杠柳新苷P和E对东方粘虫和小地老虎幼虫中肠蛋白酶活性的影响

从杀虫植物杠柳Periploca sepium Bunge根皮中分离得到的杠柳新苷P具有很高的杀虫活性, 为了探索其杀虫机理, 采用经典的昆虫蛋白酶活性测定方法, 比较研究了杠柳新苷P和无杀虫活性的杠柳新苷E对东方粘虫Mythimna separata与小地老虎Agrotis ypsilon 6龄幼虫中肠类胰蛋白酶和类胰凝乳蛋白酶活性的影响。结果表明： 对东方粘虫弱碱性类胰蛋白酶, 杠柳新苷P表现出强激活作用（酶活性为对照的3.43倍）, 激活时间可长达8 h, 而杠柳新苷E则无明显激活作用。杠柳新苷P和E对东方粘虫弱碱性类胰蛋白酶活性的影响二者差异显著（P=0.01）, 杠柳新苷P药后2, 4和8 h, 东方粘虫中肠弱碱性类胰蛋白酶的活性分别是杠柳新苷E药后的15.4, 106.8和242.7倍。酶活性测定结果还表明, 与东方粘虫相比, 小地老虎中肠类胰蛋白酶活性相对较低, 且杠柳新苷P的激活作用也较弱, 这可能是杠柳新苷P对东方粘虫具杀虫活性, 而小地老虎对其不敏感的原因之一; 另外, 杠柳新苷P和E对试虫中肠类凝乳胰蛋白酶活性均无明显影响。据此推测, 杠柳活性成分新苷P对东方粘虫中肠弱碱性类胰蛋白酶的激活作用可能是造成试虫中毒的机理之一。     

大豆胰蛋白酶抑制剂对棉铃虫幼虫消化生理和生长发育的影响

本研究根据棉铃虫Helicotverpa ormigera(Hubner)幼虫中肠蛋白酶在离体条件下对蛋白酶抑制剂的反应,选择具有较强抑制作用的大豆胰蛋白酶抑制剂,以0.21-4.2%(干重)的浓度配入幼虫人工饲料,测定了幼虫短期和长期取食这些饲料引起的中肠类胰蛋白酶、类胰凝乳蛋白酶和总蛋白酶活力的变化和生长抑制效应。短期取食抑制剂的幼虫,中肠弱碱性类胰蛋白酶活力显著增高,在4.2%。浓度下比对照高出21%;强碱性类胰蛋白酶、类胰凝乳蛋白酶和总蛋白酶活力显著降低,生长发育受到明显抑制。长期取食低浓度(0.84%)抑制剂的幼虫,弱碱性类胰蛋白酶和类胰凝乳蛋白酶活力显著增高,强碱性类胰蛋白酶活力显著降低。总蛋白酶活力变化不显著;长期取食高浓度(4.2%)抑制剂的幼虫,强碱性类胰蛋白酶和总蛋白酶活力显著降低,其它酶活力变化不显著。抑制剂随浓度的增高对幼虫生长的抑制作用加强,但浓度高于0.84%后,抑制强度的变化减小。据此作者认为,蛋白酶抑制剂对昆虫抗营养效应在于它对蛋白酶的激活和抑制作用,从而导致各种蛋白酶间的协调性破坏,昆虫消化过程受阻,影响生长发育。     

Effects of a fat body extract on larval midgut cells and growth of lepidoptera

Treatment with fat body extract (FBX) from pupae of the tobacco hornworm, Manduca sexta, caused mortality in larvae of two pest lepidopterans, the gypsy moth, Lymantria dispar, and the cotton leafworm, Spodoptera littoralis. In FBX-treated larvae, the feeding rate was depressed, causing reduced weight gain and then larval death. Their midgut showed formation of multicellular layers of midgut epidermis, indicating stem-cell hyperplasia. Hence, the integument of FBX-treated larvae had a double cuticle, indicating induction of premature molting. But radioimmunoassay measurements confirmed that the amount of ecdysteroids in FBX was too low to be responsible for the molt-inducing effects observed after treatment with FBX. With midgut stem cell cultures in vitro, addition of FBX to the culture medium stimulated cell proliferation and differentiation in a concentration-dependent manner. This effect was compared with those of insect molting hormones, ecdysone and 20-hydroxyecdysone; an ecdysteroid agonist, RH-2485; and a purified protein from FBX (multiplication factor). This article describes the mode of action of FBX and possible interplay between fat body factor(s) and insect hormones in the development and metamorphosis of the insect midgut.     

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。     

Larval esterases of the southwestern corn borer, Diatraea grandiosella: Temporal changes and specificity

Disc electrophoresis was used to examine and characterize the esterases present in the fat body, haemolymph, and midgut of last stage larvae of the southwestern corn borer, Diatraea grandiosella. Significant temporal changes were observed in the pattern of the 4 major esterases of the fat body and 3 major esterases of the haemolymph. These changing profiles presumably relate, in part, to a requirement for the degradation of juvenile hormone (JH) in preparation for metamorphosis.The binding capacity of esterases present in the larval midgut towards JH I and three JH mimics (alkyl-3,7,11-trimethyl-2,4-dodecadienoates) was also examined. The midgut of last stage nondiapausing larvae was shown to contain a carboxylesterase which bound all three JH mimics. Another esterase which bound JH I, but not the mimics, was also present. An esterase with a similar electrophoretic mobility was detected in the haemolymph and integument. Since the JH I binding esterase did not bind the JH mimics, the mimics do not appear to synergize JH by inhibiting its ester hydrolysis.     

Entomotoxic properties of Dioclea violacea lectin and its effects on digestive enzymes of Anagasta kuehniella (Lepidoptera)

Entomotoxic plant lectins have been extensively studied in the past two decades, yet the exact mechanisms underlying their toxic effects remain unknown. This study investigated the effects of Dioclea violacea lectin (DVL) on larval development in Anagasta kuehniella. Chronic exposure of larvae (from neonates to the fourth instar) demonstrated that DVL interfered with larval growth, retarding development and decreasing larval mass without affecting survival. DVL decreased trypsin-like, chymotrypsin-like, and α-amylase activities and proved resistant to proteolysis by midgut proteases up to 24 h. Shorter exposures to dietary DVL had no effect on midgut enzyme activity. Feeding fourth-instar larvae with fluorescently-labeled DVL revealed lectin binding to the peritrophic membrane.     

Characterization and comparison of midgut proteases of Bacillus thuringiensis susceptible and resistant diamondback moth (Plutellidae: Lepidoptera)

The midgut proteases of the Bacillus thuringiensis resistant and susceptible populations of the diamondback moth, Plutella xylostella L. were characterized by using protease specific substrates and inhibitors. The midgut contained trypsin-like proteases of molecular weights of 97, 32, 29.5, 27.5, and 25 kDa. Of these five proteases, 29.5 kDa trypsin-like protease was the most predominant in activation of protoxins of Cry1Aa and Cry1Ab. The activation of Cry1Ab protoxin by midgut protease was fast (T(1/2) of 23-24 min) even at a protoxin:protease ratio of 250:1. The protoxin activation appeared to be multi-step process, and at least seven intermediates were observed before formation of a stable toxin of about 57.4 kDa from protoxin of about 133 kDa. Activation of Cry1Aa was faster than that of Cry1Ab on incubation of protoxins with midgut proteases and bovine trypsin. The protoxin and toxin forms of Cry proteins did not differ in toxicity towards larvae of P. xylostella. The differences in susceptibility of two populations to B. thuringiensis Cry1Ab were not due to midgut proteolytic activity. Further, the proteolytic patterns of Cry1A protoxins were similar in the resistant as well as susceptible populations of P. xylostella.     

TMOF-like factor controls the biosynthesis of serine proteases in the larval gut of Heliothis virescens.

Proteolytic enzyme biosynthesis in the midgut of the 4th instar larva of Heliothis virescens is cyclical. Protease activity increases immediately after the molt from the 3rd to the 4th instar larvae and declines just before the molt into the 5th instar. Characterization of the midgut proteases using soybean tryspin inhibitor (SBTI) Bowman Birk Inhibitor (BBI) 4-(2-aminoethyl)benzensulfonylfluoride (AEBSF) and N-tosyl-L-phenylalanine chloromethylketone (TPCK) indicate that protease activity is mostly trypsinlike (80%) with a small amount of chymotrypsinlike activity (20%). Injections of late 3rd and 4th instar larval hemolymph into H. virescens larvae inhibited tryspin biosynthesis in the larval midgut. Similar results were obtained when highly purified 4th instar larval hemolymph that crossreacted with Aea-TMOF antisurm using ELISA was injected into 2nd instar larvae. Injections of Aea-TMOF and its analogues into 2nd instar, and Aea-TMOF alone into 4th instar larvae stopped trypsin biosynthesis 24 and 48 h after the injections, respectively. Injections of 4th instar H. virescens larval hemolymph into female Aedes aegypti that took a blood meal stopped trypsin biosynthesis and egg development. These results show that the biosynthesis of trypsin-like enzymes in the midgut of a lepidoptera is modulated with a hemolymph circulating TMOF-like factor that is closely related to Aea-TMOF. Arch.     

Adaptation of Spodoptera exigua (Lepidoptera: Noctuidae) to barley trypsin inhibitor BTI-CMe expressed in transgenic tobacco

Nicotiana tabacum plants were transformed with the cDNA of barley trypsin inhibitor BTI-CMe under the control of the 35S CaMV promoter. Although the transgene was expressed and the protein was active in the homozygous lines selected, growth of Spodoptera exigua (Lepidoptera: Noctuidae) larvae reared on transgenic plants was not affected. The protease activity in larval midgut extracts after 2 days feeding on transformed tobacco leaves from the highest expressing plant showed a reduction of 25% in the trypsin-like activity compared to that from insects fed on non-transformed controls. The susceptibility of digestive serine-proteases to inhibition by BTI-CMe was confirmed by activity staining gels. This decrease was compensated with a significant induction of leucine aminopeptidase-like and carboxipeptidase A-like activities, whilechymotrypsin-, elastase-, and carboxipeptidase B-like proteases were not affected.     

Sequestration of host plant carotenoids in the larval tissues of Helicoverpa zea

To determine the cause of the unique yellow coloration in mandibular glands of soybean-fed Helicoverpa zea larvae, the accumulation of carotenoids in various tissues of last instar larvae fed soybean, cotton and tomato foliage was quantified. Five carotenoids were detected in the foliage of all host plants but at significantly different concentrations. Xanthophylls rather than carotenes were most likely to accumulate in larval tissues. Carotenoids accumulated at different rates and some were significantly affected by larval diet. Highest levels of carotenoid accumulation, notably lutein, were detected in the testes, followed by midgut epithelium, fat body and integument. The midgut epithelium contained the greatest and the testes the least diversity of carotenoid types. Low levels of lutein were detected in both labial and mandibular glands. Tomato foliage had the highest carotenoid content and caterpillar tissues fed these leaves often had the highest amounts of carotenoid. However, the accumulation of carotenoids did not protect larvae from antibiotic effects of tomato foliage because these caterpillars had the highest mortality and slowest growth rates of all the three host plants. Transport and absorption of lipid and oxidative stress may be some reasons for differential carotenoid accumulation.     

Bioinsecticidal activity of Talisia esculenta reserve protein on growth and serine digestive enzymes during larval development of Anticarsia gemmatalis

Plants synthesize a variety of molecules to defend themselves against an attack by insects. Talisin is a reserve protein from Talisia esculenta seeds, the first to be characterized from the family Sapindaceae. In this study, the insecticidal activity of Talisin was tested by incorporating the reserve protein into an artificial diet fed to the velvetbean caterpillar Anticarsia gemmatalis, the major pest of soybean crops in Brazil. At 1.5% (w/w) of the dietary protein, Talisin affected larval growth, pupal weight, development and mortality, adult fertility and longevity, and produced malformations in pupae and adult insects. Talisin inhibited the trypsin-like activity of larval midgut homogenates. The trypsin activity in Talisin-fed larvae was sensitive to Talisin, indicating that no novel protease-resistant to Talisin was induced in Talisin-fed larvae. Affinity chromatography showed that Talisin bound to midgut proteinases of the insect A. gemmatalis, but was resistant to enzymatic digestion by these larval proteinases. The transformation of genes coding for this reserve protein could be useful for developing insect resistant crops.     

JNK MAP kinase is involved in the humoral immune response of the greater wax moth larvae Galleria mellonella

We investigated the participation of MAP kinases in the response of Galleria mellonella larvae to immune challenge. JNK MAP kinase was activated in fat body 10-15 min after LPS injection in vivo. The level of JNK activation was time- and LPS dosage-dependent. JNK MAP kinase isolated from cell-free extract of fat bodies dissected from immune stimulated larvae phosphorylated c-Jun protein in vitro. The activity of Gm JNK kinase was abolished in the presence of the JNK specific inhibitor SP600125. Our data indicate a correlation between JNK phosphorylation and induction of antimicrobial activity in the insect hemolymph after immune stimulation. Hemolymph from larvae pre-treated with JNK specific inhibitor SP600125 showed a reduced level of antibacterial activity after LPS injection. JNK inhibition by SP600125 abolished antibacterial activity of the in vitro culture of G. mellonella fat body. Finally, we also show a correlation between JNK-dependent immune response of G. mellonella larvae and elevated temperature.     

Edin Expression in the Fat Body Is Required in the Defense Against Parasitic Wasps in Drosophila melanogaster

The cellular immune response against parasitoid wasps in Drosophila involves the activation, mobilization, proliferation and differentiation of different blood cell types. Here, we have assessed the role of Edin (elevated during infection) in the immune response against the parasitoid wasp Leptopilina boulardi in Drosophila melanogaster larvae. The expression of edin was induced within hours after a wasp infection in larval fat bodies. Using tissue-specific RNAi, we show that Edin is an important determinant of the encapsulation response. Although edin expression in the fat body was required for the larvae to mount a normal encapsulation response, it was dispensable in hemocytes. Edin expression in the fat body was not required for lamellocyte differentiation, but it was needed for the increase in plasmatocyte numbers and for the release of sessile hemocytes into the hemolymph. We conclude that edin expression in the fat body affects the outcome of a wasp infection by regulating the increase of plasmatocyte numbers and the mobilization of sessile hemocytes in Drosophila larvae.     

Granzyme K activates protease-activated receptor-1

Granzyme K (GrK) is a trypsin-like serine protease that is elevated in patients with sepsis and acute lung inflammation. While GrK was originally believed to function exclusively as a pro-apoptotic protease, recent studies now suggest that GrK may possess other non-cytotoxic functions. In the context of acute lung inflammation, we hypothesized that GrK induces pro-inflammatory cytokine release through the activation of protease-activated receptors. The direct effect of extracellular GrK on PAR activation, intracellular signaling and cytokine was assessed using cultured human lung fibroblasts. Extracellular GrK induced secretion of IL-6, IL-8 and MCP-1 in a dose- and time-dependent manner in lung fibroblasts. Heat-inactivated GrK did not induce cytokine release indicating that protease activity is required. Furthermore, GrK induced activation of both the ERK1/2 and p38 MAP kinase signaling pathways, and significantly increased fibroblast proliferation. Inhibition of ERK1/2 abrogated the GrK-mediated cytokine release. Through the use of PAR-1 and PAR-2 neutralizing antibodies, it was determined that PAR-1 is essential for GrK-induced IL-6, IL-8 and MCP-1 release. In summary, extracellular GrK is capable of activating PAR-1 and inducing fibroblast cytokine secretion and proliferation.     

Bioinsecticidal activity of Archidendron ellipticum trypsin inhibitor on growth and serine digestive enzymes during larval development of Spodoptera litura

The roles of serine proteases involved in the digestion mechanism of the cutworm Spodoptera litura (Lepidoptera: Noctuidae) were examined (in vitro and in vivo) following feeding of plant protease inhibitors. A trypsin inhibitor from Archidendron ellipticum (AeTI) was purified by ammonium sulfate fractionation, ion-exchange chromatography and size-exclusion chromatography (HPLC) and its bioinsecticidal properties against S. litura were compared with Soybean Kunitz trypsin inhibitor (SBTI). AeTI inhibited the trypsin-like activities of the midgut proteases of fifth instar larvae of S. litura by over 70%. Dixon plot analysis revealed competitive inhibition of larval midgut trypsin and chymotrypsin by AeTI, with an inhibition constant (K(i)) of 3.5x10(-9) M and 1.5x10(-9) M, respectively. However, inhibitor kinetics using double reciprocal plots for both trypsin and chymotrypsin inhibitions demonstrated a mixed inhibition pattern. Feeding experiments conducted on different (neonate to ultimate) instars suggested a dose-dependent decrease for both the larval body weight as well as % survival of larva fed on diet containing 50, 100 and 150 microM AeTI. Influence of AeTI on the larval gut physiology indicated a 7-fold decrease of trypsin-like protease activity and a 5-fold increase of chymotrypsin-like protease activity, after being fed with a diet supplemented with 150 microM AeTI. This study suggests that although the early (1st to 3rd) larval instars of S. litura are susceptible to the trypsin inhibitory action of AeTI, the later instars may facilitate the development of new serine proteases, insensitive to the inhibitor.     

Cockroach midgut peptides that regulate cell proliferation, differentiation, and death in vitro

Summary The number of insect midgut cells is maintained homeostatically in vivo and in vitro. However, during starvation, the midgut shrinks and the rate of cell replacement appears to be suppressed. When they undergo metamorphosis, the internal organs of insects are drastically remodeled by cell proliferation, differentiation, and apoptotic processes, and the net number of cells usually increases. An extract of 1650 midguts ofPeriplaneta americana was fractionated by highperformance liquid chromatography (HPLC) to obtain the peptides that regulate these processes. The HPLC fractions were tested for myotropic activity in the foregut and for effects on cell proliferation or loss in primary cultures of larvalHeliothis virescens midgut cells and in a cell line derived from the last-instar larval fat body ofMamestra brassicae. Some fractions stimulated midgut stem cell proliferation and differentiation, while others caused loss of differentiated columnar and goblet cells. Other fractions stimulated cell proliferation in the larval fat body cells. Mention of products in this article does not imply endorsement by the U.S. Department of Agriculture.     

蛋白酶抑制剂对绿豆象幼虫中肠蛋白酶活性的影响

为明确蛋白酶抑制剂对绿豆象幼虫中肠蛋白酶活性的影响,采用室内人工接虫和生化测定的方法,研究了在离体条件和饲喂条件下4种蛋白酶抑制剂对绿豆象幼虫中肠蛋白酶的抑制作用,并测定了绿豆象幼虫取食不同含量的绿豆胰蛋白酶抑制剂(MBTI)的人工绿豆后,其中肠内总蛋白酶、类胰蛋白酶和类胰凝乳蛋白酶活性的变化.结果表明:在离体条件下,供试4种蛋白酶抑制剂对绿豆象幼虫总蛋白酶、类胰蛋白酶和类胰凝乳蛋白酶活性均有明显的抑制作用,且浓度越大,抑制效果越显著,其中以20μg·mL^-1的MBTI对3种酶活性的抑制效果最强,3种酶活性分别比对照降低了62.5%、41.2%和38.7%,而卵粘蛋白抑制剂(OI)抑制效果最弱.绿豆象幼虫取食含不同抑制剂的人工绿豆后,中肠内3种酶活性也均受到一定的抑制作用,取食后随龄期的延长,3种酶活性有所升高但仍显著低于对照,且以MBTI的抑制作用最强.当绿豆象幼虫取食不同含量MBTI的人工绿豆后,随MBTI含量的增加,对总蛋白酶活性和类胰蛋白酶活性的抑制作用均逐渐增强,但对类胰凝乳蛋白酶活性的抑制作用并不显著,只有当MBTI含量达20%时,对类胰凝乳蛋白酶活性才表现出明显的抑制作用.     

N-β-Alanyldopamine levels and synthesis in integument and other tissues of Manduca sexta (L.) during the larval-pupal transformation

N-β-Alanyldopamine (NBAD) and other diphenols in tissues of the fifth larval instar of the tobacco hornworm, Manduca sexta (L.), were analyzed by HPLC with electrochemical detection. NBAD accumulated in the integument during the intermolt feeding period, with maximal levels in the wandering stage (6 mmol/g). It then declined to a low level during apolysis and endocuticle digestion, while hemolymph NBAD increased during the same interval to a peak concentration (3 mM) shortly before pupal ecdysis. Trachea and foregut contained lesser amounts of NBAD (0.5 mmol/g), perhaps associated with cuticle, whereas fat body, muscle, midgut and hindgut had 0.1 mmol/g or less. Dopamine (DA), N-acetyldopamine and 3,4-dihydroxyphenylalanine (DOPA) were at least 10-fold less abundant than NBAD in the integument. NBAD synthase, which catalyzes the formation of NBAD from DA and β-alanine, was assayed in both integument and fat body. Highest activity was detected in the integument, where two peaks were observed, one at day 3 near the end of larval feeding and the other at day 9 as pupal cuticle tanning was initiated. Fat body enzyme was substantially less and was detected only in the pharate pupa. Maximal NBAD synthesis by integument cultured in vitro was dependent upon DA supplementation of at least 1.4 mM. 20-Hydroxyecdysone did not alter NBAD synthesis in vitro in either the integument or the fat body, even though injection of this hormone into isolated larval abdomens induced synthesis and/or transport of integumental NBAD back into the hemolymph. The rate-limiting steps in the NBAD biosynthetic pathway appear to be the production of DOPA and DA, because β-alanine occurs in the hemolymph at relatively high levels throughout larval-pupal development.