Biological activity of Dolichos biflorus L. trypsin inhibitor against lepidopteran insect pests.
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Abstract:
Protease inhibitors confer resistance in plants against insect pests by inhibiting larval gut proteases. Cultivars of Dolichos biflorus were screened for their inhibitory activity against midgut proteases of Pieris brassicae larvae. Seed extracts of developing and germinating seeds of HPK4 cultivar inhibited larval gut proteases of Spodoptera littoralis efficiently. Neonate larvae of P. brassicae fed on cabbage leaf discs coated with 0.025-2.50 mg protein (seed extract) resulted in 10-80% larval mortality and significantly reduced leaf area eaten and faecal matter as compared to control. The treated larvae had 40% less soluble proteins per mg faecal matter and there was similar decline in midgut proteases of treated larvae (@ 2.5 mg protein) compared to untreated ones after 5 days. The LC50 and LT50 value was calculated to be 1.05 mg/leaf disc and 4.8 days (2.5 mg protein), respectively for neonate larvae of P. brassicae. Significant reduction in egg hatching (75%) was observed in egg mass treated with 5.3 mg of crude inhibitor protein of mature seeds. This could be due to the inhibition of proteases involved in the hydrolysis of egg chorion proteins. The studies demonstrated the insecticidal activity of D. biflorus seed extracts.Keywords:
Dolichos
Spodoptera littoralis
Pieris brassicae
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Protease inhibitors confer resistance in plants against insect pests by inhibiting larval gut proteases. Cultivars of Dolichos biflorus were screened for their inhibitory activity against midgut proteases of Pieris brassicae larvae. Seed extracts of developing and germinating seeds of HPK4 cultivar inhibited larval gut proteases of Spodoptera littoralis efficiently. Neonate larvae of P. brassicae fed on cabbage leaf discs coated with 0.025-2.50 mg protein (seed extract) resulted in 10-80% larval mortality and significantly reduced leaf area eaten and faecal matter as compared to control. The treated larvae had 40% less soluble proteins per mg faecal matter and there was similar decline in midgut proteases of treated larvae (@ 2.5 mg protein) compared to untreated ones after 5 days. The LC50 and LT50 value was calculated to be 1.05 mg/leaf disc and 4.8 days (2.5 mg protein), respectively for neonate larvae of P. brassicae. Significant reduction in egg hatching (75%) was observed in egg mass treated with 5.3 mg of crude inhibitor protein of mature seeds. This could be due to the inhibition of proteases involved in the hydrolysis of egg chorion proteins. The studies demonstrated the insecticidal activity of D. biflorus seed extracts.
Dolichos
Spodoptera littoralis
Pieris brassicae
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Larval midgut extracts from rosaceous branch borer, Osphranteria coerulescens, were assayed for protease activity in the presence of protease inhibitors in order to determine the dominant protease types. The maximum total proteolytic activity in the midgut extract using azocasein as substrate was observed at pH 8, suggesting the presence of serine proteases. Also, the highest activity using azocasein as substrate was revealed at temperature 55°C. The presence of serine proteases was confirmed by their noticeable inhibition due to specific inhibitor, PMSF. The significant inhibition of tryptic activity by the inhibitors compared to chymotrypsin showed a considerable presence of trypsin in the midgut. Also, inhibition assays and zymogram analysis pointed to the presence of metalloprotease in the digestive system of O. coerulescens. The kinetic parameters of trypsin-like proteases, using BAPNA as substrate at pH 8 and temperature 25°C, indicated that the Km and Vmax values of trypsin in the gut of last instar larvae were 0.69±0.01 mM and 560±2 nmol.min -1 mg 1 protein, respectively. Inhibition assays and zymogram analysis showed that only small amounts of chymotrypsin and cysteine proteases were present in the digestive system of O. coerulescens, with metalloproteases and trypsin being dominant.
Zymography
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The role played by the gut juice of insects in the infective process of insect viruses was examined. Analysis of larval gut extract of Heliothis armigera by SDS-PAGE revealed protease activity associated with components of molecular weights 48,000 and 94,000. Proteases were found to be associated with occlusion bodies and virions of both nuclear polyhedrosis virus (NPV) and cytoplasmic polyhedrosis virus (CPV) infecting H. armigera. CPV occlusion bodies were dissolved by gut juice extract at pH 8.0, trypsin and chymotrypsin at pH 8.0, and carbonate-chloride solution at pH 10.5. Trypsin treatment was selective for occlusion bodies of CPV at pH 8.0, whereas solutions more alkaline than pH 10.0 without added enzymes were adequate to digest NPV occlusion bodies. This property was used to identify and separate the two types of viruses from a mixed infection. Gut extract proteases have characteristics similar to those of trypsin.
Nuclear Polyhedrosis Virus
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To clarify the effects of protease inhibitors on the activities of midgut proteases in Callosobruchus chinensis (L.) larvae, the inhibitory effects of four kinds of protease inhibitors on the activities of midgut proteases of C. chinensis larvae were examined in vitro and in feeding conditions with indoor artificial insect inoculation. The activities of total protease, trypsin-like enzyme and chymotrypsin-like enzyme in the midgut of C. chinensis larvae fed on artificial mung bean that contained different contents of mung bean types in inhibitor (MBTI) were examined. The results showed that those four protease inhibitors had significant inhibitory effects on the activities of total protease, trypsin-like enzyme and chymotrypsin-like enzyme in C. chinensis larvae. The inhibitory effect was more significant when the concentration was higher. Among those inhibitors, the inhibitory effect of 20 μg·mL-1 MBTI on three kinds of enzyme activities was the strongest, with reduction of 62.5%, 41.2% and 38.7%, respectively. Ovomucoid inhibitor (OI) had the lowest inhibitory effect. The activities of three enzymes in the midgut were also inhibited by C. chinensis larvae fed with artificial mung bean containing different inhibitors. The activities of three enzymes increased with the prolongation of the instar after feeding, but they were significantly lower than that in control. The inhibitory effect of MBTI was the strongest. The inhibitory effect on the activities of total protease and trypsin-like enzyme was gradually enhanced with the increases of MBTI contents when C. chinensis larvae were fed with artificial mung bean with different contents MBTI. The inhibitory effect on chymotrypsin-like enzyme activity was not significant. When the content of MBTI was up to 20%, the activity of chymotrypsin-like enzyme was obviously inhibited.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20 μg·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.
Callosobruchus chinensis
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Trypsin inhibitor
Protease inhibitor (pharmacology)
Zymography
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Plants activate direct and indirect defences in response to insect egg deposition. However, whether eggs can manipulate plant defence is unknown. In Arabidopsis thaliana, oviposition by the butterfly Pieris brassicae triggers cellular and molecular changes that are similar to the changes caused by biotrophic pathogens. In the present study, we found that the plant defence signal salicylic acid (SA) accumulates at the site of oviposition. This is unexpected, as the SA pathway controls defence against fungal and bacterial pathogens and negatively interacts with the jasmonic acid (JA) pathway, which is crucial for the defence against herbivores. Application of P. brassicae or Spodoptera littoralis egg extract onto leaves reduced the induction of insect-responsive genes after challenge with caterpillars, suggesting that egg-derived elicitors suppress plant defence. Consequently, larval growth of the generalist herbivore S. littoralis, but not of the specialist P. brassicae, was significantly higher on plants treated with egg extract than on control plants. In contrast, suppression of gene induction and enhanced S. littoralis performance were not seen in the SA-deficient mutant sid2-1, indicating that it is SA that mediates this phenomenon. These data reveal an intriguing facet of the cross-talk between SA and JA signalling pathways, and suggest that insects have evolved a way to suppress the induction of defence genes by laying eggs that release elicitors. We show here that egg-induced SA accumulation negatively interferes with the JA pathway, and provides an advantage for generalist herbivores.
Pieris brassicae
Spodoptera littoralis
Jasmonic acid
Defence mechanisms
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Abstract The rice green semi looper, Naranga aenescens Moore (Lepidoptera: Noctuidae) causes severe damage to rice fields in Eastern Asia and Middle East. We demonstrate that two types of serine proteases are active in the midgut of the third instar larvae of N. aenescens , but trypsin‐like proteases are considerably more active than chymotrypsin‐like proteases. To develop better control strategies, purification and biochemical characterization of a major trypsin‐like digestive protease from the midgut of the third instar larvae of N. aenescens was achieved by gel filtration and anion exchange chromatography. After the final purification step, the enzyme was purified 9.62‐fold with a recovery of 16.1% and a specific activity of 4.12 U/mg protein and a molecular mass of approximately 88.5 kDa. Biochemical characterization indicated that the purified protease had highest activity at pH 10 and 30°C and was stable for up to 6 h under those conditions. Divalent cations, especially Ca 2+ , Mg 2+ , and Cu 2+ , increased the enzyme activity and synthetic inhibitors that target trypsin‐like activity caused a significant reduction in caseinolytic activity. These data may be used to develop inhibitors that decrease the damage of N. aenescens to rice cultivars in the field. © 2011 Wiley Periodicals, Inc.
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Abstract Since the first observation that plants contained protease inhibitors, as identified by their ability to inhibit vertebrate enzymes, it has been postulated that the presence of these substances was related to their phytoprotective abilities. However the following assumptions (1) that phytophagous insects use trypsin, and (2) that ingested inhibitors disrupt digestive proteolysis in insects, have not been adequately tested. Identification of non-tryptic enzymes, cathepsin B, D, and H in phytophagous Coleoptera and unique trypsin-like enzymes in Lepidoptera, indicates insect proteases may differ from their vertebrate counterparts. Putative inhibitor proteins inhibited vertebrate trypsin and chymotrypsin in vitro but had no effect on the trypsin- or chymotrypsin-like activity from the insect midgut. Feeding experiments with the European corn borer, Ostrinia nubilalis (Hübner), indicate that ingestion of inhibitors may not disrupt digestive proteolysis in vivo and the vertebrate trypsin inhibitor in corn may be ineffective as a phytoprotective strategy for this insect. Limitations and implications of ingested inhibitors for future pest control may depend on the origin of the inhibitor, as well as the insect's response.
Proteolysis
Ostrinia
Protease inhibitor (pharmacology)
Trypsin inhibitor
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