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.     

Adipokinetic hormones control amylase activity in the cockroach (Periplaneta americana) gut

This study examined the biochemical characteristics of α‐amylase and hormonal (adipokinetic hormone: AKH) stimulation of α‐amylase activity in the cockroach (Periplaneta americana) midgut. We applied two AKHs in vivo and in vitro, then measured resultant amylase activity and gene expression, as well as the expression of AKH receptor (AKHR). The results revealed that optimal amylase activity is characterized by the following: pH: 5.7, temperature: 38.4 °C, K_m (Michaelis–Menten constant): 2.54 mg starch/mL, and V_max (maximum reaction velocity): 0.185 μmol maltose/mL/min. In vivo application of AKHs resulted in significant increase of amylase activity: by two‐fold in the gastric caeca and 4–7 fold in the rest of the midgut. In vitro experiments supported results seen in vivo: a 24‐h incubation with the hormones resulted in the increase of amylase activity by 1.4 times in the caeca and 4–9 times in the midgut. Further, gene expression analyses reveal that AKHR is expressed in both the caeca and the rest of the midgut, although expression levels in the former were 23 times higher than levels in the latter. A similar pattern was found for the amylase (AMY) gene. Hormonal treatment did not affect the expression of either gene. This study is the first to provide evidence indicating direct AKH stimulation of digestive enzyme activity in the insect midgut, supported by specific AKHR gene expression in this organ.     

Cloning and characterization of the adipokinetic hormone receptor from the cockroach Periplaneta americana

Cockroaches have long been used as insect models to investigate the actions of biologically active neuropeptides. Here, we describe the cloning and functional expression in Chinese hamster ovary cells of an adipokinetic hormone (AKH) G protein-coupled receptor from the cockroach Periplaneta americana. This receptor is only activated by various insect AKHs (we tested eight) and not by a library of 29 other insect or invertebrate neuropeptides and nine biogenic amines. Periplaneta has two intrinsic AKHs, Pea-AKH-1, and Pea-AKH-2. The Periplaneta AKH receptor is activated by low concentrations of both Pea-AKH-1 (EC50, 5 x 10(-9)M), and Pea-AKH-2 (EC50, 2 x 10(-9)M). Insects can be subdivided into two evolutionary lineages, holometabola (insects with a complete metamorphosis during development) and hemimetabola (incomplete metamorphosis). This paper describes the first AKH receptor from a hemimetabolous insect.     

杠柳杀虫组分F3-28对东方粘虫和小地老虎幼虫中肠消化酶活性的影响

F3-28是从杀虫植物杠柳Periploca sepium Bunge根皮中分离的一个具有杀虫活性的馏分,其主要成分为杠柳苷A、杠柳苷E和杠柳苷X。为了探索F3-28的初始作用部位, 为深入研究其作用机理奠定基础, 本研究采用经典的昆虫消化酶活性测定方法, 比较了F3-28对小地老虎Agrotis ypsilon和东方粘虫Mythimna separata 5龄幼虫消化酶系活性的影响。结果表明：对F3-28不敏感的小地老虎幼虫摄食F3-28后,其中肠蛋白酶、淀粉酶、脂肪酶的活性无显著变化。对F3-28敏感的东方粘虫幼虫摄食F3-28后2,4,8,10,12,24,48 h, 其中肠总蛋白酶活性分别为对照组的0.76,2.53,1.45,1.88,1.54,1.46,1.70倍,且和药物浓度呈依赖关系; 类胰蛋白酶的活性分别为对照组的1.60, 1.75,1.60,1.12,1.39,1.16,1.15倍（以BAPNA为底物）或1.68,1.95,1.53,1.26,1.15,1.13,1.14倍（以TAME为底物）,且和药物浓度呈依赖关系; 类胰凝乳蛋白酶活性分别为对照组的0.50,1.66,1.44,1.18,1.54,1.08和1.03 倍,但和药物浓度无依赖关系; 淀粉酶的活性分别为对照组的1.60,1.35,1.27,1.31,1.23和1.20 倍,但和药物浓度无依赖关系;对脂肪酶活性无明显影响。这些结果说明,杠柳杀虫活性组分F3-28的作用机理可能涉及对试虫中肠蛋白酶的激活,特别是对类胰蛋白酶的激活。     

Effect of taurine on serum trehalase activity in the American cockroach,Periplaneta americana

Injection of adult cockroaches with 8 nmol taurine reduces serum trehalase activity. The maximal inhibition of serum trehalase activity caused by taurine is similar to the inhibition observed in vivo at 1 h following excitation. The serum of taurine-treated insects contains active and inactive forms of the enzyme with activation of the inactive form achieved by trypsinization and by increasing the ionic strength of the assay mixture for trehalase activity.     

Effect of corazonin and crustacean cardioactive peptide on heartbeat in the adult American cockroach (Periplaneta americana)

Changes in the frequency of cardiac pulsations have been monitored in the decapitated body of adult P. americana before and 5 h after the injections of [Arg(7)]-corazonin and CCAP, using newly invented touch-free, noninvasive optocardiographic methods. Relatively large dosages of these peptides (10(-6) M concentrations in the body) had no effect on the rate of the heartbeat beyond the Ringer control limits. It has been concluded, therefore, that Corazonin and CCAP, which are currently cited in the literature as "the most potent cardiostimulating peptides" in insects, have no effect on the physiological regulation of cardiac functions in the living body.     

Localization of corazonin in the nervous system of the cockroach Periplaneta americana

Antisera raised to the cardioactive peptide corazonin were used to localize immunoreactive cells in the nervous system of the American cockroach. Sera obtained after the seventh booster injection were sufficiently specific to be used for immunocytology. They recognized a subset of 10 lateral neurosecretory cells in the protocerebrum that project to, and arborize and terminate in the ipsilateral corpus cardiacum. They also reacted with bilateral neurons in each of the thoracic and abdominal neuromeres, a single dorsal unpaired median neuron in the suboesophageal ganglion, an interneuron in each optic lobe, and other neurons at the base of the optic lobe, in the tritocerebrum and deutocerebrum. The presence of corazonin in the abdominal neurons and the lateral neurosecretory cells was confirmed by HPLC fractionation of extracts of the abdominal ganglia, brains and retrocerebral complexes, followed by determination of corazonin by ELISA, which revealed in each tissue a single immunoreactive peak co-eluting with corazonin in two different HPLC systems. Antisera obtained after the first three booster injections recognized a large number of neuroendocrine cells and neurons in the brain and the abdominal nerve cord. However, the sera from the two rabbits reacted largely with different cells, indicating that the majority of this immunoreactivity was due to cross-reactivity. These results indicate that the production of highly specific antisera to some neuropeptides may require a considerable number of booster injections.     

Bloodmeal digestion in the midgut of Phlebotomus papatasi and Phlebotomus langeroni

Abstract. Bloodmeal digestion in midguts of the sandflies Phlebotomus papatasi and Phlebotomus langeroni (Diptera: Psychodidae) was investigated in optimized assays to detect general protease, trypsin and aminopeptidase activities using synthetic substrates. Optimal activity occurred at pH 8-9 for all enzymes examined in both species. Protease activity peaked at 24-34h post human bloodmeal in midguts of P.papatasi and 34-48h in P'.langeroni; all endo- and exoprotease activities were completed by 50 h in P.papatasi compared to 72 h in P. langeroni. Hydrolysis of two chymotrypsin substrates was <2% of trypsin activity in both species. Aminopeptidase activity was associated mainly with the midgut wall, whereas trypsin activity was confined to the midgut lumen. A feature of digestion in P.langeroni was the high level of aminopeptidase recorded within 10h of the bloodmeal.     

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.     

Effect of static load on motor behavior of the cockroach Periplaneta americana

Effect of static load on activity of motor centers controlling motor activity (walking, flight) was studied in the American cockroach Periplaneta americana L. It has been established that under effect of load on the animal body the relative excitability of these centers increases. A suggestion is put forward about the presence of common neuronal elements in the generator networks providing motor acts in the American cockroach; a role of afferent systems in control of excitability of loco-motor centers functioning in the regime of static load is shown.     

不同饵料对史氏鲟仔鱼消化酶活性的影响

用活饵料(水丝蚓)、商品饵料、添加消化酶制剂的商品饵料、活饵料与商品饵料交替使用(对照组)来投喂10～40日龄(DPH,Day＿past＿hatch)的史氏鲟仔鱼。其中对照组仔鱼体内蛋白酶活性和商品饵料组及添加消化酶组的差异极显著(P<0.01),与活饵料组差异不显著(P>0.05),活饵料组仔鱼蛋白酶活性与商品饵料组差异极显著(P<0.01),与添加消化酶组差异显著(P<0.05),商品饵料组和添加消化酶组之间仔鱼的蛋白酶活性差异不显著(P>0.05);各饵料组仔鱼的淀粉酶活性差异均不显著(P>0.05);活饵料组和对照组仔鱼的脂肪酶活力与商品饵料及添加消化酶组仔鱼的脂肪酶活性差异极显著(P<0.01),在这两组之间,仔鱼的脂肪酶活性差异均不显著(P>0.05)。     

Starvation suppresses cell proliferation that rebounds after refeeding in the midgut of the American cockroach, Periplaneta americana

Starvation affects behavior, development, metabolism, reproduction, and longevity in almost all animals including insects. In the American cockroach, Periplaneta americana, we investigated the effect of starvation on organ size and cell proliferation activity of the midgut, over a period of one month, using anti-bromodeoxyuridine (BrdU), and anti-phospho-histone H3 antibodies. Under starvation conditions, the midgut became clear and fragile while its length and diameter were reduced. Both the rate of BrdU-uptake in the nucleus and the mitotic activity shown by anti-phospho-histone H3 antibody decreased under long starvation up to half that of the continuously fed control. Refeeding restored BrdU-uptake and mitosis that overshot the fed control. When casein, starch, or cooking oil was fed as representative nutrient sources to the starved cockroaches, all restored BrdU-uptake, but non-nutrient, talc, did not. This study supports the hypothesis that P. americana has a homeostatic mechanism to regulate the cell population of the midgut epithelium according to changes in the nutritional environment.     

Distribution of digestive proteinases in the alimentary tract of the european corn borer Ostrinia nubilalis (lepidoptera: Pyralidae)

The distribution of digestive proteinases in either the anterior and posterior midgut or between the midgut epithelium and ectoperitrophic and endo-peritrophic spaces in the midgut were examined in the European corn borer, Ostrinia nubilalis. Trypsin, chymotrypsin, elastase, and aminopeptidase activities were the same in the anterior and posterior halves of the midgut. Of the total aminopeptidase activity, 95% was located in the midgut epithelium, and 90% of the trypsin, 97% of chymotrypsin, and 93% of the elastase activity were found in the midgut lumen. Trypsin, measured by hydrolysis of benzoyl-L-arginine ethyl ester, and chymotrypsin levels were significantly higher in the ectoperitrophic space compared to the endoperitrophic space. Digestion in the midgut is proposed to be sequential with tryptic digestion occurring in the endoperitrophic space. Ingested protein is digested further in the ectoperitrophic space by the action of elastase, chymotrypsin, and a second trypsin. Final digestion occurs by an intracellular aminopeptidase. © 1995 Wiley-Liss, Inc.     

昆虫中肠液性质对苏云金芽孢杆菌伴孢晶体毒力的影响

综述了昆虫中肠液性质对苏云金芽孢杆菌Bacillus thuringiensis伴孢晶体毒力的影响。中肠液的酸碱度和蛋白酶是影响伴孢晶体溶解与原毒素活化的两大因素。中肠液的酸碱度不仅影响到伴孢晶体的溶解速度,还影响到各种蛋白酶的活性表现;而蛋白酶则直接参与了原毒素的活化,其组成与活性影响着原毒素的活化速度和杀虫专一性。因中肠液蛋白水解能力过高而导致原毒素的过度降解是某些昆虫对苏云金芽孢杆菌低度敏感的主要原因,而中肠液对原毒素活化能力的降低则与昆虫抗性的形成有关。此外,中肠液的沉淀作用及其它生理生化特性也影响着原毒素毒力的正常发挥。     

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.     

美洲大蠊肠道耐热碱性蛋白酶产生菌筛选及其酶特性

昆虫肠道菌是重要的微生物资源.为拓展微生物酶资源菌种来源,本研究以含Na2 CO3和脱脂奶粉的培养基从美洲大蠊Periplaneta americana(L.)肠道中筛选产碱性蛋白酶的嗜碱细菌,进行形态学和16S rDNA序列鉴定,并分析温度、pH和Na2 CO3对菌株生长的影响;采用福林-酚法和SDS-PAGE酶谱法...     

Neural influence on corpus allatum activity and egg maturation in starved virgin Periplaneta americana

ABSTRACT. Of sixty-seven adult female Periplaneta americana (L.) isolated when less than 8h old and kept without food at 25–30°C in LD 12:12, only six produced oothecae during 25–35 days. Growth of the basal oocytes was retarded, and maximal oocyte volume, only one-third of that in fed virgins, was achieved after about 12 days. By day 14, 60% of the basal oocytes were being resorbed in the starved females, and corpora lutea were usually all that remained beyond day 20. Oocyte growth was potentiated in starved, virgin females by severing either of the two nervi corporis allati 1 (NCA 1), and oothecae were then produced in 45–80% of cases. Sham-operated controls oviposited in fewer than 27% of the trials. Because oocyte maturation was prevented by extirpating both corpora allata (CA), but not when the glands were replaced, and because the juvenile hormone analogue, ZR 515, was highly effective in causing the starved cockroaches to produce oothecae, the starvation-induced reproductive failure probably reflects diminished hormone production by the CA. The most likely consequence of severing NCA 1 is de-repression of juvenile hormone production. The directness of the neural influence was shown by removing the one denervated CA, in which case stimulation of oogenesis was minimal even though the contralateral innervated gland was present. The incidence of ootheca production was not enhanced by transecting the NCA 2, which suggests that the CA of starved cockroaches are not inhibited via this pathway.     

Regulation of digestive enzyme activity in the larvae of Catopsilia crocale (Lepidoptera)

Amylase and invertase activity in the midgut tissue of the lepidopterous larvae Catopsilia crocale was studied in relation to the developmental stage and food availability. The enzyme activity increased significantly with advancing age of the larva. A direct positive correlation between food intake and enzyme activity was observed. A possible relationship between feeding and the regulation of enzyme activity is discussed.     

取食臭椿不同部位对沟眶象和臭椿沟眶象成虫消化解毒酶的影响

沟眶象Eucryptorrhynchus scrobiculatus和臭椿沟眶象E.brandti是取食单一寄主臭椿Ailanthus altissima不同部位的钻蛀性害虫。二者成虫羽化后交配之前均需补充营养,且取食臭椿不同部位(叶柄、1年生枝、2～3年生枝、主干)对其雌成虫生殖系统发育的影响差异显著。就臭椿沟眶象雌成虫而言,只有取食主干才能够产卵;而对沟眶象雌成虫来说,则只有取食2～3年生枝的无法产卵。本研究使用臭椿的4种不同部位(叶柄、1年生枝、2～3年生枝、主干)饲喂这两种象甲成虫,利用ELISA试剂盒测定两种象甲的3种解毒酶(谷胱甘肽转移酶、羧酸酯酶和细胞色素P450)和3种消化酶(胰蛋白酶、淀粉酶、脂肪酶)的酶活性。结果表明,两种象甲中后肠的6种酶活性在取食不同寄主部位后发生显著性差异。其中,臭椿沟眶象雌成虫取食主干显著降低其中后肠的CarE酶活性,沟眶象雌成虫取食2～3年生枝能显著提高中后肠的CarE酶活性,同时降低中后肠AMY酶活性。本文明确了寄主植物不同部位对这两种象甲成虫中后肠消化解毒酶活性的影响,分析了两种象甲初羽化雌成虫补充营养造成生殖系统发育差异的原因,对后续...     

The proteases and the protease inhibitor in the midgut of Leucophaea maderae

The caseinolytic enzymes of the midgut lumina and epithelia of Leucophaea were purified through precipitation by 60% saturated (NH₄)₂SO₄, followed by gel permeation on Sephadex G-200 and subsequent DEAE anionexchange chromatography. At least four peaks with enzyme activity were eluted from anionexchange chromatography columns. Gregarines of the midgut lumen apparently do not contribute to the caseinolytic activity within the midgut. Elution profiles of lumen and epithelial enzymes were nearly identical. The same enzymes were identified in the lumina of epithelial microsomal vesicles. This allows the conclusion that these enzymes are produced by the midgut epithelia.Practically all protease activity of the midgut was found in the posterior half, both in the lumen and epithelium. Feeding stimulated protease production primarily in the posterior midgut. The pH optimum of the proteases lay between 9.0 and 9.5 which was closely matched by the observed pH of the posterior midgut where most of the activity is seen. The anterior midgut pH was determined to be around 8.0.The anterior midgut of Leucophaea contained a heatstable protease inhibitor with characteristics of a competitive inhibitor. This inhibitor was precipitable by 60% saturated (NH₄)₂SO₄ and eluted from a Sephadex G-200 column more or less together with the proteases. From a DEAE anionexchange column it was eluted by 0.8 M NaCl, i.e. after the main portion of the proteases. The biological significance of the protease inhibitor in the anterior portion of the midgut is obscure.