Bacillus Thuringiensis Subspecies Israelensis Research Articles

EVALUATION OF THERMAL FOGGER FOR EFFECTIVENESS OF LARVICIDE-ADULTICIDE MIXTURE AGAINST AEDES AEGYPTI

A hand-held thermal fogger was evaluated to spray a mixture of adulticide (Aqualuer 20-20® containing permethrin and piperonyl butoxide) and a biological larvicide (VectoBac®12AS, containing Bacillus thuringiensis subspecies israelensis). Adulticide was applied at maximum label rate of 37 mL/ha while larvicide rates were 0.58, 1.76, 2.64, and 3.50 L/ha (Maximum label rate 2.34 L/ha). Two rows, 7.6 m apart, of bioassay cages containing susceptible 25, 5-7 days old female Aedes aegypti were used to assess effectiveness of the adulticide. Along with each cage were two 473 mL empty plastic cups at 0 and 1.3 m above ground to collect the larvicide. Two control cages and two empty cups were placed upwind in the field for five minutes and removed before the spray. The study was conducted in the evening with five separated replications of a rate. The exposed cages and cups were retrieved from the field and brought back to the laboratory for further observations and tests to determine mortality at 24 and 48 h, respectively. All the adults in all treatment cages and all tests died within 24 h of the spray application. In the lab, larval mortality was assessed by introducing Ae. aegypti larvae, where mortality decreased with increasing distance from the spray line and increased with increasing application rate. The larval mortality at all distances was strongly correlated with application rate (R2 = 0.98). The highest rate used produced 64 -95% larval mortality at all distances. The results indicate that Aqualuer 20-20 and VectoBac 12AS can be applied simultaneously as a mixture achieving complete adult control and considerable larval control.

Investigation of low-cost media for Bacillus thuringiensis subspecies israelensis

Bacillus thuringiensis subspecies israelensis (Bti) has been used as a mosquito control agent in agricultural field and residences. The production of Bti by conventional medium was expensive. The objective of this study was to investigate low-cost media for Bti’s production with a cost analysis of the medium formulated. Soybean Casein Digest Medium (TSB) was used as a conventional medium to compare with Saberi medium at room temperature (approximately 21.5˚C) on static and agitation (by obituary shaker at 100 rpm). Saberi medium was also modified to vary the percentage of nitrogen and carbon source to observe the growth rate and sporulation, compared to the Saberi medium. The growth of Bti in TSB with agitation for 24 hours yielded the highest growth rate. Sporulation of Bti in TSB with agitation at room temperature was 6 days which was faster than Bti in the Saberi medium. Modified Saberi nitrogen source and carbon source medium could not support the growth of Bti due to an imbalance in the percentage of nitrogen source and carbon source. Determination of sugar utilization showed sugar in the medium was utilized mostly in the exponential phase of Bti, from 3 hours to 6 hours. The price of TSB was 76.80 Thai baht per liter while Saberi medium’s cost was 20.43 Thai baht per liter. In this study, the Saberi medium may not be able to replace TSB in terms of growth rate and period of sporulation. Further study will be needed to optimize the media that use agro-industrial wastes as ingredients following the concept of circular economy and sustainability.

Combining DL-Methionine and Bacillus thuringiensis Subspecies israelensis: Prospects for a Mosquito Larvicide.

Simple SummaryWith the increasing threat that mosquito borne diseases pose to public health, the demand for environmentally sustainable pesticides has been increasing in recent years. Pesticides that target the larval stage (i.e., larvicides) are particularly useful for controlling mosquito populations as they strike at the source. Currently, Bacillus thuringiensis subspecies israelensis (BTI) is a commonly used mosquito larvicide but some studies show signs of resistance development. DL-methionine is an essential amino acid that has mosquito larvicidal capabilities, while also having minimal negative effects on non-target organisms in laboratory experiments. In this study, our objective was to evaluate the effect of these two larvicides individually and together at reducing mosquito survival. We found that while DL-methionine was more toxic to Anopheles quadrimaculatus than Aedes aegypti, the opposite was true for BTI. Additionally, when the combination was tested against An. quadrimaculatus larvae at higher concentrations the active ingredients were complementary and the effect was equal to both ingredients alone. However, the active ingredients were antagonistic when tested against Ae. aegypti larvae. These findings are important as they show the potential for DL-methionine and the combination of DL-methionine with BTI to be used as a larvicide against Anopheles mosquitoes, which are responsible for transmitting malaria.Mosquito larvicides can reduce mosquito populations at the source, potentially decreasing biting rates and pathogen transmission. However, there is a growing need for mosquito larvicides that are environmentally sustainable. Bacillus thuringiensis subspecies israelensis (BTI) is a naturally occurring bacterium commonly used as a larvicide to manage mosquito populations. Methionine is an essential amino acid that has demonstrated toxic properties against larval mosquitoes in laboratory experiments, while having minimal effects on non-target organisms. The goal of this study was to evaluate the potential for a novel combination larvicide by testing for compatibility between these two active ingredients. We began by determining the lethal concentration values (LCs) of BTI and DL-methionine against Anopheles quadrimaculatus Say and Aedes aegypti Linnaeus (Diptera: Culicidae) larvae. These bioassays were conducted in glass jars and mortality was observed 48 h post-treatment. We found that while DL-methionine was more toxic to An. quadrimaculatus than Ae. aegypti, the opposite was true for BTI. Then, we used these LCs to conduct bioassays with a combination of BTI and DL-methionine to determine the relationship between the two active ingredients when used against An. quadrimaculatus and Ae. aegypti larvae. The findings of this study demonstrate that BTI and DL-methionine have the potential to be complementary due to their additive properties at higher concentrations and effect levels when tested against An. quadrimaculatus. However, an antagonistic relationship was detected at the concentrations tested with Ae. aegypti. These results are encouraging and imply that a DL-methionine or BTI/DL-methionine combination larvicide could be used in management of Anopheles species.

The Challenges of Microbial Control of Mosquito-Borne Diseases Due to the Gut Microbiome.

Mosquitoes are one of the deadliest animals on earth because of their ability to transmit a wide range of human pathogens. Traditional mosquito control methods use chemical insecticides, but with dwindling long-term effectiveness and negative effects on the environment, microbial forms of control have become common alternatives. The insecticide Bacillus thuringiensis subspecies israelensis (Bti) is the most popular of these alternatives, although it can also have direct effects on lowering environmental biodiversity and indirect effects on food-web relationships in the ecosystems where it is deployed. In addition, microbial control agents that impede pathogen development or transmission from mosquito to human are under investigation, including Wolbachia and Asaia, but unexpected interactions with mosquito gut bacteria can hinder their effectiveness. Improved characterization of mosquito gut bacterial communities is needed to determine the taxa that interfere with microbial controls and their effectiveness in wild populations. This mini-review briefly discusses relationships between mosquito gut bacteria and microbial forms of control, and the challenges in ensuring their success.

A long-lasting biological larvicide against the dengue vector mosquito Aedes albopictus.

Aedes albopictus is the primary vector of mosquito-borne diseases, including dengue and chikungunya, in China. The management of vector mosquitoes is the primary strategy for the control of such infectious diseases. The gravid Ae. albopictus prefers to skip-oviposit its eggs into different small water containers, and the management of these breeding places is critical for mosquito control. Bacillus thuringiensis subspecies Israelensis (Bti) is a useful biological larvicide, but the effective period of the currently available commercial product is relatively short. This study aimed to develop a long-lasting formulation of Bti to control the dengue vector mosquito Ae. albopictus. Water-soluble polyethylene glycols and water-insoluble hexadecanol were mixed with Bti to develop the long-lasting formulation Bti-BLOCK, based on the solid dispersion technique. The controlled release of Bti-BLOCK and its effect on Ae. albopictus were assayed in the laboratory and in the field. The results showed that Bti toxins were slowly released from Bti-BLOCK into the water and maintained at an effective dose for at least 6 months. Bti-BLOCK caused high mortality during the immature stage (>90%) and achieved full inhibition during pupation (100%). The efficacy lasted at least 12 weeks in the laboratory and 6 weeks in the field. Furthermore, we confirmed an 89% reduction in Ae. albopictus density and a reduction in the R0 of dengue to a low-risk level after 6 months of open-field interventions. We developed a long-lasting biological larvicide, Bti-BLOCK, which displayed very good efficacy in the control of the dengue vector mosquito Ae. albopictus.

The persistence and ecological impacts of a cyanobacterium genetically engineered to express mosquitocidal Bacillus thuringiensis toxins.

BackgroundThe cyanobacterium Anabaena PCC 7120#11 has been genetically engineered to act as a delivery vehicle for Bacillus thuringiensis subspecies israelensis mosquitocidal toxins. To address ecological concerns about releasing this genetically engineered microorganism into the environment for mosquito larva control, the persistence and ecological impacts of PCC 7120#11 was evaluated using multi-species, standardized aquatic microcosms.MethodsThe microcosms were set up as described in ASTM E1366-02 (Standard Practice for Standardized Aquatic Microcosms: Fresh Water), with a few modifications. The treatment group microcosms were inoculated with PCC 7120#11 and key water quality parameters and non-target effects were compared between the treatment and control groups over a period of 35 days.ResultsPCC 7120#11 decreased from a concentration of 4.50 × 106 cells/ml (at inoculation) to 1.32 × 103 cells/ml after 4 weeks and larvicidal activity against third instar larvae of Anopheles arabiensis was only evident for two weeks after treatment. Both treatment and the interaction of treatment and time had a significant effect on nitrate, phosphate and photosynthetic microorganism concentrations. Treatment with PCC 7120#11 caused a temporary spike in ammonia in the microcosms a week after treatment, but the concentrations were well below acute and chronic criteria values for ammonia in freshwater ecosystems. Cyprinotus vidua concentrations were not significantly different between PCC 7120#11 and control microcosms. In PCC 7120#11 microcosms, Daphnia pulex concentrations were significantly lower than control concentrations between days 18 and 25. By the end of the experiment, none of the measured variables were significantly different between the treatment groups.ConclusionsThe standard aquatic microcosm experiments provided more data on the ecological impacts of PCC 7120#11 than single-organism assessments would have. On the basis of the relatively minor, short-term effects that PCC 7120#11 had on water quality parameters and non-target invertebrates, further evaluation of PCC 7120#11 for use in integrated vector management is warranted.

Evidence of Formosan Subterranean Termite Group Size and Associated Bacteria in the Suppression of Entomopathogenic Bacteria, Bacillus thuringiensis subspecies israelensis and thuringiensis

Abstract The Formosan subterranean termite, Coptotermes formosanus Shiraki, was studied for its ability to suppress two entomopathogenic bacteria, Bacillus thuringiensis subspecies israelensis (Bti) and thuringiensis (Btt). Different group sizes (50, 25, 10, and no termites [control]) of C. formosanus were placed on well-grown Bti or Btt agar plates. On day 1, the diameters of Bti and Btt colonies in the three treatments containing termites were significantly smaller than in the controls. The diameters of Bti and Btt colonies in the 50-termite treatment were significantly smaller than in the 10-termite treatment. However, neither was significantly different from the 25-termite treatment. This group sized dependent suppression was even more distinct on day 2. On day 5, inhibitory zones were observed in all three treatments containing termites where Bti or Btt colonies originally grew. The Bti and Btt cells from these inhibitory zones regenerated on new plates after transfer from 25- and 10-termite treatments as did the controls, but no regeneration was observed after transfer from 50-termite treatment. Results show that the presence of C. formosanus can suppress the growth of Bti and Btt and the suppression effect enhanced with increased of group size. Moreover, antagonistic tests show that natural bacteria carried by termites play a role in the suppression of Bti and Btt.

Activity of a Brazilian Strain of Bacillus thuringiensis israelensis Against the Cotton Boll Weevil Anthonomus grandis Boheman (Coleoptera: Tenebrionidae)

A Brazilian Bacillus thuringiensis subspecies israelensis, toxic to Diptera, including mosquitoes, was found also to show toxicity to the coleopteran boll weevil Anthonomus grandis Boheman at an equivalent level to that of the standard coleopteran-active B. thuringiensis subspecies tenebrionis T08017. Recombinant B. thuringiensis strains expressing the individual Cyt1Aa, Cry4Aa, Cry4Ba and Cry11Aa toxins from this strain were assessed to evaluate their potential contribution to the activity against A. grandis, either alone or in combination. Whilst individual toxins produced mortality, none was sufficiently potent to allow calculation of LC50 values. Combinations of toxins were unable to attain the same potency as the parental B. thuringiensis subsp. israelensis, suggesting a major role for other factors produced by this strain.

Combined molecular dynamics and continuum solvent studies of the pre-pore Cry4Aa trimer suggest its stability in solution and how it may form pore

Cry4Aa toxin is one of the highly specific mosquito-larvicidal proteins produced by the bacterium Bacillus thuringiensis subspecies israelensis. It is thought to form pores in the larval midgut membrane that cause membrane leakage and subsequent insect death. Therefore, Cry4Aa and other Cry toxins have been used as efficient and safe bacterial insecticides to control the disease-carrying mosquitoes such as Aedes, Anopheles, and Culex. However, we still do not clearly understand how Cry toxins kill mosquito-larvae at molecular details. Recent electron crystallographic images of Cry4Ba toxin, another toxin closely related to Cry4Aa toxin, have suggested that the protein forms trimer in aqueous solution and in lipid monolayer. Moreover, the unit cell of X-ray crystal structure of Cry4Ba toxin has been shown to be trimeric. In this study, we constructed the first full-atom structural model of Cry4Aa trimer using the trimeric unit cell structure of Cry4Ba toxin as a template and then used the methods of molecular dynamics (MD) and molecular mechanics combined with Poisson-Boltzmann and surface area (MM-PBSA) to show that the trimeric structure of Cry4Aa toxin is stable in 150 mM KCl solution on 10 ns timescale. The results reveal that Cry4Aa toxins use polar amino acid residues on α-helices 3, 4, and 6 to form trimer and suggest that the proteins form trimer to reduce their non-polar interactions with surrounding water. Based on the obtained trimeric structure of Cry4Aa toxins, we propose that pore formation of Cry toxins may involve a 90°-hairpin rotation during the insertion of their three α4-α5 hairpins into the membrane. This process may be mediated by water and ions.PACS Codes: 87.15.ap, 87.15.bk, 87.14.ep

Specific Targeting to Murine Myeloma Cells of Cyt1Aa Toxin from Bacillus thuringiensis Subspecies israelensis

Multiple myeloma is currently an incurable cancer of plasma B cells often characterized by overproduction of abnormally high quantities of a patient-specific, clonotypic immunoglobulin "M-protein." The M-protein is expressed on the cell membrane and secreted into the blood. We previously showed that ligand-toxin conjugates (LTC) incorporating the ribosome-inactivating Ricin-A toxin were very effective in specific cytolysis of the anti-ligand antibody-bearing target cells used as models for multiple myeloma. Here, we report on the incorporation of the membrane-disruptive Cyt1Aa toxin from Bacillus thuringiensis subsp. israelensis into LTCs targeted to murine myeloma cells. Proteolytically activated Cyt1Aa was conjugated chemically or genetically through either its amino or carboxyl termini to the major peptidic epitope VHFFKNIVTPRTP (p87-99) of the myelin basic protein. The recombinant fusion-encoding genes were cloned and expressed in acrystalliferous B. thuringiensis subsp. israelensis through the shuttle vector pHT315. Both chemically conjugated and genetically fused LTCs were toxic to anti-myelin basic protein-expressing murine hybridoma cells, but the recombinant conjugates were more active. LTCs comprising the Cyt1Aa toxin might be useful anticancer agents. As a membrane-acting toxin, Cyt1Aa is not likely to induce development of resistant cell lines.

Fine Mapping and DNA Sequence of Replication Functions of Bacillus thuringiensis Plasmid pTX14-3

pTX14-3 is a 7.5-kb cryptic plasmid isolated from a Bacillus thuringiensis subspecies israelensis strain. Like many other small plasmids in gram-positive bacteria, pTX14-3 replicates via a single-stranded DNA intermediate. The nucleotide sequence of the replication region was determined and an open reading frame of 636 base pairs encoding a protein necessary for plasmid replication was identified by deletion analysis. No significant homology was found between this open reading frame and those encoding replication proteins identified on other plasmids isolated from gram-positive bacteria, nor could we find any homology to plus origins from other single-stranded DNA plasmids. Consequently, it seems that the replicon of pTX14-3 belongs to a new family of replicons in the group of single-stranded DNA plasmids. The sequence of the single-strand origin (i.e., the minus origin) responsible for the conversion of single-stranded plasmid DNA to double-stranded plasmid DNA was also determined. A partial homology between the minus origin of pTX14-3 and the Bacillus subtilis plasmid pBAA1 was identified. A previously identified locus that suppresses formation of high molecular weight multimers was also minimized and sequenced.

Structural features of crystal-forming proteins produced by Bacillus thuringiensis subspecies israelensis

Entomocidal crystals produced by Bacillus thuringiensis ssp. israelensis are formed by two proteins with molecular masses of 130 and 28 kDa, whereas the protein with a molecular mass of 70 kDa appears as a result of 130 kDa protein limited proteolysis by admixtures of bacterial proteinases in the course of its dissolution. The comparison of the N-terminal sequences of the protein with molecular mass of 70 kDa (Met-Glu-Asn-Xaa-Pro-Leu-Asp-Thr-Leu-Ser-Ile-Val-Asn-Glu-Thr-Asp) and that of 28 kDa (Met-Glu-Asn-Leu-Asn-[Phe]-[Trp]-Pro-Leu-Gln-Asp-Ile-Lys-Val-Asn-Pro) reveals only marginal similarity between them (only 4 identical residues among 16 aligned). Both B. thuringiensis israelensis crystal-forming proteins appear hardly related to those contained in the crystal produced by other B. thuringiensis subspecies, e.g. kurstaki. It might be concluded that at least some of the entomocidal proteins found in the crystalline inclusion bodies of various B. thuringiensis subspecies revealed rather strong variations in their primary structures that facilitate their adaptation to different hosts. Bacillus thuringiensis ssp. israelensis δ-Endotoxin Entomocidal crystal Insecticide Mosquito

Molecular cloning and the nucleotide sequence of the Mr28 000 crystal protein gene ofBacillus thuringiensissubsp.israelensis

The Mr 28.000 crystal protein gene of Bacillus thuringiensis subspecies israelensis has been cloned into pBR322 as part of a 9.7 kb HindIII fragment. From hybridization experiments of recombinant p425 DNA with B.t. subspecies israelensis RNA from different stages of growth it was concluded that transcription of the gene is restricted to early sporulation stages. Nucleotide sequence analysis revealed the presence of a large open reading frame with a coding capacity of 249 amino acids (Mr 27.340). Nuclease S1 mapping demonstrated that transcription starts 44 nucleotides upstream of the initiation codon. A Shine-Dalgarno sequence (AAGGAG) was found 10 nucleotides upstream of the translation startpoint. At the 3'-end of the gene a complex secondary structure was found immediately after the stop-codon. Despite the presence of these regulation signals only limited expression in E. coli was detected. This can be explained by assuming that B.t. subsp. israelensis promotor sequences are poorly recognized by E. coli RNA polymerase.