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.     

Compartmentalization of the digestive process in Abracris flavolineata (Orthoptera: Acrididae) adults

Carbohydrases predominate in the crop and their pH optima agree with pH prevailing in crop contents. Major amounts are also found in caecal contents. Aminopeptidase and trypsin are active mainly in the caeca, where they predominate in cells and contents, respectively. Aminopeptidase is partly membrane-bound. Except for trehalase, salivary glands display negligible amounts of digestive enzymes. The specific activity of digestive enzymes is high in all midgut cells and the enzyme molecules do not differ among gut compartments, as judged by polyacrylamide gel electrophoresis. Thus, it is probable that digestive enzymes are synthesized and secreted by all midgut cells (mainly in caeca) and then passed forward into the crop. Digestive enzymes are found in hindgut in concentrations similar to those in ventricular contents and, since they are stable in gut contents, they are likely excreted at a rate similar to undigested food. The data support the hypothesis that carbohydrate and protein are digested mainly in crop and caecal lumina, respectively, with part of the final digestion of proteins occurring at the surface of caecal cells. The peculiar features of the digestion of A. flavolineata grasshoppers, including the lack of midgut countercurrent fluxes, are thought to be derived from putative Polyneoptera ancestors.     

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

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

The flow and fate of digestive enzymes in the field cricket,Gryllus bimaculatus

The flow of enzymes, the ratio of bound to unbound enzymes, and their inactivation in the cricket Gryllus bimaculatus was studied. The digestive enzymes are forced forward into the crop by caecal contraction and then they are mixed with freshly chewed food and saliva, forming a crop‐chyme. This chyme is blended by crop peristalsis, and periodic opening of the preproventricular valve (PPV) allows posterior movement into the proventriculus and further into the midgut. The contraction of the crop is modulated by Grybi‐AST and Grybi‐SK peptides, which are partially secreted by the caecal endocrine cells. Most of the aminopeptidase and the four disaccharidases examined are membrane bound (62–80%); the remaining (20–38%) as well all trypsin, chymotrypsin, lipase, and amylase are secreted free into the caecal lumen. Cricket trypsin loses only 30% of its activity in 4 h and very little thereafter. The presence of digestive products in the lumen appears to retard further trypsin autolysis. Cricket trypsin digests 42% of the chymotrypsin, 37% of the lipase, and 45% of the amylase in the caecal fluids over 24 h in vitro no significant difference. Without Ca ion amylase was almost completely digested. About 50% of the membrane bound and free aminopeptidase was digested in the caecal lumen, and about 30–38% of the bound and free maltase. This loss of digestive enzyme activity is possible, because enzyme secretion rates are high, the unbound enzymes are effectively recycled, and the time of nutrient passage is short.     

Control of the release of digestive enzymes in the caeca of the cricket Gryllus bimaculatus

Abstract In Gryllus bimaculatus, more digestive enzymes (amylase, trypsin, aminopeptidase) are secreted in the caecum of fed crickets than in unfed crickets, but the enzymes are released continuously at a basal rate in unfed animals. The rate of synthesis of the enzymes appears to parallel their rate of release. Digestive enzymes are released in response to a specific ratio of nutrients, although a high nutrient component in the food does not necessarily induce a high digestive enzyme release for that component. Rinsed flat‐sheet preparations of the caecum are incubated with specific nutrients (carbohydrates and proteins) and various concentrations of a neuropeptide (type‐A allatostatin), which affects generally the basal rates of secretion. Both maltose and glucose increase the release of amylase in vitro, but starch produces an inhibition of amylase release at lower concentrations. Bovine serum albumin (BSA), peptone and a mixture of amino acids have almost no effect on the release of aminopeptidase or carboxypeptidase, and only low concentrations of peptone increase trypsin release. High concentrations of both BSA and peptone strongly inhibit trypsin activity, perhaps by excess substrate binding to the trypsin active site. The allatostatin Grybi‐AST 5 elevates the release of amylase in vitro, but not of trypsin or aminopeptidase, in 2‐day‐old fed females. In the caeca from 1‐day‐old unfed crickets, both amylase and the trypsin release are stimulated in the presence of AST 5. The paracrine AST 5 is probably released from the gut endocrine cells and binds to the enzyme‐producing caecal cells.     

扩头蔡白蚁肠道蛋白的鉴定

【目的】本研究旨在分析比较扩头蔡白蚁Tsaitermes ampliceps工蚁前中肠和后肠及其内容物的蛋白构成和表达差异,挖掘降解木质纤维素的相关酶和蛋白。【方法】通过扩头蔡白蚁工蚁的前中肠和后肠及其内容物蛋白的双向电泳,对高表达或高差异表达的47个蛋白点进行MALDI-TOF/MS测序,并进行生物信息学分析。【结果】测序分析发现,扩头蔡白蚁肠道及其内容物蛋白中有结构蛋白13个、调节蛋白9个、白蚁代谢相关蛋白10个、微生物代谢相关蛋白7个。经PD Quest分析发现,在前中肠和后肠有11个蛋白均高表达;仅在前中肠表达的蛋白有12个,主要是白蚁代谢相关蛋白和调节蛋白;仅在后肠表达的蛋白有8个,主要是微生物代谢相关蛋白。整个肠道内参与木质纤维素降解的相关酶有5个,分别是白蚁自身分泌的内源性纤维素酶,细菌产生的内切-β-1,4-葡聚糖酶和过氧化物歧化酶以及原生动物产生的GH11。【结论】白蚁对木质纤维素食物的降解主要在前中肠,后肠对降解产物进一步降解并进行微生物生长代谢。这些降解产物和微生物菌体蛋白为白蚁的肛哺提供营养成分。     

Enzyme activities and pH variations in the digestive tract of gilthead sea bream

Two investigations were carried out with 150 g gilthead sea bream Sparus aurata to determine the relative activity of six digestive enzymes (pepsin, trypsin, chymotrypsin, carboxypeptidase A, carboxypeptidase B and amylase) and the pH variation in the lumen of different parts of the gut of fish fed one or two meals per day. Pepsin activity was found exclusively in the stomach, whereas activities of the other enzymes studied were found in all regions of the gut, including the stomach. The lack of localization of enzyme production in the digestive tract of S. aurata is similar to many other species as reported in the literature. The pH variations found in the different regions of the gut could be explained by general digestive physiology following the flow of digesta along the digestive tract. The range of pHs recorded in the various regions of the gut were generally outside the cited optima for many digestive proteases in this species.     

Control of the release of digestive enzymes in the larvae of the fall armyworm,Spodoptera frugiperda

There is a basal level of enzyme activity for trypsin, aminopeptidase, amylase, and lipase in the gut of unfed larval (L6) Spodoptera frugiperda. Trypsin activity does not decrease with non‐feeding, possibly because of the low protein levels in plants along with high amino acid requirements for growth and storage (for later reproduction in adults). Therefore, trypsin must always be present so that only a minimal protein loss via egestion occurs. Larvae, however, adjust amylase activity to carbohydrate ingestion, and indeed amylase activity is five‐fold higher in fed larvae compared to unfed larvae. Gut lipase activity is low, typical of insects with a high carbohydrate diet. A flat‐sheet preparation of the ventriculus was used to measure the release of enzymes in response to specific nutrients and known brain/gut hormones in S. frugiperda. Sugars greatly increase (>300%) amylase release, but starch has no effect. Proteins and amino acids have little or no effect on trypsin or aminopeptidase release. The control of enzyme release in response to food is likely mediated through neurohormones. Indeed, an allatostatin (Spofr‐AS A5) inhibits amylase and trypsin, and allatotropin (Manse‐ AT) stimulates amylase and trypsin release. Spofr‐AS A5 also inhibits ileum myoactivity and Manse‐AT stimulates myoactivity. The epithelial secretion rate of amylase and trypsin was about 20% of the amount of enzyme present in the ventricular lumen, which, considering the efficient counter‐current recycling of enzymes, suggests that the secretion rate is adequate to replace egested enzymes. © 2009 Wiley Periodicals, Inc.     

Final digestion of starch in Musca domestica larvae. Distribution and properties of midgut α-d-glucosidases and glucoamylase

There are low-Mr (72,700) and high-Mr (330,000) soluble α-glucosidase activities in the hind-midgut of Musca domestica that can be isolated by ultracentrifugation. The low-Mr α-glucosidase is less stable and less inhibited by Tris than is the high-Mr α-glucosidase, and occurs mainly in hind-midgut contents, whereas the high-Mr α-glucosidase is found only in hind-midgut cells. Subcellular fractionation of hind-midgut cells showed that the high-Mr α-glucosidase is associated mainly with brush-borders, from where it is set free by freezing and thawing. The low-Mr α-glucosidase is recovered chiefly in the soluble fraction of the cell. The data suggest that the high-Mr and the low-Mr α-glucosidase occur mainly tightly and loosely bound to the cell glycocalyx, respectively. Based on subcellular fractionation, ultracentrifugation, and thermal inactivation data, there is only one molecular species of α-glucosidase and glucoamylase, which are solubilized by Triton X-100 from hind-midgut cell microvillar membranes. The results suggest that starch digestion is accomplished stepwise by luminal amylase, then by membrane-bound glucoamylase, and finally by glycocalyx-associated α-glucosidase and membrane-bound α-glucosidase. Luminal α-glucosidase probably digests ingested oligomaltodextrins and, since it is significantly excreted, it may also be involved in extracorporeal digestion.     

The digestive enzymes and acidity of the pellets regurgitated by raptors

The activity of six digestive enzymes (amylase, chitinase, trypsin, chymotrypsin, carboxypeptidase A, pepsin) was examined in the water-soluble contents of the pellets egested by ten species of raptors (kestrel, saker, lanner, goshawk, barn owl, tawny owl, little owl, long-eared owl, African great-owl and steppe eagle). All the enzymes studied were present in the pellets from these birds, except for chitinase which was not detected in the pellets of the goshawk and the steppe eagle, and amylase and carboxypeptidase absent in the material egested by the lanner. The origin of the enzymes studied was examined. Pancreatic enzymes, which are present in the pellets, arise from a reflux of intestinal fluid into the stomach. The importance of this phenomenon is discussed. The acidity of the pellets was measured. Relations existing between the type of food, characteristics of the pellet and the digestive process in raptors are analysed. The evolutionary advantage of pellet egestion is discussed.     

Immobilization of enzymes on chitin and chitosan

During the last few years, d-glucose isomerase, glucoamylase, β-d-galactosidase (lactase), β-d-glucosidase, d-glucose oxidase, AMP deaminase, urease, pronase, subtilisin, trypsin, papain, alkaline phosphatase, acid phosphatase, pepsin, chymotrypsin and lysozyme have been immobilized on chitin and on some of its derivatives, mainly with glutaraldehyde. The preparation and performances of the immobilized enzymes are described.     

Insectivore to frugivore: ontogenetic changes in gut morphology and digestive enzyme activity in the characid fish Brycon guatemalensis from Costa Rican rain forest streams

Brycon guatemalensis , a Neotropical characid fish, consumes an entirely terrestrial diet, shifting from eating insects as juveniles to fruits and leaves as adults. Juvenile and larger‐sized fish collected in the Rio Puerto Viejo at the La Selva Biological Station in Costa Rica were studied to test the hypotheses that, with ontogeny, (1) relative gut length increases, (2) pyloric caeca arrangement and number remain unchanged and (3) pepsin, trypsin and lipase activities decrease, while α‐amylase activity increases. These hypotheses were mainly supported in that larger fish had longer guts, unchanged pyloric caeca arrangement but fewer caeca, and, at both environmental and standard temperatures for the enzyme assays, lower pepsin and trypsin activities but higher α‐amylase activities than the juveniles. Only lipase, among the digestive enzymes, exhibited the unexpected outcome of either not differing significantly in activity (per g of tissue) between juveniles and larger fish or being significantly higher (per mg of protein) in larger fish. The overall results support the view that B. guatemalensis is specialized morphologically and biochemically to function first as a carnivore and then as a herbivore during its life history.     

Digestive morphophysiology of Gryllodes sigillatus (Orthoptera: Gryllidae)

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

A quantitative study of some digestive enzymes in the rabbitfish, Siganus canaliculatus and the sea bass, Lates calcarifer

Digestive enzyme distribution and activity in the digestive tracts of the rabbitfish, Siganus canaliculatus and the sea bass, Lates calcarifer were studied. Quantitative determinations of digestive enzymes in the guts of both fishes showed that they were capable of digesting carbohydrates and proteins in their diet. The carbohydrases, amylase, laminarinase, maltase, sucrase and trehalase were detected in the rabbitfish; their activities being mainly in the stomach, intestine and pyloriccaeca. Amylase, maltase, trehalase and chitinase activities were recorded in the gut of the sea bass, primarily in the intestine and the pyloriccaeca. Their activities were significantly lower than those in the rabbitfish. Proteases (pepsin, chymotrypsin, elastase, leucine aminopeptidase and trypsin) were found in both the rabbitfish and the sea bass. Pepsin activity however, was higher in the sea bass; while trypsin and chymotrypsin activities were higher in the rabbitfish. The activities of the various digestive enzymes in both fishes are discussed in relation to their feeding habits.     

Enzyme-forming function of digestive glands after administration of polypeptide C, copepsidyl and panintestine

Polypeptide C (molecular weight 2640 Dalton) extracted from artificial gastric juice), copepsidyl (containing high dosage of pepsin) and panintestine (polyenzyme drug) are studied for their effect on the activity of digestive enzymes of glandular gastric element pancreas and small intestine of rats. It is established that all mentioned drugs stimulate enzymogenesis in the analyzed organs. The activity of pepsin increases in homogenates of gastric mucosa, the activity of trypsin, total proteinases, carboxypeptidase and amylase grows in pancreas homogenates, and that of leucineaminopeptidase--in small intestine.     

A comparative study of allergenic and potentially allergenic enzymes fromDermatophagoides pteronyssinus,D. farinae andEuroglyphus maynei

The presence of the enzymatically active allergens equivalent toDer p I (cysteine protease),Der p III (serine protease) and amylase in extracts ofDermatophagoides pteronyssinus, D. farinae andEuroglyphus maynei was determined using appropriate enzymatic techniques. Biochemical equivalents of all three allergens were present in each extract studied. Studies also showed that the mite extracts contained a variety of other biochemically active enzymes including trypsin, chymotrypsin, carboxypeptidase A and B, glucoamylase and lysozyme. Marked differences in the relative concentrations of some of these enzymes in different mite extracts were observed, particularly trypsin and carboxypeptidase A. The enzymes were physicochemically similar to equivalent enzymes from vertebrate and invertebrate sources. Chromatofocusing studies of faecal extracts derived fromD. pteronyssinus andD. farinae showed that several isoforms of each enzyme were present. The data indicated that there were more trypsin isoforms, with pI over a wider range, in extracts prepared fromD. pteronyssinus. Proteases and carbohydrases were also found in extracts prepared from faecally enriched material suggesting that they were endoperitrophic and associated with mite digestion. The data suggest that not only are the group I, III and amylase allergens a consistent feature of most pyroglyphid dust mites but also that other proteases and carbohydrases present in mite faeces are allergenic.     

The physiological role of the peritrophic membrane and trehalase: Digestive enzymes in the midgut and excreta of starved larvae of Rhynchosciara

Amylase, cellulase, trehalase, aminopeptidase and trypsin were determined using the midgut and trehalose using the haemolymph of starved and of subsequently fed larvae of Rhynchosciara americana. Midgut trehalase activity decreases steadily during starvation and increases again on feeding, whereas haemolymph trehalose titres remain constant, suggesting that trehalase is a true digestive enzyme. The decrease in amylase, cellulase and trypsin activity in the midgut during starvation is of the same order as that recovered from the excreta. Since this finding is exactly what one would expect if enzyme production stops in response to starvation, this supports the hypothesis that synthesis that synthesis of these enzymes is controlled. The excretion rate of amylase, cellulase and trypsin is very low in comparison to their activity inside the peritrophic membrane and the travel time of the food bolus through the gut. It is proposed that the peritrophic membrane separates two extracellular sites for digestion as an adaptation to conserve secreted enzymes. This could be accomplished by the existence of an endo-ectoperitrophic circulation of the enzymes involved in the initial attack on the food and by restricting to the ectoperitrophic fluid the enzymes which participate only in intermediary digestion of food.     

Ultrastructure of the digestive tract in Acarus siro (Acari: Acaridida)

The gut of the mite Acarus siro is characterized on the ultrastructural level. It consists of the foregut (pharynx, esophagus), midgut (ventriculus, caeca, colon, intercolon, postcolonic diverticula, postcolon), and hindgut (anal atrium). The gut wall is formed by a single-layered epithelium; only regenerative cells are located basally and these have no contact with the lumen. Eight cell types form the whole gut: (i) simple epithelial cells forming fore- and hindgut; (ii) cells that probably produce the peritrophic membrane; (iii) regenerative cells occurring in the ventriculus, caeca, colon, and intercolon; (iv) spherite cells and (v) digestive cells forming the ventriculus and caeca; (vi) colonic cells and (vii) intercolonic cells; and (viii) cells forming the walls of postcolonic diverticula and postcolon. Spherite and digestive cells change in structure during secretory cycles, which are described and discussed. The cycle of spherite, colonic, and intercolonic cells is terminated by apoptosis. Ingested food is packed into a food bolus surrounded by a single homogeneous peritrophic membrane formed by addition of lamellae that subsequently fuse together. The postcolonic diverticula serve as a shelter for filamentous bacteria, which also are abundant in the intercolon.     

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

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