RNA interference of β1 integrin subunit impairs development and immune responses of the beet armyworm, Spodoptera exigua

Integrin is a cell surface protein that is composed of α and β heterodimer and mediates cell interaction with extracellular matrix or other cells including microbial pathogens. A full length cDNA sequence (2862 bp) of a β1 subunit integrin (βSe1) was cloned from the beet armyworm, Spodoptera exigua. Phylogenetic analysis showed that βSe1 was clustered with other insect β integrin subunits with the highest amino acid sequence identity (98.3%) to β1 of Spodoptera litura. Structural analysis of the deduced amino acid sequence indicated that βSe1 possessed all functional domains known in other insect β1 integrins. RT-PCR analysis showed that βSe1 was expressed in all developmental stages and all tested tissues of S. exigua. Its expression was further upregulated in hemocytes by injections of various microbes from quantitative RT-PCR analysis. Injection of double-stranded βSe1 RNA (dsRNA^βSe1) into late instar S. exigua suppressed βSe1 expression and resulted in significant reduction in pupal weight. The dsRNA^βSe1 injection significantly impaired hemocyte-spreading and nodule formation of S. exigua in response to bacterial challenge. Furthermore, oral ingestion of dsRNA^βSe1 induced reduction of βSe1 expression in midgut and resulted in significant mortality of S. exigua during immature development. These results suggest that βSe1 plays crucial roles in performing cellular immune responses as well as larval development in S. exigua.     

Interactions between the tobacco budworm,Heliothis virescens,and the δ-endotoxin produced by the HD-1 isolate of Bacillus thuringiensis var. kurstaki: Relationship between length of exposure to the toxin and survival

Larvae of the tobacco budworm, Heliothis virescens, which were fed ad libitum for 24, 48, and 72 hr on a diet treated with various levels of the δ-endotoxin produced by the HD-1 isolate of Bacillus thuringiensis var. kurstaki and then transferred to an untreated diet, showed an unexpected capacity to recover from the effects of the toxin, although, as the length of exposure increased, the capacity decreased. Observations on larvae held to emergence indicated that recovery from the toxin was complete. X-ray studies using Ba²⁺ incorporated into the diet showed that, although the toxin paralyzed the midgut of the treated animals, many animals recovered after the toxin was removed, with food once again passing through the gut.     

Lipases in the preparation of β-blockers

β-Adrenergic blocking agents are pharmaceutical products used for the treatment of hypertension and angina pectoris. These β-blockers can be prepared in optically active form by conventional crystallization procedures, by asymmetric chiral synthesis and by using stereoselective enzymes.     

Seasonal abundance of two armyworm species, Spodoptera exitgua (Hübner) and Spodoptera litura (F.) in the Philippines

The two armyworm species Spodoptera exigua (Hübner) and S. litura (F.) are Important pests on several agricultural crops in the tropics. Knowledge about species abundance is important for the development of integrated pest management strategies. During 1998 and 1999 population densities were recorded weekly using pheromone traps at San Leonardo and Munoz (Province Nueva Ecija, 150 km north of Metro Manila) on the Philippine island Luzon. Furthermore, meteorological conditions and natural enemies were observed. Both armyworm species were present year-round at both observation sites. No differences in numbers of male insects trapped in pheromone traps between seasons were found for S. litura. Contrary, numbers of male S. exigua trapped during rainy season was significant higher than during dry season. Rainfall explained between 30 and 40% of trapped moths and has to be considered when monitoring population density with pheromone traps. Parasitism rates were below 5% in both years and for both species and can not explain the high variation observed in population densities.     

Molecular cloning and characterization of the α-glucosidase II from Bombyx mori and Spodoptera frugiperda

The α-glucosidase II (GII) is a heterodimer of α- and β-subunits and important for N-glycosylation processing and quality control of nascent glycoproteins. Although high concentration of α-glucosidase inhibitors from mulberry leaves accumulate in silkworms (Bombyx mori) by feeding, silkworm does not show any toxic symptom against these inhibitors and N-glycosylation of recombinant proteins is not affected. We, therefore, hypothesized that silkworm GII is not sensitive to the α-glucosidase inhibitors from mulberry leaves. However, the genes for B. mori GII subunits have not yet been identified, and the protein has not been characterized. Therefore, we isolated the B. mori GII α- and β-subunit genes and the GII α-subunit gene of Spodoptera frugiperda, which does not feed on mulberry leaves. We used a baculovirus expression system to produce the recombinant GII subunits and identified their enzyme characteristics. The recombinant GII α-subunits of B. mori and S. frugiperda hydrolyzed p-nitrophenyl α-d-glucopyranoside (pNP-αGlc) but were inactive toward N-glycan. Although the B. mori GII β-subunit was not required for the hydrolysis of pNP-αGlc, a B. mori GII complex of the α- and β-subunits was required for N-glycan cleavage. As hypothesized, the B. mori GII α-subunit protein was less sensitive to α-glucosidase inhibitors than was the S. frugiperda GII α-subunit protein. Our observations suggest that the low sensitivity of GII contributes to the ability of B. mori to evade the toxic effect of α-glucosidase inhibitors from mulberry leaves.     

Importance of spores,crystals, and δ-endotoxins in the pathogenicity of different varieties of Bacillus thuringiensis in Galleria mellonella and Pieris brassicae

Physical methods were used to produce spores containing impurities of 0.02–0.05% crystals and crystals containing impurities of 0.001–0.01% spores from cultures of Bacillus thuringiensis. In Galleria mellonella larvae, these preparations from varieties galleriae, aizawai, and wuhanensis were only moderately active compared to 1:1 mixtures of spores and crystals. Spores of an acrystalliferous aizawai mutant were inactive and did not contain a polypeptide of the same size as the potent M_r 138000 δ-endotoxin present in spores and crystals of all three wild-type strains. Thus, this polypeptide probably contributed to the moderate activity of wild-type spores. Spore impurities in the crystal preparation were killed by γ irradiation without harming the crystals. The crystals without live spores were virtually inactive (LC₅₀s, ca. 10¹⁰ crystals/g insect food). Addition of 10³ spores to 10⁸ crystals/g food (0.001% spores) increased the mortality of larvae from 0 to 36%, and addition of 10⁴ spores (0.01% spores) killed 64% larvae. Thus, the addition of low levels of spores increased the potency of crystals in G. mellonella from virtually zero to moderate levels, suggesting that the live spore impurities in the crystal preparations were responsible for the observed moderate potency of crystals before γ irradiation, a view supported by a reduction of potency of crystal preparations following admixture of streptomycin to the insect food. In contrast to the results with G. mellonella, crystals were ca. 30 times as active as spores in Pieris brassicae larvae. Many authors have found crystals purified by physical methods to be highly active in a range of lepidopterous hosts. The present work indicates that the role of the spore impurities in these species may need further investigation. Absence of live spores of B. thuringiensis may impair the control of some insect species feeding on spore-free products and on microorganisms or plants into which endotoxins have been introduced by genetic manipulation.     

Antigenic similarity between the β-subunits of the ATPases of a bacterium,a yeast and a higher plant

Antiserum raised against the β-subunit of wheat (Triticum aestivum) chloroplast ATPase cross-reacts with a 51000 protein located in the membrane fraction of Escherichia coli. The differential solubility of this polypeptide after chloroform treatment of unc⁺ and uncD409 strains indicates that this cross-reacting polypeptide is the bacterial β-subunit of ATPase. Thus a high degree of conservation of antigenic determinant sites exists between a bacterial β-subunit and the β-subunit of a monocot. This conservation also seems to extend to the β-subunit of mitochondrial ATPase of yeast (Saccharomyces cerevisiae).     

Relationship between Poly- β-hydroxybutyrate Production and δ-endotoxin for Bacillus thuringiensis var. kurstaki

A linear relationship between total solid concentration (TSC), δ-endotoxin production [Cry = 0.2795(TSC)−0.2472, R² = 0.8644] and poly-β-hydroxybutyrate (PHB) accumulation [PHB = 0.1327(TSC) + 0.3974, R² = 0.9877] in Bacillus thuringiensis var. kurstaki HD-73 was observed. A similar correlation between δ-endotoxin and PHB accumulation [Cry = 2.1573(PHB)−1.1248, R² = 0.9181] was found. A minimum PHB accumulation of 0.52 mg l⁻¹ was required before the onset of δ-endotoxin production. Revisions requested 28 September 2005 and 4 November 2005; Revisions received 28 October 2005 and 1 February 2006     

Host specificity of the Bacillus thuringiensis δ-endotoxin toward Lepidopteran species: Spodoptera littoralis Bdv. and Pieris brassicae L

Crystals from several strains of Bacillus thuringiensis among the first 10 serotypes were tested for their effectiveness (in terms of LC₅₀ or LD₅₀) in killing Spodoptera littoralis larvae. The δ-endotoxin obtained from the isolates aizawai 7–29 and entomocidus 601 was found to be the most active against S. littoralis; lethal doses were considerably lower than those obtained with the berliner 1715 strain but were nevertheless 10 times higher than that of this last strain against Pieris brassicae. Interestingly, entomocidus crystals were active toward both Lepidopteran species. Protein fractions with molecular mass ranging from 60 to 70 kDa, obtained by dissolving crystals with gut proteases from the two insect species, proved to be active when delivered by intrahemocoel injection. Based on the use of mild detergents such as Triton N-101, sodium cholate, or both, conditions for stabilizing the activity of the solubilized fractions are reported. Under these conditions only, the molecules of toxin thus obtained were as active against S. littoralis larvae as the native crystals, whatever the route of administration and whatever the enzymes used. The same fractions induced different responses in P. brassicae larvae, which manifested a much higher sensitivity to the proteolysis-activated molecules, particularly after intrahemocoel injection, thus suggesting differences between the two insect species as regards the nature of toxic effects. The results clearly illustrate two different patterns in activity spectrum among Lepidopteran species and they also indicate that structural characteristics of the protoxin are predominant factors in determining host specificity.     

Crystallization of α- and β-carotene in the foregut of Spodoptera larvae feeding on a toxic food plant

In the animal kingdom, carotenoids are usually absorbed from dietary sources and transported to target tissues. Despite their general importance, the uptake mechanism is still poorly understood. Here we report the “red crop” phenomenon, an accumulation of ??- and ??-carotene in crystalline inclusions in the enlarged foregut of the polyphagous Spodoptera larvae feeding on some potentially toxic plant leaves. The carotene crystals give the insect foregut a distinctive orange-red color. The crystals are embedded in a homogenous lawn of the bacterium Enterococcus casseliflavus, but the carotene seems to be selectively taken from the food plant. Caterpillars which fail to develop these carotene crystals exhibit a high mortality or fail to develop to adulthood. The crystallization of carotene and the enlargement of the foregut thus appears to manifest a multiple-step physiological adaptation of the insects to toxic food plants.     

Interactions between k-Casein and β-Lactoglobulin: Effects of Anions on Covalent Stabilization of the Complex

Structure breaking anions, trichloroacetate ( — TCA~) and thiocyanate ( — SCN~), significantly increased the interaction between 7c-casein (K-C) and /Mactoglobulin (B-Lg) to give the covalently stabilized K-C/B-Lg tetrameric (A4) complex, whereas “structure making” anions, chloride ( — Cl^-) and especially sulfate ( —SO4), reduced it. The reactivity of K-C with B-Lg in the presence of these anions followed the order, — TCA~ > — SCN~ > — Cl” > — SO4 (100mm). The percentile distribution of the covalent bonded K-C/B-Lg A4 complex after 720 s of heating at 70°C in — TCA~ (lOOmm) was one order greater than it was in — SO^- During storage of the heated K- C/B-Lg mixture, the B-Lg A3/K-C interaction in — TCA~ was rapid and uneffected by the holding temperature, whereas in — SO4, the reaction rate was inversely related to the temperature, being apparently controlled by the relative concentration of monomeric K-C.     

Formation of Multilayers at the Interface of Oil-in-Water Emulsion via Interactions between Lactoferrin and β-Lactoglobulin

The interactions between negatively charged β-lactoglobulin and the positively charged lactoferrin at the droplet surface to form a multi-protein surface layer were examined. Addition of lactoferrin to the aqueous phase of emulsions formed with β-lactoglobulin at pH 7.0 caused an increase in the ζ-potential of emulsion droplets, and the ζ-potential became positive as the concentration of added lactoferrin was higher than 1% in the system. It is found that lactoferrin binds to adsorbed β-lactoglobulin at droplet surface probably via electrostatic interactions. The amount of lactoferrin at interface increased with increasing the concentration of added lactoferrin, but it decreased with a decrease in the pH. No lactoferrin was observed at interface at pH 3 and 4. By contrast, when β-lactoglobulin was added in the emulsions formed with lactoferrin at pH 7.0, the ζ-potential of emulsions changed from positive to negative as the concentration of added β-lactoglobulin increased. The amount of β-lactoglobulin at surface increased correspondingly with increasing the concentration of added β-lactoglobulin. However, in this case, β-lactoglobulin remained bound at interface even at pH 3 and 4 where both lactoferrin and β-lactoglobulin are positively charged. The association of lactoferrin or β-lactoglobulin with the surface proteins that have oppositely charge is probably mainly through electrostatic interactions between the two proteins. It appears that alternative layers of these proteins could be created at the droplet surface.     

δ-endotoxin activity and spore production in batch and fed-batch cultures of Bacillus thuringiensis

Summary The toxicity and the spore count of batch and fed batch cultures of Bacillus thuringiensis var. israelensis were studied. Spore counts reached in both batch and fed batch cultures were as high as those reported in the literature, but the levels of toxicity found in the latter were about one order of magnitude lower than those attained in batch cultures. Avoiding restricted cultures might be necessary to reach high titres of -endotoxin, which are essential if a good product is intended. Furthermore, spore count might not be a good parameter to predict insecticidal activity of Bacillus thuringiensis cultures.     

Protein and carbohydrate moieties of a preparation of β-lactamase II

1. A crystalline preparation of beta-lactamase II has been separated into two moieties by gel filtration on a column of Sephadex G-100. 2. The first moiety consisted mainly of carbohydrate and showed virtually no beta-lactamase activity. 3. The second moiety was a protein of molecular weight 22500, which was enzymically active. 4. The protein moiety, like the original protein-carbohydrate complex, required Zn(2+) for beta-lactamase activity. It did not differ significantly from the complex in its behaviour to a number of cephalosporin substrates, but was less stable to heat than the complex. 5. About 30% of the total beta-lactamase activity was lost when the protein-carbohydrate complex was separated into the two moieties. This activity was regained when the protein and carbohydrate moieties were mixed, but the mixture did not show the heat stability of the original complex.     

Cloning and expression of a β-galactosidase gene of Bacillus circulans

A gene of β-galactosidase from Bacillus circulans ATCC 31382 was cloned and sequenced on the basis of N-terminal and internal peptide sequences isolated from a commercial enzyme preparation, Biolacta(?). Using the cloned gene, recombinant β-galactosidase and its deletion mutants were overexpressed as His-tagged proteins in Escherichia coli cells and the enzymes expressed were characterized.     

Cloning and Expression of a β-Galactosidase Gene of Bacillus circulans

A gene of β-galactosidase from Bacillus circulans ATCC 31382 was cloned and sequenced on the basis of N-terminal and internal peptide sequences isolated from a commercial enzyme preparation, Biolacta^®. Using the cloned gene, recombinant β-galactosidase and its deletion mutants were overexpressed as His-tagged proteins in Escherichia coli cells and the enzymes expressed were characterized.     

Investigation of the Relationship between the Lectin Activity of Azospirilla and Their α-Glucosidase, β-Glucosidase,and β-Galactosidase

The activities of -glucosidase, -glucosidase, and -galactosidase were studied during the isolation and purification of lectins from Azospirillum brasilenseSp7 and Azospirillum lipoferum59b cells. These enzymatic activities were revealed in crude extracts of surface proteins, protein fraction precipitated with ammonium sulfate or ethanol–acetone mixture, and protein fraction obtained by gel filtration on Sephadex G-75. The distribution of the enzymes between different protein fractions varied for the azospirilla studied. The cofunction of the A. brasilenseSp7 lectin and -galactosidase on the cell surface is assumed. A strong interaction between the A. lipoferum59b lectin and glucosidases was revealed. The lectin from A. lipoferum59b may possess saccharolytic activity.