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.     

Ultrastructural and biochemical basis of the digestion of nectar and other nutrients by the moth Erinnyis ello

The midgut epithelium of adult Erinnyis ello L. (Lepidoptera: Sphingidae) forms ramified villus-like folds. The epithelial cells exhibit long microvilli and narrow and sinuous basal channels studded with particles. These morphological features are thought to be involved in the absorption of nutrients present in low concentration in food. Enzymatic assays in E. ello adults showed the presence of amylase in salivary glands, and the occurrence of aminopeptidase, -fructosidase, - and -glucosidase, trehalase and trypsin in the midgut. Calcium differential precipitation of midgut homogenates showed that aminopeptidase and -glucosidase are integral proteins of the microvillar cell membranes, whereas the other enzymes are soluble or partly soluble and partly membrane bound. Detergent-solubilized aminopeptidase (pH optimum 7.5) sediments as a protein with Mr 120000, whereas trehalase (pH optimum 6.5), as a protein with Mr 95000. The activities of amylase (pH optimum 5.5), -glucosidase (pH optimum 5.0) and trypsin (pH optimum 9.5) were not sufficiently high to be further characterized, whereas -fructosidase and membrane-bound -glucosidase were characterized elsewhere. The data suggest that nectar-feeding E. ello adults are able to some extent to digest and absorb starch and proteins, in addition to nectar sugars. Initial digestion is considered to occur in midgut lumen and final digestion on the surface of midgut cells under the action of microvillar enzymes.

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Résumé L'épithélium de l'intestin moyen des adultes de E. ello L; (Lépido., Sphingidae) est replié en villosités. Les cellules épithéliales présentent de longs microvilli et d'étroits canaux sinueux basaux garnis de particules. Ces caractères morphologiques peuvent être interprétés comme impliquant l'absorption d'aliments présents en faible concentration dans la nourriture. Des études enzymatiques ont révélé la présence d'amylase dans les glandes salivaires, et la découverte d'aminopeptidase, de -fructosidase, d'- et -glucosidases, de tréhalase et de trypsine dans l'intestin moyen. La précipitation calcique différentielle d'homogénats de l'intestin moyen a montré que l'aminopeptidase et l'-glucosidase sont des protéines appartenant aux membranes microvillaires cellulaires, tandis que les autres enzymes sont entièrement ou partiellement solubles et partiellement liées aux membranes. L'aminopeptidase solubilisée par un détergent (pH optimum: 7,5) sédimente comme une protéine de Mr 120000, tandis que la tréhalase (pH optimum: 6,5) comme une protéine de Mr 95000. Les activités de l'amylase (pH optimum: 5,5), de la -glucosidase (pH optimum 5,0) et de la trypsine (pH optimum 9,5) n'étaient pas suffisantes pour être caractérisées, tandis que la -fructosidase et l'-glucosidase liée à la membrane ont été caractérisées ailleurs. Les résultats suggèrent que les adultes E. ello consommateurs de nectar sont capables de digérer et d'absorber une certaine quantité d'amidon et de protéines, en plus des sucres du nectar. La digestion doit débuter dans la lumière de l'intestin moyen pour s'achever à la surface des cellules intestinales sous l'action des enzymes des microvilli.

     

Effect of weaning terms and protein deficit in rat pup nutrition on activities of digestive enzymes

Restriction of protein in nutrition of rat pups weaned at different terms has been found to produce changes in activities of digestive enzymes (maltase, alkaline phosphatase, aminopeptidase M, and glycyl-L-leucine dipeptidase) in the small and large intestine both at once after cessation of nutrition with low-protein diet for 10 days and 4 months later. In adult animals after the earlier or later weaning there are observed not only a decrease or increase of the enzyme activities, but also a different type of distribution of the alkaline phosphatase activity along the small intestine, which is more pronounced in the lately weaned rats. Thus, disturbance of metabolic programming of enzyme systems of the small and large intestine due to a change of quality of nutrition in early ontogenesis depends on terms of weaning of animals.     

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.     

Salivary digestive enzymes of the wheat bug, Eurygaster integriceps (Insecta: Hemiptera: Scutelleridae)

The digestive enzymes from salivary gland complexes (SGC) of Eurygaster integriceps, and their response to starvation and feeding were studied. Moreover, digestive amylases were partially purified and characterized by ammonium sulfate precipitation and gel filtration chromatography. The SGC are composed of two sections, the principal glands and accessory glands. The principal glands are further divided into the anterior lobes and posterior lobes. The SGC main enzyme was α-amylase, which hydrolyzed starch better than glycogen. The other carbohydrases were also present in the SGC complexes. Enzymatic activities toward mannose (α/β-mannosidases) were little in comparison to activities against glucose (α/β-glucosidases) and galactose (α/β-galactosidases), the latter being the greatest. Acid phosphatase showed higher activity than alkaline phosphatase. There was no measurable activity for lipase and aminopeptidase. Proteolytic activity was detected against general and specific protease substrates. Activities of all enzymes were increased in response to feeding in comparison to starved insects, revealing their induction and secretion in response to feeding pulse. The SGC amylases eluted in four major peaks and post-electrophoretic detection of the α-amylases demonstrated the existence of at least five isoamylases in the SGC. The physiological implication of these findings in pre-oral digestion of E. integriceps is discussed.     

中华真地鳖中肠主要消化酶的活性研究

以中华真地鳖EupolyphagasinensisWalker为研究材料,测定人工饲养和野生地鳖虫在不同生长阶段消化酶的活性以及温度及pH对人工饲养地鳖虫中肠消化酶活性的影响。结果表明,在地鳖虫生长发育过程中,蛋白酶和脂肪酶活性随发育而逐渐增强,淀粉酶活性却随发育而逐渐减弱。在低龄若虫、高龄若虫和成虫阶段,人工饲养地鳖虫蛋白酶活力比野生地鳖虫低,人工饲养地鳖虫淀粉酶和脂肪酶活力比野生地鳖虫高;在30～60℃的范围内,人工饲养地鳖虫蛋白酶、淀粉酶的适宜温度范围为40～50℃,脂肪酶的适宜温度范围为35～45℃;蛋白酶、淀粉酶和脂肪酶的适宜pH范围分别为6.5～7.5,5.6～6.4和7.5～8.5。     

北草蜥几种消化酶活力比较

应用酶学分析法测定了越冬后北草蜥胃、肠组织中蛋白酶、淀粉酶、纤维素酶的活力。结果表明 ,不同年龄、性别的北草蜥同一组织中消化酶活力有显著差异 ;不同地理种群的北草蜥同一组织中消化酶活力有显著差异 ;不同消化酶在北草蜥同一组织中的活力有显著差异 ;在北草蜥不同的组织中同一消化酶的活力有显著差异。说明北草蜥消化酶的活力与年龄、性别、部位和地理环境等因素有关 ,受食物组成、能量需求和遗传等因素的影响 ,产生了不同的酶活力和分布。这也说明生物长期适应环境 ,形成了不同的代谢水平     

Comparative study of digestive enzymes of squid (Todarodes pacificus) viscera after supercritical carbon dioxide and organic solvent extraction

Three major classes of digestive enzymes of squid viscera were characterized following extraction of oil by supercritical carbon dioxide (SCO₂) and organic solvent, n-hexane. Squid viscera were extracted at temperature, 35∼45°C and pressure, 15∼25 MPa for 2.5 h by SCO₂ with a constant flow rate of 22 g/min. Oil extraction yield increased with the increasing of extraction pressure and temperature. The highest oil extracted residues of squid viscera were used for characterization of digestive enzymes. The activities of protease, lipase, and amylase were highest in n-hexane treated squid viscera samples and lowest in SCO₂ treated samples. The crude extracts of SCO₂ and n-hexane treated squid viscera samples showed almost same optimum pH and pH stability for each of the digestive enzymes. The optimum temperature of protease, lipase, and amylase were found to almost similar in SCO₂ and n-hexane treated samples. But the thermal stability for each digestive enzyme in SCO₂ treated squid viscera were slightly higher than that of n-hexane treated squid viscera. Studies using SDS-PAGE showed no significant differences in protein patterns of the crude extracts of untreated and SCO₂ and n-hexane treated squid viscera indicating no denaturation of proteins.     

Ultrastructural and biochemical aspects of digestion in the imagoes of the flyRhynchosciara americana

The midgut of adultRhynchosciara 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 inR. americana adults revealed that amylase occurs in salivary glands and midgut, whereas aminopeptidase, α-glucosidases and trypsin occur only in the midgut, mainly in the ventriculus. There is a soluble (Mr 105000) and a membrane-bound aminopeptidase (solubilized form, Mr 110000). Soluble α-glucosidase inactivates easily and could not be characterized, whereas membrane-bound α-glucosidases were resolved after solubilization into three molecular species (Mr 186000, 105000 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 thatR. 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.     

The transformation of Saperda calcarata (Coleoptera: Cerambycidae) into a cellulose digester through the inclusion of fungal enzymes in its diet

Summary The larvae of the aspen borer, Saperda calcarata, which feed on the inner bark and sapwood of living aspen stems, are unable to digest cellulose. However, they can be transformed into cellulose digesters by adding the active cellulase complex of the fungus, Penicillium funiculosum to their diet. S. calcarata larvae are preadapted to exploit the digestive potential of ingested microbial enzymes. We argue that ingested fungal enzymes may be responsible for cellulose digestion in many, perhaps most or even all, cellulose digesting cerambycid beetles.     

Properties of larval and imaginal membrane-bound digestive enzymes from Trichosia pubescens

Trichosia pubescens larval midgut ceca cells display in their plasma membranes α-glucosidases (M_r 95,000; pH_o 5.5; K_m 5.7 mM; K_i for TRIS 8.9 mM), trehalases (M_r 69,000; pH_o 5.3; K_m 0.92 mM; K_i for TRIS 57 mM), and aminopeptidases (M_r 95,000; pH_o 8.7; K_m 0.19 mM) which are solubilized by Triton X-100. The enzymes were purified by electrophoresis and used to raise antibodies in a rabbit. T. pubescens imaginal midgut cells display in their plasma membranes an α-glucosidase (M_r 156,000; pH_o 5.8; K_m 2.3 mM; K_i for TRIS 0.2 mM), a trehalase (M_r 93,000; pH_o 5.5; K_m 0.72 mM; K_i for TRIS 45.5 mM), and an aminopeptidase (M_r 210,000; pH_o 9.0; K_m 0.47 mM). Antiserum produced against the larval enzymes shows no precipitation arc when tested by double immunodiffusion or by immunoelectrophoresis with Triton X-100-solubilized membrane proteins from imaginal midguts. Otherwise, a similar test showed that larval midgut cecal enzymes and larval ventriculus enzymes display complete immunological identity. The data suggest that, despite the fact the larval and imaginal aminopeptidase, α-glucosidase, and trehalase probably have similar functions, the genes coding for them in larvae and imagoes must differ.     

Activities of digestive enzymes in rats kept on standard or excessive breast feeding and on low-protein diet at once after weaning

Activities of digestive enzymes (maltase, alkaline phosphatase, aminopeptidase M, and glycyl-L-leucine dipeptidase) in small and large intestine, liver, and kidney were studied in rats of different ages kept at the period of lactation under conditions of the standard (8 individuals per litter) and low (3 individuals) number of pups per litter. The low-protein diet for 10 days at once after weaning was found to change the mass of organs and their digestive enzyme activities in all studied rat groups. The revealed changes were more prominent in rats kept under conditions of excessive breast feeding. In adult animals of this group, distribution of the alkaline phosphatase activity along the small intestine differed from that in control rats. The obtained results seem to confirm that any disturbance of the nutrition quality in early ontogenesis leads to disturbance of the «metabolic programming of enzyme systems» of digestive organs.     

Inhibitory effect of proteinaceous seed extract of three Iranian wheat cultivars on Eurygaster integriceps (Sunn pest) digestive enzymes

Abstract

To investigate interaction between proteinaceous extracts of three Iranian wheat cultivars and digestive enzymes of Sunn pest, a population of adult insects was collected in summer from a wheat field located in Borkhar Region, Isfahan, Iran. Seed proteins were extracted by 0.15?M NaCl solution and partially purified using ammonium sulphate. Spectrophotometric assays were implemented to determine enzyme activities. Results indicated cultivar- and dose-dependent efficacy of seed protein extracts. Inhibition percentages of α-amylase, α-glucosidase, β-glucosidase and proteolytic activities at concentration of 32?μg/mL and saturation percentage of 70% ammonium sulphate were 54, 37, 25 and 59% by Hamoon, 16, 28, 18 and 19% by Karkheh and 15, 16, 15 and 23% by Dena, respectively. Zymography also confirmed the effect of inhibitors on α-amylase activity. Among wheat cultivars, Hamoon has the highest biological activity against Sunn pest major digestive enzymes and could contribute towards the development of new insect pest control strategies.     

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.     

Feeding and digestive activity of cyclopoid copepods in active diapause

Three different methods were used to compare the feeding patterns of active cyclopoids and cyclopoids in active diapause from different habitats. First, we offered ¹⁴C- labeled algal detritus to stage III, stage IV (two different physiological groups), and adult females of Acanthocyclops vernalis from temporary water-bodies of the Volga Delta. Second, we compared the functional responses of active and actively diapausing cyclopoids preying on copepod nauplii. Stage III, stage IV (various physiological groups), stage V, adult males and females of Acanthocyclops viridis, Cyclops kolensis, C. abyssorum sevani from the Volga Delta, Lake Sevan (Armenia), and Mozhaisk Reservoir (Moscow District) were tested. Finally, we examined the activity of digestive enzymes (proteases) of CIV of C. kolensis from Mozhaisk Reservoir at different temperatures and temperature acclimation regimes. All three approaches used have led to similar conclusions. Actively diapausing cyclopoids were capable of feeding on both plant and animal prey; however, their feeding rates and digestive enzyme activity were 2–8 times lower than those of active cyclopoids of similar size. At the initial stage of active diapause the feeding rates were comparable with those of active cyclopoids then declined to a low level during approximately two weeks. Transfer of cyclopoids undergoing winter diapause from ambient temperature (4 °C) to warmer water (20 °C) resulted in dramatic increase of proteases activity only if the acclimation period was relatively long (two weeks).     

Seasonal changes in the digestive enzyme levels of the amphipod Corophium volutator (Pallas) in relation to diet

The diet of the amphipod Corophium volutator (Pallas) living on extensive intertidal mud flats in the Bay of Fundy, eastern Canada, was investigated by direct and indirect methods. Microscopic observation of gut contents revealed macrophyte detritus but few diatom frustules. Laboratory cultures showed good survival on a diet of benthic diatoms, while on a diet of Spartina detritus survival was poor, although significantly better than the survival of starved animals. The ratios of gut enzyme activities, amylase to protease and amylase to laminarinase, changed markedly with the season. In summer the ratio was similar to that of animals cultured on a diet of benthic diatoms; in spring it resembled that of animals cultured on Spartina detritus, while in winter it was similar to that of starved animals. It is concluded that in summer, rapid growth is made possible by feeding on benthic diatoms, while Spartina detritus is an inferior food source which aids survival at other times of the year.     

pH值对中国龙虾消化酶活力的影响

采用酶学分析方法研究了pH对中国龙虾胃蛋白酶、类胰蛋白酶、淀粉酶、纤维素酶和脂肪酶活力的影响。结果表明,在设定的pH范围内,中国龙虾各消化酶的活力均随着pH的升高呈现先升后降的变化趋势。其中,胃、肠、肝胰腺内胃蛋白酶最适pH均为2.2,类胰蛋白酶最适pH分别为8.8-9.2、8.4、8.8,淀粉酶最适pH分别为7.0、7.0、7.4,纤维素酶最适pH分别为4.2、4.2-4.6、5.4,脂肪酶最适pH分别为7.2-7.6、7.2、6.8-7.2。同时测得中国龙虾胃、肠、肝胰腺内的生理pH分别为5.33、6.93、6.60。中国龙虾的消化酶活力存在器官特异性。在最适pH下,胃蛋白酶活力顺序为胃>肠>肝胰腺,类胰蛋白酶、纤维素酶、脂肪酶的活力顺序均为肝胰腺>肠>胃,淀粉酶的活力顺序为肠>肝胰腺>胃。     

Cyclic growth and activity of pancreatic enzymes in alevins of Arctic charr (Salvelinus alpinus L.)

Growth and digestive enzymes were studied in alevins of Arctic charr raised on six commercial diets that differed in protein source and level. There were links between diet composition (protein quality), fish performance and proteolytic and amylase enzyme activities. Trypsin and amylase activities were up to 10-fold higher in charr fed high protein (65–70%) diets than in fish fed live feed. A depression of pancreaticenzyme activities was noticeable when charr were fed starter diets in which fish meal was partially replaced by soybean meal. Specific growth rates (SGR) showed a cyclic pattern during early ontogeny of charr and SGR was highly correlated with food conversion efficiency. Activities of pancreatic enzymes also demonstrated a cyclic pattern but the periodicities of the growth cycle (50.6 days) and those of the enzymes differed. We hypothesize that both photostimulation and feedback responses might be involved in pancreatic responses in fish, which result in cyclic growth patterns.     

Distribution and localization of the digestive laminarinases in animals

The presence of laminarinases in the digestive system of a number of invertebrate and vertebrate species has been investigated. The enzymes were detected in coelenterates, annelids, sipunculids, bryozoans, molluscs, arthropods (crustaceans and insects), echinoderms, tunicates and fishes. Since it could be demonstrated that, in fishes, at least, laminarinases are secreted by the intestinal mucosa rather than by associated gut bacteria, an attempt was made to correlate the distribution of these digestive enzymes with the feeding habits of the animals. The correlation is particularly evident among fishes where in the genus Leuciscus carnivores lack laminarinases in contrast to omnivores. From an evolutionary point of view, we can postulate that the possession of laminarinases is ancient in Metazoa and has been either kept or lost by more evolved taxa depending on their feeding specialization, as has already been demonstrated for chitinases and cellulases.     

Effects of frontal ganglion removal and starvation on activity and distribution of six gut enzymes in Locusta

A study has been made on the effects of frontal ganglion removal and starvation on the activities and distribution of α- and β-glucosidase, α- and β-galactosidase, trehalase, and ‘trypsin’ in various regions of the alimentary canal of adult locusts. Both treatments resulted in a reduction in the amount of enzyme activity. In addition, the distribution of enzyme activity was changed by comparison with the operated control insects; the foregut of starved and operated animals showing a smaller proportion of the overall gut enzyme activity. The results are discussed in relation to the control of enzyme secretion.