DIGESTION IN ADULT FEMALES OF THE LEAF‐FOOTED BUG Leptoglossus zonatus (HEMIPTERA: COREIDAE) WITH EMPHASIS ON THE GLYCOSIDE HYDROLASES α‐AMYLASE, α‐GALACTOSIDASE,AND α‐GLUCOSIDASE

The leaffooted bug, Leptoglossus zonatus (Hemiptera: Coreidae) is an emerging pest of several crops around the World and up to now very little is known of its digestive system. In this article, glycoside hydrolase (carbohydrase) activities in the adult midgut cells and in the luminal contents of L. zonatus adult females were studied. The results showed the distribution of digestive carbohydrases in adults of this heteropteran species in the different intestinal compartments. Determination of the spatial distribution of α‐glucosidase activity in L. zonatus midgut showed only one major molecular form, which was not equally distributed between soluble and membrane‐bound isoforms, being more abundant as a membrane‐bound enzyme. The majority of digestive carbohydrases were found in the soluble fractions. Activities against starch, maltose and the synthetic substrate NPαGlu were found to show the highest levels of activity, followed by enzymes active against galactosyl oligosaccharides. Based on ion‐exchange chromatography elution profiles and banding patterns in mildly denaturing electrophoresis, both midgut α‐amylases and α‐galactosidases showed at least two isoforms. The data suggested that the majority of carbohydrases involved in initial digestion were present in the midgut lumen, whereas final digestion of starch and of galactosyl oligosaccharides takes place partially within the lumen and partially at the cell surface. The complex of carbohydrases here described was qualitatively appropriate for the digestion of free oligosaccharides and oligomaltodextrins released by α‐amylases acting on maize seed starch granules.     

Carbohydrases and carbohydrate digestion in the gut of Locusta migratoria

The digestion of various carbohydrates and synthetic substrates by the gut of Locusta migratoria was analysed quantitatively. Maltose, starch, and sucrose were found to be hydrolysed most rapidly, whereas the splitting of cellobiose, trehalose, lactose, and melecitose took place at much slower rates.The absolute carbohydrase activities in foregut and midgut are nearly equal. However, specific activities are much higher in the foregut. Only low activities were found in extracts from the hindgut and salivary glands. The latter show a pattern of sugar splitting which is different from that found in gut preparations.The distribution of carbohydrase activities between the epithelia and lumina of the foregut, midgut, and hindgut and between soluble and particulate fractions were studied. The midgut epithelium is shown to have a particularly high content of enzymes, although some carbohydrases are rather active also in the epithelium of the hindgut. During hunger periods the relative enzymatic activities of the epithelium are distinctly increased.The isolation and purification of the carbohydrases were attempted and a partial separation of individual enzymes was obtained by gel-filtration. These results indicate the presence of at least seven distinct carbohydrases in the locust gut. The molecular weights of the enzymes were estimated by gel-filtration, and K_M values and pH-optima are reported.     

Effects of rearing density on larval growth and activity of digestive enzymes in Lymantria dispar L. (Lepidoptera: Lymantriidae)

Density dependent responses of 4th, 5th and 6th instar gypsy moth larvae were studied at the level of larval mass, midgut loading and activities of three digestive enzymes (alpha-amylase, trypsin and leucine aminopeptidase). High density significantly reduced larval mass while midgut loading (expressed as relative midgut mass) did not change except in the 5th instar where it was increased at high density. Specific amylase and leucine aminopeptidase activities were not affected by crowding. Specific trypsin activity was on average higher in crowded than in isolated larvae. High density also affected the correlations between midgut protein content and activities of two proteolytic enzymes suggesting differences in regulatory mechanisms of insect digestion. The importance of these changes for survival under stressful conditions is discussed.     

Salivary digestive enzymes of the wheat bug, Eurygaster integriceps (Insecta: Hemiptera: Scutelleridae)

The digestive enzymes from salivary gland complexes (SGC) of Eurygaster integriceps, and their response to starvation and feeding were studied. Moreover, digestive amylases were partially purified and characterized by ammonium sulfate precipitation and gel filtration chromatography. The SGC are composed of two sections, the principal glands and accessory glands. The principal glands are further divided into the anterior lobes and posterior lobes. The SGC main enzyme was α-amylase, which hydrolyzed starch better than glycogen. The other carbohydrases were also present in the SGC complexes. Enzymatic activities toward mannose (α/β-mannosidases) were little in comparison to activities against glucose (α/β-glucosidases) and galactose (α/β-galactosidases), the latter being the greatest. Acid phosphatase showed higher activity than alkaline phosphatase. There was no measurable activity for lipase and aminopeptidase. Proteolytic activity was detected against general and specific protease substrates. Activities of all enzymes were increased in response to feeding in comparison to starved insects, revealing their induction and secretion in response to feeding pulse. The SGC amylases eluted in four major peaks and post-electrophoretic detection of the α-amylases demonstrated the existence of at least five isoamylases in the SGC. The physiological implication of these findings in pre-oral digestion of E. integriceps is discussed.     

The effect of adipokinetic hormone on midgut characteristics in Pyrrhocoris apterus L. (Heteroptera)

Digestive processes and the effect of adipokinetic hormone (Pyrap-AKH) on the amount of nutrients (lipids, proteins, and carbohydrates), and on the activity of digestive enzymes (lipases, peptidases, and carbohydrases) were studied in the midgut of the firebug, Pyrrhocoris apterus. The analyses were performed on samples of anterior (AM), middle (MM) and posterior (PM) midgut parts. The results revealed that the digestion of lipids, carbohydrates and proteins take place in the acidic milieu. The Pyrap-AKH treatment increased significantly the level of lipids and proteins in the midgut, and also the level of triacylglycerols (TGs) predominantly in the AM, and the level of diacylglycerols (DGs) in the MM. The increase was not uniform for all present TG and DG species - those containing the linoleic fatty acid were predominant. No hormonal effect on lipase activity was recorded, while peptidase and glucosidase activity was increased in the MM and PM. All these facts indicate that the Pyrap-AKH probably stimulates digestion by more intensive food ingestion or turnover, and perhaps by the stimulation of metabolite absorption; the activation of digestive enzymes seems to be secondary or controlled by other mechanisms.     

Archives of insect biochemistry and physiology guide for authors

Several carbohydrases and glycosidases from the alimentary cancal and/or salivary glands of feeding larvae of mayetiola destructor have been identified. Pectinase activity was identified in the midgut and may be present in the salivary glands. No endocellulase activity was found in larvae; however, hemicellulase activity was detected in extract of larvae. Amylase activity was present in midguts from feeding larvae and at a low level in extract of salivary glands. Amylases detected in the midgut showed mobilities during polyacrylamide gel electrophoresis similar to the two major amylases in tissues of the insect's host plant. The possibility exists that Hessian fly larvae utilize amylases obtained from their host plant in the digestion of starch. The major glycosidases detected in the midgut lumen of larve were: α-D-glucosidase and α-D-and β-D-galactosidase. The role of these enzymes in the feeding process of Hessian fly larvae is discussed as well as their potential role in feeding damage to wheat.     

Responses of midgut amylases of Helicoverpa armigera to feeding on various host plants

Midgut digestive amylases and proteinases of Helicoverpa armigera, a polyphagous and devastating insect pest of economic importance have been studied. We also identified the potential of a sorghum amylase inhibitor against H. armigera midgut amylase. Amylase activities were detected in all the larval instars, pupae, moths and eggs; early instars had lower amylase levels which steadily increased up to the sixth larval instar. Qualitative and quantitative differences in midgut amylases of H. armigera upon feeding on natural and artificial diets were evident. Natural diets were categorized as one or more members of legumes, vegetables, flowers and cereals belonging to different plant families. Amylase activity and isoform patterns varied depending on host plant and/or artificial diet. Artificial diet-fed H. armigera larvae had comparatively high amylase activity and several unique amylase isoforms. Correlation of amylase and proteinase activities of H. armigera with the protein and carbohydrate content of various diets suggested that H. armigera regulates the levels of these digestive enzymes in response to macromolecular composition of the diet. These adjustments in the digestive enzymes of H. armigera may be to obtain better nourishment from the diet and avoid toxicity due to nutritional imbalance. H. armigera, a generalist feeder experiences a great degree of nutritional heterogeneity in its diet. An investigation of the differences in enzyme levels in response to macronutrient balance and imbalance highlight their importance in insect nutrition.     

Biochemical characterization of α-amylase of the Sunn pest, Eurygaster integriceps

The α‐amylase in the midgut and salivary glands of Eurygaster integriceps was isolated and characterized. The specific activity of α‐amylase in the midgut was 1.77 U/mg protein and in the salivary glands was 1.65 U/mg protein. Sodium dodecylsulfate electrophoresis showed that both midgut and salivary glands contain isozymes. Only a trace amount of α‐amylase activity was detected in the first nymphal stage (0.19 U/mg protein), whereas α‐amylase activity was highest in the third nymphal stage (1.21 U/mg protein). The results show that α‐amylase activity in the immature stages increase constantly to the third instar stage. There was no significant difference in enzyme activity between the third, fourth and fifth nymphal stages and adults. The optimum pH and temperature for the enzyme activity was determined to be 6.5 and 35°C, respectively. The enzyme activity was inhibited by addition of ethylenediaminetetraacetic acid, urea, sodium dodecylsulfate and Mg²⁺, but NaCl and KCl enhanced enzyme activity.     

Biochemical characterization of digestive α-amylase, α-glucosidase and β-glucosidase in pistachio green stink bug,Brachynema germari Kolenati (Hemiptera: Pentatomidae)

The pistachio green stink bug, Brachynema germari, has 3–5 generations per year and causes severe damages to pistachio crops in Iran. Physiological digestive processes, such as digestive carbohydrases, can be used to design new strategies in IPM programs for controlling this pest. The enzyme α-amylase digests starch during the initial stage of digestion. Complete breakdown of carbohydrates takes place in the midgut where α- and β-glucosidic activities are highest. Alpha-amylase and α- and β-glucosidase activities were found in the midgut and salivary glands of pistachio green stink bug adults. Overall enzyme activities were significantly higher in the midgut than in salivary glands. The highest α-amylase and α- and β-glucosidase activities were in section v3, whereas the lowest activities were in section v4. V_max was higher and K_m was lower in the midgut than in the salivary glands for these enzymes. In the pistachio green stink bug, the optimal pH was pH 5–6.5 and the optimal temperature was 30 °C to 35 °C for these enzymes. Alpha-amylase activity in the midgut and salivary glands decreased as the concentrations of MgCl₂, EDTA and SDS increased. Enzyme activities in both midgut and salivary glands increased in the presence of NaCl, CaCl₂, and KCl. NaCl had a negative effect on alpha-amylase extracted from salivary glands.     

Pancreatic and intestinal carbohydrases are matched to dietary starch level in wild passerine birds

Evolutionary shifts in diet composition are presumably accompanied by simultaneous changes in digestive physiology. The adaptive modulation hypothesis predicts that activities of digestive enzymes should match the relative levels of their substrates in an animal's diet so that available membrane space and synthetic energy are not wasted on enzymes in excess of need. However, previous studies on captive passerine birds showed high intraspecific phenotypic flexibility only in proteases but not in carbohydrases in response to varying diet composition. In this study, we measured the activities of pancreatic, intestinal, and hepatic enzymes in six wild-caught passerine species. We predicted that if the adaptive modulation hypothesis holds during evolutionary shifts in diet composition in birds, then mass-specific activities of digestive enzymes should be correlated positively with the content of their relevant substrates in species' diets. Whereas mass-specific activities of proteases (aminopeptidase-N, trypsin, chymotrypsin, alanine aminotransferase) were not correlated with estimated dietary protein content, mass-specific activities of all studied carbohydrases (amylase, maltase, sucrase) were positively correlated with estimated dietary starch content. We conclude that activities of carbohydrases but not proteases are evolutionarily matched to diet composition in passerine birds. We hypothesize that the need for nitrogen and essential amino acids can prevent the evolution of a low activity of proteases, even in species feeding on a low-protein diet.     

Digestive morphophysiology of Gryllodes sigillatus (Orthoptera: Gryllidae)

The evolution of the digestive system in the Order Orthoptera is disclosed from the study of the morphophysiology of the digestive process in its major taxa. This paper deals with a cricket representing the less known suborder Ensifera. Most amylase and trypsin activities occur in crop and caeca, respectively. Maltase and aminopeptidase are found in soluble and membrane-bound forms in caeca, with aminopeptidase also occurring in ventriculus. Amaranth was orally fed to Gryllodes sigillatus adults or injected into their haemolymph. The experiments were performed with starving and feeding insects with identical results. Following feeding of the dye the luminal side of the most anterior ventriculus (and in lesser amounts the midgut caeca) became heavily stained. In injected insects, the haemal side of the most posterior ventriculus was stained. This suggested that the anterior ventriculus is the main site of water absorption (the caeca is a secondary one), whereas the posterior ventriculus secretes water into the gut. Thus, a putative counter-current flux of fluid from posterior to anterior ventriculus may propel digestive enzyme recycling. This was confirmed by the finding that digestive enzymes are excreted at a low rate. The fine structure of midgut caeca and ventriculus cells revealed that they have morphological features that may be related to their involvement in secretion (movement from cell to lumen) and absorption (movement from lumen to cell) of fluids. Furthermore, morphological data showed that both merocrine and apocrine secretory mechanisms occur in midgut cells. The results showed that cricket digestion differs from that in grasshopper in having: (1) more membrane-bound digestive enzymes; (2) protein digestion slightly displaced toward the ventriculus; (3) midgut fluxes, and hence digestive enzyme recycling, in both starved and fed insects.     

Prey digestion in the midgut of the predatory bug Podisus nigrispinus (Hemiptera: Pentatomidae)

Pre-oral digestion is described as the liquefaction of the solid tissues of the prey by secretions of the predator. It is uncertain if pre-oral digestion means pre-oral dispersion of food or true digestion in the sense of the stepwise bond breaking of food polymers to release monomers to be absorbed. Collagenase is the only salivary proteinase, which activity is significant (10%) in relation to Podisus nigrispinus midgut activities. This suggests that pre-oral digestion in P. nigrispinus consists in prey tissue dispersion. This was confirmed by the finding of prey muscles fibers inside P. nigrispinus midguts. Soluble midgut hydrolases from P. nigrispinus were partially purified by ion-exchange chromatography, followed by gel filtration. Two cathepsin L-like proteinases (CAL1 and CAL2) were isolated with the properties: CAL1 (14.7 kDa, pH optimum (pHo) 5.5, km with carbobenzoxy-Phe-Arg-methylcoumarin, Z-FR-MCA, 32 μM); CAL2 (17 kDa, pHo 5.5, km 11 μM Z-FR-MCA). Only a single molecular species was found for the other enzymes with the following properties are: amylase (43 kDa, pHo 5.5, km 0.1% starch), aminopeptidase (125 kDa, pHo 5.5, km 0.11 mM l-Leucine-p-nitroanilide), α-glucosidase (90 kDa, pHo 5.0, km 5mM with p-nitrophenyl α-d-glucoside). CAL molecular masses are probably underestimated due to interaction with the column. Taking into account the distribution of hydrolases along P. nigrispinus midguts, carbohydrate digestion takes place mainly at the anterior midgut, whereas protein digestion occurs mostly in middle and posterior midgut, as previously described in seed- sucker and blood-feeder hemipterans.     

Activity of digestive enzymes of thick-billed murre (Uria lomvia) and common murre (U. aalga) invaded by cestodes

Activities of digestive enzymes (proteases, carbohydrases, acid and alkaline phosphatases) are determined in intestinal mucosa of the thick-billed and common murres Comparative analysis of the obtained results is performed for non-infected and for birds infested by cestodes. It has been established that at invasion by cestode Alcataenia armillaris (Cestoda: Tetrabothriidae), activities of carbohydrase and alkaline phosphatase in intestinal mucosa of the thick-billed murre decreases. Parasitizing of cestodes Tetrabothrius jaegerskieldi (Cestoda: Tetrabothriidae) in intestine of the common murre induces a decrease of saccharase activity. There is studied kinetics of desorption of enzymes from digestive-transport surfaces of the bird intestine. Peculiarities of firmness of enzyme fixation are established on the surface of intestinal mucosa of invaded murres. According to the obtained data, a decrease of the carbohydrase activities in intestine of infested murres is likely to be due to absorption of a part of enzymes hydrolyzing carbohydrates on the surface of cestodes.     

Potential of the bean α‐amylase inhibitor αAI‐1 to inhibit α‐amylase activity in true bugs (Hemiptera)

True bugs (Hemiptera) are an important pest complex not controlled by Bt‐transgenic crops. An alternative source of resistance includes inhibitors of digestive enzymes, such as protease or amylase inhibitors. αAI‐1, an α‐amylase inhibitor from the common bean, inhibits gut‐associated α‐amylases of bruchid pests of grain legumes. Here we quantify the in vitro activity of α‐amylases of 12 hemipteran species from different taxonomic and functional groups and the in vitro inhibition of those α‐amylases by αAI‐1. α‐Amylase activity was detected in all species tested. However, susceptibility to αAI‐1 varied among the different groups. α‐Amylases of species in the Lygaeidae, Miridae and Nabidae were highly susceptible, whereas those in the Auchenorrhyncha (Cicadellidae, Membracidae) had a moderate susceptibility, and those in the Pentatomidae seemed to be tolerant to αAI‐1. The species with αAI‐1 susceptible α‐amylases represented families which include both important pest species but also predatory species. These findings suggest that αAI‐1‐expressing crops have potential to control true bugs in vivo.     

Carbohydrate digestion in Lutzomyia longipalpis' larvae (Diptera - Psychodidae)

Lutzomyia longipalpis is the principal species of phlebotomine incriminated as vector of Leishmania infantum, the etiological agent of visceral leishmaniasis in the Americas. Despite its importance as vector, almost nothing related to the larval biology, especially about its digestive system has been published. The objective of the present study was to obtain an overview of carbohydrate digestion by the larvae. Taking in account that phlebotomine larvae live in the soil rich in decaying materials and microorganisms we searched principally for enzymes capable to hydrolyze carbohydrates present in this kind of substrate. The principal carbohydrases encountered in the midgut were partially characterized. One of them is a α-amylase present in the anterior midgut. It is probably involved with the digestion of glycogen, the reserve carbohydrate of fungi. Two other especially active enzymes were present in the posterior midgut, a membrane bound α-glucosidase and a membrane bound trehalase. The first, complete the digestion of glycogen and the other probably acts in the digestion of trehalose, a carbohydrate usually encountered in microorganisms undergoing hydric stress. In a screening done with the use of p-nitrophenyl-derived substrates other less active enzymes were also observed in the midgut. A general view of carbohydrate digestion in L. longipalpis was presented. Our results indicate that soil microorganisms appear to be the main source of nutrients for the larvae.     

Activity, release and flow of digestive enzymes in the cricket, Gryllus bimaculatus

Abstract.  In the cricket Gryllus bimaculatus , proventricular pressure forces a nutrient fluid from the ground food-brei through a sieve at the base of the caecae, and the combination of secreted enzymes and water causes a rapid inflation of the caecae on the first day after imaginal ecdysis. The ceacal region of the midgut is the primary site for the secretion of digestive enzymes. Proteases and amylase flow from the caecae into the mostly empty crop on day 1, and carbohydrate and protein digestion starts as soon as food is present (6 h). Thereafter, much of the amylase activity (but not protease) in the crop is synthesized and released by the crop tissues themselves. Regurgitating proteases and amylases from the caecae into the crop after day 1 is most likely accomplished by temporarily halting proventricular peristalsis and allowing the caecal muscles to contract, forcing caecal contents, including enzymes, forward. The total activity of digestive enzymes in the caecae is virtually identical in 2-day-old fed and unfed females, indicating little or no secretagogue (prandial) regulation of enzyme secretion. Most of the digestive enzymes in the ventricular endoperitrophic space may originate from the mucus dragged from the caecae. Lipase activity is low in all gut regions in both starved and fed females. Head ligation or injection of trypsin modulating oostatic factor, allatostatin A or B fails to indicate any involvement of nerves or hormones in the release of digestive enzymes in the caecae. Gryllus bimaculatus appears to secrete digestive enzymes continuously, and a considerable loss of enzymes may occur at certain times through egestion.     

Biochemical characterization of digestive α‐d‐glucosidase and β‐d‐glucosidase from labial glands and midgut of wheat bug Eurygaster maura (Hemiptera: Scutelleridae)

The wheat bug Eurygaster maura (Hemiptera: Scutelleridae) is a potential pest of wheat and barley in Iran and other countries. Two major digestive enzymes of this insect, α‐d ‐glucosidase and β‐d ‐glucosidase, have been investigated. The midgut has four distinct regions including the first ventriculus (V1), second ventriculus (V2), third ventriculus (V3) and fourth ventriculus (V4). The study showed that the first three regions of the wheat bug midgut were acidic (pH 5.5–6), the fourth region of the midgut and hindgut pH were slightly acidic (pH 6.5–6.9) and the salivary gland (labial gland) pH was determined to be somewhat acidic (pH 5–5.5). Enzyme assay showed that α‐ and β‐glucosidase activity is present in both midgut and salivary glands of adult E. maura. The specific activities of midgut α‐ and β‐glucosidase were 11.2 and 10.8 mU/mg protein, respectively. The specific activities of these enzymes in salivary glands were 3.06 and 2.73 mU/mg protein, respectively. Optimum temperature and pH values for glucosidases were determined to be 30–35°C and 5, respectively. Glucosidases of the midgut were more stable than salivary glucosidases at 35°C. Evaluating enzymatic kinetic parameters showed that glucosidases of the midgut had more affinity as well as more velocity than that of salivary glands.     

Morphology and preliminary enzyme characterization of the salivary glands from the predatory bug Podisus nigrispinus (Heteroptera: Pentatomidae)

Podisus nigrispinus (Dallas) is a common predator in agricultural and natural systems in Neotropical America. Its feeding strategy involves extra-oral digestion and to better understand this process its salivary glands were extracted and subjected to morphological and preliminary enzyme characterization. The salivary glands of P. nigrispinus are formed by a pair of main and accessory gland complexes. The main salivary glands are further divided into an anterior and a posterior lobe. The compartmentalization of the salivary gland complex is likely to be important for the production, activation and release of the digestive enzymes used in the extra-oral digestion of prey items. Proteases and lipase, important digestive enzymes involved in zoophagy, were detected in the salivary glands of P. nigrispinus. The prevailing trypsin-like protease activity was characterized by using the serine-protease substrate N-alpha-benzoyl-L-Arg-p-nitroanilidine (L-BApNA) and the trypsin inhibitors tosyl-L-lysine chloromethyl ketone (TLCK) and benzamidine. The KM value obtained for trypsin-like activity was 1.57 mm and the different peaks of optimum pH and temperature activity suggest the presence of multiple forms of this enzyme in P. nigrispinus. Detection of amylase activity in the salivary glands of this predator suggests its ability to digest starch and obtain nutrients from plants, which may have adaptative value under prey scarcity.     

Activity of digestive enzymes of thick-billed guillemot (<Emphasis Type="Italic">Uria lomvia</Emphasis>) and common guillemot (<Emphasis Type="Italic">U. aalge</Emphasis>) invaded by cestodes

Activities of digestive enzymes (proteases, carbohydrases, acid and alkaline phosphatases) are determined in intestinal mucosa of the thick-billed and common guillemots. Comparative analysis of the obtained results is performed for non-infested and for birds infested by cestodes. It has been established that at infestation by cestode Alcataenia armillaris (Cestoda: Tetrabothriidae), activities of carbohydrase and alkaline phosphatase in intestinal mucosa of the thick-billed guillemot decreases. Parasitizing of cestodes Tetrabothrius jaegerskieldi (Cestoda: Tetrabothriidae) in intestine induces a decrease of saccharase activity. There is studied kinetics of desorption of enzymes from digestive-transport surfaces of the bird intestine. Peculiarities of firmness of enzyme fixation are established on the surface of intestinal mucosa of invaded guillemots. According to the obtained data, a decrease of the carbohydrase activities in intestine of infested guillemots is likely to be due to absorption of a part of enzymes hydrolyzing carbohydrates on the surface of cestodes.     

Digestive enzymes in larvae of the leaf cutting ant, Acromyrmex subterraneus (Hymenoptera: Formicidae: Attini)

The digestive physiology and biochemistry of larvae of the leaf-cutting ant Acromyrmex subterraneus were investigated here. The activity of digestive enzymes was evaluated in the labial glands, midgut epithelium (soluble and particulate fractions), and in the lumen contents, separated into endo and ectoperitrophic regions. Enzymes with high levels of activity were partially characterised using chromatography and electrophoresis techniques. Microscope observations were carried out and the anatomy of the larval digestive tract was described here for the first time. Larvae fed with pH indicator solutions showed the anterior portion of the midgut to be acidic and the posterior portion neutral to alkaline, indicating that the pH of the different regions of the midgut could optimise certain enzyme activities, whilst inhibiting others. The flow rate of the intestinal contents was also evaluated in larvae fed with a dye solution. The slow flow rate is probably due to closure of the rear end of the larval midgut. No compartmentalisation of digestive enzymes acting on oligosaccharides and disaccharides in the ectoperitrophic space and on polysaccharides in the endoperitrophic space was observed here, which could also be related to the closure of the midgut. The digestive physiology of these larvae is therefore similar to ancestral Holometabola, a paradox when considering the highly evolved nature of these insects. The larval midgut demonstrated a large diversity of enzyme activities with high levels of alpha-amylase, alpha-mannosidase, chitinase, alpha-glucosidase, beta-glucosidase and proteinase. High levels of chitinase and amylase activities were detected in the labial glands of larvae. The enzyme profile reflected the necessity of the larvae to degrade the fungal substrate, their sole diet, and a probable source of some of the digestive enzymes detected here. When compared to adults, the larvae had a greater diversity and higher levels of enzyme activity, highlighting their importance as the "digestive caste" of the colony.