Na+/K+-ATPase are located on apical and basal membranes, respectively.
The plasma membrane V-ATPase of M. sexta is well characterized (Harvey et al. 1998). When feeding ceases in preparation for a larval-larval moult, downregulation of the V-ATPase is thought to be achieved by reversible dissociation of the peripheral ATP-hydrolysing complex from the membrane-bound H+-translocating complex (Sumner et al. 1995). Expression of V-ATPase genes is also downregulated at this time, under the control of ecdysteroids (Reineke et al. 2002). This economy is necessary because 10 per cent of larval ATP production is consumed by midgut K+ transport, that is, by the V-ATPase.
The pH of the midgut lumen varies with phylo-geny and feeding ecology, and extreme alkalinity occurs in several orders besides Lepidoptera (Clark 1999; Harrison 2001). Extreme pH has complex effects on the activity of ingested allelochemicals (Section 2.4.3 and see Appel 1994). For caterpillars, a disadvantage of high gut pH is that it facilitates activation of Bt toxin (Dow 1984). The midgut of mosquito larvae is highly alkaline, probably through similar molecular mechanisms, and this characteristic might provide a basis for disease vector control just as it has for control of agricultural pests (Harvey et al. 1998). Insect acid-base physiology was reviewed by Harrison (2001).
Gut absorption was reviewed by Turunen (1985). Absorption includes transport across both apical and basal membranes, but there is more information on apical mechanisms. These are usefully studied by using purified plasma membranes which form sealed vesicles containing fluid of known composition, and can be pre-loaded with ions or amino acids (Sacchi and Wolfersberger 1996).
Leucine absorption in the midgut of Philosamia cynthia larvae (Lepidoptera, Saturniidae) has been well studied, and a model for leucine uptake by columnar cells is shown in Fig. 2.7. Goblet and columnar cells cooperate in ionic homeostasis and absorption of nutrients: the V-ATPase and K+/ nH+ antiporter on the apical membrane of the goblet cells energize K+-amino acid symporters on the microvilli of columnar cells. Amino acid transport is coupled to the movement of K+ down its electrochemical gradient from lumen to cell. This contrasts with the Na+-cotransport system of vertebrates and some insects (e.g. cockroaches), involving the basolateral Na+/K+-ATPase and apical transport proteins in the same cell (Sacchi and Wolfersberger 1996). The affinity of the sym-porter for Na+ is about 18 times that for K+, but since the luminal K+ concentration is 200 times higher (Fig. 2.7), most amino acid absorption is K+-dependent (Sacchi and Wolfersberger 1996). Transport of neutral amino acids has received the most attention, and this model seems to be generally applicable to other lepidopteran larvae. Neutral amino acid-K+ symport is effective over most of the pH range found in M. sexta midgut, but occurs mostly in the posterior third of the midgut where luminal pH is less extreme (Sacchi and Wolfersberger 1996). The latter review of amino acid absorption in insect midgut was updated by Wolfersberger (2000), including molecular studies of the symporters involved. The literature remains unbalanced in favour of large caterpillars, but the focus has shifted from P. cynthia to Bombyx mori and M. sexta, which can be reared throughout the year on artificial diets. Several absorption mechanisms are evident in midguts of larval B. mori: the neutral amino acid-K+ symport described above, and a less selective uniport system which facilitates diffusion of amino acids (Giordana et al. 1998; Leonardi et al. 1998). Another uniport system transports the dibasic amino acids arginine and lysine (Casartelli et al. 2001). The cDNA encoding a K+-amino acid symporter from M. sexta midgut has been isolated and cloned, and the deduced amino acid sequence shows homology to mammalian amino acid transporters (Castagna et al. 1998).
Absorption of lipids requires solubilization in the layer of water adjacent to the absorptive cells, but the details are poorly understood in insects (Turunen and Crailsheim 1996). Nothing is known about the possible role of fatty acid transporters in the apical membrane of midgut cells (Arrese et al. 2001). After absorption, fatty acids are converted to diacylglycerols in midgut cells and released to a haemolymph lipoprotein called lipophorin: this is a transport protein which acts as a reusable shuttle and delivers diacylglycerols and other lipids to various tissues (Arrese et al. 2001). In the fat body the diacylglycerols are converted to triacylglycerols for storage, and in the larva of M. sexta they can be 30 per cent of the wet mass of this tissue. Lipophorin also transports diacylglycerols from fat body to flight muscles during sustained flight. Ryan and van der Horst (2000) recently reviewed lipid mobilization (in response to adipokinetic hormone) and lipid transport in relation to flight. These aspects of lipid biochemistry in insects, which are being used as a model system for comparison with vertebrates, seem to be better known than digestion and absorption.
Glucose transporters such as the well known Na+-glucose cotransporters have been intensively studied in vertebrates, although little is known about their equivalents in insects (but see Andersson Escher and Rasmuson-Lestander 1999). Evidence summarized by Turunen and Crailsheim (1996) suggests that glucose transport is passive in most insects: transport is unaffected by metabolic inhibitors, depends on concentration gradient, and fructose and unmetabolized 3-O-methylglucose are transported at the same rate as glucose. Crailsheim (1988) found that 3-O-methylglucose injected into the haemolymph of honeybees became equally distributed between midgut lumen and haemo-lymph in 30 min. It is assumed that fructose transport across the gut wall is also passive, and fructose is then converted to glucose by hexokinase and phosphoglucoisomerase (Bailey 1975). In the fat body, trehalose is synthesized from glucose via hexose phosphates (also intermediates in glycogen synthesis). Like the transport disaccharide of plants (sucrose), it is a non-reducing sugar and less reactive than glucose (Candy et al. 1997). Treherne (1958) first showed the conversion of labelled glucose and fructose to trehalose, and pointed out its significance in maintaining a steep concentration gradient for absorption of monosaccharides. Water absorption from the midgut would also increase this concentration gradient (Turunen and Crailsheim 1996). Absorption of sugars is fast and complete. For example, female mosquitoes that have been flown to exhaustion will resume continuous flight within a minute of starting to feed on glucose solution (Nayar and Van Handel 1971). Unfed honeybees given labelled glucose incorporate it into trehalose within 2 min of feeding and there is no loss of label in the excreta (Gmeinbauer and Crailsheim 1993).
These last examples concern insects with initially empty crops. Crop-emptying is in fact, the limiting process for absorption of monosaccharides in insects, and its control has been attributed variously to the osmolality or sugar concentrations of food or haemolymph. Recently the control of crop-emptying has been carefully investigated in honeybees (Apis mellifera carnica), using unrestrained bees trained to collect defined amounts of sucrose solution (Roces and Blatt 1999; Blatt and Roces 2001, 2002). As in other insect species, crop-emptying rates measured by volume were inversely related to food concentration. Conversion to sugar transport rates showed that sugar left the crop at a constant rate, independent of food concentration but corresponding closely with the metabolic rate of the bees (Fig. 2.8). It is well known that the metabolic rate of honeybees depends on the reward rate at the food source (Moffat and Nunez 1997; see also Chapters 3 and 6). Haemolymph sugar homeostasis was maintained under all conditions except those involving dilute food and a high metabolic rate (induced by cold); haemolymph trehalose concentration then decreased. Crop-emptying is, therefore, adjusted to the energy demands of the bee, mediated by the trehalose concentration of its haemolymph.
We conclude this section on midgut physiology by considering phenotypic flexibility and whether there are reversible changes in gut surface area and absorption capacity depending on demand, as have been demonstrated in vertebrates (Diamond 1991; Weiss et al. 1998). Increased gut size helps to compensate for reduced food quality in grasshoppers (Yang and Joern 1994). Larval M. sexta reared on low protein diet allocate more tissue to midgut, although they still grow more slowly (Woods 1999). Woods and Chamberlin (1999) measured proline transport in the posterior midgut of M. sexta and found no response to dietary history (Fig. 2.9). They used flat sheet preparations bathed by asymmetrical salines designed to resemble in vivo conditions, and measured fluxes of 14C-labelled L-proline, which was transported from lumen to haemolymph 15 times faster than in the reverse direction. However, leucine transport in brush border membrane vesicles from the midgut of B. mori (Lepidoptera, Bombycidae) is increased in starved larvae (Leonardi et al. 2001). In contrast to data from vertebrates (e.g. Weiss et al. 1998) showing upregulation of gut function in response to increased substrate levels, the compensatory responses described above in insects suggest
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