Expression of Binary Toxin Genes in the Mosquito-Colonizable Bacteria, <Emphasis Type="Italic">Bacillus cereus</Emphasis>, Leads to High Toxicity Against <Emphasis Type="Italic">Culex quinquefasciatus</Emphasis> Larvae

Two B. cereus strains, Ae10 and Cx5, isolated from mosquito larval guts, were transformed with a recombinant plasmid, pBS373, harboring binary toxin genes from Bacillus sphaericus 2297. Immunoblotting analysis clearly revealed the production and presence of the 51-kDa toxin protein in both strains. Two recombinant B. cereus strains Ae10 and Cx5 showed very high toxicity against C. quinquefasciatus larvae. Since both strains have a close relationship with the mosquito larvae in the native environment and are capable of recolonizing in the guts of mosquito larvae, these strains can be considered promising new hosts for an effective delivery of mosquito-larvicidal toxins.     

New mosquitocidal strains from Ghana belonging to serotypes H3, H6 and H48 of Bacillus sphaericus

Summary Seven bacterial isolates from Ghana, IAB 763, IAB 769-1, IAB 769-2, IAB 774, IAB 871, IAB 872, IAB 881, are characterized as Bacillus sphaericus strains highly toxic to mosquito larvae. Most of them belong to serotype H6, except for IAB 881 and IAB 872, which belong pesrespectively to serotypes H3 and H48. Phenotypic characters of all these strains are identical to those of strains 2362 (serotype H5) and IAB 59 (serotype H6), used for comparison. Five strains out of seven produce final whole cultures and alkali-solubilized toxins, which have very high potency against Culex pipiens larvae. Their larvicidal power is similar to that of strains 2362 and IAB 59. By using polyclonal antibodies raised against 42- and 56-kDa toxic polypeptides of strain 2362, Western-blot of the alkali-solubilized toxins of these new five strains showed homologies. It is the first time that strains belonging to serotypes H3 and H48 have been found pathogenic to mosquito larvae, thus increasing to eight the number of toxic serotypes of B. sphaericus. Correspondence to: I. Thiery     

Use of a single procedure for selective enrichment, isolation, and identification of plasmid-bearing virulent Yersinia enterocolitica of various serotypes from pork samples.

Many selective enrichment methods for the isolation of Yersinia enterocolitica from foods have been described. However, no single isolation procedure has been described for the recovery and identification of various plasmid-bearing serotypes. A single improved procedure for selective enrichment, isolation, identification, and maintenance of plasmid-bearing virulent serotypes of Y. enterocolitica from pork samples was developed. Enrichment at 12 degrees C in Trypticase soy broth containing yeast extract, bile salts, and Irgasan was found to be an efficient medium for the recovery of plasmid-bearing virulent strains of Y. enterocolitica representing O:3; O:8; O:TACOMA; O:5, O:27; and O:13 serotypes. MacConkey agar proved to be a reliable medium for the isolation of presumptive colonies, which were subsequently confirmed as plasmid-bearing virulent strains by Congo red binding and low calcium response. Further confirmation by multiplex PCR employed primers directed at the chromosomal ail and plasmid-borne virF genes, which are present only in pathogenic strains. The method was applied to pig slaughterhouse samples and was effective in isolating plasmid-bearing virulent strains of Y. enterocolitica from naturally contaminated porcine tongues. Strains isolated from ground pork and tongue expressed plasmid-associated phenotypes and mouse pathogenicity.     

Persistence and efficacy of Beauveria brongniartii strains applied as biocontrol agents against Melolontha melolontha in the Valley of Aosta (northwest Italy)

AIMS: To monitor and select genetically characterized strains of Beauveria brongniartii to be used as microbiological control agents against Melolontha melolontha in different climatic conditions of the Valley of Aosta (northwest Italy). METHODS AND RESULTS: Molecular random amplified polymorphic DNA markers allowed monitoring of five B. brongniartii strains (C2, F, K2, N3 and W2) in field trials. Ten sites were chosen at Joven?an, Saint-Pierre and Quart areas, where a mixture of the five strains colonizing rye kernels was applied to the soil of each M. melolontha infested site. Growth, persistence and virulence on M. melolontha larvae of five fungal strains were evaluated in two subsequent 24-month studies. Beauveria brongniartii grew best at the Joven?an sites. Not only did strain F persist better than the other strains in most soil samples but it was also the most virulent strain. Strain F was isolated the most frequently from infected M. melolontha larvae recovered from the test sites. A general decrease in the larvae rate was detected in the test field soil. CONCLUSIONS: Strain F of B. brongniartii was better than other strains in growth, persistence and virulence against M. melolontha larvae in the test site soil. SIGNIFICANCE AND IMPACT OF THE STUDY: Results obtained from preliminary field trials support the use of strain F as a biological control agent against M. melolontha in the Valley of Aosta even if further targeted studies are still necessary.     

Isolation of Bacillus thuringiensis from intertidal brackish sediments in mangroves

Intertidal brackish sediments in mangroves were examined for isolation of Bacillus thuringiensis strains with novel toxicity spectra. A total of 18 B. thuringiensis isolates were recovered from eight sediment samples (36.4%) out of 22 samples tested. The frequency of B. thuringiensis was 1.3% among the colonies of Bacillus cereus/B. thuringiensis group. While five isolates were allocated to the four H serogroups, the majority of the isolates were serologically untypable or untestable. Two isolates belonging to the serovar israelensis/tochigiensis (H14/19) exhibited strong toxicities against larvae of the mosquito, Culex pipiens molestus, and mammalian cells (sheep erythrocyte and two human cancer cell lines) in vitro. The other 16 isolates showed no toxicity against the mosquito and mammalian cells. None of the isolates showed larvicidal activity against the diamondback moth, Plutella xylostella. Strong lectin activities against sheep erythrocytes were associated with two serologically untestable isolates and an H3 isolate.     

Diversity of Bacillus thuringiensis strains from Colombia with insecticidal activity against Spodoptera frugiperda (Lepidoptera:Noctuidae)

AIMS: To identify and characterize Bacillus thuringiensis strains highly toxic to Spodoptera frugiperda, and to explore the genetic diversity of such strains. METHODS AND RESULTS: The insecticidal activity of 1100 strains of B. thuringiensis from Colombian soil samples was assayed against first instar S. frugiperda larvae, and 32 active strains were found. After a second bioassay evaluation, the eight most potent strains were selected for further characterization, which included crystal protein profiles determined by polyacrylamide gel electrophoresis, plasmid profile, plasmid restriction patterns, cry gene composition, qualitative determination of beta-exotoxin production, random amplified polymorphic DNA, serotyping, and toxicity to S. frugiperda. All Colombian strains contained cry1Aa, cry1Ab, cry1Ac, cry1B, cry1C and cry1D genes. However, PCR profiles of the Colombian strains suggested the presence of variants of the cry1 genes. Serotyping indicated that these strains belong to the kurstaki, thuringiensis, canadiensis and indiana subspecies. Interestingly, three strains belonging to different serotypes and subspecies were found in the same soil sample, and toxicity ranged between 11 and 976 ng cm(-2) of diet. CONCLUSIONS: It has been shown that B. thuringiensis strains belonging to different serotypes and displaying variable potency to S. frugiperda larvae can be found in the same soil sample. SIGNIFICANCE AND IMPACT OF THE STUDY: The results obtained indicate that some of the B. thuringiensis strains studied could be of interest for further development for S. frugiperda control programmes.     

Insecticidal activity of Bacillus laterosporus

Strains of Bacillus laterosporus demonstrated pathogenic activity for second-instar larvae of the mosquito, Culex quinquefasciatus, but failed to demonstrate detectable pathogenicity against larvae of the cabbage looper, Trichoplusia ni. Of 29 strains of the bacterium screened, 16 displayed pathogenicity for mosquito larvae. One of the most pathogenic strains, NRS 590, also demonstrated pathogenic activity for larvae of the mosquito, Aedes aegypti, and for larvae of the black fly, Simulium vittatum. The pathogenicity for Culex larvae was associated with the cell mass rather than with the culture supernatant. A suspension of ultraviolet irradiation-killed cells demonstrated no loss in pathogenic activity, an indication that the pathogenicity is toxin mediated. The toxic substance produced by NRS 590 was found to be resistant to heating at 96°C for 10 min. The toxin was not associated with the heat-resistant, bacterial endospore or with the associated paraspore since a suspension consisting primarily of spores was not toxic to mosquito larvae. Toxic activity in stationary phase cells of NRS 590 was associated with the cell's particulate fraction rather than with the soluble fraction.     

Marginal cross-resistance to mosquitocidal Bacillus thuringiensis strains in Cry11A-resistant larvae: presence of Cry11A-like toxins in these strains

Culex quinquefasciatus mosquito larvae resistant to the Cry11A toxin showed marginal cross-resistance to the multiple toxin crystals from B. thuringiensis subsp. israelensis and also to toxin crystals from three other mosquitocidal strains, i.e. B. thuringiensis subsp. fukuokaensis, subsp. jegathesan, and subsp. kyushuensis. Cross-resistance patterns of the Cry11A-resistant larvae to mosquitocidal strains of B. thuringiensis together with the immunological screening using antisera raised against Cry11A indicated the presence of Cry11A-like toxins in these strains and could be used as a screening tool for the identification of novel toxins. The Cry11A-resistant larvae had significantly less resistance to the Cry11B toxin from B. thuringiensis subsp. jegathesan. The occurrence of cytolytic toxins in all of these mosquitocidal strains partially explains the marginal cross-resistance observed with multiple toxin crystals since each of these crystals also contains cytolytic toxins.     

Characterization and pathogenic evaluation of Bacillus thuringiensis and Bacillus sphaericus isolates from Argentinean soils

Eighty soil samples of different origin (from urban, agricultural, forested and horticultural areas) which had not previously been treated with bioinsecticides, were collected and examined to investigate the presence of Bacillus thuringiensis and B. sphaericus. From a total of 1473 bacterial isolates examined by differential staining techniques and growth on nutrient agar with the addition of penicillin and streptomycin, 31 (2.1%) strains of Bacillus sphaericus and 25 (1.6%) strains of Bacillus thuringiensis were isolated. These strains were tested for their pathogenicity against Diptera (Culex quinquefasciatus) and Lepidoptera (Anticarsia gemmatalis and Spodoptera frugiperda). Seven strains of Bacillus thuringiensis subspecies kurstaki were found to be pathogenic to Spodoptera frugiperda and twenty-two strains showed a pathological effect against Anticarsia gemmatalis. None of the strains of Bacillus thuringiensis nor the Bacillus sphaericus investigated, showed pathogenic activity against Culex quinquefasciatus. The strains of Bacillus thuringiensis were characterized serologically as belonging to six serotypes (darmstadiensis, entomocidus, kurstaki, muju, sotto and xianguangiensis). One strain seemed to be a new serotype. The electrophoretic profiles of the strains of Bacillus thruringiensis showed bands of 130 kDa similar to those found in strains pathogenic against Lepidoptera. Some physicochemical characteristics were also studied in the soil samples, in order to relate them to the presence or absence of these Bacillus species.     

Evaluation of the usefulness of selected virulence markers for the identification of virulent Yersinia enterocolitica strains. I. Phenotypic markders associated with Plasmid pYV

The species Yersinia enterocolitica includes either pathogenic or non-pathogenic strains. Therefore it is necessary to differentiate virulent bacilli from other. It is well known that pathogenic strains of Y. enterocolitica bearing virulence associated plasmid called pYV, which could be demonstrated by its isolation or detected by the presence of specific, phenotypic properties directly related with this plasmid. The aim of the presented paper was to check the ability of some phenotypic virulence markers associated with pYV, to detection of pathogenic Y. enterocolitica strains. In the presented work 152 (130 carrying pYV) clinical strains of Y. enterocolitica O3 isolated mainly from stool were examined for the presence of phenotypic virulence markers such as: calcium dependency, Congo-red binding, autoagglutination and agglutination with Mangifera indica extract. Both first features were detected parallel, on the same plate, using CRMOX (Congo-red, Magnesium Oxalate) agar. The detection of the tested markers in the examined strains was compared with the presence of virulence plasmid. The obtained results confirmed the observations done by other authors that Y. enterocolitica strains, in which bacilli bearing the virulence plasmid predominate, exhibit all tested phenotypic properties whereas the plasmid-cured isogenic strains show no one of these features. Therefore all the tested markers could be useful for detection of virulent Y. enterocolitica strains directly isolated from patients. The most useful virulence markers in bacteriological study seems to be calcium dependency and Congo-red binding, examined together by the use of CRMOX agar, because they confirm the presence of the virulence plasmid by parallel detection of two physiologically different features associated with this plasmid. In addition CRMOX agar allows for the examination rough strains while agglutination tests do not.     

Mosquito active strains of Bacillus sphaericus isolated from soil and mud samples collected in Israel

Bacteria of typical Bacillus sphaericus appearance were isolated from mud and soil samples taken from mosquito breeding sites in Israel. Five isolates, 2613, 2615, 2619, 2620, and 2631, all belonging to Phage Group 3, were highly active against Culex pipiens larvae. The most toxic isolates recovered, 2615 and 2631, had calculated ITU values of approximately 1500 ITU/mg, compared with 1000 ITU/mg for the B. sphaericus RB-80 reference standard. Isolates belonging to Phage Group 4 were of significantly lower toxicity when assayed against Culex larvae and exhibited a high variability in their toxicity. In this survey, B. sphaericus strains toxic to mosquito larvae were recovered only from the desert regions of southern Israel. The isolates in Phage Group 3 were all recovered from the central Negev region of Israel. Material taken from sources close to the Dead Sea produced isolates belonging to Phage Group 4.     

The development and assessment of DNA and oligonucleotide probes for the specific detection of Bacillus anthracis

Two DNA probes and a number of oligonucleotide probes were designed from the virulence factor genes of Bacillus anthracis. These probes were tested for specificity against 52 B. anthracis strains and 233 Bacillus strains encompassing 23 other species. A rapid slot blotting technique was used for screening the large numbers of isolates involved. All probes tested appeared to be specific for B. anthracis under high stringency conditions. These probes could differentiate between virulent and avirulent strains. The probes were also applied to the detection of B. anthracis in routine environmental and clinical samples. A non-radioactive hybridization and detection system based on digoxigenin-11-dUTP was developed.     

球形芽孢杆菌Mtx1蛋白和苏云金杆菌Cyt1Aa晶体蛋白的协同作用

采用常规的生物测定方法确定了纯化的球形芽孢杆菌(Bacillus sphaericus)的缺失信号肽的97kDa营养期杀蚊毒素(Mosquitocidal toxin 1,Mtx1)蛋白和苏云金芽孢杆菌(Bacillus thuringiensis)27.3kDa的Cyt1Aa晶体蛋白对致倦库蚊(Culex quinquefasciatus)幼虫的杀虫活性。结果表明Mtx1和Cyt1Aa不同比例的混合物对致倦库蚊的毒力比单独毒素蛋白高,经统计分析表明两毒素蛋白对目标蚊幼虫具有明显的协同作用。在LC98处理浓度下,Mtx1和Cyt1Aa按3∶1混合的混合物LT50值比单独Mtx1的提前了6.36h。表明Cyt1Aa和Mtx1对致倦库蚊具有协同毒杀作用,提高对目标蚊虫的毒力、缩短半致死时间。该结果为深入研究Mtx1和Cyt1Aa的杀蚊作用方式奠定了基础,同时为其在蚊虫防治中的应用提供了新的思路和方法。     

Identification of Metarhizium strains highly efficacious against Aedes,Anopheles and Culex larvae

Entomopathogenic fungi, such as Metarhizium anisopliae and Beauveria bassiana, have been shown to be efficacious in killing mosquito larvae of different mosquito species. The current study compared the pathogenicity and efficacy of two formulations of three fungal strains against different instars of three mosquito species with the aim of identifying the most virulent strain for use under field conditions. Three strains of Metarhizium, ARESF 4556, ARSEF 3297 and V275, were assayed against early (L_2?3) and late (L_3–4) instar larvae of Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus. Two formulations of the fungi were tested, dry conidia and aqueous suspensions (i.e. ‘wet’ conidia). Effects of all combinations of conidia, mosquito species, instar, fungal strain and concentration on mosquito mortality were analysed using Cox regression and Kaplan–Meier analyses. Strain ARSEF 4556 was more virulent than ARSEF 3297 and V275, with LT₅₀ values ranging from 0.3 to 1.1 days, with Anopheles and Culex being more susceptible than Aedes. Early and late instars were equally susceptible independent of species. Although the formulation did influence mortality rates, both ‘wet’ and ‘dry’ conidia applications were highly effective in killing mosquito larvae. Viable spores were more efficacious than heat killed spores. The latter did cause mortality but only at high concentrations. Metarhizium sp. has proved to be effective in reducing survivability of all larval stages of Aedes, Anopheles and Culex under laboratory conditions. Aedes larvae were generally more tolerant than Anopheles and Culex irrespective of fungal strain.     

Screening of selected ethnomedicinal plants from South Africa for larvicidal activity against the mosquito Anopheles arabiensis

ABSTRACT: BACKGROUND: This study was initiated to establish whether any South African ethnomedicinal plants (indigenous or exotic), that have been reported to be used traditionally to repel or kill mosquitoes, exhibit effective mosquito larvicidal properties. METHODS: Extracts of a selection of plant taxa sourced in South Africa were tested for larvicidal properties in an applicable assay. Thirty 3rd instar Anopheles arabiensis larvae were exposed to various extract types (dichloromethane, dichloromethane/methanol) (1:1), methanol and purified water) of each species investigated. Mortality was evaluated relative to the positive control Temephos (Mostop; Agrivo), an effective emulsifiable concentrate larvicide. RESULTS: Preliminary screening of crude extracts revealed substantial variation in toxicity with 24 of the 381 samples displaying 100% larval mortality within the seven day exposure period. Four of the high activity plants were selected and subjected to bioassay guided fractionation. The results of the testing of the fractions generated identified one fraction of the plant Toddalia asiatica as being very potent against the An. arabiensis larvae. CONCLUSION: The present study has successfully identified a plant with superior larvicidal activity at both the crude and semi pure fractions generated through bio-assay guided fractionation. These results have initiated further research into isolating the active compound and developing a malaria vector control tool.     

Cyt1A of Bacillus thuringiensis delays evolution of resistance to Cry11A in the mosquito Culex quinquefasciatus

Insecticides based on Bacillus thuringiensis subsp. israelensis have been used for mosquito and blackfly control for more than 20 years, yet no resistance to this bacterium has been reported. Moreover, in contrast to B. thuringiensis subspecies toxic to coleopteran or lepidopteran larvae, only low levels of resistance to B. thuringiensis subsp. israelensis have been obtained in laboratory experiments where mosquito larvae were placed under heavy selection pressure for more than 30 generations. Selection of Culex quinquefasciatus with mutants of B. thuringiensis subsp. israelensis that contained different combinations of its Cry proteins and Cyt1Aa suggested that the latter protein delayed resistance. This hypothesis, however, has not been tested experimentally. Here we report experiments in which separate C. quinquefasciatus populations were selected for 20 generations to recombinant strains of B. thuringiensis that produced either Cyt1Aa, Cry11Aa, or a 1:3 mixture of these strains. At the end of selection, the resistance ratio was 1,237 in the Cry11Aa-selected population and 242 in the Cyt1Aa-selected population. The resistance ratio, however, was only 8 in the population selected with the 1:3 ratio of Cyt1Aa and Cry11Aa strains. When the resistant mosquito strain developed by selection to the Cyt1Aa-Cry11Aa combination was assayed against Cry11Aa after 48 generations, resistance to this protein was 9.3-fold. This indicates that in the presence of Cyt1Aa, resistance to Cry11Aa evolved, but at a much lower rate than when Cyt1Aa was absent. These results indicate that Cyt1Aa is the principal factor responsible for delaying the evolution and expression of resistance to mosquitocidal Cry proteins.     

Microbial combinatorics: a simplified approach for isolating insecticidal bacteria

Bacteria can control pest insects that damage food crops, vector diseases and defoliate trees. Conventionally, isolation of these bacteria has been from soil and sporadically from dead insects. A simplified approach for isolating insecticidal bacteria from soil using the target insect as the selective agent was employed in this study. Instead of isolating single strains of bacteria from soil and testing each individual strain for insect toxicity, mixtures of bacteria present in each soil sample were tested together directly for toxicity using Manduca sexta (Linnaeus) (Lepidoptera: Sphingidae) as a model insect. Thirty-five soil suspensions or bacterial suspensions of the 40 suspensions tested killed at least one M. sexta larva. All but one bacterial culture isolated from dead larvae and retested for toxicity, killed at least one M. sexta larva. Nineteen bacterial strains isolated from larvae killed in the first test, were identical to the bacteria fed to the retested larvae. Of the 19 strains isolated, 14 were identified by 16S rDNA sequencing as belonging to the Bacillus cereus group including three strains that formed crystals that were identified as B. thuringiensis. Of the three other spore-forming strains, two were identified as psychrotrophic B. weihenstephanensis and the third as Lysinibacillus fusiformis. Two others were identified as Enterococcus faecalis. This approach, microbial combinatorics, reduces the number of insects necessary for toxicity screening and associated time and resources compared to conventional methods that first isolate bacteria and then individually test for toxicity as well as a means of discovery of new pathogens using the insect as the selective agent.     

Expression of the mosquitocidal toxins of Bacillus sphaericus and Bacillus thuringiensis subsp. israelensis by recombinant Caulobacter crescentus, a vehicle for biological control of aquatic insect larvae.

In the quest for effective control of mosquitoes, attention has turned increasingly to strains of the bacteria Bacillus sphaericus and Bacillus thuringiensis subsp. israelensis, which produce potent toxins with specific mosquitocidal activities. However, sedimentation of the bacterial spores limits the duration of effective control after field application of these bacilli. We describe here the cloning of genes encoding the 51.4- and 41.9-kDa toxins from B. sphaericus 2297, the 100-kDa toxin from B. sphaericus SSII-1, and the 130-kDa toxin from B. thuringiensis subsp. israelensis into the broad-host-range plasmid pRK248 and the transfer of these genes for expression in Caulobacter crescentus CB15. The recombinant C. crescentus cells were shown to be toxic to mosquito larvae. Caulobacter species are ubiquitous microorganisms residing in the upper regions of aquatic environments and therefore provide the potential for prolonged control by maintaining mosquitocidal toxins in larval feeding zones.     

Expression of the mosquitocidal toxins of Bacillus sphaericus and Bacillus thuringiensis subsp. israelensis by recombinant Caulobacter crescentus, a vehicle for biological control of aquatic insect larvae.

In the quest for effective control of mosquitoes, attention has turned increasingly to strains of the bacteria Bacillus sphaericus and Bacillus thuringiensis subsp. israelensis, which produce potent toxins with specific mosquitocidal activities. However, sedimentation of the bacterial spores limits the duration of effective control after field application of these bacilli. We describe here the cloning of genes encoding the 51.4- and 41.9-kDa toxins from B. sphaericus 2297, the 100-kDa toxin from B. sphaericus SSII-1, and the 130-kDa toxin from B. thuringiensis subsp. israelensis into the broad-host-range plasmid pRK248 and the transfer of these genes for expression in Caulobacter crescentus CB15. The recombinant C. crescentus cells were shown to be toxic to mosquito larvae. Caulobacter species are ubiquitous microorganisms residing in the upper regions of aquatic environments and therefore provide the potential for prolonged control by maintaining mosquitocidal toxins in larval feeding zones.     

Cellular fatty acid analysis as a potential tool for predicting mosquitocidal activity of Bacillus sphaericus strains.

Gas-liquid chromatography of fatty acid methyl esters and numerical analysis were carried out with 114 Bacillus sphaericus strains. Since only two clusters harbored mosquitocidal strains, this technique could be developed in screening programs to limit bioassays on mosquito larvae. It also allows differentiation of highly homologous strains.