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.     

Phenotypic changes and the fate of digestive enzymes during induction of amber disease in larvae of the New Zealand grass grub (Costelytra zealandica)

Amber disease of the New Zealand grass grub Costelytra zealandica (Coleoptera: Scarabaeidae) is caused by ingestion of pADAP plasmid carrying isolates of Serratia entomophila or Serratia proteamaculans (Enterobacteriaceae) and causes infected larvae to cease feeding and clear their midgut to a pale amber colour where midgut serine protease activities are virtually eliminated. Using bacterial strains and mutants expressing combinations of the anti-feeding (afp) and gut clearance (sep) gene clusters from pADAP, we manipulated the disease phenotype and demonstrated directly the relationship between gene clusters, phenotype and loss of enzyme activity. Treatment with afp-expressing strains caused cessation of feeding without gut clearance where midgut protease activity was maintained at levels similar to that of healthy larvae. Treatment with strains expressing sep-genes caused gut clearance followed by a virtual elimination of trypsin and chymotrypsin titre in the midgut indicating both the loss of pre-existing enzyme from the lumen and a failure to replenish enzyme levels in this region by secretion from the epithelium. Monitoring of enzymatic activity through the alimentary tract during expression of disease showed that loss of serine protease activity in the midgut was matched by a surge of protease activity in the hindgut and frass pellets, indicating a flushing and elimination of the midgut contents. The blocking of enzyme secretion through amber disease appears to be selective as leucine aminopeptidase and α-amylase were still detected in the midgut of diseased larvae.     

Partial purification and characterization of midgut leucyl aminopeptidase of Morimus funereus (Coleoptera: Cerambycidae) larvae

Exopeptidases of Morimus funereus larvae were partially purified and characterized. Specific leucyl aminopeptidase (LAP) activity was increased eight-fold by gel filtration of the crude midgut extract. The partially purified LAP had a molecular mass greater than 100 kDa with pH optima from 7.0-9.0 and no strict substrate specificity. M. funereus LAP preferentially hydrolyzed p-nitroanilides with hydrophobic amino acids in the active site, with a K(m) for leucine-p-nitroanilide of 0.21 mM. Zymogram analysis of an electropherogram obtained by native polyacrylamide gel electrophoresis revealed four enzymatically active proteinases using leucine-p-nitroanilide and methionine-p-nitroanilide as substrates and two enzymatically active proteinases using lysine-p-nitroanilide as a substrate. Although the optimal temperature of LAP activity was 40 degrees C, the enzyme was active over a broad temperature range from 2 to 60 degrees C. Among a number of inhibitors tested, heavy metals and 1,10-phenanthroline completely inhibited the enzyme, while methanol, ethanol and EGTA stimulated somewhat LAP activity.     

Constitutive Activation of the Midgut Response to Bacillus thuringiensis in Bt-Resistant Spodoptera exigua

Bacillus thuringiensis is the most effective microbial control agent for controlling numerous species from different insect orders. The main threat for the long term use of B. thuringiensis in pest control is the ability of insects to develop resistance. Thus, the identification of insect genes involved in conferring resistance is of paramount importance. A colony of Spodoptera exigua (Lepidoptera: Noctuidae) was selected for 15 years in the laboratory for resistance to Xentari™, a B. thuringiensis-based insecticide, reaching a final resistance level of greater than 1,000-fold. Around 600 midgut ESTs were analyzed by DNA-macroarray in order to find differences in midgut gene expression between susceptible and resistant insects. Among the differentially expressed genes, repat and arylphorin were identified and their increased expression was correlated with B. thuringiensis resistance. We also found overlap among genes that were constitutively over-expressed in resistant insects with genes that were up-regulated in susceptible insects after exposure to Xentari™, suggesting a permanent activation of the response to Xentari™ in resistant insects. Increased aminopeptidase activity in the lumen of resistant insects in the absence of exposure to Xentari™ corroborated the hypothesis of permanent activation of response genes. Increase in midgut proliferation has been proposed as a mechanism of response to pathogens in the adult from several insect species. Analysis of S. exigua larvae revealed that midgut proliferation was neither increased in resistant insects nor induced by exposure of susceptible larvae to Xentari™, suggesting that mechanisms other than midgut proliferation are involved in the response to B. thuringiensis by S. exigua larvae.     

Digestive enzymes associated with the glycocalyx,microvillar membranes and secretory vesicles from midgut cells of Tenebrio molitor larvae

Acetylglucosaminidase, amylase, cellobiase and maltase are more active in anterior midgut cells, whereas aminopeptidase, carboxypeptidase and trypsin are more active in posterior midgut cells of Tenebrio molitor larvae. Differential centrifugation of midgut homogenates prepared in saline (or mannitol) isotonic buffered solutions revealed that aminopeptidase is associated with membranes, which occur in subcellular fractions displaying many microvilli. Carboxypeptidase, trypsin and the carbohydrases are mostly found in the soluble fraction, although significant amounts sediment together with cell vesicles. Data on differential calcium precipitation of midgut homogenates and on partial ultrasound disruption of midgut tissue suggest that aminopeptidase is a microvillar enzyme and that the digestive enzymes recovered in the soluble fraction of cells are loosely bound to the cell glycocalyx. About 5% of the non-absorbable dye amaranth fed to T. molitor larvae remains in the midgut tissue after rinsing. Most dye was recovered in the soluble fraction of midgut cells. This provided further support for the hypothesis that the digestive enzymes found in the soluble fraction are actually extracellular and that the true intracellular enzymes are those associated with cell vesicles. The results suggest that the carbohydrases are secreted by exocytosis from the anterior midgut and carboxypeptidase and trypsin from the posterior midgut.     

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.     

Purification and properties of major midgut leucyl aminopeptidase of Morimus funereus (Coleoptera, Cerambycidae) larvae

The major leucyl aminopeptidase (LAP) from the midgut of Morimus funereus larvae was purified and characterised. Specific LAP activity was increased 292-fold by purification of the crude midgut extract. The purified enzyme had a pH optimum of 7.5 (optimum pH range 7.0-8.5) and preferentially hydrolysed p-nitroanilides containing hydrophobic amino acids in the active site, with the highest V(max)/K(M) ratio for leucine-p-nitroanilide (LpNA). Among a number of inhibitors tested, the most efficient were 1,10-phenanthroline having a K(i) value of 0.12 mM and cysteine with K(i) value of 0.31 mM, while EGTA stimulated LAP activity. Zn(2+), Mg(2+) and Mn(2+) all showed bi-modal effects on LAP activity (activated at low concentrations and inhibited at high concentrations). The purified LAP (after gel filtration on Superose 6 column) had molecular mass of 400 kDa with an isoelectric point of 6.2. Sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed one band of 67 kDa, suggesting that the enzyme is a hexamer. Six peptide sequences from protein band were obtained using ESI/MS-MS analysis. Comparison of the obtained peptide sequences with the EMBL-EBI sequence analysis toolbox and the BLASTP database showed a high degree of identity with other insect aminopeptidases.     

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.     

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.     

Toward the physiological basis for increased Agrotis ipsilon multiple nucleopolyhedrovirus infection following feeding of Agrotis ipsilon larvae on transgenic corn expressing Cry1Fa2

Larvae of the black cutworm, Agrotis ipsilon Hufnagel, were more susceptible to infection by A. ipsilon multiple nucleopolyhedrovirus (AgipMNPV: Baculoviridae) after feeding on Herculex^® I, a transgenic corn hybrid expressing the Bacillus thuringiensis (Bt)-derived toxin Cry1Fa2 compared to larvae fed on isoline corn. We investigated the physiological basis for increased susceptibility to virus infection following exposure to Herculex^® I by analyzing the midgut pH, gut protease activity and peritrophic matrix structure which are important factors for both Bt toxin action and baculovirus infection. No significant treatment differences were found in the pH of anterior midgut, central midgut or posterior midgut in larvae fed Herculex^® I or isoline diets. Analysis of soluble and membrane-associated gut proteinase activities from larvae fed Herculex^® I or isoline diets indicated that membrane-associated aminopeptidase activity and soluble chymotrypsin-like proteinase activity were significantly lower in Herculex^® I -fed larvae compared to isoline-fed larvae. The number and relative molecular masses of soluble chymotrypsin-like proteinases did not differ. Baculoviruses were not susceptible to in vitro degradation by bovine chymotrypsin, suggesting that chymotrypsin degradation of baculovirus occlusion-derived virus did not result in reduced infection of larvae fed on isoline diet. Scanning electron micrographs of the peritrophic matrices of Herculex^® I -fed larvae and isoline-fed larvae indicated that Herculex^® I did not result in damage to the peritrophic matrix that could facilitate subsequent baculovirus infection. Additional research is required to further delineate the physiological basis for enhanced baculovirus infection following exposure to sublethal doses of Bt toxins.     

Partial purification and characterization of Helicoverpa armigera (Lepidoptera: Noctuidae) active aminopeptidase secreted in midgut

We report the partial purification to apparent homogeneity of a soluble aminopeptidase (EC 3.4.11.1) from midgut of Helicoverpa armigera larvae, which preferentially degraded Leucine p-nitroanilide (LpNA). After midgut isolation, extraction and precipitation of soluble proteins with acetone, proteins were purified in two consecutive steps including gel filtration and ion-exchange chromatographies. Aminopeptidase activity was increased 8.95 fold after gel filtration chromatography. The purified enzyme appeared as single band with a molecular mass of ～ 112 kDa in SDS-PAGE, with a pH optimum of 7.0. Zymogram analysis revealed two enzymatically active proteinases using LpNA as substrate. The optimal temperature of aminopeptidase activity was 50–60 °C. The enzyme was characterized as metalloprotease as it was strongly inhibited by 1,10 phenanthroline. Strong inhibition was also being observed using the specific aminopeptidase inhibitor bestatin. Heavy metal ions, EDTA and cysteine strongly inhibited the enzyme, while Ca^+ 2, Mn^+ 2 and Mg^+ 2 somewhat stimulated aminopeptidase activity. Besides LpNA, the purified aminopeptidase also cleaved with decreasing activity ApNA, VpNA and BApNA. Study could be helpful to understand the mechanism of action of N-terminal degrading enzymes and also important is to further study the differential interaction of Bacillus thuringiensis cry insecticidal toxin with midgut receptor of insects.     

Distribution of digestive proteinases in the alimentary tract of the european corn borer Ostrinia nubilalis (lepidoptera: Pyralidae)

The distribution of digestive proteinases in either the anterior and posterior midgut or between the midgut epithelium and ectoperitrophic and endo-peritrophic spaces in the midgut were examined in the European corn borer, Ostrinia nubilalis. Trypsin, chymotrypsin, elastase, and aminopeptidase activities were the same in the anterior and posterior halves of the midgut. Of the total aminopeptidase activity, 95% was located in the midgut epithelium, and 90% of the trypsin, 97% of chymotrypsin, and 93% of the elastase activity were found in the midgut lumen. Trypsin, measured by hydrolysis of benzoyl-L-arginine ethyl ester, and chymotrypsin levels were significantly higher in the ectoperitrophic space compared to the endoperitrophic space. Digestion in the midgut is proposed to be sequential with tryptic digestion occurring in the endoperitrophic space. Ingested protein is digested further in the ectoperitrophic space by the action of elastase, chymotrypsin, and a second trypsin. Final digestion occurs by an intracellular aminopeptidase. © 1995 Wiley-Liss, Inc.     

Regeneration of midgut epithelial cell in the silkworm, Bombyx mori, infected with viruses

In the larvae of the silkworm, Bombyx mori, the regeneration of midgut cells infected with a cytoplasmic polyhedrosis virus (CPV), a flacherie virus (FV), and a small DNA virus (SDV) was studied. Large numbers of newly developed cells appeared in the CPV-infected part of the midgut epithelium just before larval molt, and along with their development, the CPV-infected old columnar cells were discharged into the midgut lumen during the molt. On the other hand, in the uninfected portion of the midgut only a few cells developed, and no columnar cells were discharged. Similarly, the marked replacement of midgut epithelial cells during larval molt was also observed in larvae infected with CPV + FV. In the larvae infected with CPV + SDV, the columnar cells lost their regenerative ability, and because of the exfoliation of infected columnar cells, the midgut epithelium consisted mainly of uninfected goblet cells at a late stage of infection. The degree of epithelial regeneration varied with the silkworm strain and the dosage of the virus.     

Recycling of urea associated with the host plant urease in the silkworm larvae,Bombyx mori

Urea concentration and urease activity in the midgut content were compared between larvae of the silkworm, Bombyx mori fed an artificial diet and those fed fresh mulberry leaves. A considerable amount of urea was found in the midgut content of the both larvae, however it was significantly lower in the larvae fed fresh mulberry leaves than in the larvae fed the artificial diet; average urea concentrations in the midgut content of the larvae fed fresh mulberry leaves and the artificial diet were 2.9 and 4.6 &mgr;mol/g, respectively. Urea in the midgut content seems to be secreted from the insect itself since the amount of urea in both diets were negligibly small. Urease activity was detected only in the midgut content of the larvae fed fresh mulberry leaves but not in other tissues of the larvae. On the other hand, no urease activity was detected in the midgut content of the larvae fed the artificial diet. Subsequently, to elucidate the role of mulberry leaf urease in the midgut lumen, larvae that had been reared on the artificial diet were switched to fresh mulberry leaves. The diet switch caused a rapid decrease in urea concentration in the midgut content and an increase in ammonia concentration in the midgut content, suggesting that secreted urea could be hydrolyzed to ammonia by mulberry leaf urease in the midgut lumen. Furthermore, to investigate the physiological significance of mulberry leaf urease on urea metabolism of the silkworm, (15)N-urea was injected into the hemocoel, and after 12 h the larvae were dissected for (15)N analysis. A considerable amount of (15)N was found to be incorporated into the silk-protein of the larvae fed fresh mulberry leaves, but there was little incorporation of (15)N into the silk-protein of the larvae fed the artificial diet. These data indicate that urea is converted into ammonia by the action of mulberry leaf urease in the midgut lumen and used as a nitrogen source in larvae fed mulberry leaves.     

Peptidases in Drosophila melanogaster. I. Characterization of dipeptidase and leucine aminopeptidase activities

Four major peptidases of Drosophila melanogaster have been described and distinguished by their electrophoretic mobilities, molecular weights, net electrical charges, and substrate specificities. The previously described leucine aminopeptidase, LAP D, consists of at least two isozymes, designated here LAP P and LAP G. In pupae most LAP activity results from LAP P (pupal); in larvae and adults, in contrast, most LAP activity results from LAP G (gut). These two LAPs may be separated by electrophoresis in the presence of the nonionic detergent Triton X-100. A specific assay for LAP P, which exploits the large difference between the net electrical charge of LAP P and that of LAP G, is described. The activity levels of two dipeptidases, Dip A and Dip B, were high in all the postembryonic stages examined. Specific assays for Dip A and Dip B were used to show that for each of these isozymes, the activity in an adult is proportional to gene dosage.     

Morphometry of the midgut epithelium of Diatraea saccharalis Fabricius, 1794 (Lepidoptera) parasitized by Cotesia flavipes Cameron, 1891 (Hymenoptera)

The morphometric study of the midgut in Diatraea saccharalis (Lepidoptera) larvae parasitized by the Cotesia flavipes (Hymenoptera) showed that there was significant increase in the columnar, goblet and regenerative cells and their nuclei; the midgut lumen diameter and the epithelial height were also increased in the parasitized larvae. The multivariate analysis showed that parasitism affected the columnar cell only in the posterior region, and the goblet cells along the midgut length (anterior and posterior regions).     

Analyses of α-amylase and α-glucosidase in the malaria vector mosquito,Anopheles gambiae,as receptors of Cry11Ba toxin of Bacillus thuringiensis subsp. jegathesan

Bacillus thuringiensis subsp. jegathesan produces Cry11Ba crystal protein with high toxicity to mosquito larvae. The Cry11Ba toxicity is dependent on its receptors on mosquito larval midgut epithelial cells. Previously, a cadherin-like protein (AgCad2), aminopeptidase (AgAPN2) and alkaline phosphatase (AgALP1) were reported to be involved in regulation of Cry11Ba toxicity on Anopheles gambiae larvae. Here, the cDNAs encoding α-amylase (AgAmy1) and α-glucosidase (Agm3) were cloned from A. gambiae larva midgut. Both are glycophosphatidylinositol (GPI) anchored proteins on brush border membranes (BBMV). Immunohistochemistry revealed their localization on different regions of the larval midgut. AgAmy1 and Agm3 bound Cry11Ba with high affinity, 37.6 nM and 21.1 nM respectively. Cry11Ba toxicity against A. gambiae larvae was neutralized by both AgAmy1 and Agm3. The results provide evidence that both AgAmy1 and Agm3 function as receptors of Cry11Ba in A. gambiae.     

Ontogenetic patterns and genetic variation in Anopheles (Anopheles) intermedius Chagas, 1908 and Anopheles (Anopheles) mattogrossensis Lutz & Neiva, 1911 (Diptera: Culicidae) in the Brazilian amazon

Changes in the expression of genes were observed during development in populations of Anopheles (Anopheles) intermedius and Anopheles (Anopheles) mattogrossensis. Esterase showed seven zones of activity: EST1 was present in all developmental stages of both species; EST2 was observed only in larvae of A. intermedius and larvae and pupae of A. mattogrossensis, with greater activity in pupae; EST3 and EST5 were present in all developmental stages, with greater intensity in larvae; EST4 and EST6 showed weak activity in larvae of A. mattogrossensis and was not found in A. intermedius. Leucine aminopeptidase showed four zones of activity, of which LAP1 and LAP2 were found in all stages of A. intermedius, with highest activity in larvae, and in larvae only of A. mattogrossensis. LAP3 was detected in all stages of A. mattogrossensis and in larvae only of A. intermedius. LAP4 was detected only in larvae and pupae of A. mattogrossensis, with greater intensity in pupae. alpha-Glycerophosphate dehydrogenase showed a single zone of activity, detected in older fourth-instar larvae and becoming more intense from the pupal stage onwards.     

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.