Brain-midgut short neuropeptide F mechanism that inhibits digestive activity of the American cockroach, Periplaneta americana upon starvation

Immunohistochemical reactivity against short neuropeptide F (sNPF) was observed in the brain-corpus cardiacum and midgut paraneurons of the American cockroach, Periplaneta americana. Four weeks of starvation increased the number of sNPF-ir cells in the midgut epithelium but the refeeding decreased the number in 3h. Dramatic rises in sNPF contents in the midgut epithelium and hemolymph of roaches starved for 4 weeks were confirmed by ELISA. Starvation for 4 weeks reduced α-amylase, protease and lipase activities in the midgut of P. americana but refeeding restored these to high levels. Co-incubation of dissected midgut with sNPF at physiological concentrations inhibited α-amylase, protease and lipase activities. sNPF injection into the hemocoel led to a decrease in α-amylase, protease and lipase activities, whereas PBS injection had no effects. The injection of d-(+)-trehalose and l-proline into the hemocoel of decapitated adult male cockroaches that had been starved for 4 weeks had no effect on these digestive enzymes. However, injection into the hemocoel of head-intact starved cockroaches stimulated digestive activity. Injection of d-(+)-trehalose and l-proline into the lumen of decapitated cockroaches that had been starved for 4 weeks increased enzymes activities and suppressed sNPF in the midgut. Our data indicate that sNPF from the midgut paraneurons suppresses α-amylase, protease and lipase activities during starvation. Injection of d-(+)-trehalose/l-proline into the hemocoel of head-intact starved cockroach decreased the hemolymph sNPF content, which suggests that sNPF could be one of the brain factors, demonstrating brain-midgut interplay in the regulation of digestive activities and possibly nutrition-associated behavioral modifications.     

The effect of adipokinetic hormones on the activity of digestive enzymes

The role of the adipokinetic hormone (AKH) in the control of protease, amylase and lipase activities is examined using the cockroach Periplaneta americana and the fruit fly Drosophila melanogaster as model species. The effects of Peram‐CAH‐I and ‐II on the activity of cockroach digestive enzymes in the gastric caeca and midgut are measured both in vivo and in vitro. The results show the activity of proteases, amylases and lipases in both parts of the gut: amylase activity is higher in the gastric caeca than in the midgut; lipase activity presents the opposite trend; and protease activity is similar in both organs. The applied hormones stimulate the activity of all digestive enzymes, although this stimulation is not uniform; AKHs affect enzymes selectively, and in some cases unequally, in the gastric caeca and midgut. No substantial differences between Peram‐CAH‐I and ‐II stimulation are recorded. The in vitro results demonstrate that AKH stimulates digestive enzyme activity directly. In agreement with the cockroach results, enzymatic activity in D. melanogaster larvae producing nonfunctional AKH is lower than that in the larvae with ectopically expressed Akh gene, where enzyme activity reaches or even exceeds that of the controls. Overall, the results demonstrate the active role of AKHs in the stimulation of digestive enzyme activity in insects.     

Effects of an allatostatin and a myosuppressin on midgut carbohydrate enzyme activity in the cockroach Diploptera punctata

Neuropeptides of the cockroach allatostatin (AST) family are known for their ability to inhibit the production of juvenile hormone by the corpora allata of cockroaches. Since their discovery, they have also been shown to modulate myotropic activity in a range of insect species as well as to act as neurotransmitters in Crustaceans and possibly in insects. The midgut of cockroaches contains numerous endocrine cells, some of which produce AST whereas others produce the FMRFamide-related peptide, leucomyosuppressin (LMS). We have determined if ASTs and LMS are also able to influence carbohydrate-metabolizing enzyme activity in the midgut of the cockroach, Diploptera punctata. Dippu-AST 7 stimulates activity of both invertase and alpha-amylase in a dose-dependent fashion in the lumen contents of ligatured midguts in vitro, but not in midgut tissue, whereas the AST analog AST(b)phi2, a cyclopropyl-ala, hydrocinnamic acid analog of Dippu-AST 6, has no effect. Leucomyosuppressin also stimulates enzyme activity in lumen contents only, although the EC50 is considerably greater than for Dippu-AST. Dippu-AST is also able to inhibit proctolin-induced contractions of midgut muscle, and this action had already been described for LMS [18]. Thus, in this organ, AST and LMS have at least two distinct physiological effects.     

环氧苍耳素Ⅰ对菜青虫中肠消化酶和羧酸酯酶活性的影响

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

Characterization and distribution of digestive proteases of the stalk corn borer,Sesamia nonagrioides Lef. (Lepidoptera: Noctuidae)

Larval midgut extracts from the noctuid Sesamia nonagrioides Lef. were assayed for protease activity. Total proteolytic activity, as measured by azocasein hydrolysis, showed a pH optimum in the range 10.0 to 11.5, suggesting a digestive system based largely on serine-like proteases. The ability of midgut extracts to hydrolyze specific synthetic substrates, the elucidation of the pH at which maximal hydrolysis occurs, and their sensitivity to protease inhibitors confirmed the presence of the serine endoproteases: trypsin, chymotrypsin, and elastase; and the exopeptidases: carboxypeptidase A, carboxypeptidase B, and leucine aminopeptidase. The distribution of these digestive proteases along the gut sections and among the different midgut regions was examined. All types of endoproteases and exopeptidases were mainly located in the midgut, with less than 5% of the activity in the foregut and hindgut. When the two halves of the midgut were compared, all proteolytic activities were higher in the anterior portion of the midgut. Trypsin, chymotrypsin, elastase, and carboxypeptidase B activities were mainly located in the endoperitrophic space of the midgut, with some activity in the ectoperitrophic space, whereas aminopeptidase and carboxypeptidase A activities were preferentially located in the midgut epithelium. © 1996 Wiley-Liss, Inc.     

Effects of entomopathogenic bacterium Photorhabdus temperata infection on the digestive enzymes of Diatraea saccharalis (Lepidoptera: Crambidae) larvae

This study investigated the effects of Photorhabdus temperata infection on the activities of digestive enzymes of the sugarcane stalk borer Diatraea saccharalis. Non-infected D. saccharalis larvae present a major alpha-amylase, several proteinases, three sucrose hydrolases and two alpha-glucosidases in their midgut. Analysis of these hydrolases by electrophoresis and "in gel" assays showed that the activities of all enzymes decreased following infection, with an initial decline observed 12 h after infection. The activities of alpha-glucosidases decreased by 50% twelve hours after infection, whereas, at this time, the alpha-galactosidase activities decreased by 70%. Interestingly, the animals died 48 h after infection, but approximately 5% of all the enzymes tested remained active in the midgut following host death. At this time, most of the cultivable native intestinal bacteria had died.     

Effect of natural toxins and adipokinetic hormones on the activity of digestive enzymes in the midgut of the cockroach Periplaneta americana

This study examined the effect of two natural toxins (a venom from the parasitic wasp Habrobracon hebetor and destruxin A from the entomopathogenic fungus Metarhizium anisopliae), and one pathogen (the entomopathogenic fungus Isaria fumosorosea) on the activity of basic digestive enzymes in the midgut of the cockroach Periplaneta americana. Simultaneously, the role of adipokinetic hormones (AKH) in the digestive processes was evaluated. The results showed that all tested toxins/pathogens elicited stress responses when applied into the cockroach body, as documented by an increase of AKH level in the central nervous system. The venom from H. hebetor showed no effect on digestive enzyme activities in the ceca and midgut in vitro. In addition, infection by I. fumosorosea caused a decrease in activity of all enzymes in the midgut and a variable decrease in activity in the ceca; application of AKHs did not reverse the inhibition. Destruxin A inhibited the activity of all enzymes in the midgut but none in the ceca in vitro; application of AKHs did reverse this inhibition, and no differences between both cockroach AKHs were found. Overall, the results demonstrated the variable effect of the tested toxins/pathogens on the digestive processes of cockroaches as well as the variable ability of AKH to counteract these effects.     

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

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

Effects of Diets on Hydrolase Activities Localized in the Brush Border Membrane of the Small Intestine in Rats

This study was undertaken to investigate the response of sucrase (SA), alkaline phosphatase (ALP), and leucine aminopeptidase (LAP) activities localized in the brush border membrane of the small intestine to ingested nutrients. In rats previously meal-fed on carbohydrate-free diets, the maximal increase in SA activity after the administration of a 18 % casein and high sucrose diet (HSD) occurred 24 hr after the beginning of the 12-hr period of HSD-feeding, but ALP and LAP activities reached the maximal levels 12 hr after the HSD administration and then rapidly declined. These changes in three enzyme activities were similar to those after HSD-feeding following a 2-day fast, but those were not observed after giving a carbohydrate-free and high fat diet. The increases in these enzyme activities after giving HSD were not observed entirely after giving HSD containing 0.5% concanavalin A, which was undigestible and preferentially bound mitotically active cells of the small intestine. From these findings, it can be suggested that the adaptive response of these enzyme activities to ingested nutrients, as sucrose and casein, was produced on the luminal surface of the small intestine, especially on the immature cells.     

The inhibitory effects of berry polyphenols on digestive enzymes

The evidence for the effect of polyphenol components of berries on digestive enzymes is reviewed. Anthocyanins inhibit alpha-glucosidase activity and can reduce blood glucose levels after starch-rich meals, a proven clinical therapy for controlling type II diabetes. Ellagitannins inhibit alpha-amylase activity and there is potential for synergistic effects on starch degradation after ingestion of berries such as raspberries and strawberries, which contain substantial amounts of ellagitannins and anthocyanins. A range of berry polyphenols (e.g. flavonols, anthocyanidins, ellagitannins and proanthocyanidins) can inhibit protease activities at levels which could affect protein digestion in the gastrointestinal tract. In contrast, potential for the inhibition of gastrointestinal lipase activity, a proven therapeutic target for the control of obesity through reduced fat digestion, may be limited to proanthocyanidins. Taking into account the manifold possible synergies for inhibition of starch, protein and/or lipid digestion by the spectrum of polyphenol components present within berry species, the inhibition of digestive enzymes by dietary polyphenols may represent an under-reported mechanism for delivering some of the health benefits attributed to a diet rich in fruit and vegetables.     

Comparison of alpha-amylase and protease activities of a zoophytophagous and two phytozoophagous Heteroptera

To better understand the nature of facultative phytophagy in the zoophytophagous Geocoris punctipes (Say), and facultative zoophagy in phytozoophagous Lygus hesperus (Knight) and Lygus lineolaris (Palisot de Beauvois), we compared the activities of both the starch digesting enzyme alpha-amylase and of general proteases in these species. The alpha-amylases and proteases were demonstrated in L. hesperus, L. lineolaris and G. punctipes. The presence of alpha-amylase in the salivary gland complexes of G. punctipes indicates a disposition of this species toward utilization of nutrients that can be derived only from plants, either directly from ingestion of plant macromolecules or from second-hand ingestion of plant material from the digestive system of their prey. The alpha-amylase activity in G. punctipes was much less than those of phytozoophagous L. hesperus and L. lineolaris. The relative importance of amylolytic activity and proteolytic activity is also discussed.     

Nutrient control of release of pancreatic enzymes in yellowtail (Seriola quinqueradiata): involvement of CCK and PY in the regulatory loop

Cholecystokinin (CCK) and neuropeptide Y (NPY)-related peptides are key regulators of pancreatic enzyme secretion in vertebrates. CCK stimulates enzyme secretion whereas peptide Y (PY), a NPY-related peptide, plays an antagonistic role to that of CCK. In fish, very little is known about how different nutrients affect the synthesis of CCK and PY in the digestive tract, and the mechanism by which CCK and PY actually regulate digestive enzyme secretion is not well understood. In order to determine how different nutrients stimulate the synthesis of CCK and PY in yellowtail (Seriola quinqueradiata), CCK and PY mRNA levels in the digestive tract were measured after oral administration of a single bolus of either phosphate-buffered saline (PBS: control), starch (carbohydrate), casein (protein), oleic acid (fatty acid) or tri-olein (triglyceride). In addition, in order to confirm the synthesis and secretion of digestive enzymes, the mRNA levels and enzymatic activities of three digestive enzymes (lipase, trypsin and amylase) were also analyzed. Casein, oleic acid and tri-olein increased the synthesis of lipase, trypsin and amylase, while starch and PBS did not affect the activity of any of these enzymes. CCK mRNA levels rose, while PY mRNA levels were reduced in fish administered casein, oleic acid and tri-olein. These results suggest that in yellowtail, CCK and PY maintain antagonistic control of pancreatic enzyme secretion after intake of protein and/or fat.     

A amylase activity of nymphal stages of sunn pest, Eurygaster integriceps Puton (Hemiptera: Scutelleridae)

Wheat production in Iran has changed substantially over the past one or two decades with development of higher-yielding cultivars and improved methods of planting. Sunn pest, Eurygaster integriceps (Heteroptera: Pentatomidae), is the most important cereal pest in Iran. Sunn pest like other insect pests of wheat lives on a polysaccharide-rich diet and depends to a large extent on effectiveness of their alpha-amylases for survival. alpha-amylase (1-4-alpha-D-glucan glucanohydrolase) hydrolyses starch, and related polysaccharides by randomly cleaving internal alpha-1,4-glucosidic linkages and has a major role in the utilization of polysaccharides. The recent increase in study of insect digestive enzymes seems to make sense in the realization that the gut is the major interface between the insect and its environment. Hence, an understanding of digestive enzyme function is essential when developing methods of insect control such as the use of enzyme inhibitor's and transgenic plants to control phytophagous insects. The aim of the current study is to identify and characterize alpha-amylase activity in order to gain a better understanding of its digestive physiology, which hopefully will lead to new strategies of the insect control. In order to analyze a-amylase activity adult and different nymphal stages were collected from wheat field from Karaj area and midgut complex from these individuals were dissected under a light microscope in ice-cold saline buffer (0.006M NaCl). After homogenization in buffer, homogenate was centrifuged at 15000 g for 20 min at 4 degrees C. The supernatant was pooled and stored at -20 degrees C for subsequent analysis. alpha-amylase activity was assayed by the dinitrosalicylic acid (DNS) procedure using soluble starch as substrate (starch 1%). Our result showed that enzyme activities in different nymphal stages (first, second, third, fourth and fifth stadium) were 0.19, 0.78, 1.21, 1.23, 1.25 units/mg protein, respectively.     

Urotensin I-like immunoreactivity in the midgut endocrine cells of the insects Gryllus bimaculatus and Periplaneta americana

Summary The occurrence of urotensin I (UI)-like immunoreactivity is demonstrated in the midgut of the cricket Gryllus bimaculatus and the cockroach Periplaneta americana. The immunoreactivity is restricted to endocrine cells dispersed in the epithelium of the midgut. The UI-positive endocrine cells are numerous in the cricket, but much fewer in the cockroach. These UI-positive cells are different from bovine pancreatic polypeptide (BPP)-immunoreactive cells which represent a dominant cell type in the midgut of both insects. This study suggests the possibility that the neuropeptide UI or a closely related substance may also occur in animals other than fishes.     

Maltose adaptive mutant of heterotrophic marine bacterium, Alteromonas espejiana Bal-31: changes in the growth and the induction of extracellular alpha-amylase

Growth of the heterotrophic marine bacterium, Alteromonas espejiana Bal-31 was inhibited in the presence of sucrose, maltose and even glucose, but not with starch. Extracellular alpha-amylase was induced with a lag phase of 2 h in the presence of starch. In contrast, cell growth of the S2a mutant was not affected by the addition of maltose, and starch was ineffective in the induction of extracellular alpha-amylase in this mutant. Activity of extracellular alpha-amylase was induced from the S2a mutant with a 4-h lag phase in the presence of maltose, and the high level of enzyme activity was maintained for at least 24 h. Activity of alpha-amylase induced by both wild type starch and S2a mutant maltose cultures were mainly observed in extracellular locations. This activity could be stopped by tetracycline treatment, indicating that enzyme induction was dependant on gene expression and not on enzyme protein secretory mechanisms. Our results showed that the mutation in S2a changed the growth and the modulation of the specific alpha-amylase in response to carbon nutrients.     

Characterization of the proteases in the midgut of the xylophagous larvae of Oemona hirta (Coleoptera: Cerambycidae)

Abstract  The protein digestive capability of the larvae of the longhorn beetle ( Oemona hirta , Coleoptera: Cerambycidae, Fabricius, 1775) was investigated. This species feeds only on wood where there is a high proportion of vascular tissue. The pH of the midgut, the major digestive organ, was alkaline and protein hydrolysis was maximal at alkaline pH. Use of specific synthetic peptide substrates showed that the major protease activities were the endopeptidases, trypsin and chymotrypsin-like activity, and the exopeptidase, leucine aminopeptidase and the pH curves corresponded to that with protein substrate. Studies using a range of serine protease inhibitors as well as specific inhibitors of metalloproteases, cysteine proteases and aspartate proteases confirmed a serine protease-based digestive system similar to earlier reports of sapwood-feeding Cerambycids. Control of these insect pests using protease inhibitors is discussed.     

Biochemical characterization of digestive α-amylase, α-glucosidase and β-glucosidase in pistachio green stink bug,Brachynema germari Kolenati (Hemiptera: Pentatomidae)

The pistachio green stink bug, Brachynema germari, has 3–5 generations per year and causes severe damages to pistachio crops in Iran. Physiological digestive processes, such as digestive carbohydrases, can be used to design new strategies in IPM programs for controlling this pest. The enzyme α-amylase digests starch during the initial stage of digestion. Complete breakdown of carbohydrates takes place in the midgut where α- and β-glucosidic activities are highest. Alpha-amylase and α- and β-glucosidase activities were found in the midgut and salivary glands of pistachio green stink bug adults. Overall enzyme activities were significantly higher in the midgut than in salivary glands. The highest α-amylase and α- and β-glucosidase activities were in section v3, whereas the lowest activities were in section v4. V_max was higher and K_m was lower in the midgut than in the salivary glands for these enzymes. In the pistachio green stink bug, the optimal pH was pH 5–6.5 and the optimal temperature was 30 °C to 35 °C for these enzymes. Alpha-amylase activity in the midgut and salivary glands decreased as the concentrations of MgCl₂, EDTA and SDS increased. Enzyme activities in both midgut and salivary glands increased in the presence of NaCl, CaCl₂, and KCl. NaCl had a negative effect on alpha-amylase extracted from salivary glands.     

Characterization of digestive proteolytic activity in Lygus hesperus Knight (Hemiptera: Miridae)

The tarnished plant bug, Lygus hesperus Knight, is a pest that causes considerable economic losses to vegetables, cotton, canola, and alfalfa. Detailed knowledge of its digestive physiology will provide new opportunities for a sustainable pest management approach to control this insect. Little is known about the different protease class contributions to the overall digestion of a specific protein. To this end, the proteolytic activities in female adult L. hesperus salivary gland and midgut homogenates were quantified over a range of pH's and time points, and the contribution of different classes of proteases to the degradation of FITC-casein was determined. In the salivary gland, serine proteases were the predominant class responsible for caseinolytic activity, with the rate of activity increasing with increasing pH. In contrast, both aspartic and serine proteases contributed to caseinolytic activity in the midgut. Aspartic protease activity predominated at pH 5.0 and occurred immediately after incubation, whereas serine protease activity predominated at pH 7.5 after a 9h delay and was resistant to aprotinin. The salivary serine proteases were distinctly different from midgut serine proteases, based on the tissue-specific differential susceptibility to aprotinin and differing pH optima. Collectively, the caseinolytic activities complement one another, expanding the location and pH range over which digestion can occur.     

Substrate specificity in the control of digestive enzymes in larvae of the black carpet beetle

When starved larvae of the black carpet beetle, Attagenus megatoma, were fed selected diets, increases in proteolytic, trypsin, and chymotrypsin activity were correlated with total midgut protein and not with the amount of food consumed. Although larvae initially consumed more of a starch diet than of 2 diets that contained added protein, total protease activity in these larvae was minimal. Starch-fed larvae and larvae fed a casein-sucrose diet had a consistently higher level of sucrase activity than larvae fed an all-casein diet. These total results support a secretagogue mechanism for control of digestive enzyme synthesis in insects. In addition, the absence of parallel stimulation of different digestive enzymes by a single substrate (starch) indicated nutrient class specificity in the control of inducible midgut enzymes in this species.     

Production and Characteristics of Raw-Starch-Digesting alpha-Amylase from a Protease-Negative Aspergillus ficum Mutant

Mutational experiments were carried out to decrease the protease productivity of Aspergillus ficum IFO 4320 by using N-methyl-N'-nitro-N-nitrosoguanidine. A protease-negative mutant, M-33, exhibited higher alpha-amylaseactivity than the parent strain under submerged culture at 30 degrees C for 24 h. About 70% of the total alpha-amylase activity in the M-33 culture filtrate was adsorbed onto starch granules. The electrophoretically homogeneous preparation of raw-starch-adsorbable alpha-amylase (molecular weight, 88,000), acid stable at pH 2, showed intensive raw-starch-digesting activity, dissolving corn starch granules completely. The preparation also exhibited a high synergistic effect with glucoamylase I. A mutant, M-72, with higher protease activity produced a raw cornstarch-unadsorbable alpha-amylase. The purified enzyme (molecular weight, 54,000), acid unstable, showed no digesting activity on raw corn starch and a lower synergistic effect with glucoamylase I in the hydrolysis of raw corn starch. The fungal alpha-amylase was therefore divided into two types, a novel type of raw-starch-digesting enzyme and a conventional type of raw-starch-nondigesting enzyme.