Investigations on cellular defense reactions with Galleria mellonella against Bacillus thuringiensis

In contrast to a great number of foreign particles (bacteria and inorganic materials), cells of some strains of Bacillus thuringiensis are not phagocytosed in the first hours after injection into the hemocoel of Galleria mellonella. This phenomenon is not caused by the production of β-exotoxin or exoenzymes, because heat-killed cells are not phagocytosed and the phagocytosis of latex particles is not prevented by the presence of living B. thuringiensis. The phagocytosis of heat-killed B. thuringiensis subtoxicus can be encouraged by treatment of the cells and by simultaneous injection of latex particles. A factor stimulating the phagocytosis is discussed. It is induced by the injection of phagocytosable latex particles into the hemocoel but not by injection of living or killed B. thuringiensis subtoxicus.     

Immunological activation of phagocytic cells in Galleria mellonella

A phagocytosis-stimulating activity against the normally nonphagocytosable cells of Bacillus thuringiensis subtoxicus develops in the hemolymph of larvae of Galleria mellonella at different periods after injection with readily phagocytosable latex beads. The phagocytosis-stimulating factor can be transferred into new animals with the cell-free hemolymph of treated larvae. It is not detectable in the hemolymph of normal larva but is present in the supernatant of homogenates of the skin. The fractionation of activated hemolymph on Sephadex shows that the active substance has a low molecular weight. It appears to have a biological effect in cellular defense reactions as in the case of lymphokine in vertebrates.     

Fate of Bacillus thuringiensis strains in different insect larvae

In favorable conditions Bacillus thuringiensis spores germinate and vegetative cells multiply, whereas in unfavorable conditions Bacillus thuringiensis sporulates and produces insecticidal crystal proteins. The development of B. thuringiensis strains was investigated in the larvae of insects belonging to the orders Lepidoptera and Diptera. Bacillus thuringiensis strains able to kill the insects did not always multiply in cadavers. Strains with no specificity to kill the insect sometimes multiplied when the insects were killed mechanically. These results indicate that some insect larvae represent an environment that favors the germination of B. thuringiensis spores and the multiplication of vegetative cells; however, there was no correlation between the toxin specificity and the specificity of the host.     

BACILLUS THURINGIENSIS SPECIFIC TO SCARABAEID BEETLES: A REVIEW

Abstract  Since the first report of Bacillus sotto by Ishiwata in 1901, thousands of related papers about Bacillus thuringiensis have been documented. In the field of biocontrol of insect pests by this bacterium, after the initial discovery of several B. thuringiensis isolates specific for lepidopteran insects, the isolation of B. thuringiensis israelensis , specific to dipteran larvae by Goldberg and Margalit, and B. thuringiensis tenebrionis , specific to some group of coleopteran insects by Krieg et al . were epoch making advances. In 1992, Ohba et al . isolated B. thuringiensis ja ponensis strain Buibui, which was specific to only scarabaeid larvae. This isolate is the main target of our discussion in this review. These discoveries by which not only B. thuringiensis sciences, but also applied biological control strategies have been enriched, which inspirit us to screen novel isolates.
On the other hand, the fields of molecular biology and biochemistry studies on the structural elucidation of toxin proteins and mechanism of action have also tremendously progressed. But the complete mechanism has yet to be solved. For instance, interaction between receptor proteins locating on the plasma membrane of insect midgut epithelial cells and insecticidal proteins has not been fully sketched- In a way, this field is still in chaos. Addressing these exciting and enigmatic subjects will eventually lead to the construction of sustainable agriculture in the 21st century.     

对金龟子具有杀虫专一性的苏云金芽孢杆菌综述(英文)

自从 190 1年Ishiwata首次报导猝倒菌以来 ,已有数千篇有关苏云金芽孢杆菌的论文相继发表。在苏云金芽孢杆菌 (Bt)的害虫生物防治领域中首次发现对鳞翅目昆虫有杀虫专一性的Bt菌株以后 ,又有了划时代的进展。Goldberg和Margalit发现了杀双翅目幼虫的以色列亚种 (subsp .isrealensis) ,以及Kreig等人发现了对某些鞘翅目幼虫有毒的拟步行甲亚种 (sbusp .tenebrionis)。 1992年 ,Ohba等分离到了日本亚种(subsp .japonensis)对金龟子有专一性杀虫作用的菌株Buibui。该菌株及与之有关的研究进展即是本文讨论的重点。这些发现不仅丰富了苏云金芽孢杆菌科学 ,也丰富了生物防治的应用策略。另一方面 ,在毒素蛋白结构及其杀虫机制的分子生物学和生物化学领域也已获巨大进展。但对杀虫机制尚未完全明了。例如 ,对杀虫蛋白与中肠上皮细胞原生质膜上的受体蛋白的相互作用就知之甚少。这一领域在某种程度上还是众说纷纭。展论这些令人兴奋而又难解的课题将最终促成二十一世纪可持续农业的建立。     

Extracellular alkaline ribonuclease of Bacillus thuringiensis var. subtoxicus]

Intraspecific selection of Bacillus thuringiensis strains producing extracellular alkaline ribonucleases was carried out. Subtoxicus subspecies with increased expression of the enzyme was detected. A method was developed to isolate preparative amounts of homogeneous extracellular RNase of B. thuringiensis var. subtoxicus. The physico-chemical and catalytic properties of the enzyme was studied and compared with extracellular RNases of others Bacillus species. The conclusion about the structural and evolutional conservation of Bacillus extracellular RNases was drawn.     

Gene knockout demonstrates that vip3A contributes to the pathogenesis of Bacillus thuringiensis toward Agrotis ipsilon and Spodoptera exigua

Vip3A is an 89-kDa protein secreted by Bacillus thuringiensis during vegetative growth. To determine the importance of Vip3A for the insect pathogenicity of B. thuringiensis the vip3A gene was deleted from strain HD1, yielding strain HD1Deltavip3A. Compared with HD1, strain HD1Deltavip3A was one-fourth as toxic to Agrotis ipsilon larvae and less than one-tenth as toxic to Spodoptera exigua larvae. When streptomycin was included in the S. exigua diet the toxicity of HD1Deltavip3A was approximately half that of HD1. Addition of HD1 spores increased the toxicity of purified Cry1 protein more than 600-fold against S. exigua, whereas addition of HD1Deltavip3A spores increased toxicity of Cry1 protein approximately 10-fold. These results demonstrate that an important component of B. thuringiensis insecticidal activity against S. exigua is the synthesis of Vip3A protein by B. thuringiensis cells after ingestion of spores and crystal proteins by insect larvae.     

Stimulation of NO synthase activity in the immune-competent lepidopteran Estigmene acraea hemocyte line.

In contrast to the vertebrate immune system, nearly nothing is known about the immunological role of nitric oxide (NO) in invertebrates. This study provides evidence of the presence of a NO synthase (NOS) activity in an immune-competent, macrophage-like insect hemocyte line, previously established from larvae of the lepidopteran insect Estigmene acraea. As proven by photometric determination of nitroblue tetrazolium reduction after cell fixation, the E. acraea cells possess NADPH diaphorase (NADPHd) activity. This NADPH diaphorase activity was NADPH dependent, not inhibitable by superoxide dismutase, influenced by extracellular addition of L-arginine, and inhibited in a dose-dependent manner by the specific NOS inhibitor Nomega-monomethyl-L-arginine. Furthermore, the NADPH diaphorase activity was stimulated within 30 min by the addition of insect pathogenic bacteria (Bacillus thuringiensis var. kurstaki, Photorhabdus luminescens), bacterial lipopolysaccharide, and silica beads. In activated E. acraea cell suspensions strongly increased amounts of L-citrulline and enhanced levels of total nitrite/nitrate (as NO derivates) can be determined. This is the first report on stimulable NOS activity in insect hemocytes.     

Toxicity of Bacillus thuringiensis and protoplast fusant against Spodoptera litura (F.)

U.S. PUNTAMBEKAR, S.N. MUKHERJEE AND P.K. RANJEKAR. 1995. The octopine-positive tumorogenic intergeneric hybrid (AB0242) derived by protoplast fusion between Agrobacterium tumefaciens and Bacillus thuringiensis was found to be toxic towards the lepidopteran larvae of Spodoptera litura . Twenty-four-hour-grown cells of AB0242 showed 80% mortality, whereas a spore suspension of the parent B. thuringiensis , grown for 72 h, exhibited 93% mortality when tested against the neonate larvae of the test insect.     

Ribonuclease from Bacillus thuringiensis var. subtoxicus. Gene structure and biosynthesis regulation

The gene for extracellular guanyl-specific ribonuclease of Bacillus thuringiensis var. subtoxicus (RNase Bth), a close homologue of the B. intermedius RNase (binase), was completely sequenced. Analysis of nucleotide sequences in the regions adjoining RNase genes revealed an identical organization of the chromosomal loci of RNase Bth and binase. Growth characteristics of the Bacillus thuringiensis var. subtoxicus strain and its synthesis of RNase were studied. It was shown that the exogenous inorganic phosphate inhibits the biosynthesis of RNase. At the same time, actinomycin D in low doses stimulates the enzyme synthesis. Comparative analysis of the influence of inorganic phosphate and actinomycin D on the biosynthesis of RNAse Bth and binase suggests a possibility of coincidence of regulatory pathways of synthesis of these enzymes.     

一个调控抗菌肽表达的小菜蛾肽聚糖识别蛋白(PxPGRP-SA)

【目的】肽聚糖识别蛋白(peptidoglycan recognition proteins,PGRPs)是昆虫免疫系统中一类重要的模式识别蛋白。本研究旨在阐明经苏云金芽孢杆菌Bacillus thuringiensis侵染后,小菜蛾Plutella xylostella PGRP-SA基因(命名为Px PGRP-SA)在体内的表达模式和对抗菌肽基因的表达调控。【方法】本研究利用实时荧光定量PCR(qRT-PCR)技术分析B.thuringiensis侵染小菜蛾幼虫后Px PGRP-SA的转录模式,通过RNAi技术结合抗血清封闭实验检测Px PGRP-SA对小菜蛾抗菌肽基因的表达调控作用。【结果】qRT-PCR检测表明,小菜蛾4龄幼虫在注射具有活性的B.thuringiensis 6 h后,Px PGRP-SA在脂肪体和血细胞中表达量迅速上升,其中脂肪体中的表达量在注射24 h后达到高峰,而在血细胞中的表达量在18 h后达到高峰。RNAi沉默小菜蛾4龄幼虫Px PGRP-SA的转录后,可显著降低小菜蛾脂肪体中cecropin,moricin-2,lysozyme和defensin 4个抗菌肽基因及Dorsal和Sptzle基因的mRNA转录水平;注射anti-Px PGRP-SA封闭小菜蛾体内Px PGRP-SA的活性后,也可降低小菜蛾脂肪体中4个抗菌肽基因的mRNA转录水平;Px PGRP-SA转录沉默后,同时导致添食B.thuringiensis的小菜蛾幼虫的存活率明显降低。【结论】Px PGRP-SA参与了小菜蛾体内抗菌肽cecropin,moricin-2,lysozyme和defensin基因的表达调控,并在免疫防御B.thuringiensis的侵染过程中起了重要的作用。     

Inhibition by theophylline of phagocytosis in Acanthamoeba castellanii.

Theophylline inhibited the phagocytosis of latex beads by Acanthamoeba castellanii (Neff). The cells recovered the ability to engulf beads after 1-2 hr of exposure to theophylline. Cells which have been exposed to 25 mM theophylline for a period of inhibition and recovery were not inhibited further by incubation with a fresh medium containing the same concentration of theophylline. However, the medium in which the cells recovered was as effective as a fresh medium in inhibiting phagocytosis in a fresh batch of cells, suggesting that the development of insensitivity to theophylline inhibition resides with the cells themselves. Dibutyryl cyclic AMP also inhibited bead uptake.     

苏云金杆菌营养期杀虫蛋白的研究

营养期杀虫蛋白 (vegetativeinsecticidalproteins ,VIPs)是苏云金杆菌 (Bacillusthuringiensis,Bt)在对数生长中期分泌的一类新型杀虫毒蛋白。VIPs主要分为VIP1、VIP2和VIP3三种。VIP1和VIP2构成二元毒素 ,对鞘翅目叶甲科的昆虫具有杀虫特异性 ;而VIP3对鳞翅目昆虫具有较广谱的杀虫活性。VIP1和VIP2的杀虫作用机理还不清楚 ;VIP3通过诱发细胞凋亡 ,最终导致昆虫死亡 ,这种作用机理与Bt杀虫晶体蛋白的作用机理完全不同 ,这为筛选新的杀虫活性物质提供了新的思路。vip基因现已被应用于转基因杀虫植物的构建 ,得到高效抗虫的多价转基因玉米。此外 ,VIPs嵌合蛋白的构建、vip及其融合基因导入其它许多宿主微生物等方面的研究也具有诱人的潜在应用前景。     

Fibronectin-coated beads are endocytosed by cells and align with microfilament bundles

Carboxylated latex beads coated with fibronectin bind to the surfaces of NIL8 hamster cells. Similar beads coated with bovine serum albumin, gelatin or normal rabbit immunoglobulin do not bind to the cells, whereas beads coated with polylysine or rabbit anti-hamster immunoglobulin bind, as do fibronectin-coated beads. Surface-bound beads are endocytosed by the cells. This endocytosis is inhibited by N-ethyl maleimide or low temperatures. The endocytosed beads form arrays aligned with the microfilament bundles inside the cells and after extended incubations (18–24 h) become concentrated in the perinuclear region. These results suggest that, after phagocytosis of large particles, the endocytic vesicles align with microfilament bundles, as previously reported for coated pits involved in receptor-mediated endocytosis. The system described here offers possibilities for the analysis of membrane and transmembrane interactions involved in cell-substratum binding and phagocytosis.     

Toxicity of Bacillus thuringiensis Spo Cr Mutants for the European Corn Borer Ostrinia nubilalis

Inclusion bodies isolated from Spo Cr mutants of Bacillus thuringiensis were toxic for larvae of the European corn borer. Probit analysis revealed comparable toxicity between wild-type crystals (isolated from B. thuringiensis subsp. kurstaki) and crystals produced from two spore-free mutants of the same subspecies. Death of the larvae was due to starvation, presumably through delta-endotoxin-induced gut paralysis. Inclusion bodies pretreated with alpha-chymotrypsinogen were equally as toxic as native crystals for the insect larvae.     

Inhibition by Theophylline of Phagocytosis in Acanthamoeba castellanii*

SYNOPSIS. Theophylline inhibited the phagocytosis of latex beads by Acanthamoeba castellanii (Neff). The cells recovered the ability to engulf beads after 1–2 hr of exposure to theophylline. Cells which have been exposed to 25 mM theophylline for a period of inhibition and recovery were not inhibited further by incubation with a fresh medium containing the same concentration of theophylline. However, the medium in which the cells recovered was as effective as a fresh medium in inhibiting phagocytosis in a fresh batch of cells, suggesting that the development of insensitivity to theophylline inhibition resides with the cells themselves. Dibutyryl cyclic AMP also inhibited bead uptake.     

Phagocytic Activity of FRTL-5 Rat Thyroid Follicular Cells as Measured by Ingestion of Fluorescent Latex Beads

A rat thyroid cell line (FRTL-5) was used to study the phagocytic activity of thyroid follicular cells using fluorescent latex beads and flow cytometric analysis. Morphologic studies demonstrated that latex beads were engulfed and located within cytoplasmic vacuoles of thyrocytes. Flow cytometric evaluation of cell suspensions revealed high levels of fluorescence in cells engulfing latex beads. Using thyrotropin (TSH) as a stimulator of thyroid function and human interleukin-1β as an inhibitor, protocols were established for measuring the effects of these substances on either basal or TSH-induced phagocytosis. Cells exposed to latex beads over time in basal (0H) or TSH-containing medium had an increase in time-dependent phagocytic activity which was maximal after 24 or 8 h, respectively. Treatment of FRTL-5 cells with either a stimulator or an inhibitor revealed maximal change in phagocytic activity after 72 h as measured by the percentage of phagocytic cells as well as the mean fluorescence intensity. Phagocytic activity and iodide trapping by FRTL-5 cells were qualitatively similar in both sensitivity and magnitude of change in the assays used in this study. Phagocytosis of fluorescent latex beads represents a sensitive nonradioactive assay of thyrocyte function whose regulation is similar to iodide trapping.