Digestive peptidases in Tenebrio molitor and possibility of use to treat celiac disease

Digestion in Tenebrio molitor larvae occurs in the midgut, where there is a sharp pH gradient from 5.6 in the anterior midgut (AM) to 7.9 in the posterior midgut (PM). Accordingly, digestive enzymes are compartmentalized to the AM or PM. Enzymes in the AM are soluble and have acidic or neutral pH optima, while PM enzymes have alkaline pH optima. The main peptidases in the AM are cysteine endopeptidases presented by two to six subfractions of anionic proteins. The major activity belongs to cathepsin L, which has been purified and characterized. Serine post‐proline cleaving peptidase with pH optimum 5.3 was also found in the AM. Typical serine digestive endopeptidases, trypsin‐like and chymotrypsin‐like, are compartmentalized to the PM. Trypsin‐like activity is due to one cationic and three anionic proteinases. Chymotrypsin‐like activity consists of one cationic and four anionic proteinases, four with an extended binding site. The major cationic trypsin and chymotrypsin have been purified and thoroughly characterized. The predicted amino acid sequences are available for purified cathepsin L, trypsin and chymotrypsin. Additional sequences for putative digestive cathepsins L, trypsins and chymotrypsins are available, implying multigene families for these enzymes. Exopeptidases are found in the PM and are presented by a single membrane aminopeptidase N‐like peptidase and carboxypeptidase A, although multiple cDNAs for carboxypeptidase A were found in the AM, but not in the PM. The possibility of the use of two endopeptidases from the AM – cathepsin L and post‐proline cleaving peptidase – in the treatment of celiac disease is discussed.     

Digestive enzyme compartmentalization and recycling and sites of absorption and secretion along the midgut of Dermestes maculatus (Coleoptera) larvae

Bostrichiformia is the less known major series of Coleoptera regarding digestive physiology. The midgut of Dermestes maculatus has a cylindrical ventriculus with anterior caeca. There is no cell differentiation along the ventriculus, except for the predominance of cells undergoing apocrine secretion in the anterior region. Apocrine secretion affects a larger extension and a greater number of cells in caeca than in ventriculus. Ventricular cells putatively secrete digestive enzymes, whereas caecal cells are supposed to secrete peritrophic gel (PG) glycoproteins. Feeding larvae with dyes showed that caeca are water-absorbing, whereas the posterior ventriculus is water-secreting. Midgut dissection revealed a PG and a peritrophic membrane (PM) covering the contents in anterior and posterior ventriculus, respectively. This was confirmed by in situ chitin detection with FITC-WGA conjugates. Ion-exchange chromatography of midgut homogenates, associated with enzymatic assays with natural and synthetic substrates and specific inhibitors, showed that trypsin and chymotrypsin are the major proteinases, cysteine proteinase is absent, and aspartic proteinase probably is negligible. Amylase and trypsin occur in contents and decrease along the ventriculus; the contrary is true for cell-membrane-bound aminopeptidase. Maltase is cell-membrane-bound and predominates in anterior and middle midgut. Digestive enzyme activities in hindgut are negligible. This, together with dye data, indicates that enzymes are recovered from inside PM by a posterior-anterior flux of fluid outside PM before being excreted. The combined results suggest that protein digestion starts in anterior midgut and ends in the surface of posterior midgut cells. All glycogen digestion takes place in anterior midgut.     

Digestive enzymes in midgut cells,endo-and ectoperitrophic contents,and peritrophic membranes of Spodoptera frugiperda (lepidoptera) larvae

In the midgut of Spodoptera frugiperda larvae, subcellular fractionation data suggest that aminopeptidase and part of amylase, carboxypeptidase A, dipeptidase, and trypsin are bound to the microvillar membranes; that major amounts of soluble dipeptidase, cellobiase, and maltase are trapped in the cell glycocalyx; and finally that soluble carboxypeptidase, amylase, and trypsin occur in intracellular vesicles. Most luminal acetylglucosaminidase is soluble and restricted to the ectoperitrophic contents. Aminopeptidase occurs in minor amounts bound to membranes both in the ectoperitrophic contents and incorporated in the peritrophic membrane. Amylase, carboxypeptidase A, and trypsin are found in minor amounts in the ectoperitrophic contents (both soluble and membrane-bound) and in major amounts in the peritrophic membrane with contents. Part of the activities recovered in the last mentioned contents corresponds to enzyme molecules incorporated in the peritrophic membrane. The results suggest that initial digestion is carried out in major amounts by enzymes in the endoperitrophic space and, in minor amounts, by enzymes immobilized in the peritrophic membrane. Intermediate and final digestion occur at the ectoperitrophic space or at the surface of midgut cells. The results also lend support to the hypothesis that amylase and trypsin are derived from membrane-bound forms, are released in soluble form by a microapocrine mechanism, and are partly incorporated into the peritrophic membrane. © 1994 Wiley-Liss, Inc.     

Spatial organization of digestion,secretory mechanisms and digestive enzyme properties in Pheropsophus aequinoctialis (Coleoptera: Carabidae)

Aminopeptidase (soluble form M_r 110,000), carboxypeptidase A (soluble form M_r 47,000), maltase (a dimer composed of two identical M_r 60,000 subunits) and trypsin (two charge isomers with M_r 34,000) are found in major amounts in the crop and midgut tissue, whereas amylase (a trimer of three identical M_r 18,000 subunits) and cellobiase (a trimer of three identical M_r 27,000 subunits) occur mainly in the crop and midgut contents. Subcellular fractions of midgut cells were obtained by conventional homogenization, followed by differential centrifugation or differential calcium precipitation. The results suggest that part of the aminopeptidase and carboxypeptidase A activity is bound to microvilli, that major amounts of trypsin and maltase are trapped in the cell glycocalyx and finally that soluble aminopeptidase, amylase and cellobiase occur in intracellular vesicles. The data support the hypothesis that most protein and carbohydrate digestion takes place in the crop under the action of enzymes passed forward from the midgut, after being secreted by exocytosis. Nevertheless, part of the intermediate and final digestion occurs at the surface of the midgut cells. The peculiar features of the digestion of P. aequinoctialis beetles, including their partly fluid peritrophic membranes, are thought to be derived from putative Coleoptera ancestors.     

The digestive system of the “stick bug” Cladomorphus phyllinus (Phasmida,Phasmatidae): A morphological,physiological and biochemical analysis

This work presents a detailed morphofunctional study of the digestive system of a phasmid representative, Cladomorphus phyllinus. Cells from anterior midgut exhibit a merocrine secretion, whereas posterior midgut cells show a microapocrine secretion. A complex system of midgut tubules is observed in the posterior midgut which is probably related to the luminal alkalization of this region. Amaranth dye injection into the haemolymph and orally feeding insects with dye indicated that the anterior midgut is water-absorbing, whereas the Malpighian tubules are the main site of water secretion. Thus, a putative counter-current flux of fluid from posterior to anterior midgut may propel enzyme digestive recycling, confirmed by the low rate of enzyme excretion. The foregut and anterior midgut present an acidic pH (5.3 and 5.6, respectively), whereas the posterior midgut is highly alkaline (9.1) which may be related to the digestion of hemicelluloses. Most amylase, trypsin and chymotrypsin activities occur in the foregut and anterior midgut. Maltase is found along the midgut associated with the microvillar glycocalix, while aminopeptidase occurs in the middle and posterior midgut in membrane bound forms. Both amylase and trypsin are secreted mainly by the anterior midgut through an exocytic process as revealed by immunocytochemical data.     

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.     

Plasma membrane-associated amylase and trypsin: Intracellular distribution of digestive enzymes in the midgut of the cassava hornworm,Erinnyis ello

Differential centrifugation of homogenates of midgut cells prepared in isotonic solutions has been carried out and hydrolase and enzyme marker activities have been determined in the isolated fractions. α- and β-Glucosidase and trehalase seem to occur loosely associated with large structures, from which they are set free by homogenizing in water. They are also found in the cytosolic fraction. Aminopeptidase activity follows that of alkaline phosphatase, whereas that of amylase and trypsin occur mainly among particulate fractions. Part of the amylase present in the particulate fractions seems to correspond to soluble amylase bound by membranes. The enrichment factor for alkaline phosphatase and aminopeptidase in microvilli purified from midgut cells is 4 and for amylase 1.5 Amylase and trypsin are only partly released from a membrane fraction after several washings in different media, including ultracentrifugation in a discontinuous glycerol gradient. About 50% of the amylase and trypsin are solubilized from the membranes by treatment with Triton X-100. The results support the proposal that intermediate and final digestion in Erinnyis ello occur under the action of glycocalyx-associated hydrolases (α- and β-glucosidase and trehalase) and of plasma membrane-bound enzymes (aminopeptidase, and perhaps also amylase and trypsin).     

Apocrine secretion of amylase and exocytosis of trypsin along the midgut of Tenebrio molitor larvae

Amylase and trypsin were purified from Tenebrio molitor midgut larvae and used to raise antibodies in a rabbit. A Western blot of T. molitor midgut homogenates, after sodium dodecyl sulfate-polyacrylamide gel electrophoresis using amylase and trypsin antisera, showed only bands co-migrating with the purified enzymes. The antisera were used to localize the enzymes by immunofluorescence and immunogold labeling. Amylase occurs in a few regularly disposed anterior midgut cells. Non-amylase-secreting anterior midgut cells are proposed to be water-absorbing cells based on morphology and dye movements. Amylase is found inside vesicles originating from Golgi areas that seem to fuse together before their release along with the now disorganized apical cytoplasm (apocrine secretion). Trypsin precursors are observed inside small vesicles near the apical plasma membrane of posterior midgut cells, suggesting an exocytic mechanism of secretion, followed by putative trypsin activation. Apocrine secretion is thought to be an adaptation to enhance the dispersion of secretory vesicle contents released from a water-absorbing epithelium, whereas exocytosis is an efficient secretory mechanism in a water-secreting epithelium.     

Regional distribution and substrate specificity of digestive enzymes involved in terminal digestion in Musca domestica hind-midguts.

One membrane-bound alpha-glucosidase and two soluble alpha-glucosidases were isolated from homogenates of the hind-midgut, the main digestive region in Musca domestica larvae. The membrane-bound alpha-glucosidase and the low-Mr soluble alpha-glucosidase hydrolyze maltopentaose better than maltose, maltotriose, and maltotetraose, the reverse being true for the high-Mr soluble alpha-glucosidase. A membrane-bound glucoamylase previously described in Musca domestica midgut was shown by gradient centrifugation and dialysis against EDTA to result from the combined action of an amylase and an alpha-glucosidase. The determination of amylase, alpha-glucosidases, soluble and membrane-bound carboxypeptidase A, membrane-bound aminopeptidase and dipeptidase along the tissue and luminal contents of the hind-midgut is described. The data support a proposal concerned with how starch and protein are digested in Musca domestica larval hind-midguts and where and how midgut glycosidases and peptidases are secreted.     

Ultrastructure and immunolocalization of digestive enzymes in the midgut of Podisus nigrispinus (Heteroptera: Pentatomidae)

The predatory stinkbug Podisus nigrispinus has been utilized in biological control programs. Its midgut is anatomically divided into anterior, middle and posterior regions, which play different roles in the digestive process. We describe the midgut ultrastructure and the secretion of digestive enzymes in the midgut of P. nigrispinus. Midguts were analyzed with transmission electron microscopy and the digestive enzymes amylase, cathepsin L, aminopeptidase and α-glucosidase were immunolocalized. The ultrastructural features of the digestive cells in the anterior, middle and posterior midgut regions suggest that they play a role in digestive enzyme synthesis, ion and nutrient absorption, storage and excretion. The digestive enzymes have different distribution along the midgut regions of the predator P. nigrispinus. Amylase, aminopeptidase and α-glucosidase occur in three midgut regions, whereas cathepsin L occurs in the middle and posterior midgut regions. The anterior midgut region of P. nigrispinus seems to play a role in water absorption, the middle midgut may be involved in nutrient absorption and the posterior midgut region is responsible for water transport to the midgut lumen.     

Digestive proteinases of larvae of the corn earworm, Heliothis zea: characterization, distribution, and dietary relationships.

Proteinases and peptidases from the intestinal tract of fifth-instar larvae of Heliothis (= Helicoverpa) zea (Boddie) (Lepidoptera:Noctuidae) were characterized based on their substrate specificity, tissue of origin, and pH optimum. Activity corresponding to trypsin, chymotrypsin, carboxypeptidases A and B, and leucine aminopeptidase was detected in regurgitated fluids, midgut contents, and midgut wall. High levels of proteinase activity were detected in whole midgut homogenates, with much lower levels being observed in foregut and salivary gland homogenates. In addition, enzyme levels were determined from midgut lumen contents, midgut wall homogenates, and regurgitated fluids. Proteinase activities were highest in the regurgitated fluids and midgut lumen contents, with the exception of leucine aminopeptidase activity, which was found primarily in the midgut wall. Larvae fed their natural diet of soybean leaves had digestive proteinase levels that were similar to those of larvae fed artificial diet. No major differences in midgut proteinase activity were detected between larvae reared under axenic or xenic conditions, indicating that the larvae are capable of digesting proteins in the absence of gut microorganisms. The effect of pH on the activity of each proteinase was studied. The pH optima for the major proteinases were determined to be pH 8.0-8.5 for trypsin, when tosyl-L-arginine methyl ester was used as the substrate; and pH 7.5-8.0 for chymotrypsin, when benzoyl-L-tyrosine ethyl ester was used as the substrate.     

Distribution,properties, and functions of midgut carboxypeptidases and dipeptidases from Musca domestica larvae

Dipeptidase and carboxypeptidase A activities were determined in cells and luminal contents of the fore-, mid-, and hind-midgut of Musca domestica larvae. Dipeptidase activity was found mainly in hind-midgut cells, whereas carboxy-peptidase activity was recovered in major amounts in both cells and in luminal contents of hind-midguts. The subcellular distribution of dipeptidase and part of the carboxypeptidase A activities is similar to that of a plasma membrane enzyme marker (aminopeptidase), suggesting that these activities are bound to the microvillar membranes. Soluble carboxypeptidase A seems to occur both bound to secretory vesicles and trapped in the cell glycocalyx. Based on density-gradient ultracentrifugation and thermal inactivation, there seems to be only one molecular species of each of the following enzymes (soluble in water or solubilized in Triton X-100): membrane-bound dipeptidase (pH optimum 8.0; Km 3.7 mM GlyLeu, M_r 111,000), soluble carboxypeptidase (pH optimum 8.0; Km 1.22 mM N-carbobenzoxy-glycyl-L-phenylalanine (ZGlyPhe), M_r45,000) and membrane-bound carboxypeptidase (pH optimum 7.5, Km 2.3 mM ZGlyPhe, M_r58,000). The results suggest that protein digestion is accomplished sequentially by luminal trypsin and luminal carboxypeptidase, by membrane-bound carboxypeptidase and aminopeptidase, and finally by membrane-bound dipeptidase.     

Nature of the anchors of membrane-bound aminopeptidase,amylase, and trypsin and secretory mechanisms in Spodoptera frugiperda (Lepidoptera) midgut cells

Spodoptera frugiperda larvae have a microvillar aminopeptidase and both soluble and membrane-bound forms of amylase and trypsin. Membrane-bound aminopeptidase is solubilized by glycosyl phosphatidylinositol-specific phospholipase C (GPI-PLC) and detergents, suggesting it has a GPI anchor. Membrane-bound trypsin is not affected by GPI-PLC, although it is solubilized by papain and by different detergents. Membrane-bound amylase is similar to trypsin, although once solubilized in detergent it behaves as a hydrophilic protein. Musca domestica trypsin antiserum cross-reacts with only one polypeptide from S. frugiperda midgut. With this antiserum, trypsin was immunolocalized in the anterior midgut cells at the microvillar surface and on the membranes of secretory vesicles found in the apical cytoplasm and inside the microvilli. The data suggest that in this region trypsin is bound to the secretory vesicle membrane by a hydrophobic anchor. Vesicles migrate through the microvilli and are discharged into the lumen by a pinching-off process. Trypsin is then partly processed to a soluble form and partly, still bound to vesicle membranes, incorporated into the peritrophic membrane. In posterior midgut cells, trypsin immunolabelling is randomly distributed inside the secretory vesicles and at the microvilli surface, suggesting exocytosis. Amylase probably follows a route similar to that described for trypsin in anterior midgut, although membrane-bound forms (peptide anchor) solubilize apparently as a consequence of a pH increase inside the vesicles.     

The 3D structure and function of digestive cathepsin L-like proteinases of Tenebrio molitor larval midgut

Cathepsin L-like proteinases (CAL) are major digestive proteinases in the beetle Tenebrio molitor. Procathepsin Ls 2 (pCAL2) and 3 (pCAL3) were expressed as recombinant proteins in Escherichia coli, purified and activated under acidic conditions. Immunoblot analyses of different T. molitor larval tissues demonstrated that a polyclonal antibody to pCAL3 recognized pCAL3 and cathepsin L 3 (CAL3) only in the anterior two-thirds of midgut tissue and midgut luminal contents of T. molitor larvae. Furthermore, immunocytolocalization data indicated that pCAL3 occurs in secretory vesicles and microvilli in anterior midgut. Therefore CAL3, like cathepsin L 2 (CAL2), is a digestive enzyme secreted by T. molitor anterior midgut. CAL3 hydrolyses Z-FR-MCA and Z-RR-MCA (typical cathepsin substrates), whereas CAL2 hydrolyses only Z-FR-MCA. Active site mutants (pCAL2C25S and pCAL3C26S) were constructed by replacing the catalytic cysteine with serine to prevent autocatalytic processing. Recombinant pCAL2 and pCAL3 mutants (pCAL2C25S and pCAL3C26S) were prepared, crystallized and their 3D structures determined at 1.85 and 2.1 Å, respectively. While the overall structure of these enzymes is similar to other members of the papain superfamily, structural differences in the S2 subsite explain their substrate specificities. The data also supported models for CAL trafficking to lysosomes and to secretory vesicles to be discharged into midgut contents.     

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.     

Activity cycles and the control of four digestive proteinases in the posterior midgut of Rhodnius prolixus Stål (Hemiptera: Reduviidae)

Proteinase levels in the posterior midgut of fifth-instar and adult Rhodnius prolixus follow a cyclic pattern after ingestion of the bloodmeal. In the fifth instar, cathepsin B showed two peaks: the first occurred 6 days after ingestion and the second at the time of ecdysis. Cathepsin D, cathepsin B and lysosomal carboxypeptidase B reached maximal levels 6 days after ingestion. At this time the highest levels of these proteinases were found in mated females, the lowest in males and intermediate levels in virgin females. Maximal levels of aminopeptidase occurred later than catheptic enzymes, and the decline, after maximal levels were achieved, was much more gradual.Catheptic-proteinase levels within the posterior midgut in fifth-instar larvae and adults correlated positively with the amount of protein contained in this gut region. This indicates that production of these proteinases is controlled by a secretagogue mechanism. Aminopeptidase levels were controlled in a different manner. The mated state or sex of adults altered the proteinase levels by changing the amount of protein that was passed into the digestive midgut from the crop.     

Characterization and cDNA cloning of midgut carboxypeptidases from Trichoplusia ni

Carboxypeptidase A and carboxypeptidase B activities from the midgut of Trichoplusia ni larvae were characterized. In the T. ni larval midgut, the primary digestive carboxypeptidase activity was attributed to carboxypeptidase A, which was eight times more active than carboxypeptidase B. Both the midgut carboxypeptidase A and carboxypeptidase B exhibited maximal activities at pH 8.0-8.5 and were similarly susceptible to inhibition by potato carboxypeptidase inhibitor and phenanthroline. The midgut carboxypeptidase activities were analyzed in T. ni larvae fed on various diet sources and the results indicated that midgut carboxypeptidase activities per milligram of gut were similar regardless of the amount of dietary proteins or amino acids. However, midgut carboxypeptidase A activity was significantly higher in larvae exposed to soybean trypsin inhibitor and was significantly lower in larvae fed on broccoli foliage. From the T. ni larval midgut, five putative carboxypeptidase cDNAs were cloned, demonstrating that midgut carboxypeptidase activities are composed of multiple carboxypeptidase types. Sequence analysis indicated that the midgut carboxypeptidases were produced as secreted proenzymes which could be activated after removal of an N-terminal activation fragment by a trypsin. Two cloned cDNAs are predicted to code for carboxypeptidase A and one cDNA is predicted to code for a putative carboxypeptidase B. The other two cDNAs are highly similar to carboxypeptidase A and carboxypeptidase B in sequences, but their activity was not predictable.     

Spatial organization of digestion in the larval and imaginal stages of the sciarid fly Trichosia pubescens

Aminopeptidase, amylase, cellulase and trypsin are found in major amounts in the midgut lumen, whereas alkaline phosphatase, cellobiase, α-glucosidase, maltase and trehalase occur mainly in the midgut tissue of Trichosia pubescens larvae. Cellulase and a part of the amylase seem to be derived from the fungi the larvae eat. Based on the molecular weights of the enzymes which pass and of those which do not pass through the peritrophic membrane, it is possible to estimate the peritrophic membrane pores as having diameters of 7.5–8.0 nm. Purification and assays of microvillar enzymes from different larval midgut regions suggest that alkaline phosphatase, cellobiase, α-glucosidase, maltase and trehalase are bound to the plasma membrane chiefly of midgut caeca cells. The results support the hypothesis that digestion starts in the endoperitrophic space under the action of amylase, cellulase and trypsin, goes on in the ectoperitrophic space by amylase, cellulase and aminopeptidase and is completed through the catalytic action of plasma membrane-bound hydrolases. The data lead to the conclusion that the spatial organization in T. pubescens larvae is identical to that of another Sciarid fly (Rhynchosciara americana) despite the finding that the midgut trehalase is bound to the plasma membrane in T. pubescens and soluble in R. americana. With metamorphosis salivary amylase appears, α-glucosidase, trehalase and maltase increase, and the other midgut hydrolases decrease or even disappear. This is in accordance with the fact that the larvae feed on decaying plants and fungi and the imagoes feed on nectar.     

Secretion of beta-glycosidase by middle midgut cells and its recycling in the midgut of Tenebrio molitor larvae

There are four β-glycosidases (βgly1, βgly2, βgly3, and βgly4) in Tenebrio molitor midgut larvae. βgly1 and βgly2 have identical kinetic properties, and differ in a few amino acid residues. Purified βgly1 was used to raise antibodies in a rabbit. The resulting antiserum recognizes in a Western blot only βgly1 and βgly2 in midgut tissue homogenates and contents. An immunocytochemical study carried out using confocal fluorescence and immunogold techniques showed that βgly1+βgly2 are secreted by exocytosis mainly from the distal part of the second third of T. molitor midguts. This is the first immunocytochemical study of an insect digestive enzyme that does not have polymers as substrates. Enzyme assays with 0.3 mM amygdalin, a condition that detects only βgly1+βgly2, revealed that most of those β-glycosidases are found in the lumen of anterior and middle midgut. This supports the hypothesis that a countercurrent flux of fluid occurs in T. molitor midgut that is able to carry βgly1 and βgly2 to anterior midgut, in agreement with the enzyme recycling mechanism thought to occur in most insects.     

A digestive prolyl carboxypeptidase in Tenebrio molitor larvae

Prolyl carboxypeptidase (PRCP) is a lysosomal proline specific serine peptidase that also plays a vital role in the regulation of physiological processes in mammals. In this report, we isolate and characterize the first PRCP in an insect. PRCP was purified from the anterior midgut of larvae of a stored product pest, Tenebrio molitor, using a three-step chromatography strategy, and it was determined that the purified enzyme was a dimer. The cDNA of PRCP was cloned and sequenced, and the predicted protein was identical to the proteomic sequences of the purified enzyme. The substrate specificity and kinetic parameters of the enzyme were determined. The T. molitor PRCP participates in the hydrolysis of the insect's major dietary proteins, gliadins, and is the first PRCP to be ascribed a digestive function. Our collective data suggest that the evolutionary enrichment of the digestive peptidase complex in insects with an area of acidic to neutral pH in the midgut is a result of the incorporation of lysosomal peptidases, including PRCP.