strength is provided by the meshwork of chitin fibrils, while permeability properties are determined by pore diameters in the gel-like matrix. Labelled dextrans with diameters ranging from 21 to 36 nm penetrate the peritrophic matrix of several species of Lepidoptera and Orthoptera (Barbehenn and Martin 1995), so size exclusion does not explain impermeability to digestive enzymes or to tannins, and other properties of the matrix may be involved.

The importance of midgut compartments in the efficient, sequential breakdown of food was first demonstrated in Rhynchosciara americana (Diptera, Sciaridae) by assaying enzyme activities in different luminal compartments and midgut tissue (Terra et al. 1979). It is also thought that counter-current flux of fluid assists in both the absorption of nutrients and the recycling of digestive enzymes. Countercurrent fluxes may result from fluid secretion in the posterior midgut or the anterior movement of primary urine from the Malpighian tubules, the result being that fluid moves in an anterior direction outside the pertriophic matrix, and is absorbed in the anterior midgut or caecae. The anatomical differences vary with phylogeny. The evidence for compartmentalization of digestive processes in the major insect orders has been thoroughly reviewed by Terra and colleagues (Terra 1990; Terra and Ferreira 1994; Terra et al. 1996b). Countercurrent movement of gut fluids occurs in Orthoptera, but only in animals deprived of food (Dow 1981), and it is considered unlikely in continuously feeding caterpillars (Dow 1986; Woods and Kingsolver 1999). Terra and colleagues argue that phylogeny is more important than feeding habits in determining the enzymes present and the organization of digestion, and it is possible that most insect species possess a full complement of digestive enzymes, although the relative amounts vary with diet (Terra et al. 1996a). The fast gut passage rates of many insects suggest potential costs in terms of digestive enzyme loss, but countercurrent fluxes may displace enzymes anteriorly and aid in their recycling (Terra et al. 1996b).

2.3.2 Gut physicochemistry of caterpillars

Conditions in the gut lumen can vary dramatically among insect herbivores (Appel 1994). The most extreme conditions (and most expensive to maintain) are found in caterpillars. Dow (1984) recorded pH values over 12, the highest known in any biological system, in the anterior and middle regions of caterpillar midgut (Fig. 2.6). The large volume of the midgut compartment suggests substantial acid-base transport to regulate this extreme pH. Ion transport in the midgut of M. sexta has been studied intensively using electrophysio-logical techniques, initially because of its potent K+-transporting ability (Klein et al. 1996). Manduca midgut is a model tissue (the frog skin of invertebrates), possessing the advantages of large size, commercial availability of insects and synthetic diet, and ease of making in vitro preparations. Caterpillar midgut was also the first animal tissue in which proton pumps were identified and found to energize secondary active transport (Wieczorek et al. 1991). Vacuolar-type proton ATPases (V-ATPases) are highly conserved enzymes located in bacterial, yeast, and plant plasma membranes but are now also known to occur in many animal plasma membranes (Harvey et al. 1998; Wieczorek et al. 1999). They are coupled to antiporters: in caterpillar midgut the V-ATPase is coupled to a K+/2H+ antiporter to produce what was long assumed to be a primary K+ pump (Harvey et al. 1998). The V-ATPase is confined to the apical membrane of the goblet cells (shown by

Insect Caterpillar Midgut

Figure 2.6 Midgut pH profile for four larval lepidopterans, with values for food and faeces shown for comparison.

Note: In all cases, haemolymph pH was 6.7. Species: circles, Acherontia atropos (Sphingidae); triangles, Manduca sexta (Sphingidae); squares, Lichnoptera felina (Noctuidae); and asterisks, Lasiocampa quercus callunae (Lasiocampidae). Mean ± SE, n = 4.

Figure 2.6 Midgut pH profile for four larval lepidopterans, with values for food and faeces shown for comparison.

Note: In all cases, haemolymph pH was 6.7. Species: circles, Acherontia atropos (Sphingidae); triangles, Manduca sexta (Sphingidae); squares, Lichnoptera felina (Noctuidae); and asterisks, Lasiocampa quercus callunae (Lasiocampidae). Mean ± SE, n = 4.

immunohistochemistry using plasma membrane fractions) and is responsible for a large lumenpositive apical voltage in excess of 150 mV. It serves to alkalinize the midgut lumen, maintaining favourable pH conditions for enzyme activity, and energizes amino acid uptake via K+-amino acid symport (see below). Alkalinization results from the stoichiometry of the K+/2H+ antiporter and the high voltage generated by the V-ATPase, the result being net K+ secretion and net H+ absorption (Wieczorek et al. 1999). A model of midgut transport processes is presented in Fig. 2.7.

Leaf-eating caterpillars have a diet very low in Na+, and their low haemolymph Na+ concentration precludes use of a sodium pump as primary energizer (goblet cells lack any detectable Na+/K+-ATPase). However, carnivorous and some herbivorous insects may use the Na+/K+-ATPase to drive absorption of fluid and organic solutes, as vertebrates do (Klein et al. 1996). In both cases secondary processes are coupled to a primary ion transport ATPase, but the V-ATPase and

[Leu] = 0.58 mM [Na+] = 4.6 mM [K+] = 24 mM pH =6.8

Lumen

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