Sucrose hydrolases from the midgut of the sugarcane stalk borer Diatraea saccharalis

A beta-fructosidase (EC 3.2.1.26) was isolated from the midgut of larval sugar cane stalk borer Diatraea saccharalis by mild-denaturing electrophoresis and further purified to near homogeneity by gel filtration. beta-Fructosidase hydrolysed sucrose, raffinose and the fructosyl-trisaccharide isokestose, but it had no activity against maltose, melibiose and synthetic substrates for alpha-glucosidases. Two other sucrose hydrolases, one resembling a alpha-glucosidase (EC 3.2.1.20) and the other one active specifically against sucrose (sucrase) were detected in the larval midgut of D. saccharalis. All three sucrose hydrolases were associated with the midgut epithelium of larval D. saccharalis. Relative molecular mass (M(r)) of the beta-fructosidase was estimated around 45,000 (by gel filtration). The other two sucrose hydrolases had M(r) of 54,000 (alpha-glucosidase) and 59,000 (sucrase). The pH optima of the sucrose hydrolases were 5-10 for both alpha-glucosidase and sucrase and 7-8 for beta-fructosidase. Considering V(max)/K(m) ratios, beta-fructosidase preferentially cleaves isokestose rather than raffinose and sucrose. In order to evaluate the possible contribution of microorganisms isolated from the midgut to the pool of sucrose hydrolases, washed midgut epithelia were homogenised and plated onto appropriate media. Seven bacterial and one yeast species were isolated. None of the sucrose hydrolases extracted from the microorganisms corresponded to the enzymes isolated from midgut tissue homogenates. This result suggests that the major sucrose hydrolases found in the midgut of larval D. saccharalis were probably produced by the insect themselves not by the gut microflora.     

Sugar digestion in mosquitoes: identification and characterization of three midgut alpha-glucosidases of the neo-tropical malaria vector Anopheles aquasalis (Diptera: Culicidae)

Dietary carbohydrates provide an important source of energy for flight, and contribute to longevity and fecundity of mosquitoes. The most common sugar mosquitoes ingest is sucrose, and digestion of this substance is carried out mainly by alpha-glucosidases. In the current work, we tested the efficiency of sucrose on Anopheles aquasalis female diet. The best longevity (days) was reached when sugar was available in the diet, whereas most only blood fed females were dead 6 days after emergence. Three alpha-glucosidase isoforms were detected in the adult female midgut, named alphaGlu1, alphaGlu2 and alphaGlu3. These are acidic alpha-glucosidases with optima pH around pH 5.5. alphaGlu1 and alphaGlu2 are present in both secreted and membrane-bound forms, whereas alpha-Glu3 only in anchored to membranes. The alpha-glucosidase activity is concentrated mainly in the posterior midgut (70%), both in non-fed or 10% sucrose fed females. The single form of these alpha-glucosidases seemed to be approximately 70 kDa polypeptides, although alphaGlu2 is presented in >or=600 kDa self-aggregates. Km values of alphaGlu1, alphaGlu2 and alphaGlu3 differed significantly from each other, supporting the statement that three alpha-glucosidases are produced in the female midgut. Together, all data suggest that sugar is an essential component of A. aquasalis female diet. In addition, alpha-glucosidases are synthesized in the same place where sucrose is digested and absorbed, the midgut.     

Toxic impact of ingested Jatropherol-I on selected enzymatic activities and the ultrastructure of midgut cells in silkworm, Bombyx mori L.

Abstract:  Jatropherol-I, a phorbol-type diterpene from Jatropha curcas seeds was found highly toxic to third instars silkworm larvae after ingestion with LC₅₀ values 0.5793, 0.2197 and 0.1578 mg/ml at 48, 72 and 120 h respectively. The acute toxicity was associated with changes in activities of several midgut enzymes and pathological changes in midgut epithelial cells. Jatropherol-I caused various fluctuations in activities of different midgut enzymes in third instars larvae of silkworm. Compared with controls, both esterase and carboxyliesterase showed significantly depressed activities at 3 h ( t -test, P < 0.05) and increased activities at 24 h and rapidly decreased activities at 48 h after ingestion of 0.125 and 0.25 mg/ml of Jatropherol-I. Jatropherol-I induced two (E4 and E5) and depressed one (E2) of midgut esterase isozymes analysed 3 h after ingestion. There was no significantly different glutathione S-transferase activity between most of treated silkworm and control ( t -test, P > 0.05). Activities of acetylcholinesterase fluctuated weakly in treated silkworm in 48 h. Jatropherol-I induced a gradual decline in midgut protease activities in silkworm with an obvious dose- and time-dependent effect. Jatropherol-I also caused pathological changes in midgut cells, especially in their endoplasmic reticulum. They were noted slight dilatation on 12 h exposure, while extreme dilatation, vesiculations and shedding of ribosome from membrane were observed in endoplasmic reticulum after a longer time exposure. Other pathological changes in microvilli, lysosome, mitochondria and chromatin were also observed in midgut cell of treated silkworm.     

Ingestion of the epoxide hydrolase inhibitor AUDA modulates immune responses of the mosquito,Culex quinquefasciatus during blood feeding

Epoxide hydrolases (EHs) are enzymes that play roles in metabolizing xenobiotic epoxides from the environment, and in regulating lipid signaling molecules, such as juvenile hormones in insects and epoxy fatty acids in mammals. In this study we fed mosquitoes with an epoxide hydrolase inhibitor AUDA during artificial blood feeding, and we found the inhibitor increased the concentration of epoxy fatty acids in the midgut of female mosquitoes. We also observed ingestion of AUDA triggered early expression of defensin A, cecropin A and cecropin B2 at 6 h after blood feeding. The expression of cecropin B1 and gambicin were not changed more than two fold compared to controls. The changes in gene expression were transient possibly because more than 99% of the inhibitor was metabolized or excreted at 42 h after being ingested. The ingestion of AUDA also affected the growth of bacteria colonizing in the midgut, but did not affect mosquito longevity, fecundity and fertility in our laboratory conditions. When spiked into the blood, EpOMEs and DiHOMEs were as effective as the inhibitor AUDA in reducing the bacterial load in the midgut, while EETs rescued the effects of AUDA. Our data suggest that epoxy fatty acids from host blood are immune response regulators metabolized by epoxide hydrolases in the midgut of female mosquitoes, inhibition of which causes transient changes in immune responses, and affects growth of microbes in the midgut.     

Digestive enzymes activity in larvae of Cameraria ohridella (Lepidoptera: Gracillariidae)

This article presents the activity of carbohydratases and proteases in the midgut of Cameraria ohridella larvae--an oligophagous pest whose preferred feeding is horse chestnuts leaves. Optimal media pH of the assayed enzymes were similar to those of other Lepidopterans. Relatively high amylase activity, as well as maltase and sucrase activities, indicates that starch and sucrose are the main digested saccharides. Trehalase activity was similar to that described in other Lepidopterans. Activities of glycosidases were significantly lower than those of disaccharidases what suggests that neither cellulose nor glycosides are important for C. ohridella. Trypsin is the main endoprotease of this pest. Like in other leaf-eaters carboxypeptidase activity was higher than that of aminopeptidase. The activity of the majority of examined enzymes increased in the following successive pest generations, which could be explained by the decreased nutritional value of older leaves. Probably this phenomenon in hydrolases activity in Cameraria is a nonspecific mechanism present at this stage of co-evolution of the horse chestnut and its pest.     

Comparative enzyme histochemical study on the visceral yolk sac endoderm in the rat in vivo and in vitro

Summary Histochemical study of the visceral yolk-sac endoderm of the rat was performed in vitro (whole-embryo culture for 24, 48 and 72 h explanted at 9.5 days of gestation) and in vivo (10.5, 11.5 and 12.5 days of gestation) in order to compare the distribution and activity of various enzymes involved in the digestion and energy metabolism in both systems. It was shown that, both in vitro and in vivo -glytamyltransferase and dipeptidylpeptidase IV are demonstrable in the apical cell membranes (membranebound hydrolases), while acid phosphatase, dipeptidylpeptidases I, II and acid -galactosidase are concentrated in the supranuclear vacuoles (lysosomal hydrolases), and cytoplasmic lactate dehydrogenase and mitochondrial enzymes (succinate dehydrogenase, NAD-dependent isocitrate dehydrogenase, cytochrom oxidase) are localized in the whole cytoplasm and mainly in the apical cytoplasm, respectively, of the visceral yolk-sac epithelium. In vivo, the activity of all enzymes increased until 12.5 days, but in vitro, this activity increased only until 48 h after the start of culture (corresponding to 11.5 days in vivo). Comparison of the yolk sacs at 10.5 and 11.5 days in vivo with those after 24 and 48 h in vitro showed that the activities of all the investigated enzymes were almost identical. Yolk sacs which were cultured for 72 h showed lower activities of lysosomal and mitochondrial enzymes than those at 12.5 days in vivo. It is concluded that the digestive function and energy metabolism of the visceral yolk-sac epithelium are almost identical in vitro and in vivo at 10.5 and 11.5 days.Supported by the Deutsche Forschungsgemeinschaft 541/1-1)     

Molecular cloning and insecticidal effect of Inga laurina trypsin inhibitor on Diatraea saccharalis and Heliothis virescens

Native Inga laurina (Fabaceae) trypsin inhibitor (ILTI) was tested for anti-insect activity against Diatraea saccharalis and Heliothis virescens larvae. The addition of 0.1% ILTI to the diet of D. saccharalis did not alter larval survival but decreased larval weight by 51%. The H. virescens larvae that were fed a diet containing 0.5% ILTI showed an 84% decrease in weight. ILTI was not digested by the midgut proteinases of either species of larvae. The trypsin levels were reduced by 55.3% in the feces of D. saccharalis and increased by 24.1% in the feces of H. virescens. The trypsin activity in both species fed with ILTI was sensitive to the inhibitor, suggesting that no novel proteinase resistant to ILTI was induced. Additionally, ILTI exhibited inhibitory activity against the proteinases present in the larval midgut of different species of Lepidoptera. The organization of the ilti gene was elucidated by analyzing its corresponding genomic sequence. The recombinant ILTI protein (reILTI) was expressed and purified, and its efficacy was evaluated. Both native ILTI and reILTI exhibited a similar strong inhibitory effect on bovine trypsin activity. These results suggest that ILTI presents insecticidal properties against both insects and may thus be a useful tool in the genetic engineering of plants.     

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.     

Cortisone-evoked decrease of acid-β-galactosidase, β-glucuronidase, N-acetyl-β-glucosaminidase and arylsulphatase in the ileum of suckling rats

Changes of activity of intestinal acid beta-galactosidase, beta-glucuronidase, N-acetyl-beta-glucosaminidase and arylsulphatase were studied in suckling rats treated with cortisone (5mg/100g body wt. daily, started on day 9 postnatally) and compared with changes in control animals. Specific activities were not changed within the first 72h, but all enzymes decreased similarly 96h after the first injection. Total activities per ileum and animal were not changed within the first 48h, but within 72h a significant decrease was observed. Calculation of the rate of decrease of the hydrolases studied in cortisone-treated animals shows that it proceeds faster than the rate of renewal of enterocytes in this period.     

转基因杨树对杨小舟蛾体内三种保护酶活力的影响

用转Bt单基因（FB56）和转Bt＋CpTI双价基因（D18）杨树叶片饲喂杨小舟蛾Micromelalopha troglodyta (Graeser),采用酶活力测定方法研究了转基因杨树对幼虫中肠和血淋巴三种保护酶（SOD、CAT 和POD）活力的影响。结果表明,幼虫中肠SOD活力显著升高,而CAT和POD活力受到了显著抑制。用FB56处理24 h,杨小舟蛾幼虫中肠SOD活力增加了41.7%,与对照有显著差异;而用D18处理,SOD活力增加了25.3%,与对照差异不显著。用FB56处理24 h,中肠CAT活力受到显著抑制,36 h后抑制能力不强;而用D18处理,CAT活力全程受到显著抑制。用FB56和D18处理36 h,中肠POD活力均受到显著抑制,分别比对照下降70.1%和898%。转基因杨树通过扰乱幼虫中肠SOD、CAT和POD三种保护酶的动态平衡,使虫体内自由基清除遇到了障碍,从而对其产生了毒害作用。转双价基因杨树对幼虫中肠保护酶的毒害作用大于转单基因杨树。用2种转基因杨树叶片饲喂杨小舟蛾幼虫后其血淋巴SOD和POD活力均没有受到显著影响,但CAT活力均显著高于对照,转基因杨树对血淋巴保护酶系统的影响小于对中肠保护酶系统的影响。     

Effect of bacterial infection on antioxidant activity and lipid peroxidation in the midgut of Galleria mellonella L. larvae (Lepidoptera, Pyralidae)

Bacillus thuringiensis is one of the most widely used sources of biorational pesticides, as well as a key source of genes for transgenic expression to provide pest resistance in plants. In this study the effect of Bacillus thuringiensis ssp. galleriae (Bt) infection on the activity of superoxide dismutase (SOD), glutathione S-transferase (GST), catalase (CAT), concentrations of oxidated and reduced thiols (RSSR/RSH) and malondialdehyde (MDA) was tested in the midgut of Galleria mellonella larvae. We found that Bt infection resulted in increased activities of SOD, GST, malondialdehyde and RSSR/RSH ratio the first day after inoculation. However, catalase activity decreased on the first and following days after bacterial infection by Bt. Our results confirm the hypothesis that Bt infection increases the level of oxidative stress in the larval midgut. In light of this study, it seems possible that oxidative damage contributes to cell death in the midgut during bacteriosis.     

A specific acid alpha-glucosidase in lamellar bodies of the human lung

In the present investigation, we have demonstrated that three lysosomal-type hydrolases, alpha-glucosidase, alpha-mannosidase and a phosphatase, are present in lamellar bodies isolated from adult human lung. The hydrolase activities that were studied, all showed an acidic pH optimum, which is characteristic for lysosomal enzymes. The properties of acid alpha-glucosidase in the lamellar body fraction and that in the lysosome-enriched fraction were compared. Using specific antibodies against lysosomal alpha-glucosidase from human placenta, two alpha-glucosidases could be distinguished in the lamellar body fraction: one with a high affinity to the antibodies as found in the lysosome-enriched fraction and another with a much lower affinity. Both forms showed an acidic pH optimum. The same heterogeneity of alpha-glucosidase in the lamellar body fraction could be observed using immobilized concanavalin A. The lectin was able to precipitate nearly all alpha-glucosidase activity of the lysosome-enriched fraction. In contrast, 30% of the alpha-glucosidase activity in the lamellar body fraction was not precipitable. Furthermore, the lamellar body alpha-glucosidase with the low antibody affinity could not be bound to concanavalin A. The results suggest that lamellar bodies contain at least two acid alpha-glucosidases: one similar to the lung lysosomal alpha-glucosidase, and another lamellar body-specific isoenzyme with a different immunoreactivity and lectin affinity. The lamellar body-specific alpha-glucosidase should prove useful as a lamellar body-specific marker enzyme.     

环氧苍耳素Ⅰ对菜青虫中肠消化酶和羧酸酯酶活性的影响

【目的】探明苍耳Xanthium sibiricum活性物质--环氧苍耳素Ⅰ对菜青虫的作用机理。【方法】采用饲喂法处理4龄菜青虫Pieris rapae, 测试从苍耳中分离提纯的环氧苍耳素Ⅰ（为倍半萜内酯类物质）对菜青虫中肠蛋白酶、 淀粉酶和羧酸酯酶活性的影响。【结果】环氧苍耳素Ⅰ对蛋白酶活性的抑制最强, 处理后12, 24和48 h, 菜青虫中肠蛋白酶抑制率分别为20.95%,29.38%和50.06%; 其次为淀粉酶, 抑制率分别为11.89%,39.01%和31.92%。同时,中肠羧酸酯酶活性在处理后12 h与对照之间无显著变化, 24 h 时活性被显著抑制, 而48 h时活性却明显高于对照。【结论】环氧苍耳素Ⅰ对昆虫中肠消化酶活性的抑制, 可能是引起昆虫表现取食抑制和生长发育不良的重要原因之一。     

Consumption of food and spatial organization of digestion in the cassava hornworm, Erinnyis ello

Fifth-instar Erinnyis ello larvae eat 2.1 times their own weight per day of Euphorbia pulcherrima leaves, with a coefficient of digestibility of 45% and an efficiency of food conversion into tissue of 25%. The food takes about 150 min to go through the gut. Midgut contents have a pH of 9.3–9.8, depending on the region. Cellulase is absent from the gut in E. ello. Significant gut hydrolase activities are found only in midgut. Amylase and trypsin occur in the midgut tissue and contents and in regurgitated material, whereas aminopeptidase, α-glucosidase, β-glucosidase and trehalase are found in major amounts in the midgut tissue, in minor amounts in the midgut contents and are absent from regurgitated material. The results support the hypothesis that digestion starts in the endoperitrophic space under the action of amylase and trypsin and is largely completed in the ectoperitrophic space through the catalytic action of several oligomer and dimer hydrolases. Involvement of a membrane-bound aminopeptidase in the terminal digestion of oligopeptides cannot, at present, be excluded. The finding that less than 7% of the total amylase and trypsin are excreted, after a time identical to the passage time of the food bolus, leads to the proposal for the existence of some mechanism by which those enzymes are recovered from the undigested food before it is excreted.     

Effect of <Emphasis Type="Italic">Caryophyllaeus laticeps</Emphasis> (Cestoda,Caryophyllidea) upon activity of digestive enzymes in bream

The activities of the main digestive hydrolases were comparatively studied in bream infected and noninfected with cestodes Caryophyllaeus laticeps (Pallas, 1781). It was shown that enzyme activities are distributed in the fish intestine in an irregular manner; the gradient of protease and lipase activities along the gut is presented. Following the infection of bream by cestodes, the activities of the studied enzymes decreased and the percentages of activities of various proteinase subclasses changed. No relation between the distribution of worms along the intestine and the levels of activities of digestive hydrolases was revealed.     

Changes in Two Acid Hydrolase Levels During Cyst Differentiation of a Ciliate,Histriculus muscorum1

Cellular levels of protein and two acid hydrolases, acid phosphatase (EC 3.1.3.2) and acid proteinase, were followed during cyst differentiation, arbitrarily divided into five stages, in the ciliate Histriculus muscorum Kahl. Extracellular enzyme activities were also measured. Protein content decreased gradually during cyst differentiation. In mature cysts the protein content was ca. 60% that of stationary phase organisms. The activities of both acid hydrolases remained unchanged during stage 1 and then decreased gradually; acid proteinase decreased more rapidly. Both enzymes remained slightly active in the mature cysts. The acid proteinase activity of stage 1 was reduced by cycloheximide treatment at time zero, whereas the enzyme was no longer sensitive to the inhibitor when treated at 1.5 h (late stage 1) after the first wash with encysting medium. Acid phosphatase activity was insensitive to the inhibitor. Extracellular release of acid phosphatase increased linearly at least until stage 5, although the extracellular release of acid proteinase was not detected. Cycloheximide blocked the extracellular release of acid phosphatase after stage 1. These results suggest that de novo synthesis of acid proteinase occurs during stage 1 and that lysosomes may play an important role during early stages of cyst differentiation.     

Purification and characterization of three beta-glycosidases from midgut of the sugar cane borer,Diatraea saccharalis

Three beta-glycosidases, named betaGly1, betaGly2 and betaGly3, were isolated from midgut tissues of the sugar cane borer, Diatraea saccharalis Fabricius (Lepidoptera: Pyralidae). The three enzymes have similar Mr (58,000; 61,000; 61,000), pI (7.5, 7.4, and 7.4) and optimum pH (6.7, 6.3, and 7.2) and were resolved by hydrophobic chromatography. The beta-glycosidases prefer beta-glucosides to beta-galactosides, have four subsites for glucose binding and hydrolyse glucose-glucose beta-1,3 linkages better than beta-1, 4- or beta-1,6 linkages. betaGly1 and 2 were completely purified, whereas betaGly3 was isolated with a contaminant peptide that has no activity upon beta-glycosides.By using competing substrates, it was shown that betaGly 1 and 3 have one active site, whereas betaGly2 has two, one hydrolyzing natural and the other synthetic substrates. betaGly2 is the only D. saccharalis beta-glycosidase that can efficiently hydrolyse prunasin, the glycoside remaining after glucose removal from the plant glycoside amygdalin and that liberates the cyanogenic mandelonitrile. As shown elsewhere, betaGly2 activity is reduced when D. saccharalis is reared in amygdalin containing diets. From the results, we propose that the physiological role of betaGly 1 and 3 is the digestion of oligo- and disaccharides derived from hemicelluloses and of betaGly2 is glycolipid hydrolysis.Free energy relationships showed that D. saccharalis betaGly3 and Tenebrio molitor (Coleoptera) betaGly1 have active sites that bind similarly the transition states formed with different substrates. The same is also true for the active sites of D. saccharalis betaGly1 and T. molitor betaGly2. This suggests that active sites of similar enzymes are probably homologous, displaying nearly identical bonds between active site amino acids and substrate moieties.     

Binding of hydrolases from wheat aleurone to concanavalin A- and wheat-germ agglutinin-sepharose

We have studied the ability of hydrolases (acid phosphatase and glycosidases) from the aleurone layers of resting wheat grains to interact with Con A- and WGA-Sepharose as a way to examine their glycoprotein nature. Aliquots (6–85% depending on the enzyme) of all the enzymes interacted with Con A-Sepharose. The major part of α-mannosidase activity (85%) was present in this form. Aliquots (2–20% depending on the enzyme) of the following four enzymes, β-galactosidase, α-mannosidase, β-N-acetylglucosaminidase and acid phosphatase, interacted with WGA-Sepharose. All the enzymes were found in forms which were unable to interact with either lectin. No forms of hydrolases interacting with both lectins were found in the crude extract. The specific activities of most of the enzymes recovered from the lectin-Sepharose gels were greater than those measured in the crude extract. In particular, the highest specific activities were found for β-N-acetylglucosaminidase and β-galactosidase recovered from WGA-Sepharose. Different lectin-binding forms of hydrolases were compared with respect to pH optimum and stability under various conditions (heat and guanidine hydrochloride treatments). The lectin-binding pattern of the hydrolases released in the incubation medium by the aleurone layers was similar to that reported above for the enzymes extracted from these tissues, suggesting that none of the hydrolase forms found in the aleurone layers is selectively released during incubation of these tissues.     

Soil oxidases recovered faster than hydrolases in a 50-year chronosequence of desert revegetation

Aims

Desert characterized by alkaline soil with low organic matter and nutrients has a high soil oxidative potential. We hypothesized that oxidase activities would recover faster than hydrolases during the succession of sand-fixing community.

Methods

Sand dunes stabilized in different years, including a moving sand dune and a steppe at the southeastern fringe of the Tengger Desert, were selected to investigate restoration of extracellular enzyme activities (EEAs) in a 50-year chronosequence.

Results

Oxidases showed significantly higher activities than hydrolases at all ten studied sites and EEAs exibited a decreasing trend from catalase, phenol oxidase, sucrase, urease, alkaline phosphatase, α-Amylase to cellulase. After 50 years of revegetation, most EEAs in topsoil recovered to 50–83% of that of the steppe except for urease. Oxidase activities recovered earlier and faster than hydrolases, while hydrolases activities attained the fastest recovery at 19–25 years in the 50-year chronosequence.

Conclusions

Recovery of EEAs was modulated by the succession of the sand-fixed community: oxidases activities exhibited peak recovery rates at the stage when shrubs dominated the community, while recovery of hydrolases activities appeared to be mainly regulated by biological soil crusts and annual plants.     

Peritrophic membrane role in enhancing digestive efficiency. Theoretical and experimental models

The peritrophic membrane (PM) is an anatomical structure surrounding the food bolus in most insects. Rejecting the idea that PM has evolved from coating mucus to play the same protective role as it, novel functions were proposed and experimentally tested. The theoretical principles underlying the digestive enzyme recycling mechanism were described and used to develop an algorithm to calculate enzyme distributions along the midgut and to infer secretory and absorptive sites. The activity of a Spodoptera frugiperda microvillar aminopeptidase decreases by 50% if placed in the presence of midgut contents. S. frugiperda trypsin preparations placed into dialysis bags in stirred and unstirred media have activities of 210 and 160%, respectively, over the activities of samples in a test tube. The ectoperitrophic fluid (EF) present in the midgut caeca of Rhynchosciara americana may be collected. If the enzymes restricted to this fluid are assayed in the presence of PM contents (PMC) their activities decrease by at least 58%. The lack of PM caused by calcofluor feeding impairs growth due to an increase in the metabolic cost associated with the conversion of food into body mass. This probably results from an increase in digestive enzyme excretion and useless homeostatic attempt to reestablish destroyed midgut gradients. The experimental models support the view that PM enhances digestive efficiency by: (a) prevention of non-specific binding of undigested material onto cell surface; (b) prevention of excretion by allowing enzyme recycling powered by an ectoperitrophic counterflux of fluid; (c) removal from inside PM of the oligomeric molecules that may inhibit the enzymes involved in initial digestion; (d) restriction of oligomer hydrolases to ectoperitrophic space (ECS) to avoid probable partial inhibition by non-dispersed undigested food. Finally, PM functions are discussed regarding insects feeding on any diet.