Characterization of an Insecticidal Toxin and Pathogenicity of Pseudomonas taiwanensis against Insects

Pseudomonas taiwanensis is a broad-host-range entomopathogenic bacterium that exhibits insecticidal activity toward agricultural pests Plutella xylostella, Spodoptera exigua, Spodoptera litura, Trichoplusia ni and Drosophila melanogaster. Oral infection with different concentrations (OD = 0.5 to 2) of wild-type P. taiwanensis resulted in insect mortality rates that were not significantly different (92.7%, 96.4% and 94.5%). The TccC protein, a component of the toxin complex (Tc), plays an essential role in the insecticidal activity of P. taiwanensis. The ΔtccC mutant strain of P. taiwanensis, which has a knockout mutation in the tccC gene, only induced 42.2% mortality in P. xylostella, even at a high bacterial dose (OD = 2.0). TccC protein was cleaved into two fragments, an N-terminal fragment containing an Rhs-like domain and a C-terminal fragment containing a Glt symporter domain and a TraT domain, which might contribute to antioxidative stress activity and defense against macrophagosis, respectively. Interestingly, the primary structure of the C-terminal region of TccC in P. taiwanensis is unique among pathogens. Membrane localization of the C-terminal fragment of TccC was proven by flow cytometry. Sonicated pellets of P. taiwanensis ΔtccC strain had lower toxicity against the Sf9 insect cell line and P. xylostella larvae than the wild type. We also found that infection of Sf9 and LD652Y-5d cell lines with P. taiwanensis induced apoptotic cell death. Further, natural oral infection by P. taiwanensis triggered expression of host programmed cell death-related genes JNK-2 and caspase-3.     

Efficient polymer-mediated delivery system for thiocyclam: Nanometerization remarkably improves the bioactivity toward green peach aphids

The unscientific application of synthetic pesticides has brought various negative effects on the environment, hindering the sustainable development of agriculture. Nanoparticles can be applied as carriers to improve pesticide delivery, showing great potential in the development of pesticide formulation in recent years. Herein, a star polymer (SPc) was constructed as an efficient pesticide nanocarrier/adjuvant that could spontaneously assemble with thiocyclam or monosultap into a complex, through hydrophobic association and hydrogen bonding, respectively, with the pesticide-loading contents of 42.54% and 19.3%. This complexation reduced the particle sizes of thiocyclam from 543.54 to 52.74 nm for pure thiocyclam, and 3 814.16 to 1 185.89 nm for commercial preparation (cp) of thiocyclam. Interestingly, the introduction of SPc decreased the contact angles of both pure and cp thiocyclam on plant leaves, and increased the plant uptake of cp thiocyclam to 2.4–1.9 times of that without SPc. Meanwhile, the SPc could promote the bioactivity of pure/cp thiocyclam against green peach aphids through leaf dipping method and root application. For leaf dipping method, the 50% lethal concentration decreased from 0.532 to 0.221 g/L after the complexation of pure thiocyclam with SPc, and that decreased from 0.390 to 0.251 g/L for cp thiocyclam. SPc seems a promising adjuvant for nanometerization of both pure and cp insecticides, which is beneficial for improving the delivery efficiency and utilization rate of pesticides.     

Soaking RNAi-mediated modification of Sf9 cells for baculovirus expression system by ectopic expression of Caenorhabditis elegans SID-1

RNA interference (RNAi) is a biological phenomenon that silences the expression of genes of interest. Passive double-stranded RNA (dsRNA) uptake has been uniquely observed in Caenorhabditis elegans due to the expression of systemic RNAi defective-1 (SID-1). We report that ectopic expression of CeSID-1 endows the Sf9 cells with a capacity for soaking RNAi. Soaking the Sf9-SID1 with dsRNA corresponding to either exogenous or endogenous target genes induced a significant decrease in the amount of mRNA or protein. These results enabled us to modify the target proteins of baculovirus expression vector system in both quantities and posttranslational modifications. The current low-cost and high-efficiency RNAi system is useful for high-throughput gene function analysis and mass production of recombinant protein.     

Identification of lipopeptide xantholysins from Pseudomonas sp. DJ15 and their insecticidal activity against Myzus persicae

Myzus persicae is an important insect pest that reduces crop production worldwide. The use of pesticides for aphid control has generated much concern related to insect resistance and undesirable environmental effects. In an effort to discover new alternatives to counter M. persicae, we found that Pseudomonas isolate DJ15 produced insecticidal metabolites. To isolate the insecticidal metabolites, a cell‐free supernatant of DJ15 was extracted and subjected to bioassay‐guided chromatography. Based on the structures elucidated in instrumental analyses, the metabolites were identified as xantholysins A and B. The metabolites showed strong insecticidal activity against M. persicae with 50% mortality at levels of 13.4 and 24.6 μg/mL for xantholysins A and B, respectively. This is the first study to identify xantholysins as insecticidal metabolites against M. persicae.     

Evaluation of the insecticidal toxicity of various pesticides for Atractomorpha lata (Orthoptera: Pyrgomorphidae) control

The insecticidal activity of 38 commercialized pesticides against Atractomorpha lata nymphs in Korea was studied. Seven pesticides showed greater than 70% insecticidal activity against A. lata nymphs after 48 h of treatment, and similar results were observed for adult A. lata using the spray method. The insecticidal activity of the seven pesticides, except for thiacloprid, against Oxya sinuosa adults was similar to that against A. lata. Clothianidin, tetraniliprole, and thiacloprid showed higher toxicity when plants and insects were treated together than when insects were treated directly, indicating that pesticides have ingestion toxicity against A. lata. Clothianidin and thiacloprid have been observed to have knockdown effects on both A. lata nymphs and adults. Tetraniliprole showed 86.7% mortality until 7 days after treatment in the residual effect test of pesticide exposure under field conditions. Therefore, this study provides basic data for A. lata control; tetraniliprole was found to be an effective insecticide for the control of A. lata nymphs and adults.     

Toxicity,membrane binding and uptake of the <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis> agglutinin (SSA) in different insect cell lines

The fungal lectin purified from Sclerotinia sclerotiorum, further referred to as Sclerotinia sclerotiorum agglutinin or SSA, possesses insecticidal activity against important pest insects such as pea aphids (Acyrthosiphon pisum). This paper aims at a better understanding of its activity at cellular level. Therefore, different insect cell lines were treated with SSA. These cell lines were derived from different tissues and represent the three major orders of insects important in agriculture: CF-203 (midgut Choristoneura fumiferana, Lepidoptera), GUTAW1 (midgut, Helicoverpa zea, Lepidoptera), High5 cells (ovary, Trichoplusia ni, Lepidoptera), Sf9 (ovary cells from Spodoptera frugiperda, Lepidoptera), S2 (hemocyte, Drosophila melanogaster, Diptera), and TcA (whole body, Tribolium castaneum, Coleoptera). Although the sensitivity to SSA differs between the cell lines, SSA clearly showed toxicity in all six cell lines with median effect concentrations (EC₅₀) ranging between 9 and 42 μg/ml. An in-depth analysis of the mechanism of uptake in the cells revealed superior amounts of FITC-SSA at the membrane of CF-203 cells compared to Sf9 cells, while a similar small amount of SSA was internalized in both cell lines. Pre-incubation with the clathrin-mediated endocytosis inhibitor phenylarsine oxide inhibited the internalization of SSA into the CF-203 and Sf9 cells with a respective reduction of 6- and 1.7-fold. The data are discussed in relation to the importance of cellular uptake mechanism for SSA binding and cytotoxicity.     

Lethal and behavioral sublethal side effects of thiamethoxam on the predator Harmonia axyridis

Thiamethoxam (TMX) is one of the most effective neonicotinoid insecticides for the control of green peach aphid, Myzus persicae (Sulzer) (Hemiptera: Aphididae), and various side effects can be expected in its natural enemies. The multicolored Asian lady beetle or harlequin ladybird, Harmonia axyridis (Pallas) (Coleoptera: Coccinellidae), is an important predator against M. persicae in greenhouses and fields. In this study, we evaluated the toxicity of TMX to H. axyridis and the effect on the functional response of this predator to M. persicae through three routes of exposure. Acute toxicity bioassays indicated that LC₅₀ values of TMX on H. axyridis through direct residual contact (‘contact’), systemic application (‘systemic’), and leaf‐dip treatment (‘leaf‐dip’) were 18.99, 21.26, and 15.39 mg a.i. l^?1, respectively. The hazard quotient indicated a potential hazard of this agrochemical regardless of the exposure routes. The mortality caused by the lowest rate, 2 mg a.i. l^?1, was not significantly different compared with the control group. For the three routes of sublethal TMX exposure, the type‐II functional response was a good fit to the prey consumption of H. axyridis. Predation was most affected by leaf‐dip treatment, followed by contact and systemic treatments, which had similar effect. For all exposure routes, the predation capacity of the predator recovered quickly after transfer to untreated cabbage leaves. Thiamethoxam applied systemically was the least toxic to H. axyridis and did not affect the functional response of the predator. However, the sublethal effects of TMX through both contact and leaf‐dip application may reduce the population growth of H. axyridis and consequently impair the biological control of M. persicae by this predator. These results illustrate that the assessment of potential effects of TMX on H. axyridis is crucial to develop effective integrated pest management programs for M. persicae in China.     

Triterpene-based plant defenses

Pentacyclic triterpenes are abundant in the plant kingdom and have a wide array of pharmacological activities. They also have insect antifeedant effects and therefore apparently play a role in plant defense. In this paper, we describe the insecticidal activity of pentacyclic triterpenes of plant origin from different chemical classes on several insect pests (Spodoptera littoralis, Leptinotarsa decemlineata and Myzus persicae), their phytotoxic properties and their selective cytotoxic effects on insect-derived Sf9 and mammalian CHO cells. We also discuss the role they play in plant defense based on these activities.     

Effects of nanostructured essential oils against subterranean termites (Coptotermes gestroi)

Currently, the control of termites is mainly carried out with the use of chemical pesticides, which although effective, pose risks to the environment and to human beings. An alternative to using such chemical pesticides is natural products such as essential oils in insecticidal action. Despite the fact of being effective, essential oils are unstable, poorly soluble in water, and degrade from the action of light, oxygen even at moderate temperatures. Thus, the nanostructuring of essential oils could circumvent such problems and ensure its effectiveness. The following study aimed to assess for the first time the effects of nanostructured systems of essential oils of Cymbopogon flexuosus, Eucalyptus globulus and Melaleuca alternifolia in subterranean termites (Coptotermes gestroi). The results showed that the solid lipid nanoparticles of M. alternifolia have both repellent and insecticide action. The following study showed that nanostructuring of essential oils with insecticidal action is a promising tool in the fight against termites.     

iTRAQ analysis reveals the effect of gabD and sucA gene knockouts on lysine metabolism and crystal protein formation in Bacillus thuringiensis

Lysine metabolism plays an important role in the formation of the insecticidal crystal proteins of Bacillus thuringiensis (Bt). The genes lam, gabD and sucA encode three key enzymes of the lysine metabolic pathway in Bt4.0718. The lam gene mainly affects the cell growth at stable period, negligibly affected sporulation and insecticidal crystal protein (ICP) production. While, the deletion mutant strains of the gabD and sucA genes showed that the growth, sporulation and crystal protein formation were inhibited, cells became slender, and insecticidal activity was significantly reduced. iTRAQ proteomics and qRT-PCR used to analyse the differentially expressed protein (DEP) between the two mutant strains and the wild type strain. The functions of DEPs were visualized and statistically classified, which affect bacterial growth and metabolism by regulating biological metabolism pathways: the major carbon metabolism pathways, amino acid metabolism, oxidative phosphorylation pathways, nucleic acid metabolism, fatty acid synthesis and peptidoglycan synthesis. The gabD and sucA genes in lysine metabolic pathway are closely related to the sporulation and crystal proteins formation. The effects of DEPs and functional genes on basic cellular metabolic pathways were studied to provide new strategies for the construction of highly virulent insecticidal strains, the targeted transformation of functional genes.     

Molecular approaches for identification and construction of novel insecticidal genes for crop protection

Summary The insecticidal cry (crystal) genes from Bacillus thuringiensis (Bt) have been used for insect control both as biopesticides and in transgenic plants. Discovery of new insecticidal genes is of importance for delaying the development of resistance in target insects. The diversity of Bt strains facilitates isolation of new types of cry and vip (vegetative insecticidal protein) genes. PCR is a useful technique for quick and simultaneous screening of Bt strains for classification and prediction of insecticidal activities. PCR together with other methods of analysis such as RFLP, gene sequence determination, electrophoretic, immunological and chromatographic analysis of Cry proteins and insect bioassays for evaluation of toxicity have been employed for identification of new insecticidal proteins. Some other new approaches have also been devised. Many Bt strains with novel insecticidal genes have been found. A desired combination of Cry proteins can be assembled via site-specific recombination vectors into a recipient Bt strain to create a genetically improved biopesticide. For better pest control, the cry genes have been transferred to plants. Stacking of more than one insecticidal gene is required for resistance management in transgenic crops. Modification of Cry proteins through protein engineering for increasing the toxicity and/or the insecticidal spectrum is also a promising approach, but requires detailed understanding of the structure and function of these proteins and analysis of toxin-receptor interactions. More research into this area will provide useful insights for the design of toxins for management of insect resistance. Insecticidal genes from other bacteria and plants are also being examined for their potential for deployment in transgenic crops. Stringent implementation of resistance management is needed for maintaining the efficacy of Bt transgenic crops and deriving maximum economic and environmental benefit.     

Laboratory and field evaluations of the efficacy of a fast-killing baculovirus isolate from Spodoptera frugiperda

Three biopesticide parameters were evaluated for a fast-killing isolate (3AP2) and a wild-type isolate (Sf3) of Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV). Both isolates were evaluated for virus production using in vivo methods, for speed of kill based on bioassay of applications to glasshouse-grown and field-grown plants, and for residual insecticidal activity of unformulated virus and an encapsulating formulation to provide UV protection. Two inoculation rates comparing relative in vivo production of the isolates demonstrated 3AP2 inoculated larvae were significantly smaller than Sf3 inoculated larvae at death. At the lower inoculation rate, Sf3 inoculated larvae produced approximately twofold more occlusion bodies as the 3AP2 inoculated larvae. A model system of applications to cabbage plants and a bioassay to observe mortality of neonate S. frugiperda (J.E. Smith) after feeding on samples of treated leaves was used to evaluate speed of kill and residual insecticidal activity. The LT(50) for the 3AP2 isolate was at least 30 h less than the LT(50) for the Sf3 isolate when applied to either glasshouse-grown or field-grown plants. The spray-dried lignin encapsulating formulation provided similar benefits to both virus isolates when exposed to simulated sunlight in the laboratory and to natural sunlight in the field. For treatment applications to field grown cabbage in June, the half-life for efficacy of unformulated virus was <7.5 h compared with a half-life of >26.7 h for encapsulated virus. These results demonstrate that improved technologies can be combined to address characteristics which otherwise can limit the commercial potential of microbial-based biological insecticides.     

Rapid one-step inactivation of single or multiple genes in Escherichia coli

Gene knockout experiments are frequently performed for both fundamental and applied biological research. We developed an integration helper plasmid-based knockout system for more efficient and rapid engineering of Escherichia coli. The integration helper plasmid, pCW611, contains two recombinases that are expressed in the reverse direction by two independent inducible systems. One is Red recombinase under the control of the arabinose-inducible system to induce a recombination event by using the linear gene knockout DNA fragment, while the other is Cre recombinase, which is controlled by the isopropyl β-D -1-thiogalactopyranoside-inducible system to obtain markerless mutant strains. The time and effort required can be reduced with this system because iterative transformation and curing steps are not required. We could delete one target gene in three days by using pCW611. To verify the usefulness of this system, deletion experiments were performed to knock out four target genes individually (adhE, sfcA, frdABCD, and ackA) and two genes simultaneously for two cases (adhE–aspA and sfcA–aspA). Also, sequential deletion of four target genes (fumB, iclR, fumA, and fumC) was successfully performed to make a fumaric acid producing strain. This successfully developed and validated rapid and efficient gene manipulation system should be useful for the metabolic engineering of E. coli.     

Kinetic Properties of the Channels Formed by the Bacillus thuringiensis Insecticidal Crystal Protein Cry1C in the Plasma Membrane of Sf9 Cells

Spectrofluorimetric measurements were conducted to quantify, in real-time, membrane permeability changes resulting from the treatment of Sf9 insect cells (Spodoptera frugiperda, Lepidoptera) with different Bacillus thuringiensis Cry insecticidal proteins. Coumarin-derived CD222 and Merocyanin-540 probes were respectively used to monitor extracellular K⁺ and membrane potential variations upon Sf9 cells incubation with Cry toxins. Our results establish that Cry1C induces, after a delay, the depolarization of the cell membrane and the full depletion of intracellular K⁺. These changes were not observed upon Sf9 cells treated with Cry1A family toxins. Both the rate of the K⁺ efflux and the delay before its onset were dependent on toxin concentration. Both parameters were sensitive to temperature but only the delay was affected by pH. Cry1C-induced K⁺ efflux was inhibited by lanthanum ions in a dose-dependent manner. This study provides the first kinetic and quantitative characterization of the ion fluxes through the channels formed by a Cry toxin in the plasma membrane of a susceptible insect cell line. Received: 4 October 1999/Revised: 21 December 1999     

Validation of reference genes for quantitative real-time polymerase chain reaction assay of honeybee under various pesticide treatment conditions

In quantitative real-time polymerase chain reaction (qRT-PCR), target gene expression levels are normalized to internal reference gene(s) that are stably expressed across different conditions to determine whether they are up- or down-regulated. Therefore, it is essential to select appropriate reference gene(s) for the accurate comparison of target gene expression across different experimental conditions. Honeybee colonies can be damaged due to pesticide exposure, resulting in a decline of their population. Determination of gene expression levels is important for understanding the physiological response of honeybees to pesticide exposure. Therefore, in this study, we used qRT-PCR to analyze the expression stability of five candidate reference genes (RPS5, RPS18, GAPDH, ARF1, and RAB1a) in honeybees subjected to treatment with different dosages and exposure durations of seven pesticides (acetamiprid, imidacloprid, flupyradifurone, fenitrothion, carbaryl, amitraz, and bifenthrin) using four programs (geNorm, NormFinder, BestKeeper, and RefFinder). Subsequently, the expression levels of the target genes (PER, FOR, and EGR1) were calculated using different normalization methods and compared. Based on our collective results, we propose RPS5 as the most appropriate reference gene for the normalization of target gene expression levels in qRT-PCR assays for honeybees under various conditions of pesticide exposure, including pesticide type, exposure duration, and concentration.     

Evaluation of Nostoc Strain ATCC 53789 as a Potential Source of Natural Pesticides

The cyanobacterium Nostoc strain ATCC 53789, a known cryptophycin producer, was tested for its potential as a source of natural pesticides. The antibacterial, antifungal, insecticidal, nematocidal, and cytotoxic activities of methanolic extracts of the cyanobacterium were evaluated. Among the target organisms, nine fungi (Armillaria sp., Fusarium oxysporum f. sp. melonis, Penicillium expansum, Phytophthora cambivora, P. cinnamomi, Rhizoctonia solani, Rosellinia, sp., Sclerotinia sclerotiorum, and Verticillium albo-atrum) were growth inhibited and one insect (Helicoverpa armigera) was killed by the extract, as well as the two model organisms for nematocidal (Caenorhabditis elegans) and cytotoxic (Artemia salina) activity. No antibacterial activity was detected. The antifungal activity against S. sclerotiorum was further studied with both extracts and biomass of the cyanobacterium in a system involving tomato as a host plant. Finally, the herbicidal activity of Nostoc strain ATCC 53789 was evaluated against a grass mixture. To fully exploit the potential of this cyanobacterium in agriculture as a source of pesticides, suitable application methods to overcome its toxicity toward plants and nontarget organisms must be developed.     

Isolation and characterisation of an aryl-β-D-glucoside uptake and utilisation system ( abg ) from the gram-positive ruminal Clostridium species C. longisporum

A phosphotransferase-dependent aryl-β-glucoside uptake and utilisation system (abg) was isolated from the ruminal Clostridium (“C. longisporum”). The system is composed of three genes, abgG, abgF and abgA, and a number of regulatory regions, including terminator/antiterminator type stem-loop structures preceding the abgG and abgF genes. Similarity analysis of the proteins encoded by these genes indicated that they were responsible for the regulation of the abg system through antitermination (AbgG), the uptake and phosphorylation of aryl-β-glucosides (AbgF) and the hydrolysis of the intracellular phosphorylated glycosides (AbgA). Experimental evidence for the functions of AbgF and AbgA was obtained. Although it was not possible to demonstrate any function for AbgG, a promoter 5′ to the abgG gene was identified which was responsible for expression of the downstream genes. The abg system is remarkably similar to operons from the gram negative Enterobacteriaceae, both in the coding and non-coding regulatory regions. Received: 3 April 1997 / Accepted: 8 September 1997