Serovar Israelensis Research Articles

Toxicity of Bacillus thuringiensis d-exotoxins and b-endotoxins to Drosophila melanogaster, Ephestia kuhniella and human erythrocytes

A total of 73 Bacillus thuringiensis (Bt) strains were screened for the presence of non-hemolytic insecticidal β -exotoxin-free δ -endotoxins. Out of them, 45 Bt strains produced δ -endotoxins with specific insecticidal activity against Drosophila melanogaster and/or Ephestia kuhniella larvae. The thermostable β -exotoxin was observed only in 15 Bt strains and appeared to exhibit dual non-specific insecticidal activity against both D. melanogaster and E. kuhniella larvae and showed in vitro hemolysis for human erythrocytes. It was found that β -exotoxin was produced by Bt strains belonging to five serovars ( israelensis , kenyae , kurstaki , pakistani , and tohokuensis ) and two non-serotypable strains. This result suggests that β-exotoxin production is a strain-specific property rather than a serovar-specific property. To our knowledge, this is the first study that demonstrates β -exotoxins production association with Bt strains belonging to serovars israelensis, pakistani, and tohokuensis. The plasmid DNA profiles of some β -exotoxin producing Bt strains shared large plasmid patterns which may have the common β -exotoxin regulatory gene(s). It was found that 16 local Bt strains, 15 of which belonged to five serovars ( aizawai , israelensis , kurstaki , morrisoni , and pakistani ) and one was autoagglutinated strain, produced non-hemolytic insecticidal β -exotoxin-free δ -endotoxins. Based on random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR), the genotypic relatedness among these 16 Bt strains was investigated. The strains were grouped into two clusters. Bt strains within serovars israelensis were grouped in two subclusters, Bt strains within serovars aizawai were genomically homogeneous and clustered together, while the other serovars were grouped together in one subcluster. The autoagglutinated strain was clustered within serovar israelensis . Thus, these δ -endotoxins can be developed for the use in Bt-based insecticidal preparations. Keywords: Endotoxin, exotoxin, hemolytic, serovar, thuringiensis

Treatment of an Aedes aegypti colony with the Cry11Aa toxin for 54 generations results in the development of resistance

To study the potential for the emergence of resistance in Aedes aegypti populations, a wild colony was subjected to selective pressure with Cry11Aa, one of four endotoxins that compose the Bacillus thuringiensis serovar israelensis toxin. This bacterium is the base component of the most important biopesticide used in the control of mosquitoes worldwide. After 54 generations of selection, significant resistance levels were observed. At the beginning of the selection experiment, the half lethal concentration was 26.3 ng/mL and had risen to 345.6 ng/mL by generation 54. The highest rate of resistance, 13.1, was detected in the 54th generation. Because digestive proteases play a key role in the processing and activation of B. thuringiensis toxin, we analysed the involvement of insect gut proteases in resistance to the Cry11Aa B. thuringiensis serovar israelensis toxin. The protease activity from larval gut extracts from the Cry11Aa resistant population was lower than that of the B. thuringiensisserovar israelensis susceptible colony. We suggest that differences in protoxin proteolysis could contribute to the resistance of this Ae. aegypti colony.

Current Trends in the Control of Mosquito Vectors by Means of Biological Larvicides

Bacillus sphaericus Neide (B) and B. thuringiensis serovar israelensis (Bti) de Barjac provides effective alternatives to broad spectrum larvicides in many situations, with little or no environmental impact. Taking into account environmental benefits including safety for humans and other non-target organisms, reduction of pesticide residues in the aquatic environment, increased activity of most other natural enemies and increased biodiversity in aquatic ecosystems, their advantages are numerous. In addition to recombinant bacteria used as larvicides, research is also underway to develop transgenic algae and cyanobacteria using larvicidal endotoxins of Bti and Bs. The advent of recombinant DNA technology is now having an enormous impact on agriculture and medicine, and it is appropriate that the ability to manipulate and recombine genes with this technology be applied to improving larvicides for vector control. These new recombinant bacteria are as potent as many synthetic chemical insecticides, yet are much less prone to resistance, as they typically contain a mixture of endotoxins with different modes of action. The existing recombinants also have what can be considered disadvantageous, in that they do not show significantly improved activity against aedine and anopheline mosquitoes, in comparison to Bti. But it may be possible to overcome this limitation using some of the newly discovered mosquitocidal proteins, such as the Mtx proteins and peptides such as the trypsin-modulating oostatic factor, which could be easily engineered for high expression in recombinant bacteria. While other microbial technologies such as recombinant algae and other bacteria are being evaluated, it has yet to be shown that these are as efficacious and environment friendly as Bti and Bs. By combining the genes from a variety of organisms, it should ultimately be possible to design ‘smart’ bacteria that will seek out and kill larvae of specific vector mosquitoes. Thus, recombinant bacteria show excellent promise for development and use in operational vector control programs, hopefully within the next few years.

Evaluation of Bacillus thuringiensis serovar israelensis produced from a new culture medium for the control of the filariasis vector, Culex quinquefasciatus

The efficacy of Bacillus thuringiensis serovar israelensis (Bti) produced from a culture medium based on chicken feather extract was found to effectively control the filariasis vector, Culex quinquefasciatus for 3 weeks (>90% mortality). The results were on par with Bti produced from the conventional medium.

APLIKASI METODE ARDRA DALAM IDENTIFIKASI ISOLAT Bacillus thuringiensis ENDOGENIK SEBAGAI PENGENDALI HAMA KUBIS (Crocidolomia binotalis)

ABSTRACT Indonesian indigenous bacterial isolates of B. thuringiensis pathogenic to cabbage pest (C. binotalis) were molecularly characterized and identified using DNA fingerprinting method of ARDRA (Amplified Ribosomal DNA Restriction Analysis). Chromosomal DNA of 10 selected isolates (SLK2.3, SRNG4.2, TKO1, TK9, YPPA1, UG1A, BLPPN8.2, YWKA1, BAU3.2, LPST1) and 2 reference strains (B. thuringiensis serovar kurstaki HD1 & B. thuringiensis serovar israelensis H14) were isolated and purified by standard method. 16S rRNA genes were amplified by PCR method using universal primers of 27f and 1529r. PCR products were digested by 4 restriction endonucleases (EcoR1, HindIII, Pst1 dan HaeIII), and separated by agarose electrophoresis method to generate ARDRA profiles. Results of study showed that only ARDRA profiles generated by Hae III digestion were found to be meaningful and therefore used to identify the isolates. The ARDRA profile analysis indicated that the reference strain of B. thuringiensis serovar kurstaki HD1 could be clearly separated with B. thuringiensis serovar israelensis H14. In fact, those two strains have been widely recognized to be different in terms of their pathogenic specifity against insects. B. thuringiensis serovar kurstaki HD1 has been known to be specifically pathogenic to Lepidopteran whereas B. thuringiensis serovar israelensis H14 has been known to be specifically pathogenic to Dipteran. Key words : application, ARDRA, indigenous, B. thuringiensis, C. binotalis

Cyt1Aa protein fromBacillus thuringiensis(Berliner) serovarisraelensisis active against the Mediterranean fruit fly,Ceratitis capitata(Wiedemann)

Ceratitis capitata (Wiedemann) is one of the world's most destructive fruit pests. The aim of this study was to ascertain insecticidal activity of Bacillus thuringiensis (Berliner) delta-endotoxins to C. capitata. Among 42 selected Bacillus strains, only B. thuringiensis serovar israelensis (Bti) solubilised protoxins showed biological activity against C. capitata neonate larvae, whereas Bti spore and crystal mixture was inactive. Insecticidal activity of Bti protoxins was significantly enhanced by incubation with Culex pipiens L. gut extracts. Overdigestion of Bti protoxins with Sesamia nonagrioides (Lefebvre) gut extracts suppressed biological activity against C. capitata, and this correlated with degradation of Cyt toxins. Cyt1Aa solubilised protoxin showed the highest toxicity, LC(50) after 7 days of 4.93 microg cm(-2), while proteolytical processing of Cyt1Aa protoxins by larval gut extracts did not enhance insecticidal activity. The present study provides evidence for the first time of the insecticidal activity of a B. thuringiensis strain against C. capitata and identifies a single delta-endotoxin with potential for controlling this pest.

A novel l-isoleucine hydroxylating enzyme, l-isoleucine dioxygenase from Bacillus thuringiensis, produces (2 S,3 R,4 S)-4-hydroxyisoleucine

The unique function of 4-hydroxyisoleucine (4-HIL) is to stimulate glucose-induced insulin secretion in a glucose-dependent manner. 4-HIL is distributed only in certain kinds of plants and mushrooms, but the biosynthetic mechanism of 4-HIL has not been elucidated. Moreover, 4-HIL-producing microorganisms have not been reported. l-isoleucine ( l-Ile) hydroxylating activity producing 4-HIL was detected in a cell lysate of Bacillus thuringiensis strain 2e2 AKU 0251 obtained from the mid-late exponential phase of growth. Properties of the purified hydroxylase demonstrated that it is a α-ketoglutaric acid (α-KG) dependent l-Ile dioxygenase (IDO) and requires α-KG, ferric ion, and ascorbic acid for its maximum activity. IDO showed high stereoselectivity in l-Ile hydroxylation producing only (2 S,3 R,4 S)-4-HIL. The N-terminal 22 amino acids sequence revealed high homology to a hypothetical protein (GenBank ID: RBTH_06809) in B. thuringiensis serovar israelensis ATCC 35646. The histidine motif, which is conserved in α-KG dependent dioxygenases, is found in RBTH_06809.

Occurrence of Bacillus thuringiensis in Canopies of a Natural Lucidophyllous Forest in Japan

A total of 39 Bacillus thuringiensis isolates were recovered from 38 leaves collected from 5- to 10-m-high canopies of 8 micro-/meso-phanerophyte species in a lucidophyllous forest of Japan. B. thuringiensis-positive leaves accounted for 1.4% of a total of 2805 leaves from 15 tree species. The frequency of the organism was 0.8% among the Bacillus cereus/B. thuringiensis group. Of 39 isolates obtained, 27 (69.2%) were allocated to 11 H serovars, and 12 isolates remained unidentified: 11 were motile but lacked reactivity to the 55 reference antisera, and 1 isolate was not flagellated. Two H serovars, kurstaki (H3abc) and tohokuensis (H17), occurred predominantly on canopy phylloplanes. Larvicidal activities against Bombyx mori and/or Aedes aegypti were associated with 49% of the canopy isolates. Strong hemolysis was induced by parasporal inclusion proteins of the two isolates of serovar israelensis (H14). Hemagglutinating (lectin) activity was associated with parasporal proteins of nine isolates. There was little correlation between insecticidal activity and lectin activity.

Effects of spinosad and Bacillus thuringiensis israelensis on a natural population of Daphnia pulex in field microcosms

Spinosad, a candidate biological larvicide for mosquito control, was evaluated for its effects on a field population of Daphnia pulex, using Bacillus thuringiensis serovar israelensis (Bti) as a reference larvicide. Microcosms (125 L enclosures) were placed in a shallow temporary oligohaline marsh where D. pulex was present. Three concentrations of spinosad (8, 17 and 33 μg L −1) and two concentrations of Bti (0.16 and 0.50 μL L −1) were applied (5 replicates per concentration, including the controls). Effects of larvicides on D. pulex were evaluated after 2, 4, 7, 14 and 21 d of exposure, through measurements of abundance and individual size. Dissipation of spinosad from the water phase was rapid. Four days after treatment, residue concentration represented 11.8%, 3.9% and 12.7% of the initial exposure level for the nominal concentrations of 8, 17 and 33 μg L −1, respectively. Spinosyns A and D dissipated at similar rates. Analysis of abundance and size structure of the D. pulex population showed an impact of spinosad. Both survival and size structure were affected. However, at the lowest concentration (8 μg L −1), population recovered after the first week. In microcosms treated with Bti, the abundance of D. pulex was not affected but the size structure of the population changed after 21 d. As compared to laboratory tests, the use of in situ microcosms improved the environmental risk assessment of larvicides, taking into account the influence of environmental factors ( e.g., temperature, light, salinity) and intrinsic capacity of recovery of D. pulex under field conditions.

Investigation of the cyt gene in Bacillus thuringiensis and the biological activities of Bt isolates from the soil of China

The presence of cyt genes was investigated in 80 type strains of Bacillus thuringiensis and 143 isolates obtained from soil samples of China by PCR amplification using two pairs of primers for the cyt1 and cyt2 genes. Three type strains of serotypes H11ac, H14 and H36, eight isolates belonging to H3, H14, H18 and H21, and one isolate of unknown serotype harbored cyt genes. We also tested the cytolytic activity for mammal cells, the hemolytic activity for sheep erythrocytes and insecticidal activity against mosquitoes of five isolates that contained cyt genes but did not belong to B. thuringiensis serovar israelensis. The protein profiles of the five isolates were different from those of the type strains of B. thuringiensis serovar israelensis, and among the five isolates, only Y-5 showed mosquitocidal activity against larvae of Culex quinquefasciatus. All five of the isolates exhibited hemolytic activity, but only three could cause the cell death of A549 cells. The cytopathological changes induced by NX-4 in some A549 cells were characterized with cell-ballooning.

Biodegradation of poultry waste for the production of mosquitocidal toxins

The idea of the present study was to explore the possibility of degrading chicken feathers, discarded as environmental waste, from poultry processing industries, for bacterial culture media preparation to produce mosquitocidal toxins. For this study, we made use of the chicken feather powder (0.5%), for the preparation of bacterial culture media. It enabled the complete degradation of feather waste by the entomopathogenic bacteria ( Bacillus sphaericus and Bacillus thuringiensis serovar israelensis), thus helping to overcome the problem of dumping of poultry feather waste into the environment. The new fermentation technique also facilitated the effective storage of raw material (feathers), enabling easy transportation and convenience in handling and application. The bioremediation method thus envisaged proved to be cost effective for the preparation of culture media with high efficacy of bacterial toxin production and mosquito vector control. In conclusion, this study possesses the dual benefits of efficient biodegradation of environmental bioorganic waste (chicken feathers) and effective production of mosquitocidal biopesticides.

Bacterial Community Structure in Tree Hole Habitats of Ochlerotatus triseriatus: Influences of Larval Feeding

We investigated the bacterial community composition of tree holes in relation to the presence and absence of larvae of the mosquito Ochlerotatus triseriatus. Larvae were eliminated from a subset of natural tree holes with Bacillus thuringiensis serovar israelensis, and total bacterial numbers, slow- and fast-growing colony-forming units on minimal media, and 16S rRNA gene sequence data from water column and leaf material were obtained. Total bacterial counts did not change significantly with treatment; however, the number of slow-growing cultivable bacteria significantly increased in the absence of larvae. Sequence classifications and comparisons of sequence libraries using LIBSHUFF indicated that the elimination of larvae significantly altered bacterial community composition. Major groups apparently affected by larvae were Flavobacteriaceae, Rhodobacteraceae, Comamonadaceae, and Sphingomonadaceae. A clear dominance of Flavobacteriaceae in the water column after larval removal suggests members of this group are a major bacterial food source.

Phenotypic and genotypic features of new autoagglutinating Bacillus thuringiensis strains

A total of 28 autoagglutinating strains of Bacillus thuringiensis were isolated from different ecologic niches and distinct sites. Twenty-six strains demonstrated toxicity to mosquito larvae of Aedes aegypti and Culex quinquefasciatus. The electrophoretic protein profiles of the crystal components were studied. Twenty-three out of the 28 strains showed the same larvicidal activity and the same protein profiles as B. thuringiensis serovar israelensis. Using isoenzyme analysis (MLEE), it was observed the presence of three electrophoretic types (ETs). The mosquitocidal strains grouped into one ET. The random amplified polymorphic DNA analysis (RAPD) was evaluated using six primers, which demonstrated three different patterns for the 28 autoagglutinating strains, allowing correlation of the profiles obtained with the toxicity observed in the bioassays. The RAPD patterns for mosquitocidal strains were identical to the one of serovar israelensis. However, to strains of low toxicity, each primer generated distinctive RAPD patterns, which demonstrated that these strains belong to different serovars. Although the antigenic classification the 26 autoagglutinating strains of B. thuringiensis could not be determined by classical flagellar serotyping, MLEE and RAPD profiles proved these strains to be compatible with B. thuringiensis serovar israelensis.

New bacterial culture medium for production of mosquito pathogenic bacilli using agro-poultry industrial wastes1

A cost-effective technology has been developed to utilise bioorganic wastes as culture media, to produce mosquitocidal biopesticides, Bacillus sphaericus and B. thuringiensis serovar israelensis. The mosquitocidal spore/crystal toxins produced from the experimental medium (chicken feather waste, CFW+paddy husk waste, PHW) was higher than that of the conventional medium (Nutrient Yeast Extract Salt Medium, NYSM). The bacterial toxins produced from different media (NYSM, CFW, PHW, CFW+PHW) were bioassayed against mosquito vectors and the toxic effect was found to be significant. The use of chicken feathers and paddy husk wastes as bacterial culture media is cost-effective and economical for the production of these mosquito pathogenic bacilli.

LARVAL GUT PH PROFILE IN PESTIFEROUS CHIRONOMUS CRASSICAUDATUS AND GLYPTOTENDIPES PARIPES (CHIRONOMIDAE: DIPTERA) IN REFERENCE TO THE TOXICITY POTENTIAL OF BACILLUS THURINGIENSIS SEROVAR ISRAELENSIS

Gut pH and pH optimum of amylase were measured in 4th-stage larvae of 2 pestiferous chironomid species, Chironomus crassicaudatus and Glyptotendipes paripes. The gut pH in both species was close to neutral, varying from 6.7 to 7.4 and 6.9 to 7.6 pH units for C. crassicaudatus and G. paripes, respectively. In both species, slightly alkaline pH values were measured in the anterior part of the mesenteron, whereas slightly acidic pH values were recorded in gastric caecae and the posterior part of mesenteron. Amylase pH optimum in both chironomid species remained between pH values of 5.5 and 7, being closer to pH value 7 than 5.5. This profile was consistent with the measured values of the gut pH. The recorded larval gut pH profile in the 2 species of chironomids in the present study is much lower than gut pH reported for mosquitoes or Lepidoptera larvae. This could be the reason for the relatively much lower susceptibility of chironomid larvae to Bacillus thuringiensis serovar israelensis toxin proteins than some other nematoceran Diptera, specifically mosquitoes.

Use of feather-based culture media for the production of mosquitocidal bacteria

Abstract Human industrial activities, inevitably, generate industrial wastes, consisting of, inorganic and organic materials, discharged from factories, fisheries, poultries and food processing industries. Degrading or handling these wastes, as unused disposals, without acquiring any additional benefits has led to an idea to develop a suitable technology to utilize bio-organic wastes. Chicken feathers have been discarded in bulk as waste from poultry processing industries, poultry farms and shops, globally. They normally accumulate structural proteins (keratins) that are resistant to biodegradation. Considering the abundant supply of these feather wastes, we have successfully produced the biopesticides by culturing Bacillus sphaericus (Bs) and Bacillus thuringiensis serovar israelensis (Bti) strains to synthesize mosquitocidal toxins. Biochemical studies indicate that the mosquitocidal spore/crystal toxins produced from the experimental culture medium (chicken feather waste medium, CFWM) are similar to that of conventional medium (Nutrient Yeast Extract Salt Medium, NYSM). The bacteria produced in these media (NYSM and CFWM) were bioassayed against the mosquito vectors (Culex quinquefasciatus, Anopheles stephensi, Aedes aegypti) and the toxic effect was found to be comparable. Cost-effective analysis indicates that the use of chicken feather waste as culture medium is highly economical for the industrial production of these mosquito pathogenic bacilli. This study is, therefore, very important as it possesses the dual benefit of effective utilization of bio-organic waste materials from the environment and for the production of mosquitocidal biopesticides as well.

Comparison between highly toxic "Bacillus thuringiensis" serovar israelensis and "Bacillus sphaericus" strains against "Lutzomyia longipalpis" Lutz & Neiva 1912 (Diptera, Psychodidae, Phlebotominae) larvae

The effects of standardized concentrated spores and crystals suspensions of Bacillus thuringiensis serovar israelensis and Bacillus sphaericus against 3rd instar larvae of Lutzomyia longipalpis were recorded by means of qualitative bioassays. The experiments showed a significant difference on mortality and moulting delay between the entomopathogenic bacilli strains tested (p < 0.001). B. thuringiensis serovar israelensis LFB-Fiocruz 584 (a clone of IPS-82) was active against insect larvae assayed (62.5% ± 14.2 of mortality), however, no significant effect was observed with B. sphaericus LFBFiocruz 736 (a clone of strain 2362). The data reinforce the suggestion that B. thuringiensis serovar israelensis should be considered for further evaluation as a potential agent against L. longipalpis larvae. Key words: Bacillus, Lutzomyia longipalpis, control, bioassays.

Serological identification and insect toxicity of Bacillus thuringiensis isolated from the island Okinoerabu-jima, Japan

We examined soils of the island Okinoerabu-jima, Kagoshima Prefecture, Japan, for the occurrence of Bacillus thuringiensis. The organism occurred in 13 (65%) out of 20 soil-sampling sites, and 27 (27%) out of 100 soil samples. Among 2,680 Bacillus cereus group colonies examined, 66 were allocated to B. thuringiensis. Soils of the island provided at least 11 H-serotypes. Most of the isolates exhibited no insecticidal activities, while an isolate of serovar kurstaki (H3abc) with bipyramidal parasporal inclusions killed both dipteran and lepidopteran larvae, and another isolate of serovar colmeri (H21) with irregularly-pointed inclusions had a high mosquitocidal activity. None of the seven isolates of serovar israelensis (H14) showed mosquito larvicidal activities, while producing parasporal inclusions morphologically similar to those of the reference strain of serovar israelensis with strong mosquitocidal activities.

Low Persistence of Bacillus thuringiensis Serovar israelensis Spores in Four Mosquito Biotopes of a Salt Marsh in Southern France

We studied the persistence of Bacillus thuringiensis serovar israelensis (Bti) in a typical breeding site of the mosquito Ochlerotatus caspius in a particularly sensitive salt marsh ecosystem following two Bti-based larvicidal applications (Vectobac 12AS, 1.95 L/ha). The treated area was composed of four larval biotopes that differed in terms of the most representative plant species (Sarcocornia fruticosa, Bolboschoenus maritimus, Phragmites australis, and Juncus maritimus) and the physical and chemical characteristics of the soil. We sampled water, soil, and plants at various times before and after the applications (from spring to autumn, 2001) and quantified the spores of B. thuringiensis (Bt) and Bacillus species. The B. cereus group accounted for between 0% and 20% of all Bacillus spp. before application depending on the larval biotope. No Bti were found before application. The variation in the quantity of bacilli during the mosquito breeding season depended more on the larval biotope than on the season or the larvicidal application. More bacilli were found in soil (10(4)-10(6) spores/g) than on plant samples (10(2)-10(4) spores/g). The abundance in water (10(5) to 10(7) spores/L) appeared to be correlated to the water level of the breeding site. The number of Bti spores increased just after application, after declining; no spores were detected in soil or water 3 months after application. However, low numbers of Bti spores were present on foliage from three of the four studied plant strata. In conclusion, the larvicidal application has very little impact on Bacillus spp. flora after one breeding season (two applications).

A new black fly isolate of Bacillus thuringiensis autoagglutinating strain highly toxic to Simulium pertinax (Kollar) (Diptera, Simuliidae) larvae

Formulations containing the entomopathogenic Bacillus thuringiensis serovar israelensis strain IPS-82 has been widely applied for mosquito control around the world. Strain IPS-82 is highly active against Aedes aegypti but less active against other well-known vectors such as Culex quinquefasciatus and Simulium spp. larvae. Eighteen strains of B. thuringiensis were isolated from Simulium pertinax larvae naturally occurring in rivers of Southeast Brazil with one demonstrating special toxic effects. Simulated field tests against S. pertinax larvae showed that the native Brazilian autoagglutinating B. thuringiensis (LFB-FIOCRUZ 1035) has an LC50 at least 25 times lower than the standard IPS-82 strain. The same bacterial preparation was also tested against Ae. aegypti larvae in laboratory trials and the LC50 values obtained with LFB-FIOCRUZ 1035 were at least three times lower than the one for the IPS 82 strain. The results indicate that this strain is more toxic than the standard B. thuringiensis serovar israelensis (H14) in the two Dipteran species tested. It is noteworthy that differences between LC50 values were more pronounced in S. pertinax larvae, the source of the original isolation.