Rice cystatin: bacterial expression, purification, cysteine proteinase inhibitory activity, and insect growth suppressing activity of a truncated form of the protein.

A cDNA clone that encodes oryzacystatin, a cysteine protease inhibitor from rice, was isolated and expressed in Escherichia coli BL-21 (DE3) using an expression plasmid under the control of a T7 RNA polymerase promoter. The construct pT7OC 9b encoded a fusion protein containing 11 amino acid residues of the NH2 terminus of the bacterial protein phi 10 and 79 residues of oryzacystatin lacking 23 NH2-terminal residues of the wild-type protein. Recombinant oryzacystatin (ROC) constituted approximately 10% of the total bacterial protein mass and was purified in a single step by anion-exchange chromatography. The inhibitory activity of ROC toward papain (Ki = 3 x 10(-8) M) was comparable with that of the naturally occurring protein isolated from rice. Caseinolytic activity in midgut homogenates from seven species of stored product insects was inhibited from 18 to 85% by ROC, whereas the same activity was inhibited from 14 to 69% by the serine proteinase inhibitor phenylmethylsulfonyl fluoride. Midguts of stored product insects apparently contain both cysteine proteinases and serine proteinases, but the relative amounts vary with the species. When fed to the red flour beetle, Tribolium castaneum, 10 wt% ROC in the diet suppressed growth approximately 35% relative to that of the control group of insects.     

Effects of proteinase inhibitors on digestive proteinases and growth of the red flour beetle,Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae)

The physiology of the gut lumen of the red flour beetle, T. castaneum, was studied to determine the conditions for optimal protein hydrolysis. Although the pH of gut lumen extracts from T. castaneum was 6.5, maximum hydrolysis of casein by gut proteinases occurred at pH 4.2. The synthetic substrate N-alpha-benzoyl-DL-arginine-rho-nitroanilide was hydrolyzed by T. castaneum gut proteinases in both acidic and alkaline buffers, whereas hydrolysis of N-succinyl-ala-ala-pro-phe rho-nitroanilide occurred in alkaline buffer. Inhibitors of T. castaneum digestive proteinases were examined to identify potential biopesticides for incorporation in transgenic seed. Cysteine proteinase inhibitors from potato, Job's tears, and sea anemone (equistatin) were effective inhibitors of in vitro casein hydrolysis by T. castaneum proteinases. Other inhibitors of T. castaneum proteinases included leupeptin, L-trans-epoxysuccinylleucylamido [4-guanidino] butane (E-64), tosyl-L-lysine chloromethyl ketone, and antipain. Casein hydrolysis was inhibited weakly by chymostatin, N-tosyl-L-phenylalanine chloromethyl ketone, and soybean trypsin inhibitor (Kunitz). The soybean trypsin inhibitor had no significant effect on growth when it was bioassayed alone, but it was effective when used in combination with potato cysteine proteinase inhibitor. In other bioassays with single inhibitors, larval growth was suppressed by the cysteine proteinase inhibitors from potato, Job's tears, or sea anemone. Levels of inhibition were similar to that observed with E-64, although the moles of proteinaceous inhibitor tested were approximately 1000-fold less. These proteinaceous inhibitors are promising candidates for transgenic seed technology to reduce seed damage by T. castaneum.     

Hole density and capture of stored-product insect pests in grain probe traps

The relationship between number of holes in a grain probe trap body and capture of stored-grain pests was determined in laboratory tests using adults of the rice weevil, Sitophilus oryzae (L.), the sawtoothed grain beetle, Oryzaephilus surinamensis (L.), and the red flour beetle, Tribolium castaneum (Herbst). Polyvinylchloride (PVC) probe bodies were attached to electronic sensor heads, and insect captures were recorded electronically using an Electronic Grain Probe Insect Counter (EGPIC) system. In comparisons among PVC probe trap bodies with 60-492 holes, tested at 71 insects per kg in 2.8 kg of soft wheat in cylindrical mini-silos, sawtoothed grain beetle and rice weevil captures were directly related to number of holes in the probe trap body, but there was no relationship for red flour beetle capture. Subsequent tests were conducted comparing sawtoothed grain beetle and rice weevil captures in a PVC probe body with 210 holes over a 40-cm long trapping surface with two commercially available probe traps, a polycarbonate (Lexan) probe trap with 180 holes over a 14-cm long trapping surface and a polyethylene (WBII) probe trap with 750 holes over a 34-cm long trapping surface. The highest percentage capture of both species was in the WBII probe trap, but the 210-hole PVC probe body was as effective as the Lexan probe body for rice weevils and sawtoothed grain beetles at 71 and 17 insects per kg of wheat, respectively.     

Bioactivities of the leaf essential oil of Curcuma longa (var. ch-66) on three species of stored-product beetles (Coleoptera)

Essential oil extracted from the leaves of turmeric, Curcuma longa L., was investigated for contact and fumigant toxicity and its effect on progeny production in three stored-product beetles, Rhyzopertha dominica F. (lesser grain borer), Sitophilus oryzae L. (rice weevil), and Tribolium castaneum Herbst (red flour beetle). Oviposition-deterrent and ovicidal actions of C. longa leaf oil were also evaluated against T. castaneum. The oil was insecticidal in both contact and fumigant toxicity assays. The adults of R. dominica were highly susceptible to contact action of C. longa leaf oil, with LD50 value of 36.71 microg/mg weight of insect, whereas in the fumigant assay, adults of S. oryzae were highly susceptible with LC50 value of 11.36 mg/liter air. Further, in T. castaneum, the C. longa oil reduced oviposition and egg hatching by 72 and 80%, respectively at the concentration of 5.2 mg/cm2. At the concentration of 40.5 mg/g food, the oil totally suppressed progeny production of all the three test insects. Nutritional indices indicate >81% antifeedant action of the oil against R. dominica, S. oryzae and T castaneum at the highest concentration tested.     

Effect of d-limonene on three stored-product beetles

d-Limonene was investigated for contact and fumigant toxicity, ovicidal effects, oviposition-deterrent, development inhibition, and feeding-deterrent activities against three stored-product beetles (Coleoptera): lesser grain borer, Rhyzopertha dominica (F.); rice weevil, Sitophilus oryzae (L.); and red flour beetle, Tribolium castaneum (Herbst). Contact and fumigant toxicity decreased as larvae aged. Contact toxicity was similar for adults of the three species tested, but R. dominica was most susceptible to fumigant activity. T. castaneum oviposition decreased as concentration of d-limonene increased and d-limonene reduced oviposition up to 92.3% at the concentration of 2.14 mg/cm2. Hatching of d-limonene-treated eggs of T. castaneum was reduced by 94.5% with no subsequent larval and adult survival at 2.14 mg/cm2 concentration. A flour disc bioassay indicated 87.7 to 96.8% feeding-deterrency by d-limonene toward all three insect species tested at the highest concentration of 60.0 mg/g food. These results suggest that d-limonene can be effectively used to suppress populations of stored-product beetles.     

Efficacy of ethiprole applied alone and in combination with conventional insecticides for protection of stored wheat and stored corn

The insecticidal pyrazole ethiprole, applied at rates of 7.5 and 10.0 ppm either alone or in combination treatments with deltamethrin, piperonyl butoxide, and chlorpyrifos-methyl, was evaluated as a protectant of stored wheat and stored corn. The commodities were treated with six treatment combinations, including an untreated control, and held for 6 mo at 22, 27, or 32 degrees C and 57% RH. Bioassays were conducted monthly by exposing the rice weevil, Sitophilus oryzae (L.), and the red flour beetle, Tribolium castaneum (Herbst), on treated wheat and the maize weevil, Sitophilus zeamais (Motschulsky), and the red flour beetle on treated corn. The storage temperature of wheat did not significantly affect mortality of exposed insects (P > or = 0.05). All rice weevils were dead after 1 wk in all treatments, and no F1 adults were produced. Mortality of red flour beetles was not dependent on either chemical treatment or bioassay month, and no F1 adults were produced. The storage temperature of corn did not significantly affect mortality of exposed insects (P > or = 0.05). Mortality of maize weevils varied from 77.9 to 100% in all chemical treatments, and no F1 adults were produced. Mortality of red flour beetles was also variable among treatments and bioassay month and no F1 adults were produced. This is the first published report of a study in which pyrazoles have been evaluated against stored-grain insects.     

Effectiveness of spinosad on four classes of wheat against five stored-product insects

Spinosad is a commercial reduced-risk pesticide that is naturally derived. Spinosad's performance was evaluated on four classes of wheat (hard red winter, hard red spring, soft red winter, and durum wheats) against adults of the lesser grain borer, Rhyzopertha dominica (F.); rice weevil, Sitophilus oryzae (L.); sawtoothed grain beetle, Oryzaephilus surinamensis (L.); red flour beetle, Tribolium castaneum (Herbst); and larvae of the Indianmeal moth, Plodia interpunctella (Hübner). Beetle adults (25) or P. interpunctella eggs (50) were exposed to untreated wheat and wheat treated with spinosad at 0.1 and 1 mg (AI)/kg of grain. On all untreated wheat classes, adult beetle mortality ranged from 0 to 6%, and P. interpunctella larval mortality ranged from 10 to 19%. The effects of spinosad on R. dominica and P. interpunctella were consistent across all wheat classes. Spinosad killed all exposed R. dominica adults and significantly suppressed progeny production (84-100%) and kernel damage (66-100%) at both rates compared with untreated wheat. Spinosad was extremely effective against P. interpunctella on all wheat classes at 1 mg/kg, based on larval mortality (97.6-99.6%), suppression of egg-to-adult emergence (93-100%), and kernel damage (95-100%), relative to similar effects on untreated wheats. The effects of spinosad on S. oryzae varied among wheat classes and between spinosad rates. Spinosad was effective against S. oryzae, O. surinamensis and T. castaneun only on durum wheat at 1 mg/kg. Our results suggest spinosad to be a potential grain protectant for R. dominica and P. interpunctella management in stored wheat.     

Chronological age-grading of three species of stored-product beetles by using near-infrared spectroscopy

The accuracy of near-infrared spectroscopy (NIRS) for predicting the chronological age of adults of the rice weevil, Sitophilus oryzae (L.); the lesser grain borer, Rhyzopertha dominica (F.); and the red flour beetle, Tribolium castaneum (Herbst), three pests of stored grain, was examined. NIRS-predicted age correlated well with actual age of these three species. Age predictions in S. oryzae by using the NIRS method are not dependent upon adult sex or temperatures to which adult weevils are exposed. Results indicated that water content decreased with increasing age in rice weevil adults, and excluding wavelengths at which water absorbs NIR radiation reduced the accuracy of correct classification. Additionally, removing cuticular lipids from insects resulted in a significant decrease in classification accuracy of weevils, indicating that these compounds may be partly responsible for the ability of NIRS to differentiate young from old beetles. NIRS is a nondestructive technique that can be used to age-grade large numbers of adult stored-product beetles, information that could help to increase the accuracy of population models for these pest species.     

Response of digestive cysteine proteinases from the colorado potato beetle (Leptinotarsa decemlineata) and the black vine weevil (Otiorynchus sulcatus) to a recombinant form of human stefin A

The effects of the cystatins, human stefin A (HSA) and oryzacystatin I (OCI) on digestive cysteine proteinases of the Colorado potato beetle (CPB), Leptinotarsa decemlineata, and the black vine weevil (BVW), Otiorynchus sulcatus, were assessed using complementary inhibition assays, cystatin-affinity chromatography, and recombinant forms of the two inhibitors. For both insects, either HSA and OCI used in excess (10 or 20 μM) caused partial and stable inhibition of total proteolytic (azocaseinase) activity, but unlike for OCI the HSA-mediated inhibitions were significantly increased when the inhibitor was used in large excess (100 μM). As demonstrated by complementary inhibition assays, this two-step inhibition of the insect proteases by HSA was due to the differential inactivation of two distinct cysteine proteinase populations in either insect extracts, the rapidly (strongly) inhibited population corresponding to the OCI-sensitive fraction. After removing the cystatin-sensitive proteinases from CPB and BVW midgut extracts using OCI- (or HSA-) affinity chromatography, the effects of the insect “non-target” proteases on the structural integrity of the two cystatins were assessed. While OCI remained essentially stable, HSA was subjected to hydrolysis without the accumulation of detectable stable intermediates, suggesting the presence of multiple exposed cleavage sites sensitive to the action of the insect proteases on this cystatin. This apparent susceptibility of HSA to proteolytic cleavage may partially explain its low efficiency to inactivate the insect OCI-insensitive cysteine proteinases when not used in large excess. It could also have major implications when planning the use of cystatin-expressing transgenic plants for the control of coleopteran pests. © 1996 Wiley-Liss, Inc.     

Bioactivities of l-carvone, d-carvone, and dihydrocarvone toward three stored product beetles

The rice weevil, Sitophilus oryzae L., adults were highly susceptible by contact to l-carvone, d-carvone, and dihydrocarvone when compared with the lesser grain borer, Rhyzopertha dominica F., adults and red flour beetle, Tribolium castaneum (Herbst.). Adults of R. dominica were more susceptible than the other species to fumigant vapors of l-carvone, d-carvone, and dihydrocarvone. The three larval stages (14-, 16-, and 18-d-old) of T. castaneum progressively became more susceptible with age toward contact toxicity of three test compounds but in fumigant toxicity, 16-d-old larvae of T. castaneum were more susceptible to the three compounds. Comparison of contact and fumigant toxicity of the test compounds indicates that l-carvone and d-carvone possess 24 times more fumigant toxicity toward adults of R. dominica than its contact toxicity. Overall order of toxicity was l-carvone > d-carvone > dihydrocarvone. Egg hatching and subsequent larval and adult survival of T. castaneum were significantly reduced when the eggs of T. castaneum were treated with l-carvone, d-carvone, and dihydrocarvone. l-Carvone completely suppressed egg hatching at the concentration of 7.72 mg/cm2. Data on feeding-deterrent indices indicate the high potency of l-carvone as feeding-deterrent in order of S. oryzae adults > T. castaneum adults > R. dominica adults > T. castaneum larvae.     

Synthesis of active oryzacystatin I in transgenic potato plants

Summary Transformation of potato (Solanum tuberosum L.) with cysteine proteinase inhibitor (PI) genes represents a potential way of controlling the major insect pest Colorado potato beetle (CPB; Leptinotarsa decemlineata Say). The present study describes the Agrobacterium-mediated transformation of potato (cv. Kennebec) with an oryzacystatin I (OCI) cDNA clone linked to a CaMV 35S promoter. The transgenic plants accumulated active OCI in potato leaves, as demonstrated by the papain-inhibitory activity of transgenic plant leaf extracts. In addition to their anti-papain activity, the extracts also caused a partial but significant inhibition of CPB digestive proteinases, similar to that observed with pure inhibitors. Recombinant OCI did not alter the activity of the major potato leaf endogenous proteinases, which seemed to be of the serine-type. Therefore we suggest that the OCI cDNA can be used for the production of CPB-resistant transgenic potato plants without interfering with endogenous proteinases of these plants.Abbreviations CPB Colorado potato beetle - E-64 trans-epoxy-succinyl-L-leucylamido (4-guanidino) butane - OCI oryzacystatin I - PI proteinase inhibitor - PMSF phenylmethylsulfonyl fluoride     

Compensatory proteolytic responses to dietary proteinase inhibitors in the red flour beetle, Tribolium castaneum (Coleoptera: Tenebrionidae)

Increasing levels of inhibitors that target cysteine and/or serine proteinases were fed to Tribolium castaneum larvae, and the properties of digestive proteinases were compared in vitro. Cysteine proteinases were the major digestive proteinase class in control larvae, and serine proteinase activity was minor. Dietary serine proteinase inhibitors had minimal effects on either the developmental time or proteolytic activity of T. castaneum larvae. However, when larvae ingested cysteine proteinase inhibitors, there was a dramatic shift from primarily cysteine proteinases to serine proteinases in the proteinase profile of the midgut. Moreover, a combination of cysteine and serine proteinase inhibitors in the diet prevented this shift from cysteine proteinase-based digestion to serine proteinase-based digestion, and there was a corresponding substantial retardation in growth. These data suggest that the synergistic inhibitory effect of a combination of cysteine and serine proteinase inhibitors in the diet of T. castaneum larvae on midgut proteolytic activity and beetle developmental time is achieved through the prevention of the adaptive proteolytic response to overcome the activity of either type of inhibitor.     

Digestive proteinases of the larger black flour beetle, Cynaeus angustus (Coleoptera: Tenebrionidae)

Digestion in the larger black flour beetle, Cynaeus angustus (LeConte), was studied to identify new control methods for this pest of stored grains and grain products. The physiological pH of the larval gut, as measured with extracts in water, was approximately 6.1, and the pH for optimal hydrolysis of casein by gut extracts was 6.2 when buffers were reducing. However, under non-reducing conditions, hydrolysis of casein and synthetic serine proteinase substrates was optimal in alkaline buffer. Three major proteinase activities were observed in zymograms using casein or gelatin. Caseinolytic activity of C. angustus gut extracts was inhibited by inhibitors that target aspartic and serine proteinase classes, with minor inhibition by a cysteine proteinase inhibitor. In particular, soybean trypsin and trypsin/chymotrypsin inhibitors were most effective in reducing the in vitro caseinolytic activity of gut extracts. Based on these data, further studies are suggested on the effects of dietary soybean inhibitors of serine proteinases, singly and in combination with aspartic and cysteine proteinase inhibitors, on C. angustus larvae. Results from these studies can be used to develop new control strategies to prevent damage to grains and stored products by C. angustus and similar coleopteran pests.     

Effectiveness of recombinant soybean cysteine proteinase inhibitors against selected crop pests

Three recombinant soybean cysteine proteinase inhibitors (rSCPIs), L1, R1 and N2, were assessed for their potential to inhibit the growth and development of three major agricultural crop pests known to utilize digestive cysteine proteinases: Western corn rootworm (Diabrotica virgifera virgifera, WCR), Colorado potato beetle (Leptinotarsa decemlineata, CPB) and cowpea weevil (Callosobruchus maculatus, CW). In vitro experiments showed that cysteine proteinase activities in the crude gut extracts of the WCR, CPB, and CW were inhibited to various degrees by the three rSCPIs. Of the three rSCPIs tested, N2 was most effective in inhibiting the crude gut extract of WCR, CPB, and CW (50% inhibition at 5 x 10(-8), 5 x 10(-8), and 3 x 10(-7) M, respectively). The L1 was the least potent of the three CPIs tested, with 50% inhibition at 5 x 10(-6) M of the crude gut extracts of WCR. Results of in vivo experiments conducted to assess the effect of the three rSCPIs on the vital growth parameters of WCR, CPB and CW were consistent with results of the in vitro experiments.     

Oryzacystatin I expressed in transgenic potato induces digestive compensation in an insect natural predator via its herbivorous prey feeding on the plant

We observed recently that the rice cysteine proteinase inhibitor, oryzacystatin I (OCI) expressed in transgenic potato does not affect growth and development of the two-spotted stinkbug predator (Perillus bioculatus) via its herbivorous prey feeding on the plant. Here we monitored the inhibitory activity of recombinant OCI along this potato --> herbivore --> predator continuum, to determine if the absence of effect was associated with a digestive compensatory response of the predator following inhibition of its proteinases by the recombinant cystatin. After confirming that OCI is present in the plant, and ingested in an active form by potato beetle larvae, quantitative and electrophoretic assays allowed us to determine that the recombinant cystatin (representing about 0.8% of total soluble proteins in leaves) was entirely bound to a approximately 30-kDa target proteinase in the prey's midgut, forming a sodium dodecyl sulphate (SDS)-stable complex detected on immunoblots with an anti-OCI polyclonal antibody. Despite the apparent absence of free, residual OCI in the beetle's midgut, digestive protease activity in the predator, known to include OCI-sensitive activity, was altered negatively when the prey was fed the modified plant. This inhibitory process at the third trophic level was accompanied by a compensatory response in the predator, by which serine-type proteinases were synthesized de novo. Overall, our data suggest that the affinity between OCI and the predator's OCI-sensitive proteinases is: (i) as strong as (or stronger than) the affinity between OCI and the potato beetle 30-kDa-sensitive proteinase; and (ii) stronger than the affinity between these enzymes and the plant endogenous homologue of OCI, potato multicystatin, induced in the plant by potato beetle feeding. Our results also show that predatory organisms can adapt their digestive metabolism to the presence of plant antidigestive proteins ingested by their herbivorous preys. In a broader context, this study stresses the need to monitor the inhibitory effects of PI-expressing plants not only on the herbivorous insects targeted, but also on the organisms likely to consume these pests in the environment.     

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.     

Effectiveness of spinosad against seven major stored-grain insects on corn

In January 2005, the United States Environmental Protection Agency registered spinosad as a stored grain protectant. No referenced data on the efficacy of spinosad on corn in suppressing major stored-grain insects have been published. In this paper, we evaluated the efficacy of spinosad against seven major stored-grain insects on shelled corn in the laboratory. Insect species tested were the red flour beetle, Tribolium castaneum （Jacquelin duVal）; rusty grain beetle, Cryptolestesferrugineus （Stephens）; lesser grain borer, Rhyzopertha dominica （F.）; sawtoothed grain beetle, Oryzaephilus surinamensis （L.）; rice weevil, Sitophilus oryzae （L.）; maize weevil, Sitophilus zeamais （Motschulsky）; and Indian meal moth, Plodia interpunctella （Htibner）. Corn kernels were treated with spinosad at 0, 0. 1, 0.5, 1, and 2 active ingredient （a.i.） mg/kg for controlling the seven species. Beetle adults or P. interpunctella eggs were introduced into each container holding 100 g of untreated or insecticide-treated corn. The seven insect species survived well on the control treatment, produced 28 to 336 progeny, and caused significant kernel damage after 49 days. On spinosad-treated corn, adult mortality of C. ferrugineus, R. dominica, 0. surinamensis, S. oryzae, and S. zeamais was 〉 98% at 1 and 2 mg/kg after 12 days. Spinosad at≥ 0.5 mg/kg completely suppressed egg-to-larval survival after 21 days and egg-to-adult emergence of P. interpunctella after 49 days, whereas 16% T. castaneum adults survived at 1 mg/kg after 12 days. Spinosad at 1 or 2 mg/kg provided complete or near complete suppression of progeny production and kernel damage of all species after 49 days. Our results indicate that spinosad at the current labeled rate of 1 mg/kg is effective against the seven stored-grain insect pests on corn.     

Two distinct cystatin species in rice seeds with different specificities against cysteine proteinases. Molecular cloning, expression, and biochemical studies on oryzacystatin-II.

Oryzacystatin (oryzacystatin-I) is a proteinaceous cysteine proteinase inhibitor (cystatin) in rice seeds and is the first well defined cystatin of plant origin. In this study we isolated cDNA clones for a new type of cystatin (oryzacystatin-II) in rice seeds by screening with the oryzacystatin-I cDNA probe. The newly isolated cDNA clone encodes 107 amino acid residues whose sequence is similar to that of oryzacystatin-I (approximately 55% of identity). These oryzacystatins have no disulfide bonds, and so could be classified as family-I cystatins; however, the amino acid sequences resemble those of family-II members more than family-I members. Oryzacystatin-I and -II are remarkably distinct in two respects: 1) their specificities against cysteine proteinases; and 2) the expression patterns of their mRNAs in the ripening stage of rice seeds. Oryzacystatin-I inhibits papain more effectively (Ki 3.0 x 10(-8) M) than cathepsin H (Ki 0.79 x 10(-6) M), while oryzacystatin-II inhibits cathepsin H (Ki 1.0 x 10(-8) M) better than papain (Ki 0.83 x 10(-6) M). The mRNA for oryzacystatin-I is expressed maximally at 2 weeks after flowering and is not detected in mature seeds, whereas the mRNA for oryzacystatin-II is constantly expressed throughout the maturation stages and is clearly detected in mature seeds. Western blotting analysis using antibody to oryzacystatin-II showed that, as is the case with oryzacystatin-I, oryzacystatin-II occurs in mature rice seeds. Thus, these two oryzacystatin species are believed to be involved in the regulation of proteolysis caused by different proteinases.     

Susceptibility of last instar red flour beetles and confused flour beetles (Coleoptera: Tenebrionidae) to hydroprene.

Last instar larvae of the red flour beetle, Tribolium castaneum (Herbst), and the confused flour beetle, Tribolium confusum Jacquelin du Val, were either exposed for 8-144 h on concrete treated with 1.9 x 10(-3) mg(AI)/per cm2 hydroprene, or continually exposed on concrete treated with 9.8 x 10(-4) to 1.9 x 10(-3) mg[AI]/per cm2 hydroprene. In both tests, larvae were exposed and held at 27 or 32 degrees C and 40, 57, or 75% RH. When larvae were exposed with no food to hydroprene for different time intervals, then transferred to untreated concrete containing flour, consistent effects were produced only at 144 h. At this exposure interval, the percentage of beetles arrested in the larval stage after 3-4 wk was generally greater at 75% RH compared with 40 and 57% RH, but there were no differences between species or temperature. The percentages of dead adult red flour beetles and live adults with morphological deformities were also greatest at 75% RH, and defects were more prevalent in red flour beetles than in confused flour beetles. When larvae were continually exposed to different concentrations of hydroprene on concrete that contained flour, the percentage of arrested larvae, dead adults, and live adults of both species generally increased with concentration. There were more deleterious effects at 75% RH compared with either 40 or 57% RH, and effects were more pronounced in the red flour beetle compared with the confused flour beetle. In both experiments, temperature effects were variable and inconclusive. Results indicate that continual exposure of last instar red flour beetle and confused flour beetle to hydroprene can limit population development, but exposure intervals of >6 d may be required for maximum effectiveness.     

The amino acid sequence of a 20 kDa bifunctional subtilisin/alpha-amylase inhibitor from bran [correction of brain] of rice (Oryza sativa L.) seeds.

A 20 kDa bifunctional inhibitor of the microbial proteinase, subtilisin, and the alpha-amylase from the larvae of the red flour beetle (Tribolium castaneum) was purified from bran of rice seeds by saline extraction, precipitation with ammonium sulphate, ion-exchange chromatography on DEAE-Cellulose and Toyopearl CM-650, and preparative HPLC on Vydac C18. The complete primary structure was determined by automatic degradation of the intact, reduced and S-alkylated protein, and by manual DABITC/PITC micro-sequencing of peptides obtained from the protein following separate enzymic digestions with trypsin, pepsin, chymotrypsin, elastase and the protease from S. aureus V8. The protein sequence, which contained 176 residues, showed strong homology with similar bifunctional inhibitors previously isolated from wheat and barley which are related to the Kunitz family of proteinase inhibitors from legume seeds.