Thuringiensis Serotype Research Articles

<i>Attacus atlas</i> (L.) sericin extract as an effective UV Protectant of <i>Bacillus thuringiensis</i> serotype <i>kurstaki</i> for controlling <i>Spodoptera litura</i> (Fab.)

Bacillus thuringiensis serotype kurstaki is an entomopathogenic bacteria commonly used to control the cutworm Spodoptera litura (Fab.). However, B. thuringiensis has disadvantage of being easily degraded due to exposed sunlight. The objective of this research was to determine the effectiveness of adding A. atlas (L.) cocoon extract as UV protectant B. thuringiensis to the mortality of S. litura. This research formulated 2.5% of the original substance of A. atlas cocoon extract and B. thuringiensis serotype kurstaki strain HD-7 applied from commercial product DiPel-WP®. The formulation was exposed to sunlight for 0, 1, 2, and 3 weeks. The suspension treated for 20 individuals of first instar larvae S. litura shifted into the artificial diet using 3-5 replicates. The scanning electron microscope (SEM) method began from a sample that was vacuumed, sample coated, and observed on SEM with the electron in a certain level probe. This research showed that the mortality of S. litura decreased with the growth of S. litura. The mortality of S. litura achieved 20%-100% mortality after treatments. The A. atlas cocoon extract was effective as UV protectant B. thuringiensis for three weeks of exposure to sunlight. The SEM analysis represented that formulation of B. thuringiensis and A. atlas cocoon extract sunlight exposure for one week has harsher surface than exposed during three weeks.

A Review of the Effects of the Biopesticides Bacillus thuringiensis Serotypes israelensis (Bti) and kurstaki (Btk) in Amphibians

Insecticides are important in agriculture, to reduce human disease, and to decrease the nuisance of biting insects. Despite this, many have the potential for environmental impacts and toxicity in nontarget organisms. We reviewed data on the effects of insecticides based on toxins from Bacillus thuringiensis var. israelensis (Bti) and Bacillus thuringiensis var. kurstaki (Btk) on amphibians. The few peer-reviewed publications that are available for Bti provide variable conclusions, ranging from few observable effects to evidence of acute toxicity at high concentrations. We briefly highlight the current controversies and identify key areas for future investigation.

Evaluation of Susceptibility of Aedes caspius (Diptera: Culicidae) to Insecticides in a Potent Arboviral-Prone Area, Southern Iran.

Background:Southern part of the country is a high risk for mosquito transmitted Arboviruses. This study was carried out to determine the base line susceptibility of the Aedini mosquitoes to the WHO-recommended insecticide.Methods:Larval collection was carried out by dipping method and adult collection occurred by suction tube from January to December 2017. The adult susceptibility test was assessed to Bendiocarb 0.1%, DDT 4%, Deltamethrin 0.05%, Lambda-cyhalothrin 0.05%, Malathion 5% and, Permethrin 0.75% at different interval times as well as at discriminative dose recommended by WHO. The larval susceptibility test was occurred using Temephos and Bacillus thuringiensis serotype H-14, at different concentrations. The LT50, LT90 and LC50, LC90 values were calculated for plotting the regression line using Microsoft office Excel software ver. 2007.Results:Aedes caspius was quite resistant to DDT, Malathion, Bendiocarb and showed susceptible or tolerant to other insecticides.The LT50 and LT90 values to DDT in this species were 157.896, and 301.006 minutes, respectively. The LC50 and LC90 values of Ae. caspius to Temephos were 0.000068, and 0.000130ppm, the figures for B. thuringiensis was 111.62 and 210.2ppm, respectively.Conclusion:A routine and continuous study for monitoring and evaluation of different species of Aedes to insectides is recommend at different parts of country for decision making.

Bacillus thuringiensis (serotype 14) crystal toxins shows non-toxic effects on fingerlings freshwater fish cyprinus carpio against the mosquito larvae.

Bacillus thuringiensis (serotype 14) crystal toxins shows non-toxic effects on fingerlings freshwater fish cyprinus carpio against the mosquito larvae.

A Deep Learning-Based Automatic Mosquito Sensing and Control System for Urban Mosquito Habitats.

Mosquito control is important as mosquitoes are extremely harmful pests that spread various infectious diseases. In this research, we present the preliminary results of an automated system that detects the presence of mosquitoes via image processing using multiple deep learning networks. The Fully Convolutional Network (FCN) and neural network-based regression demonstrated an accuracy of 84%. Meanwhile, the single image classifier demonstrated an accuracy of only 52%. The overall processing time also decreased from 4.64 to 2.47 s compared to the conventional classifying network. After detection, a larvicide made from toxic protein crystals of the Bacillus thuringiensis serotype israelensis bacteria was injected into static water to stop the proliferation of mosquitoes. This system demonstrates a higher efficiency than hunting adult mosquitos while avoiding damage to other insects.

Insights into the Structure of the Spruce Budworm (Choristoneura fumiferana) Genome, as Revealed by Molecular Cytogenetic Analyses and a High-Density Linkage Map.

Genome structure characterization can contribute to a better understanding of processes such as adaptation, speciation, and karyotype evolution, and can provide useful information for refining genome assemblies. We studied the genome of an important North American boreal forest pest, the spruce budworm, Choristoneura fumiferana, through a combination of molecular cytogenetic analyses and construction of a high-density linkage map based on single nucleotide polymorphism (SNP) markers obtained through a genotyping-by-sequencing (GBS) approach. Cytogenetic analyses using fluorescence in situ hybridization methods confirmed the haploid chromosome number of n = 30 in both sexes of C. fumiferana and showed, for the first time, that this species has a WZ/ZZ sex chromosome system. Synteny analysis based on a comparison of the Bombyx mori genome and the C. fumiferana linkage map revealed the presence of a neo-Z chromosome in the latter species, as previously reported for other tortricid moths. In this neo-Z chromosome, we detected an ABC transporter C2 (ABCC2) gene that has been associated with insecticide resistance. Sex-linkage of the ABCC2 gene provides a genomic context favorable to selection and rapid spread of resistance against Bacillus thuringiensis serotype kurstaki (Btk), the main insecticide used in Canada to control spruce budworm populations. Ultimately, the linkage map we developed, which comprises 3586 SNP markers distributed over 30 linkage groups for a total length of 1720.41 cM, will be a valuable tool for refining our draft assembly of the spruce budworm genome.

Comparative Toxicity of <i>Bacillus thuringiensis</i> Berliner Strains to Larvae of Simuliidae (Insecta: Diptera)

Blackflies ( Simulium spp.) are widely distributed and can cause serious economic losses causing annoyance to humans and other animals and damage to health, agriculture and the tourist industry. In addition, they are vectors of diseases eg. onchocerciasis. One alternative to control this insect is the use of biopesticides based on Bacillus thuringiensis ( Bt ) serotype israelensis but other dipteran-active serotypes have not been tested against these insects. This study assessed the toxicity of standard Bt strains belonging to serotypes israelensis , medellin and jegathesan , known to kill dipteran insects, along a collection of Bt strains from Amazonia (a blackfly-endemic area) to find the most effective strains for Simulium control. Ninety-six strains were isolated from soil collected in the Amazon and two of these showed high toxicity against larvae of Simulium spp. The biochemical and molecular characterization of the two strains showed that both produce proteins with molecular weights of 130 kDa, 72 kDa and 30 kDa that are comparable with proteins Cry4A and Cry4B (130 kDa), Cry10 and Cry11 (72 kDa) and Cyt1 and Cyt2 (30 kDa) and corresponding genes cry4A , cry4B , cry10 , cry11 , cyt1 and cyt2 , similar to Bt israelensis , used as standard. These strains also showed similar toxicity to Bt israelensis and higher toxicity than Bt medellin and jegathesan against blackflies.

Mutagenicity of Bacillus thuringiensis exotoxin

The mutagenicity of B. thuringiensis preparations was tested in different mammalian systems. Human blood cultures were used for studying cytological effects on the cells after in vitro treatment with autoclaved supernatant on B. thuringiensis serotype 1 (producer of exotoxin), serotype 3 (non-producer) and purified exotoxin. Bone-marrow cells and cultured blood cells of rats were studied after long-term feeding with varying concentrations of supernatant of serotype 1. Cytological effects of acute exposure to lethal concentrations of exotoxin were studied in the same cell systems. In human blood cultures toxic concentrations of exotoxin induced a significant increase in the incidence of chromosomal aberrations. Also the treatment with supernatant of serotype 3 revealed a slight clastogenic effect, although no mitotic arrest could be observed. In in vivo experiments with rats no clastogenic effects could be demonstrated in bone-marrow cells. Low concentrations of serotype 1 supernatant did not affect blood metaphases of the treated animals either. Clastogenic effects appeared when the drinking water was substituted by 50 or 100% of the agent during three months. Similarly, clastogenic effects were observed in blood cells of rats given lethal doses of serotype 1 supernatant.

Mutagenicity of Bacillus thuringiensis exotoxin III. Sister chromatid exchange in rats in vivo

Sublethal doses of the autoclaved supernatant of exotoxin-producing Bacillus thuringiensis serotype 1 (“AS 1”) were given intraperitoneally to rats, which were killed 12 h later. The sister chromatid exchange (SCE) frequencies in the bone marrow cells of exotoxin-treated animals were within normal ranges (3.1–3.5 per cell); no differences between exotoxin-treated and control animals were observed. Signs of C-mitotic influences were, however, found in cells of the test animals. Previous long-term feeding experiments with exotoxin have been repeated and extended to comprise 6 months. In the bone marrow cells of treated rats, no clastogenic effects were observed. The results of the tests performed, thus, revealed no mutagenic or clastogenic potency of B. thuringiensis exotoxin. Risks of cytogenetic damage caused by exotoxin in amounts normally used to control pest insects are thus considered negligible.

Fingerprinting of Bacillus thuringiensis type strains and isolates by using Bacillus cereus group-specific repetitive extragenic palindromic sequence-based PCR analysis.

A total of 119 Bacillus thuringiensis strains (83 type strains and 26 native isolates), as well as five B. cereus group species, were analyzed by repetitive extragenic palindromic sequence-based PCR analysis (Rep-PCR) fingerprinting. Primers Bc-REP-1 and Bc-REP-2 were specifically designed according to an extragenic 26-bp repeated sequence found in the six B. cereus group genomes reported. A total of 47 polymorphic bands were detected, and the patterns varied from 5 to 13 bands in number and from 0.2 to 3.8 kb in size. Virtually each type strain showed a distinctive B. cereus (Bc)-Rep-PCR pattern, except for B. thuringiensis serovars dakota (H serotype 15 [H15]) and sotto (H4a,4b), as well as serovars amagiensis (H29) and seoulensis (H35), which shared the same patterns. As expected, serovar entomocidus (H6) and its biovar subtoxicus showed an identical pattern; similarly, serovars sumiyoshiensis (H3a,3d) and fukuokaensis (H3a,3d,3e), which share two antigenic determinants, also showed identical Bc-Rep-PCR patterns. Interestingly, serovars israelensis (H14) and malaysiensis (H36), which share several phenotypic attributes, also showed identical Bc-Rep-PCR patterns. Native, coleopteran-active strains, including the self-agglutinated LBIT-74 strain, showed Bc-Rep-PCR patterns identical or very similar to that of the tenebrionis strain. Likewise, native mosquitocidal strains (including some self-agglutinated strains) also showed patterns identical or very similar to that of the serovar israelensis IPS-82 strain. Additionally, native beta-exotoxin-producing strains from serovar thuringiensis showed patterns identical to that of the B. thuringiensis type strain. The B. cereus group-specific Bc-Rep-PCR fingerprinting technique was shown to be highly discriminative, fast, easy, and able to identify B. thuringiensis serotypes, including nonflagellar and self-agglutinated strains.

The patchwork nature of rolling-circle plasmids: comparison of six plasmids from two distinct Bacillus thuringiensis serotypes

Bacillus thuringiensis, the entomopathogenic bacteria from the Bacillus cereus group, harbors numerous extrachromosomal molecules whose sizes vary from 2 to more than 200 kb. Apart from the genes coding for the biopesticide δ-endotoxins located on large plasmids, little information has been obtained on these plasmids and their contribution to the biology of their host. In this paper, we embarked on a detailed comparison of six small rolling-circle replicating (RCR) plasmids originating from two major B. thuringiensis strains. The complete nucleotide sequences of plasmid pGI1, pGI2, pGI3, pTX14-1, pTX14-2, and pTX14-3 have been obtained and compared. Replication functions, comprising, for each plasmid, the gene encoding the Rep-protein, double-strand origin of replication ( dso), single-strand origin of replication ( sso), have been identified and analyzed. Two new families, or homology groups, of RCR plasmids originated from the studies of these plasmids (Group VI based on pGI3 and Group VII based on pTX14-3). On five of the six plasmids, loci involved in conjugative mobilization (Mob-genes and origin of transfer ( oriT)) were identified. Plasmids pTX14-1, pTX14-2, and pTX14-3 each harbor an ORF encoding a polypeptide containing a central domain with repetitive elements similar to eukaryotic collagen (Gly–X–Y triplets). These genes were termed bcol for Bacillus-collagen-like genes.

Extremely high frequency of common flagellar antigens between Bacillus thuringiensis and Bacillus cereus

Bacillus cereus isolates, recovered from natural environments of Japan, were examined for their flagellar (H) antigenicities with the reference H antisera against Bacillus thuringiensis serotypes H1–H55. Of 236 B. cereus isolates tested, 165 (70%) were agglutinated with the reference antisera available. The frequencies of seropositive isolates were: 77% in soils, 68% on phylloplanes, and 60% in animal fecal populations. Among the 45 H serogroups detected, the serovar shandongiensis (H22) was the predominant, followed by the serovars entomocidus (H6), indiana (H16), pakistani (H13), and neoleonensis (H24ab). These five H serovars were commonly distributed in the three populations from different sources.

Extremely high frequency of common flagellar antigens between Bacillus thuringiensis and Bacillus cereus.

Bacillus cereus isolates, recovered from natural environments of Japan, were examined for their flagellar (H) antigenicities with the reference H antisera against Bacillus thuringiensis serotypes H1-H55. Of 236 B. cereus isolates tested, 165 (70%) were agglutinated with the reference antisera available. The frequencies of seropositive isolates were: 77% in soils, 68% on phylloplanes, and 60% in animal fecal populations. Among the 45 H serogroups detected, the serovar shandongiensis (H22) was the predominant, followed by the serovars entomocidus (H6), indiana (H16), pakistani (H13), and neoleonensis (H24ab). These five H serovars were commonly distributed in the three populations from different sources.

Bacillus thuringiensis serotype H34 isolated from human and insecticidal strains serotypes 3a3b and H14 can lead to death of immunocompetent mice after pulmonary infection

In 1995, we isolated a strain of Bacillus thuringiensis serotype H34 from severe human tissue necrosis. This bacterium was able to induce myonecrosis in immunosuppressed mice after cutaneous infection. Its potential pathogenicity for immunocompetent hosts was investigated in a mouse model of pulmonary infection. Mice infected intranasally by a suspension containing 10 8 spores died within 8 h in a clinical toxic-shock syndrome. In the same conditions, infection with a mutant without crystalline toxin, with the supernatant from a culture containing 10 8 bacteria ml −1 and by the insecticidal strain serotypes 3a3b or H14 led to identical results. Lower inocula simply induced a local inflammatory reaction with bacterial persistence observed during the course of 10 days.

Random Amplified Polymorphic DNA Fingerprinting of Mosquito-Pathogenic and Nonpathogenic Strains of Bacillus sphaericus

Random amplified polymorphic DNA fingerprinting was used to examine 31 mosquito-pathogenic and 14 nonpathogenic strains of Bacillus sphaericus. We verified that DNA bands that migrated the same distance in an agarose gel were homologous by using PCR-generated probes made from the random amplified polymorphic DNA bands. The band patterns obtained with eight primers were analyzed by using the Jaccard coefficient and unweighted pair group with arithmetic average clustering. Pathogenic strains belonging to DNA homology group IIA were similar to strains belonging to nonpathogenic homology groups at an average level of similarity of 6.3%. Individual serotypes were clearly identified among the pathogenic strains. This suggests that there is overall genetic homogeneity among strains within serotypes. It is also consistent with the uniform toxicity pattern found for each serotype (unlike the toxin diversity found in Bacillus thuringiensis serotypes). These results, together with DNA homology data, support the proposal that a new species should be described for the pathogenic strains.

Screening by polymerase chain reaction of Bacillus thuringiensis serotypes for the presence of cryV-like insecticidal protein genes and characterization of a cryV gene cloned from B. thuringiensis subsp. kurstaki.

Polymerase chain reaction screening using cryV-specific oligonucleotides, designed to amplify the 5' half of cryV-type genes, revealed the presence of such genes in 7 of 21 Bacillus thuringiensis serotypes examined. Restriction analysis and hybridization studies indicated that these putative genes fall into at least three subclasses. The nucleotide sequence of the cryV-type gene cloned from B. thuringiensis subsp. kurstaki DSIR732 revealed an open reading frame coding for a protein of 719 amino acids, and lysates of Escherichia coli cells expressing the 81.2-kDa CryV732 protein were toxic to Epiphyas postvittana (Lepidoptera: Tortricidae).

Nucleotide sequence of gene cryIIID encoding a novel coleopteran-active crystal protein from strain BTI109P of Bacillus thuringiensis subsp. kurstaki

The nucleotide sequence of a novel insecticidal crystal protein(Cry)-encoding gene from a Bacillus thuringiensis serotype kurstaki isolate is described. The gene is related to the known coleopteran-active cryIII genes and encodes a CryIIID that is much more active against Colorado potato bettle than other CryIII.

Formulation of a flowable liquid concentrate ofBacillus thuringiensisserotype H-14 spores and crystals as mosquito larvicide

Bacillus thuringiensis serotype H-14 spores and crystals, produced in 51 fermenters, were centrifuged and resuspended in emulsified palm olein to give 3.2 x 10(11) colony forming units (cfu)/ml. The suspension was mixed with a cassava-molasses-palm olein-charcoal (CMPC-2) mixture which served as the carrier, adhesive, dispersant and protectant. The final concentration of the formulation was 3.2 x 10(9) cfu/ml. The lethal concentrations capable of killing 50% of the test population (LC50) of CMPC-2 during 0, 1 and 2 years of storage at 32 +/- 4 degrees C were 0.056, 0.058 and 0.058 mg/ml respectively as against 0.054, 0.051 and 0.054 mg/ml for the Institut Pasteur Standard-1978 (IPS-78) during the corresponding period. The chi 2 tests showed that the results were homogeneous at P = 0.05. The relative potencies of the preparations were 964.3, 879.3 and 931 International toxic units (ITU) Aedes aegypti as compared with the 1000 ITU assigned to IPS-78. At 95% confidence limits there was no significant difference between the potencies of CMPC-2 and IPS-78. Field tests showed that CMPC-2 provided between 87.5 and 100% control of natural populations of Aedes spp. and Cutex spp. Sedimentation tests showed that CMPC-2 settled markedly during storage. This, therefore, required that the product be thoroughly shaken before use.

Influence of media composition on the production of δ-endotoxin by Bacillus thuringiensis var. thuringiensis

Powders of edible leguminous seeds, greengram ( Vigna radiata) or soybean ( Glycine max), were used as the major protein source with different combinations of soluble starch and/or cane sugar molasses as the major carbohydrate source for the production of δ-endotoxin by Bacillus thuringiensis var. thuringiensis serotype 1 in submerged fermentation. The primary product (lyophilized with 6 g of lactose) yield was 8.7 to 9.1 g/liter from media with dehusked greengram powder and 9.7 to 10.3 g/liter from media with defatted soybean powder in basal medium. The toxicity of primary products was assayed against fifth-instar Bombyx mori larvae by force-feeding. The primary product from the medium containing defatted soybean powder and soluble starch gave a maximum viable spore count of 91.3 × 10 6/mg, with a corresponding potency of 35,800 IU/mg, whereas the medium containing dehusked greengram powder and cane sugar molasses gave a spore count of 49.5 × 10 6/mg, with a highest potency of 38,300 IU/mg. Either legume protein in combination with cane sugar molasses yielded primary product 2.1 to 2.4 times more potent than the U.S. standard. The combined carbohydrate source consisting of soluble starch and cane sugar molasses, irrespective of the source of protein in the media, drastically reduced δ-endotoxin production, thereby reducing the potency of the primary products compared to the U.S. standard.

Sustained-release formulations ofBacillus thuringiensis serotype H-14 and plastic formulations of Abate for long-term control of mosquito larvae

The larvicidal activity of sustained-release formulations ofBacillus thuringiensis H-14 (Bactimos briquets) and controlledrelease plastic formulations of Abate against mosquito larvae ofAedes aegypti (L.) has been evaluated. The test formulations provided continuous effective control with 90–100% larval mortality for several weeks.