Os insetos podem ser criaturas delicadas e maravilhosas. Entretanto, não se deixe iludir. Aquela borboleta linda da foto é a forma adulta de uma lagarta voraz capaz de comer 2,1 vezes o seu próprio peso em alimento por dia. Na verdade, os insetos são animais bem sucedidos e podem ser considerados os principais competidores do homem na terra, sendo responsáveis pela destruição de porcentual importante de plantações, dos alimentos armazenados, além de serem vetores de grande número de doenças que afetam o homem e os animais de criação .

Em vista disso, o esforço para controlar os insetos tem sido uma tarefa secular do homem. Várias estratégias tem sido empregadas.

Entretanto, em resposta a uma maior consciência ecológica , o processo de controle dos insetos atualmente tem sido dirigidos principalmente para o fortalecimento do próprio sistema de defesa das plantas contra os herbívoros. Com o reconhecimento de que a maior área de contato do inseto com o meio ambiente é o sistema digestivo, ênfase tem sido dada na produção de plantas transgênicas que expressam produtos que desorganizam as funções digestivas dos insetos. Dentro desse objetivo várias plantas transgênicas de interesse comercial já foram produzidas e que são resistentes a insetos.

O desenvolvimento dessa linha de pesquisa está limitado pela informação escassa em relação ao sistema digestivo dos insetos. Em conseqüência , recentemente tem havido um grande esforço na ampliação dos nossos conhecimentos sobre o sistema digestivo dos insetos envolvendo as enzimas digestivas, a sua distribuição espacial e os seus mecanismos de secreção, assim como de aspectos da biologia celular das células intestinais.

A equipe de nosso laboratório se dedica a todos esses aspectos. Para isso nosso laboratório possui equipamentos de purificação de proteínas (sistemas eletroforéticos, cromatógrafos de baixa e alta pressão, concentradores, etc...), para determinação de enzimas (espectrofotômetros, fluorimetros, etc...), para isolamento de frações celulares (sonicador, ultracentrífuga, etc...) e para clonar, expressar e mutar as enzimas que estudamos.

Em colaboração com o Centro de Microscopia Eletrônica do Instituto de Biociências da USP (Prof. Alberto F. Ribeiro) e do Laboratório Nacional de Luz Sincroton (Prof. Igor Polikarpov) estudamos, respectivamente, a biologia celular das células intestinais e a estrutura molecular de enzimas selecionadas.

Finalmente, em um esforço comum com o Prof. Márcio C. Silva Filho e Prof. José R. P. Parra da Escola Superior de Agricultura Luiz de Queiroz (ESALQ) da USP, estamos procurando usar os nossos conhecimentos no desenvolvimento de plantas transgênicas de interesse brasileiro.

• Terra W.R. ; ( 1990 ), Evolution of digestive systems of insects., Annu. Rev. Entomol.- [35 ]: 181- 200
  
  

  
  

• Terra W.R. ; Ferreira C. ; ( 1994 ), Insect digestive enzymes: properties, compartmentalization and function., Comp. Biochem. Physiol.- [109B ]: 1- 62
  
  

  
  

• Regel R. ; Matioli S.R. ; Terra W.R. ; ( 1998 ), Molecular adaptation of Drosophila melanogaster lysozymes to a digestive function., Insect Biochem. Molec. Biol.- [28 ]: 309- 319
  
  

  A lysozyme (pI 5.5) was purified to homogeneity from heated acid extracts of Drosophila melanogaster larvae, using gel filtration in a Superose column and ion-exchange chromatography in a Mono Q column. The final yield was 67%. The purified lysozyme with Mr 13 700 (determined by SDS-polyacrylamide gel electrophoresis) decreases in activity and has its pH optimum displaced towards acidic values and Kin increases as the ionic strength of the medium becomes higher. The lysozyme is resistant to a cathepsin D-like proteinase present in cyclorrhaphous Diptera and displays a chitinase activity which is 11-fold higher than that of chicken lysozyme. Microsequencing of an internal peptide of the purified lysozyme showed that this enzyme is the product of the previously sequenced Lys D gene. The results suggest that the product of the Lys P gene has pI 7.2, a pH optimum around 5 and is not a true digestive enzyme. The most remarkable sequence convergence of D, melanogaster lysozyme D and lysozymes from vertebrate foregut fermenters are serine 104 and a decrease in the number of basic amino acids, suggesting that these features are necessary for digestive function in an acid environment. Adaptive residues putatively conferring stability in an acid proteolytic environment differ between insects and vertebrates, probably because they depend on the overall three-dimensional structure of the lysozymes. A maximum likelihood phylogeny and inferences from insect lysozyme sequences showed that the recruitment of lysozymes as digestive enzymes is an ancestral condition of the flies (Diptera: Cyclorrhapha). (C) 1998 Elsevier Science Ltd. All rights reserved.

  
  

• Ferreira C. ; Torres B.B. ; Terra W.R. ; ( 1998 ), Substrate specificities of midgut beta-glucosidases from insects of different orders., Comp. Biochem. Physicol.- [119B ]: 219- 225
  
  

  beta-Glycosidase activity was determined in midgut homogenates and in electrophoretically-resolved midgut samples from the following insects: Orthoptera, Abracris flavolineata; Coleoptera, Pheropsophus aequinoctialis, Tenebrio molitor, Pyrearinus termitilluminans; Hymenoptera, Scaptotrigona bipunctata; Diptera, Rhynchosciara americana; Lepidoptera, Erinniys ello, Spodoptera frugigerda, and Diatraea saccharalis. The substrates used in the assays included disaccharides (cellobiose and lactose), synthetic aryl beta-glucosides (p-nitrophenyl beta-glucoside and p-nitrophenyl beta-galactoside), and plant glycosides (salicin and amygdalin). Orthopterans, coleopterans, and hymenopterans have in their midgut beta-galactosidases (active only on beta-galactosides) and class 2 (active only on disaccharides) and class 3 (active only on synthetic and plant glycosides) beta-glucosidases. Class 1 (active on disaccharides and on synthetic and plant glycosides) beta-glucosidases, at least in orthopterans, have putatively different sites for each activity. Dipterans have beta-galactosidases and a single class 1 beta-glucosidase. Lepidopterans have no beta-galactosidases and may have a single class 1 beta-glucosidase or class 1 and class 3 beta-glucosidases. There seems to be an evolutionary trend from multiple enzymes with different substrate specificities to a single enzyme able to hydrolyse all the beta-glycosides in the same site. (C) 1998 Elsevier Science Inc.

  
  

• Capella A. N. ; Terra W.R. ; Ribeiro A.F. ; Ferreira C. ; ( 1997 ), Cytoskeleton removal and characterization of the microvillar membranes isolated from two midgut regions of Spodoptera frugiperda (Lepidoptera). , Insect Biochem. Molec. Biol.- [27 ]: 793- 801
  
  

  Brush borders (microvilli) were isolated from Spodoptera frugiperda posterior midgut cells by using the cation differential precipitation technique. These microvilli preparations still contained some cytoskeleton elements visible by electron microscope, These elements are removed by Tris treatment followed by dilution and ultracentrifugation, Tris treatment solubilizes protein with M-r values similar to some cytoskeleton proteins, and causes an increase in the specific activity of the marker enzyme (aminopeptidase) and in the lipid-protein ratio of the purified microvillar membrane, Microvilli preparations from anterior midgut cells seem to be free from cytoskeleton. This is supported by electron microscopy and by the absence, after Tris treatment, of changes in the specific activity of the marker enzyme and in the lipid-protein ratio, The cytoskeleton of these microvilli should assemble and disassemble to permit the passage of vesicles observed migrating through them, As a result, it is likely that the cytoskeleton is not rigid and is lost during microvilli preparation by differential precipitation, In comparison with other insects, S, frugiperda purified microvillar membranes have low cholesterol, carbohydrate and lipid-protein ratios, Because S, frugiperda midgut microvillar membranes almost lack intrinsic digestive enzymes, their low lipid-protein ratios might result from a large amount of carrier or other proteins. (C) 1997 Elsevier Science Ltd, All rights reserved.

  
  

• Marana S. ; Ribeiro A.F. ; Terra W.R. ; Ferreira C. ; ( 1997 ), Ultrastructure and secretory activity of Abracris flavolineata (Orthoptera: Acrididae) midguts., J. Insect Physiol.- [43 ]: 465- 473
  
  

  The midgut of Abracris flavolineata adults comprises a ventriculus and six anteriorly placed caeca each displaying an anterior and a posterior lobe, Columnar cells in the caeca and anterior ventriculus present secretory vesicles originating from abundant Golgi areas, which seem to result (through exocytosis) in dark granules among the microvilli, A. flavolineata males were starved for 24 h, fed for 20 min at noon and dissected at 0, 1, 3 and 5 h after the meal, Enzyme assays were accomplished on crop and caecal contents and in subcellular fractions obtained from the isolated anterior caeca. Subcellular fractions putatively containing secretory vesicles were recognized, Digestive enzyme activity is usually low (amylase is high) in the secretory vesicles in starving insects, decreases 1 h after the meal, increases at 3 h, and thereafter decreases again (amylase remains constant), In caecal contents, digestive enzymes decrease at 1 h and increase at 3 h after the meal, the contrary being true for crop contents, Thus, in A. flavolineata caecal cells, digestive enzymes (beta-glucosidase is an exception) are synthesized and secreted by exocytosis in response to feeding, Also in response to feeding, digestive enzymes are transferred from caecal contents to the crop and, after about 3 h following the meal, crop-caecal dispersed material with accompanying enzymes are translocated to the caeca, where digestion ends and absorption occurs. (C) 1997 Elsevier Science Ltd.

  
  

• Silva C.P. ; Terra W.R. ; ( 1997 ), alpha-Galactosidase activity in ingested seeds and in the midgut of Dysdercus peruvianus (Hemiptera: Pyrrhocoridae)., Archs. Insect Biochem. Physiol.- [34 ]: 443- 460
  
  

  The midgut of Dysdercus peruvianus is divided into three main sections (V-1-V-3) and is linked through V-4 to the hindgut. The distribution of alpha-galactosidase activity in the different gut segments of D. peruvianus females was studied. alpha-galactosidase hydrolyzes the trisaccharide raffinose, the major carbohydrate of cotton seeds, on which the insects live. In D. peruvianus midgut alpha-galactosidase activity is mainly found in soluble fractions of V-1 contents. However, a comparison between specific activities using different alpha-galactosidase substrates in cotton seed extracts, V-1 tissue homogenate, and midgut contents suggested that the contribution of the enzymes from seeds may be very significant. Cel filtration on Sephacryl S-200 of samples from seed extracts, V-1 tissue, and V-1 contents revealed that in all samples raffinose hydrolysis is accomplished by alpha-galactosidases with similar M(r) (30,000 +/- 3,000) and dogs not involve the activity of a beta-fructosidase. Thermal inactivation studies of extracts from the three sources suggested that there was only one molecular form of the insect alpha-galactosidase and that the activity found in V-1 contents includes enzymes derived from the seed kernel. In insects fed with cotton seeds, the alpha-galactosidase activity increased in parallel with diet ingestion. In starved insects fed with tablets of sucrose plus raffinose, an increase in alpha-galactosidase activity was also observed, confirming that the insect is able to synthesize part of the gut enzyme. The results indicated that raffinose digestion starts in V-1 utilizing alpha-galactosidases derived from the seed kernel and by an additional alpha-galactosidase synthesized by insect tissues. The action of alpha-galactosidases liberates galactose and sucrose, which are sequentially hydrolyzed by the major membrane-bound alpha-glucosidase releasing glucose and fructose in V-1 and V-2 lumina. (C) 1997 Wiley-Liss, Inc.

  
  

• Ferreira C. ; Parra J.R.P. ; Terra W.R. ; ( 1997 ), The effect of dietary plant glycosides in larval midgut beta-glucosidases from Spodoptera frugiperda and Diatraea saccharalis., Insect Biochem. Molec. Biol.- [27 ]: 55- 59
  
  

  Spodoptera frugiperda development and midgut beta-glucosidase activity are not affected by salicin and amygdalin present in their diets up to a concentration of at least 0.5%, suggesting that glycoside tolerance results from aglycone detoxification. Both salicin and amygdalin affect Diatraea saccharalis development, whereas only 0.5% amygdalin causes a decrease in midgut beta-glucosidase activity. There are three electrophoretically-resolved beta-glucosidases in D. saccharalis midguts. beta-Glucosidases 1 and 3 are thought to hydrolyze amygdalin to glucose and prunasin, and beta-glucosidase 2 to hydrolyze prunasin to glucose and the cyanogenic mandelonitrile. A short (48-h) exposure to 0.5% amygdalin causes a decrease of beta-glucosidase 1 and 3, whereas a long exposure (whole larval stage) depresses beta-glucosidase 2 activity. This suggests that glycoside tolerance may result from reduction in the midgut aryl beta-glucosidase activity without affecting the glycosyl beta-glucosidase activity, which hydrolyzes cellobiose and other beta-glycans. (C) 1997 Elsevier Science Ltd.

  
  

• Terra W.R. ; ( 1996 ), Evolution and function of insect peritrophic membrane. ,Ciência e Cultura- [48 ]: 317- 324
  
  

  
  

• Silva C.P. ; Ribeiro A.F. ; Terra W.R. ; ( 1996 ), Enzyme markers and isolation of the microvillar and perimicrovillar membranes of Dysdercus peruvianus (Hemiptera: Pyrrhocoridae) midgut cells., Insect Biochem. Molec. Biol.- [26 ]: 1011- 1018
  
  

  The midgut of Dysdercus peruvianus is divided into four sections (V-1-V-4), AII the cells have microvilli ensheathed by a lipoprotein membrane (perimicrovillar membrane) extending toward the lumen as narrow tubes with dead ends. Subcellular fractionation of V-1 and V-2 tissue in isotonic and hypotonic conditions showed that alpha-glucosidase is associated with membranous structures larger than those associated with beta-glucosidase. The alpha/beta-glucosidase activity ratio is 3+/-4 in V-1 tissue and 170+/-10 in membranes recovered from the V-1 luminal contents. These membranes are resolved in sucrose gradients into low density (1.087+/-0.001g/cm(3)) Alpha-glucosidase-carrying membranes (alpha/beta-glucosidase activity ratio of 330+/-30) and high density (1.132+/-0.002g/cm(3)) beta-glucosidase-carrying-membranes. Low-density membranes have 1090+/-60 mu g lipid/mg protein and apparently are not contaminated by high-density ones (electron micrographs), SDS-polyacrylamide gel electrophoresis (SDS-PAGE) showed that membranes recovered from V-1 luminal contents are composed mainly of alpha-glucosidase-rich membranes. The data suggest that alpha-glucosidase-rich membranes are perimicrovillar membranes which may be partly lost into luminal contents on dissection, with densities and lipid/protein ratios similar to that of myelin sheaths, in accordance with previous freeze-fracture data. beta-Glucosidase-rich membranes are probably microvillar membranes with densities increased by the presence of associated portasomes. Copyright (C) 1997 Elsevier Science Ltd.

  
  

• Jordão B.P. ; Lehane M.J. ; Terra W.R. ; Ribeiro A.F. ; Ferreira C.F. ; ( 1996 ), An immunocytochemical investigation of trypsin secretion in the midgut of the stablefly, Stomoxys calcitrans., Insect Biochem. Molec. Biol.- [26 ]: 445- 453
  
  

  Musca domestica trypsin antibody cross-reacts with polypeptide bands of M(r) 25,000 and 30,000 showing proteolytic activity from Stomoxys calcitrans midgut extracts, Secretory granules from the main enzyme-secreting region, the opaque zone, stained heavily with the trypsin antibody in both unfed and blood-fed flies, Heterogeneous staining of granules suggests the unequal distribution of trypsin in secretory granules, This is also consistent with the occurrence of non-parallel secretion, which is also suggested by the possible preferential release of smaller, heavily stained secretory granules in fed flies, The predigestive, anterior midgut region responsible for rapid dehydration of the blood meal, the reservoir zone, contains a different population of secretory granules which stain heavily with trypsin antibody. This zone contains 20% of the midgut trypsin activity in unfed flies; trypsins are held here as proenzymes which are probably only activated postsecretion, In the midgut lumen of both unfed and blood-fed flies, trypsin is mainly immunolocalized in the ectoperitrophic space. Enzyme assays suggest that 5-15% of the lumenal trypsin is associated with the peritrophic matrix, The finding of intact secretory granules plus cell debris in the ectoperitrophic space of opaque and lipoid zones of blood-fed flies supports the contention that some trypsin is released by apocrine secretion in this insect. Copyright (C) 1996 Elsevier Science Ltd.

  
  

• Jordão B.P. ; Terra W.R. ; Ribeiro A.F. ; Lehane M.J. ; Ferreira C. ; ( 1996 ), Trypsin secretion in Musca domestica larval midguts: A biochemical and immunicytochemical study., Insect Biochem. Molec. Biol.- [26 ]: 337- 346
  
  

  Musca domestica larvae have trypsin in their midgut cells and contents, mainly at the posterior region, Both soluble and membrane-bound forms of trypsin are found, Membrane-bound trypsin is not affected by papain or phosphatidylinositol-specific phospholipase C, although it is solubilized with equal efficiency by detergents with high and low critical micellar concentrations, The solubilization increases at high pH values in the presence or absence of detergent, Once solubilized, membrane-bound trypsin behaves as a hydrophilic protein, M. domestica trypsin was purified to homogeneity and used to raise antibodies in a rabbit. A Western blot of M. domestica membrane-bound and soluble midgut proteins, after sodium dodecyl sulfate-polyacrylamide gel electrophoresis using trypsin antiserum, showed only bands co-migrating with trypsin, With this antiserum, trypsin was immunolocalized in the membranes of secretory and Golgi vesicles, surface of microvilli and midgut contents of M, domestica posterior midgut, The data suggest that trypsin is bound to the secretory vesicle membrane by a hydrophobic peptide anchor. Upon exocytosis, most trypsin is solubilized apparently because the neutral pH of the luminal contents causes a conformational change which hinders part of the trypsin anchor, Copyright (C) 1996 Elsevier Science Ltd.

  
  

• Terra W.R. ; Cristofoletti P.T. ; ( 1996 ), Midgut proteinases in three divergent species of Coleoptera., Comp. Biochem. Physiol.- [113B ]: 725- 730
  
  

  Cysteine proteinases have been found in some families of Coleoptera and, based on this, these enzymes were supposed to be characteristic of Coleoptera. To test this hypothesis, we studied midgut homogenates of three phylogenetically distant Coleoptera species: Tenebrio molitor (Tenebrionidae) larvae, Pyrearinus termitilluminans (Elateridae) larvae, and Pheropsophus aequinoctialis (Carabidae) adults. T. molitor display two cysteine proteinases (pHo 6.8) resolved in Superose (FPLC) with Mr 31,000 and 51,000. These enzymes are inhibited by E-64 and pHMB, are activated by EDTA + cysteine and hydrolyze benzoyl-DL-arginine-beta-naphthylamide. T. molitor enzymes differ from a cysteine proteinase (Mr 64,000 using Superose) present in the wheat meal ingested by the insect. The cysteine proteinases predominate in the anterior two thirds of T. molitor midgut, probably because they are unstable in the higher luminal pH observed in the posterior third of the midgut. P. termitilluminans and P. aequinoctialis do not display cysteine proteinases, although they have trypsins (Mr 15,000, 25,000 and 41,000 for P. termitilluminans; Mr 26,000, 33,000 and 52,000 for P. aequinoctialis) and chymotrypsins (Mr 38,000 and 25,000 for P. Aequinoctialis and Mr 15,000 for P. termitilluminans). Our results, together with literature data, suggest that cysteine proteinases occur in the Cucujiformia ancestor, which corresponds to the ancestor of most Coleoptera which ingest seeds rich in serine proteinase inhibitors.

  
  

• Terra W.R. ; Regel R. ; ( 1995 ), pH buffering in Musca domestica midguts., Comp. Biochem. Physiol.- [112A ]: 559- 564
  
  

  The M. domestica midgut displays three morphological regions with the following luminal pH values: anterior, 6.1; middle, 3.1; posterior, 6.8. Looking for enzymes that might be related to the acidification of middle midguts or to the neutralization of luminal contents in the anterior and posterior midguts, M. domestica larvae were placed on layers of 10% starch gels containing 0.1% Congo Red or 0.1% lacmoid and one of the following compounds: acetazolamide, vanadate, KSCN, NaF, ouabain, calcium acetate, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, or amiloride. None of these compounds induced alteration in anterior or posterior midgut pH, whereas acetazolamide, vanadate, NaF, and ouabain affected luminal pH in middle midgut. Ammonia and phosphate predominate in anterior and posterior midguts, chloride concentrates in middle midgut, and bicarbonate occurs in low concentration along the midgut lumen. Ouabain and vanadate cause a relative decrease of chloride in the middle midgut and an increase of ammonia mainly in the posterior midgut. The results suggest that chloride may follow the protons translocated into middle midgut contents by a type-P ATPase, similar to that found in vertebrate stomachs, which is inhibited by high intracellular concentration of Na+ Ammonia probably neutralizes the contents of the anterior and mainly posterior midguts and is secreted by mechanism probably involving an, apical Na+/K+-ATPase.

  
  

• Silva C.P. ; Ribeiro A.F. ; Gulbenkian S. ; Terra W.R. ; ( 1995 ), Organization, origin and function of the outer microvillar (perimicrovillar) membranes of Dysdercus peruvianus (Hemiptera) midgut cells., J. Insect Physiol.- [41 ]: 1093- 1103
  
  

  The midgut of Dysdercus peruvianus is divided into three main sections (V-1-V-3), and is linked through V-4 to the hindgut. V-1 cells have microvilli ensheathed by a lipoprotein membrane (perimicrovillar membrane) extending toward the lumen as narrow tubes, which may be dilated in some places forming spheres, and have dead ends, The cytoplasmic side of the microvillar membrane is studded with small particles (portasomes), putatively involved in creating ionic gradients used in the cotransport of organic compounds. The basal membrane invaginates forming narrow and ramified channels with associated mitochondria. V-2, V-3 and V-4 cells are similar to V-1 cells, except for less developed basal infoldings. V-4 cells lack particles associated to the cytoplasmic side of the microvillar membranes and morphological features associated with protein secretion, V-3 and V-4 cells show bacteria associated to the microvilli, These morphological features agree with physiological data showing that V-1 absorbs water, V-2 and V-3 absorb amino acids and no digestion occurs in V-4. alpha-Glucosidase, a perimicrovillar membrane marker, was immunolocalized in V-1 cells. The data suggest that perimicrovillar membranes are formed in Golgi areas, migrate as the internal membrane of double membrane vesicles, which finally fuse at the cell apex-the outer vesicle membrane with the microvillar membrane and the inner vesicle membrane with the perimicrovillar membrane.

  
  

• Silva C.P. ; Terra W.R. ; ( 1995 ), An alpha-glucosidase from perimicrovillar membranes of Dysdercus-peruvianus (Hemiptera: Pyrrhocoridae) midgut cells. Purification and properties., Insect Biochem. Molec. Biol.- [25 ]: 487- 494
  
  

  Dysdercus peruvianus (Hemiptera: Pyrrhocoridae) has a major alpha-glucosidase bound to perimicrovillar membranes, which are the lipoprotein membranes ensheathing the midgut cell microvillar membranes in hemipterans. The enzyme was solubilized in detergent and purified to homogeneity by means of affinity chromatography on Concanavalin A-Sepharose, ion-exchange on Mono Q and preparative polyacrylamide gel electrophoresis (PAGE). The yield, purification factor and final specific activity were, respectively: 3.5%, 50-fold, 7.6 U/mg of protein. The alpha-glucosidase is a glycoprotein with a pH optimum of 5.0. M(r) values were 61,000 (SDS-PAGE), 120,000 (gel filtration), 130,000 (ultracentrifugation), or 431,000 (electrophoresis in native conditions). The data suggest that the alpha-glucosidase occurs in vivo as dimers and, during electrophoresis, as octamers. Taking into account k(cat)/K-m ratios, the enzyme is more active on maltose than sucrose and prefers oligomaltodextrins up to maltotetraose. Using p-substituted phenyl alpha-glucosides as substrates, it was shown that aglycone leaving is rate limiting and that the reaction constant rho is negative, suggesting the intermediary formation of a carbonium ion in the reaction path. Erythritol, delta-gluconolactone and Tris are simple linear competitive inhibitors of the alpha-glucosidase. Experiments involving competition between substrates suggested that the enzyme operates in accordance with rapid-equilibrium kinetics, hydrolyzing maltose, p-nitrophenyl-alpha-D-glucoside, sucrose and turanose at the same active site. Evidence suggests that the active site may have five sub-sites.

  
  

• Marana S.R. ; Terra W.R. ; Ferreira C. ; ( 1995 ), Midgut beta-D. glucosidases from Abracris flavolineata (Orthoptera: Acrididae). Physical properties, substrate specificities and function. , Insect Biochem. Molec. Biol.- [25 ]: 835- 843
  
  

  A combination of gel filtration, ion-exchange chromatography, polyacrylamide gel electrophoresis, and heat inactivation data revealed the existence of three beta-glucosidases with M(r) 82,000 in Abracris flavolineata midgut contents: 1, a major heat-stable activity against cellobiose (cellobiase-argl beta-glucosidase); 2, a minor heat-unstable activity against p-nitrophenyl beta-D-glucoside (NP beta Glu) (aryl beta-glucosidase); 3, an activity against octyl-beta-glucoside (alkyl beta-glucosidase). The cellobiase-aryl beta glucosidase has a pH optimum of 5.5 and is more active on cellobiose and laminaribiose than on synthetic or natural aryl beta-glucosides. Experiments involving competition between substrates and the use of inhibitors suggested that cellobiase-aryl beta-glucosidase hydrolyzes cellobiose and aryl beta-glucosides at different active sites. Alkyl beta-glucosidase (pH optimum 4.8) has a sigmoidal activity-octyl beta-glucoside-concentration profile, which changes to a hyperbolic profile in the presence of excess Triton X-100, NP beta Glu, which is hydrolyzed at the same site as octyl beta-glucoside, has a hyperbolic activity-NP beta Glu-concentration profile that increases in the presence of Triton X-100. It seems that amphipathic molecules activate the alkyl beta-glucosidase, which is inactive on methyl beta-D-glucoside and is most active on C-7-C-10 alkyl-beta glucosides. The aryl beta-glucosidase activity of the cellobiase-aryl beta-glucosidase and the alkyl beta-glucosidase are probably responsible for in vivo digestion of beta 1,3-glucans and glucosylceramides, respectively. Activation by detergent-like molecules is supposed to maintain high alkyl beta-glucosidase activity only during plant cell membrane digestion, This avoids extensive hydrolysis of toxic plant beta-glucosides which may be ingested by the insects.

  
  

• Jordão B.P. ; Terra W.R. ; Ferreira C. ; ( 1995 ), Chemistry of microvillar membranes purified from brush borders isolated from the larval midgut from one Coleoptera and two Diptera species. , Insect Biochem. Molec. Biol.- [25 ]: 417- 426
  
  

  Brush-borders (microvilli) were isolated from different midgut regions of larvae of Tenebrio molitor (Coleoptera), Rhynchosciara americana (lower Diptera), and Musca domestica (higher Diptera) by differential precipitation from homogenates prepared as previously described. The microvillar preparations were treated with the chaotropic salts lithium diiodosalicylate and sodium thiocyanate in order to disrupt microvilli into microvillar membranes and core (cytoskeleton) material. Marker enzymes were extensively inactivated and there was not a selective release of cytoskeleton elements from the microvillar membranes. Insect microvillar preparations were also treated with hyperosmotic Tris buffer, then diluted, centrifuged, and the purified microvillar membranes were recovered from the resulting pellet. Specific activities of marker enzymes in purified membranes were 1.5-3.0-fold higher than in the original microvillar preparations with a final yield of about 20%. Contamination by soluble proteins was under 0.3% and by other membranes never exceeded 5%, as judged by chromatography in Sepharose 4B and sucrose gradient ultracentrifugation. In comparison with mammals, insect membranes are rich in carbohydrates (all insects studied), cholesterol (T. molitor), lipids (T. molitor), and protein (M. domestica). The densities, and the ratio of lipid to protein in the microvillar membranes of T. molitor is lower than that in the two species of Diptera. This agrees with the fact that microvillar hydrolases are more important in Diptera than in Coleoptera digestion.

  
  

• Klinkowstrom A.M. ; Terra W.R. ; Ferreira C. ; ( 1995 ), Midgut dipeptidases from Rhynchosciara americana (Diptera) larvae. Properties of soluble and membrane bound forms. , Insect Biochem. Molec. Biol.- [25 ]: 303- 310
  
  

  Dipeptidase activity in Rhynchosciara americana (Diptera: Sciaridae) is found mainly in midgut caeca cells, The caecal dipeptidase activity is partly soluble and partly membrane- bound, Differential centrifugation of midgut caeca homogenates, followed by assays of enzyme markers and dipeptidase, suggest that soluble dipeptidase is cytosolic or weakly associated with the cell glycocalyx, Membrane-bound dipeptidase is likely a microvillar enzyme, Soluble dipeptidase activity is resolved by gel filtration and ion exchange chromatography into two enzymes (M, 63,000 and 73,000), which hydrolyze both Gly-Leu and Pro-Gly, although with different efficiency, The two enzymes also differ in their stability in the presence of EDTA and degree of inhibition by phenanthroline and aminoacyl hydroxamates. Dipeptidase inhibition by phenanthroline is reversed by dialysis, Membrane-bound dipeptidase activity was solubilized by Triton X-100 and papain, Density-gradient ultracentrifugation, gel filtration, and ion-exchange chromatography suggest that there is only one detergent (M, 86,000) form of this enzyme, which is active on Gly-Leu and Pro-Gly. No activity upon Gly-Pro was found in R, americana midguts, whereas the weak activity observed upon carnosine is independent of the enzymes hydrolyzing Gly-Leu and Pro-Gly, Thus, R, americana midguts seem to have major soluble and membrane-bound dipeptide hydrolases (EC 3.4.13.11), which in contrast to the mammalian enzyme, are very active upon Pro-Gly, R, americana also has a minor carnosinase (EC 3.4.13.3).

  
  

• Klinkowstrom A.M. ; Terra W.R. ; Ferreira C. ; ( 1994 ), Aminopeptidase A from Rhynchosciara americana (Diptera) larvae midguts. Properties and midgut distribution. , Arch. Insect Biochem. Physiol.- [27 ]: 301- 315
  
  

  L-aspartic acid alpha-(beta-naphthylamide) (Asp beta NA) hydrolase activity is restricted mostly to the midgut caeca of Rhynchosciara americana larvae. The membrane-bound activity is solubilized in detergent and, after electrophoretic separation, proved to be identical to leucine p-nitroanilide (LpNA) hydrolases previously described. Differential centrifugation of midgut caeca homogenates, followed by assays of enzyme markers and aminopeptidase, suggests that the soluble Asp beta NA hydrolase is associated with the cell glycocalyx. Soluble aminopeptidases from R. americana midgut caeca are resolved into three fractions by gel electrophoresis. The slow migrating fraction hydrolyzes Asp beta NA well and displays a low activity on LpNA and proline beta-naphthylamide (Pro beta NA). Thus, this enzyme is an ami; nopeptidase A (EC 3.4.11.7). It has a pH optimum of 7.5, M(r) 117,000 (gel filtration), and is competitively inhibited by aspartate hydroxamate (K-i 0.1 mM). Nevertheless, this enzyme, in contrast to the vertebrate enzyme, is not activated by calcium ions. The aminopeptidase A seems to have a charge variant that displays an intermediate migration and is not resolved from an aminopeptidase N (enzyme very active on LpNA). These two activities are not resolved by either gel filtration or ion-exchange chromatography. The aminopeptidases N with intermediate and high migration, previously reported to be charge variants, were shown in this paper to differ in substrate specificities and in the strength with which they associate to the cell glycocalyx. (C) 1994 Wiley-Liss, Inc.

  
  

• Silva C.P. ; Terra W.R. ; ( 1994 ), Digestive and absortive sites along the midgut of the cotton seed sucker bug Dysdercus peruvianus (Hemiptera: Pyrrhocoridae). , Insect Biochem. Molec. Biol.- [24 ]: 493- 505
  
  

  The midgut of Dysdercus peruvianus is divided into three sections (V-1-V-3), which are linked through V-4 to the hindgut. The midgut contents are reducing with a pH around 5.8. D. peruvianus saliva lacks digestive enzymes and apparently is used only to suspend seed material which is ingested, distending V-1. Most midgut carbohydrase activities are found in V-1, where is also observed a alpha-mannosidase and an aminopeptidase derived from the meal. The high glycosidase and low amylase activities found in V-1 are in agreement with the major and minor cotton seed content of saccharides and starch, respectively. Water and glucose absorption occur without dietary salt ions in V-1. About 5h after the filling of V-1, V-2 is also filled. At V-2, a cysteine-proteinase (pH optimum 5.5, M(r) 53,000) predominates and amino acids are absorbed. This absorption is activated by potassium and inhibited by sodium ions. Both midgut alpha-glucosidase and cysteine proteinase activities increase on feeding. As soon as V-1 empties, V-3 is filled. After a long stay (70-90h) in V-2 and V-3, the food passes quickly through V-4 into the hindgut. The results on D. peruvianus (Hemiptera: Pentatomomorpha) and literature data on Rhodnius prolixus (Hemiptera: Cimicomorpha) are used to infer the digestive physiology characteristics of the Heteroptera ancestor, and to discuss the major adaptations which occurred when the bugs moved from a sap sucking habit into a habit which included digestion of polymers.

  
  

• Ferreira C. ; Capella A.N. ; Sitnik R. ; Terra W.R. ; ( 1994 ), Digestive enzymes in midgut cells, endo-and ectoperitrophic contents and peritrophic membranes of Spodoptera frugiperda (Lepidoptera) larvae. , Arch. Insect Biochem. Physiol.- [26 ]: 299- 313
  
  

  In the midgut of Spodoptera frugiperda larvae, subcellular fractionation data suggest that am in opeptidase 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. (C) 1994 Wiley-Liss, Inc.

  
  

• Ferreira C. ; Capella A.N. ; Sitnik R. ; Terra W.R. ; ( 1994 ), Properties of the digestive enzymes and the permeability of the peritrophic membrane of Spodoptera frugiperda (Lepidoptera) larvae., Comp. Biochem. Physiol.- [107A ]: 631- 640
  
  

  The physical and kinetic properties of several soluble and detergent-solubilized membrane-bound midgut hydrolases of Spodoptera frugiperda (Lepidoptera) were investigated. The following techniques were employed: isoelectric focusing and electrophoresis in polyacrylamide gels, density-gradient ultracentrifugation and gel filtration in Superose columns. In vivo molecular weights of the hydrolases were considered to correspond with those determined by centrifugation, which is the more gentle of the procedures employed. Using this criterion, the soluble hydrolases display the following number of sub-units: acetylglucosaminidase [pHo5 (pH optimum), Mr 123,000], 2; aminopeptidase (pHo 7.4, Mr 107,000), 2; amylase (pHo 9.6, Km for starch 0.38%, Mr 87,000), 1; carboxypeptidase A (pHo 8.0, Mr 45,000), 2; cellobiase (pHo 7.0, Mr 124,000), 2; dipeptidase (pHo 8.0, Mr 95,000), 2; maltase (pHo 5.0, Mr 150,000), 2; trypsin (pHo 7.9, Mr 51,000), 2.Amylase is not activated by chloride. A comparison between the diameters of the enzymes which pass through the peritrophic membrane (amylase, carboxypeptidase and trypsin) with that which is secreted into the lumen but do not pass through the peritrophic membrane (acetylglucosaminase) suggests that the pores of S. frugiperda larval peritrophic membrane have diameters of 7.5-8 nm.

  
  

• Lemos F.J.A. ; Ribeiro A.F. ; Terra W.R. ; ( 1993 ), A bacteria - digesting midgut-lysozyme from Musca domestica (Diptera) larval. Purification, properties and secretory mechanism., Insect Biochem. Molec. Biol.- [23 ]: 533- 541
  
  

  Two lysozymes were purified to homogeneity from heated acid extracts of Musca domestica larval midguts, using an S-Sepharose column, and a semi-preparative polyacrylamide gel electrophoresis (PAGE). The final yield was 60%. Lysozymes 1 and 2 display M(r) 22,000, determined by ultracentrifugation or electrophoresis in non-denaturing conditions, and M(r) 17,000, determined by SDS-polyacrylamide gradient gel electrophoresis. Isoelectric focusing showed the following pI values: lysozyme 1, 7.9; lysozyme 2, 8.2. Lysozyme 1 and 2 display identical kinetic properties, which include decrease in activity, displacement of the pH optimum toward acidic values and a K(m) increase as the ionic strength of the medium becomes higher. The lysozymes are resistant to a cathepsin D-like proteinase present in M. domestica midgut, and display a chitinase activity which is 6-fold higher than that of chicken lysozyme. Lysozyme immunolabeling revealed that lysozyme mainly occurs in secretory vesicles and at the outside surface of microvilli from M. domestica anterior midgut cells. The results showed that M. domestica lysozymes are similar to ruminant stomach lysozyme in being more active at acid pH values, when present in media with physiological ionic strengths, and in being resistant to an acid proteinase derived from the same animal as the lysozyme. Furthermore, the data support the assertion that M. domestica midgut lysozyme is secreted by exocytosis, partly remaining adsorbed to the cell glycocalyx.

  
  

• Schumaker T.T.S. ; Cristofoletti P.T. ; Terra W.R. ; ( 1993 ), Properties and compartmentalization of digestive carbohydrases and proteases in Scaptotrigona bipunctata (Apidae:Meliponinae) larvae., Apidologie- [24 ]: 3- 17
  
  

  Aminopeptidase (pH optimum, pH(o), 7.5; enzyme relative molecular weights, M(r) values: 1, 110000; 2, 190 000; 3, 300 000), amylase (pH(o) 5.5, M(r) values: 1, 21 000; 2, 68 000); cellobiase (pH(o) 5.5) and maltase (pH(o) 5.0, M(r) values: 1, 75 000; 2, 110 000; 3, 200 000) are found in the anterior (60-80%) and posterior (20-35%) midgut contents, with minor amounts occurring in midgut cells (2-5%). Trypsin (pH(o)) 7.0, M(r) 38 000) occurs mainly in the posterior (62%) rather than in the anterior (37%) midgut contents. Maltase 1 is more active on sucrose than on maltose, the reverse being true for the other maltases. A cysteine-proteinase (pH(o) 5.6, M(r) 79 000) was found in major amounts in the pollen grains ingested by the larvae. The results suggest that, except for a cysteine-proteinase derived from ingested pollen, all digestive enzymes originate in the midgut tissue and are most active in the luminal contents. Evidence is presented supporting the hypothesis that enzymes and nutrients diffusing through the peritrophic membrane are translocated forward by a countercurrent flux. The absence of a midgut differentiation of midgut luminal pH in S bipunctata larvae is though to be derived from putative Hymenopteran ancestors.

  
  

• Ferreira C. ; Ribeiro A.F. ; Terra W.R. ; ( 1993 ), Ultrastructural and biochemical aspects of digestion in the imagoes of the fly Rhynchosciara americana., Entomol. exp. appl.- [66 ]: 135- 143
  
  

  The midgut of adult Rhynchosciara americana Wiedemann (Diptera: Sciaridae) displays, in contrast to the midguts of other adult Diptera, two caeca connected to a ventriculus. All midgut cells exhibit long apical microvilli, and narrow and ramified basal channels with openings to the underlying space. These morphological features are thought to be involved in the absorption of nutrients from food. Enzymatic assays in R. americana adults revealed that amylase occurs in salivary glands and midgut, whereas aminopeptidase, alpha-glucosidases and trypsin occur only in the midgut, mainly in the ventriculus. There is a soluble (Mr 105 000) and a membrane-bound aminopeptidase (solubilized form, Mr 110000). Soluble alpha-glucosidase inactivates easily and could not be characterized, whereas membrane-bound alpha-glucosidases were resolved after solubilization into three molecular species (Mr 186 000, 105 000 and 84000) with different substrate specificities. The activities of trypsin (pH optimum 9.0), which was inhibited completely by soybean trypsin inhibitor, and of amylase (pH optimum 5.5), were not sufficiently high to be further characterized. The data support the assertion that R. americana adults are able, to a limited extent, to digest and absorb starch and proteins, in addition to nectar sugars. The results, supported by published data, suggest that there is an inverse correlation between the digestive enzyme activities and midgut absorptive surface in insects which has nectar as a major food.

  
  

• Terra W.R. ; ( 1988 ), Physiology and Biochemistry of insect digestion: an evolutionary perspective. , Brazil J. Med. Biol. Res.- [21 ]: 675- 734
  
  

  
  

• Terra W.R. ; Ferreira C. ; ( 1983 ), Further evidence that enzymes involved in the final stages of digestion by Rhynchosciara americana do not enter the endoperitrophic space. , Insect Biochem.- [13 ]: 143- 150
  
  

  
  

• Terra W.R. ; Ferreira C. ; ( 1981 ),The physiological role of the peritrophic membrane and trehalase: digestive enzymes in the midgut and excreta of starved larvae of Rhynchosciara americana. ,J. Insect Physiol.- [27 ]: 325- 331
  
  

  
  

• Ferreira C. ; Terra W.R. ; ( 1980 ),Intracellular distribution of hydrolases in midgut caeca cells from an insect with emphasis on plasma membrane-bound enzymes. ,Comp. Biochem. Physiol.- [66B ]: 467- 473
  
  

  
  

• Terra W.R. ; Ferreira C. ; Bianchi A.G. ; ( 1979 ),Distribution of digestive enzymes among the endo-and ectoperitrophic spaces and midgut cells of Rhynchosciara americana and its physiological significance.,J. Insect Physiol.- [25 ]: 487- 494