Characterization of the Mamestra configurata (Lepidoptera: Noctuidae) larval midgut protease complement and adaptation to feeding on artificial diet,Brassica species,and protease inhibitor

The midgut protease profiles from 5th instar Mamestra configurata larvae fed various diets (standard artificial diet, low protein diet, low protein diet with soybean trypsin inhibitor [SBTI], or Brassica napus) were characterized by one‐dimensional enzymography in gelatin gels. The gut protease profile of larvae fed B. napus possessed protease activities of molecular masses of approximately 33 and 55 kDa, which were not present in the guts of larvae fed artificial diet. Similarly, larvae fed artificial diet had protease activities of molecular masses of approximately 21, 30, and 100 kDa that were absent in larvae fed B. napus. Protease profiles changed within 12 to 24 h after switching larvae from artificial diet to plant diet and vice versa. The gut protease profiles from larvae fed various other brassicaceous species and lines having different secondary metabolite profiles did not differ despite significant differences in larval growth rates on the different host plants. Genes encoding putative digestive proteolytic enzymes, including four carboxypeptidases, five aminopeptidases, and 48 serine proteases, were identified in cDNA libraries from 4th instar M. configurata midgut tissue. Many of the protease‐encoding genes were expressed at similar levels on all diets; however, three chymoptrypsin‐like genes (McSP23, McSP27, and McSP37) were expressed at much higher levels on standard artificial diet and diet containing SBTI as was the trypsin‐like gene McSP34. The expression of the trypsin‐like gene McSP50 was highest on B. napus. The adaptation of M. configurata digestive biochemistry to different diets is discussed in the context of the flexibility of polyphagous insects to changing diet sources. Published 2010 Wiley Periodicals, Inc.     

The interactions between soybean trypsin inhibitor and delta-endotoxin of Bacillus thuringiensis in Helicoverpa armigera larva

No significant difference in larval mortality was observed when a sublethal dose of Bacillus thuringiensis (Bt) var. kurstaki HD-1 crystal was supplemented with soybean trypsin inhibitor (STI) in the artificial diet fed to Helicoverpa armigera in the laboratory, but supplementing a nonlethal dose of crystal with STI in the diet led to a pronounced reduction of larval growth. This concentration of crystal and two lower concentrations of STI alone had no significant effects on larval growth. The results of substrate-gel electrophoresis demonstrated that the proteases in the H. armigera midgut fluid responsible for the degradation of protoxin consisted of at least four proteases with molecular weights of 71, 49, 36, and 30 kDa. All four proteases could utilize casein also as the substrate. When larvae were fed with STI or Bt + STI, the proteolytic activities of the 49-kDa enzyme disappeared, and the activities of the other three enzymes were reduced. Enzyme assays also indicated that feeding larvae with diets containing Bt, STI, or Bt + STI significantly decreased the specific activities of larval general proteases and the trypsin-like enzyme. The protein concentration of midgut fluid was elevated, especially in the larvae fed on the diets containing STI and Bt + STI. Both in vitro and in vivo studies showed that the degradation of protoxin and toxin could be inhibited by soybean trypsin inhibitors, but when the incubation time was prolonged, the protoxin could be degraded completely, while the degradation of toxin was inhibited further. This suggested that the retention time of toxins in the larval midgut was extended and synergism between insecticidal crystal protein and soybean trypsin inhibitor occurred, which showed as the inhibition of H. armigera larval growth.     

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。     

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.     

Digestive proteinases of larvae of the corn earworm, Heliothis zea: characterization, distribution, and dietary relationships.

Proteinases and peptidases from the intestinal tract of fifth-instar larvae of Heliothis (= Helicoverpa) zea (Boddie) (Lepidoptera:Noctuidae) were characterized based on their substrate specificity, tissue of origin, and pH optimum. Activity corresponding to trypsin, chymotrypsin, carboxypeptidases A and B, and leucine aminopeptidase was detected in regurgitated fluids, midgut contents, and midgut wall. High levels of proteinase activity were detected in whole midgut homogenates, with much lower levels being observed in foregut and salivary gland homogenates. In addition, enzyme levels were determined from midgut lumen contents, midgut wall homogenates, and regurgitated fluids. Proteinase activities were highest in the regurgitated fluids and midgut lumen contents, with the exception of leucine aminopeptidase activity, which was found primarily in the midgut wall. Larvae fed their natural diet of soybean leaves had digestive proteinase levels that were similar to those of larvae fed artificial diet. No major differences in midgut proteinase activity were detected between larvae reared under axenic or xenic conditions, indicating that the larvae are capable of digesting proteins in the absence of gut microorganisms. The effect of pH on the activity of each proteinase was studied. The pH optima for the major proteinases were determined to be pH 8.0-8.5 for trypsin, when tosyl-L-arginine methyl ester was used as the substrate; and pH 7.5-8.0 for chymotrypsin, when benzoyl-L-tyrosine ethyl ester was used as the substrate.     

Identification of six chymotrypsin cDNAs from larval midguts of Helicoverpa zea and Agrotis ipsilon feeding on the soybean (Kunitz) trypsin inhibitor

Lepidopteran insects like Helicoverpa zea and Agrotis ipsilon produce STI-insensitive trypsins in the midgut following ingestion of dietary plant proteinase inhibitors like STI [Broadway, R. M., J. Insect Physiol. 43(9) (1997) 855-874]. In this paper, the effects of dietary STI on a related family of midgut serine proteinases, the chymotrypsins, were investigated. STI-insensitive midgut chymotrypsins were detected in larvae of H. zea and A. ipsilon feeding on diets containing 1% STI while STI-sensitive chymotrypsins were present in larvae feeding on diets containing 0% STI. These chymotrypsins were unaffected by TPCK, a diagnostic inhibitor of mammalian chymotrypsins but were fully inhibited by chymostatin. Four midgut cDNA libraries were constructed from larvae of each species fed either 0% STI or 1% STI diets. Six full-length cDNAs(1) encoding diverse preprochymotrypsins were isolated (three from H. zea and three from A. ipsilon) with certain sequence motifs that set them apart from their mammalian counterparts. Northern blots showed that some chymotrypsin mRNA were detected at higher levels while others were down-regulated when comparing insects reared on 0% STI and 1% STI diets. Southern hybridizations suggested that (like mammals) both species contained several chymotrypsin genes. A full-length chymotrypsin gene(1) from H. zea was sequenced for the first time and the presence of four introns was deduced. A first time comparison of 5' upstream regions(1) from three chymotrypsin genes and two trypsin genes of A. ipsilon indicated the presence of putative TATA boxes and regulatory elements. However a lack of consensus motifs in these upstream regions suggested the likelihood of multiple trans factors for regulation of genes encoding digestive proteinases and a complex response mechanism linked to ingestion of proteinase inhibitors.     

大豆胰蛋白酶抑制剂与棉酚或丹宁混用对棉铃虫中肠蛋白酶和生长率的影响

本文报告了大豆胰蛋白酶抑制剂(STl)与棉酚、丹宁酸单一和协同作用对棉铃虫Helicoverpa armigera(Hubner)幼虫中肠蛋白酶活性和生长速率的影响。在离体条件下,STI、棉酚和丹宁酸均对中肠蛋白酶有抑制作用,以STI的作用最强。活体试验表明,人工饲料中0.84%(干重)的S丁I对强碱性类胰蛋白酶活力有显著抑制作用;0.3%丹宁酸则对弱碱性类胰蛋白酶和总蛋白酶活力有显著抑制作用;0.3%棉酚对几种蛋白酶活力的影响均不显著。三者均能显著抑制幼虫的生长,而Sn与棉酚或丹宁酸的协同作用比三者的单独作用更能有效地抑制幼虫的生长发育和中肠蛋白酶活性。     

大豆胰蛋白酶抑制剂与棉酚或丹宁混用对棉铃虫中肠蛋白酶和生长率的影响

本文报告了大豆胰蛋白酶抑制剂(STl)与棉酚、丹宁酸单一和协同作用对棉铃虫Helicoverpa armigera(Hubner)幼虫中肠蛋白酶活性和生长速率的影响。在离体条件下,STI、棉酚和丹宁酸均对中肠蛋白酶有抑制作用,以STI的作用最强。活体试验表明,人工饲料中0.84%(干重)的S丁I对强碱性类胰蛋白酶活力有显著抑制作用;0.3%丹宁酸则对弱碱性类胰蛋白酶和总蛋白酶活力有显著抑制作用;0.3%棉酚对几种蛋白酶活力的影响均不显著。三者均能显著抑制幼虫的生长,而Sn与棉酚或丹宁酸的协同作用比三者的单独作用更能有效地抑制幼虫的生长发育和中肠蛋白酶活性。     

Inhibition of midgut protease of yellow stem borer (<Emphasis Type="Italic">Scirpophaga incertulas</Emphasis>) by cysteine protease-like inhibitor from mature jackfruit (<Emphasis Type="Italic">Artocarpus heterophyllus</Emphasis>) seed

A novel protease inhibitor was isolated and purified from the mature seeds of jackfruit (Artocarpus heterophyllus) by precipitation with ammonium sulphate, followed by DEAE-cellulose and gel filtration (Sephadex G-100) chromatography. The isolated protease inhibitor strongly inhibited papain and midgut proteases of yellow stem borer (Scipophaga incertulas) larvae, as seen by in vitro assay. The purified protease inhibitor was active over a wide range of pH with the maximum activity between pH 4 and 10. This protein was also stable up to 80°C, but the retained activity was lost at 100°C, when heated for 30 min. The molecular mass of the purified cysteine-like protease inhibitor is to be 14.50 kDa as determined by SDS-PAGE. Significant reduction in larval weight and mortality was observed, when fresh rice culms with protease inhibitor was feeded to the yellow stem borer larvae. These results may provide important information to control the yellow stem borer in rice with respect to naturally occurring insecticidal proteins. The observed differences would potentially translate into reductions in population growth of yellow stem borer, indicating a potential value of using jackfruit protease inhibitor for protecting rice plants against damage by the yellow stem borer.     

Larval development and proteolytic activity of Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae) exposed to different soybean protease inhibitors

Anticarsia gemmatalis represents a relevant factor for lowering soybean and other legume crop productivities. Protease inhibitors affect protein degradation and reduce the availability of amino acids, impairing the development and survival of insect pests. To evaluate the possible use of proteinaceous protease inhibitors in the management of this pest, the activities of midgut proteases and the growth and development of A. gemmatalis larvae exposed to soybean Bowman–Birk trypsin-chymotrypsin inhibitor (SBBI) and soybean Kunitz trypsin inhibitor (SKTI) were determined. The survival curves obtained using Kaplan–Meier estimators indicated that SKTI and SBBI stimulated larval survival. However, the development of A. gemmatalis was delayed, and prepupal weight decreased in the presence of both inhibitors. The results showed that SKTI and SBBI inhibited the trypsin-like and total proteolytic activities of larvae on the 12th day after eclosion. On the 15th day after eclosion, larvae exposed to SKTI increased the activities of trypsin and total proteases. Although SKTI and SBBI did not affect the survival of the insect, they had effects on midgut proteases in a stage wherein A. gemmatalis fed voraciously, increased the larval cycle, and decreased prepupal weight. These findings provide baseline information about the potential of proteinaceous protease inhibitors to manage the velvetbean caterpillar, avoiding chemical pesticides.     

Diet-related plasticity of the digestive proteolytic system in larvae of the Colorado potato beetle (Leptinotarsa decemlineata Say)

Quantitative and qualitative changes in digestive proteolytic activities were monitored in fourth-instar larvae of the Colorado potato beetle (Leptinotarsa decemlineata Say) subjected to three different leaf diets. Depending on the diet, the larvae exhibited variable growth rates, similar for potato (Solanum tuberosum) and eggplant (Solanum melongena) diets but lower for the tomato (Lycopersicon esculentum) diet. Interestingly, these growth rates were not associated with total protease activity in the midgut. While growth of tomato-fed insects was negligible, midgut protease activity in these insects was 1.5 and 4.2 times higher than that measured for potato- and eggplant-fed insects, respectively. As seen on gelatin-containing polyacrylamide gels, midgut extracts from insects that ingested eggplant leaves contained only a few proteinase forms, while numerous forms were observed in extracts of potato- and tomato-fed larvae. Although several forms were common to the three diets, their relative importance in the insect midgut varied. This diet-related plasticity of the digestive proteolytic system in Colorado potato beetle larvae leads one to question the potential for control approaches based on the inhibition of digestive proteases. Arch. Insect Biochem. Physiol. 36:241–250, 1997. © 1997 Wiley-Liss, Inc.     

Partial characterization of the proteolytic enzymes in the gut of the banana weevil, Cosmopolites sordidus, and effects of soybean Kunitz trypsin inhibitor on larval performance

The proteolytic enzymes in the gut of the banana weevil, Cosmopolites sordidus (Germar) (Coleoptera: Curculionidae), have been characterized. Both larvae and adults rely on a complex proteolytic system based on at least cathepsin D‐, cathepsin B‐, trypsin‐, chymotrypsin‐, leucine aminopeptidase‐, carboxypeptidase A‐, and carboxypeptidase B‐like activities. All endoproteolytic activities were higher in the anterior section of the gut, whereas the exopeptidases were evenly distributed in the anterior and middle sections, and almost no activity was detected in the posterior section. Gelatin‐containing gels confirmed the spatial organization of the proteolytic digestive process. According to this proteolytic profile, the STI (soybean Kunitz trypsin inhibitor) was tested in vivo to establish its potential as a resistance factor against C. sordidus. Newly hatched larvae fed on diets containing 0.2% (w/w) STI experience lower survival rates and display significant reductions in larval growth. Biochemical analysis carried out on guts of larvae reared on STI‐treated diet showed a reduction of trypsin‐like activity compared to that from larvae fed on control diet. This decrease was compensated with an induction of cathepsin B, whereas cathepsin D, chymotrypsin, and leucine aminopeptidase were not affected. These results are discussed as a basis for selecting appropriate inhibitors to obtain transgenic banana and plantain plants with enhanced resistance to this pest.     

Toxin-binding proteins isolated from yellow mealworm <Emphasis Type="Italic">Tenebrio molitor</Emphasis> and wax moth <Emphasis Type="Italic">Galleria mellonella</Emphasis>

A 67-kDa protein that can specifically bind the activated Cry9A endotoxin under ligand-blotting conditions was purified from midgut epithelium apical membranes of wax moth Galleria mellonella by affinity chromatography. N-Terminal amino acid sequencing enabled identification of this protein as aminopeptidase N. In similar experiments, 66- and 58-kDa proteins specific to endotoxin Cry3A were isolated from the midgut epithelium apical membranes of Tenebrio molitor larvae. Mass spectrometry showed close similarity of the 58-kDa protein to the Tenebrio molitor α-amylase.     

REVISITING RUBISCO AS A PROTEIN SUBSTRATE FOR INSECT MIDGUT PROTEASES

Gene fragments encoding the large subunit (LS) of Rubisco (RBCL) were cloned from various species of host plants of phytophagous Lepidoptera and expressed as recombinant proteins in Escherichia coli. Recombinant RBCLs were compared among each other along with casein and native Rubisco as proteinaceous substrates for measuring total midgut protease activities of fourth instar larvae of Helicoverpa armigera feeding on casein, Pieris brassicae feeding on cauliflower, and Antheraea assamensis feeding on Litsea monopetala and Persea bombycina. Cognate rRBCL (from the pertinent host plant species) substrates performed similar to noncognate rRBCL reflecting the conserved nature of encoding genes and the versatile use of these recombinant proteins. Casein and recombinant RBCL generally outperformed native Rubisco as substrates, except where inclusion of a reducing agent in the enzyme assay likely unfolded the plant proteins. Levels of total midgut protease activities detected in A. assamensis larvae feeding on two primary host species were similar, suggesting that the suite(s) of digestive enzymes in these insects could hydrolyze a plant protein efficiently. Protease activities detected in the presence of protease inhibitors and the reducing agent dithiothreitol (DTT) suggested that recombinant RBCL was a suitable protein substrate for studying insect proteases using in vitro enzyme assays and substrate zymography.     

Protein digestion in larvae of the red oak borer Enaphalodes rufulus

Abstract In the Ozark Mountains of the U.S.A., the red oak borer Enaphalodes rufulus contributes to the destruction of red oaks. To understand nutrient digestion in E. rufulus larvae, digestive proteinases are compared in both larvae fed heartwood phloem and those transferred to artificial diet. The pH of gut extracts is approximately 6.3 in the midgut and foregut and decreases to 5.5 in the hindgut region. The hydrolysis of casein by midgut extracts from E. rufulus larvae fed either artificial diet or phloem from tree sections increases in buffers greater than pH 6.19, with maximum hydrolysis being observed at pH 10.1. Casein zymogram analysis reveals two major proteinase activities in larval midgut extracts of diet‐fed larvae, with molecular masses of approximately 25 and 40–60 kDa, whereas phloem‐fed larvae have proteinase activities corresponding to 40, 45, 60, 80 and >100 kDa. Substrate analysis indicates at least one major trypsin‐like activity in both gut extracts with a molecular mass of >100 kDa, but two chymotrypsin‐like activities of approximately 25 and >200 kDa are found only in diet‐fed larvae. Inhibitors of serine proteinases are most effective in reducing the general proteolytic activity of midgut extracts from larvae fed either food source. The data indicate that serine proteinase inhibitors have the potential to reduce E. rufulus larval damage to oaks. In particular, transgenic technologies incoporating trypsin inhibitors may be effective in reducing protein digestion in phloem‐feeding larvae.