Evaluating large‐scale effects of Bacillus thuringiensis var. israelensis on non‐biting midges (Chironomidae) in a eutrophic urban lake

1. There is continued interest in controlling non‐biting midges (chironomids) in some freshwaters because of the potential nuisance caused by emergent adults. One option is to apply Bacillus thuringiensis var. israelensis (Bti), primarily used against mosquitoes and blackflies, to control benthic chironomid larvae. Chironomids are also at risk of collateral effects where Bti is used on other targets, and such instances may increase if climate change alters the incidence of dipteran‐borne diseases. However, most research on Bti effects on chironomids is available from mesocosms or ponds and might not scale‐up to larger waterbodies. 2. We present results on the effects of Bti on larval chironomids from eight experimental treatments, over 3 years, on a newly created eutrophic, urban lake of 200 ha, Cardiff Bay. 3. The first two experimental years provided limited evidence of Bti effects, with chironomid densities reduced by up to 14%. Increased scale of application and altered experimental design in the third year revealed reductions in chironomid larval densities of around 35% following Bti treatment, with suppression lasting several months. 4. These large‐scale Bti experiments – among the largest ever undertaken on chironomids – complement previous smaller‐scale experiments in illustrating how field conditions and application methods influence Bti effects on chironomid densities. Specifically, the work reveals how near‐neutral buoyancy formulations of Bti can reduce chironomid numbers in large lakes exceeding 3 m depth, but only where treatment methods avoid over‐dispersion. We advocate further evaluation to assess whether chironomids can be suppressed over longer periods using whole‐lake application without long‐term ecological implications or excessive cost. We also suggest further consideration be given to non‐buoyant Bti formulations for use in deeper lakes.     

Long lasting persistence of Bacillus thuringiensis Subsp. israelensis (Bti) in mosquito natural habitats

Background

The detrimental effects of chemical insecticides on the environment and human health have lead to the call for biological alternatives. Today, one of the most promising solutions is the use of spray formulations based on Bacillus thuringiensis subsp. israelensis (Bti) in insect control programs. As a result, the amounts of Bti spread in the environment are expected to increase worldwide, whilst the common belief that commercial Bti is easily cleared from the ecosystem has not yet been clearly established.

Methodology/Main Findings

In this study, we aimed to determine the nature and origin of the high toxicity toward mosquito larvae found in decaying leaf litter collected in several natural mosquito breeding sites in the Rhône-Alpes region. From the toxic fraction of the leaf litter, we isolated B. cereus-like bacteria that were further characterized as B. thuringiensis subsp. israelensis using PCR amplification of specific toxin genes. Immunological analysis of these Bti strains showed that they belong to the H14 group. We finally used amplified length polymorphism (AFLP) markers to show that the strains isolated from the leaf litter were closely related to those present in the commercial insecticide used for field application, and differed from natural worldwide genotypes.

Conclusions/Significance

Our results raise the issue of the persistence, potential proliferation and environmental accumulation of human-spread Bti in natural mosquito habitats. Such Bti environmental persistence may lengthen the exposure time of insects to this bio-insecticide, thereby increasing the risk of resistance acquisition in target insects, and of a negative impact on non-target insects.     

Characterization of the genes encoding the haemolytic toxin and the mosquitocidal delta-endotoxin of Bacillus thuringiensis israelensis

Summary The crystalline parasporal inclusions (crystals) of Bacillus thuringiensis israelensis (Bti), which are specifically toxic to mosquito and black fly larvae, contain three main polypeptides of 28 kDa, 68 kDa and 130 kDa. The genes encoding the 28 kDa protein and the 130 kDa protein have been cloned from a large plasmid of Bti. Escherichiacoli recombinant clones containing the 130 kDa protein gene were highly active against larvae of Aedes aegypti and Culex pipiens, while B. subtilis recombinant cells containing the 28 kDa protein gene were haemolytic for sheep red blood cells. A fragment of the Bti plasmid which is partially homologous to the 130 kDa protein gene was also isolated; it probably corresponds to part of a second type of mosquitocidal toxin gene. Furthermore, restriction enzyme analysis suggested that the 130 kDa protein gene is located on the same Bti EcoRI fragment as another kind of Bti mosquitocidal protein gene cloned by Thorne et al. (1986). Hybridization experiments conducted with the 28 kDa protein gene and the 230 kDa protein gene showed that these two Bti genes are probably present in the plasmid DNA of B. thuringiensis subsp. morrisoni (PG14), which is also highly active against mosquito larvae.     

Monitoring resistance to Bacillus thuringiensis subsp. israelensis in the field by performing bioassays with each Cry toxin separately

Bacillus thuringiensis subsp. israelensis (Bti) is increasingly used worldwide for mosquito control and is the only larvicide used in the French Rhône-Alpes region since decades. The artificial selection of mosquitoes with field-persistent Bti collected in breeding sites from this region led to a moderate level of resistance to Bti, but to relatively high levels of resistance to individual Bti Cry toxins. Based on this observation, we developed a bioassay procedure using each Bti Cry toxin separately to detect cryptic Bti-resistance evolving in field mosquito populations. Although no resistance to Bti was detected in none of the three mosquito species tested (Aedes rusticus, Aedes sticticus and Aedes vexans), an increased tolerance to Cry4Aa (3.5-fold) and Cry11Aa toxins (8-fold) was found in one Ae. sticticus population compared to other populations of the same species, suggesting that resistance to Bti may be arising in this population. This study confirms previous works showing a lack of Bti resistance in field mosquito populations treated for decades with this bioinsecticide. It also provides a first panorama of their susceptibility status to individual Bti Cry toxins. In combination with bioassays with Bti, bioassays with separate Cry toxins allow a more sensitive monitoring of Bti-resistance in the field.     

A mosquito-virulent Bacillus sphaericus in adult Simulium damnosum from Northern Nigeria

From newly emerged adult Simulium damnosum from Kaduna River, Northern Nigeria, a mosquito-virulent strain of Bacillus sphaericus was isolated. In another four cases non-virulent B. sphaericus were present. The isolate exerted a LC₅₀ of 50 spores/ml for Culex pipiens L₄-larvae and was less virulent for Anopheles stephensi (400 sp/ml) and Aedes aegypti (800 sp/ml).     

Expression of Bacillus thuringiensis mosquitocidal toxin in an antimicrobial Bacillus brevis strain

Brown blotch is a common disease of many mushrooms, especially oyster mushrooms, in Korea. Recently, we isolated a promising Bacillus brevis strain which has antimicrobial activity against Pseudomonas tolaasii, a serious mushroom pathogen. In this study, in order to confer insecticidal activity, a recombinant B. brevis strain was constructed via the expression of Bacillus thuringiensis mosquitocidal crystal protein. A B. thuringiensis expression vector (pPro11A), which contained the cry11Aa gene under the control of cry1Ac promoter, was introduced into this B. brevis strain. The recombinant B. brevis strain successfully expressed and produced rhomboidal shaped Cry11A protein, although the initiation time of expression was slower than that of B. thuringiensis Cry^-B transformant. The insecticidal and antimicrobial activities of the transformant were verified using two dipteran larvae, Aedes aegypti and Culex pipiens, and four bacteria, including P. tolaasii, respectively. These results demonstrate that the introduction of B. thuringiensis insecticidal activity to antimicrobial Bacillus strains might be a useful tool to construct dual functional Bacillus strains.     

Isolation, Characterization and Biological Role of Camelysin from Bacillus thuringiensis Subsp. israelensis

The present study reports a simple rapid method for isolating the zinc-containing metalloprotease camelysin from Bacillus thuringiensis subsp. israelensis (Bti) by extraction from intact bacterial cells with egg l-α-phosphatidylcholine containing monolamellar liposomes, followed by separation on a sucrose gradient. Characterization of the isolated camelysin revealed a molecular weight of 23 kDa and a pI of 6.2. The camelysin exhibited maximal activity against the substrate azocasein at a temperature of 37°C and pH 7.5. However, the enzyme’s activity remained high also at basic pH values (8–10). In a rich growth medium (LB), camelysin appeared at the late logarithmic phase of Bti growth and reached its maximum in the stationary phase. Camelysin was shown to activate the protoxins Cyt1Aa and Cyt2Ba produced by Bti. The hemolytic activity of Cyt1Aa increased from 40 to 70% and that of Cyt2Ba from 6 to 50% in the presence of 50% (w/w) camelysin. It is concluded that these protoxins can be activated not only by insect gut proteases, but also by the endogeneous metalloprotease camelysin of the Bti bacterium.     

Bacillus thuringiensis in Fecal Samples from Greenhouse Workers after Exposure to B. thuringiensis-Based Pesticides

In a study of occupational exposure to Bacillus thuringiensis, 20 exposed greenhouse workers were examined for Bacillus cereus-like bacteria in fecal samples and on biomonitoring filters. Bacteria with the following characteristics were isolated from eight individuals: intracellular crystalline inclusions characteristic of B. thuringiensis, genes for and production of B. cereus enterotoxins, and positivity for cry11 as determined by PCR. DNA fingerprints of the fecal isolates were identical to those of strains isolated from the commercial products used. Work processes (i.e., spraying) correlated with the presence of B. thuringiensis in the fecal samples (10² to 10³ CFU/g of feces). However, no gastrointestinal symptoms correlated with the presence of B. thuringiensis in the fecal samples.     

Cytotoxicity and anticancer studies of Bacillus cereus and Bacillus pumilus metabolites targeting human cancer cells

The metabolites of bacteria Bacillus cereus and Bacillus pumilus isolated from soil samples in Shimoga region, Karnataka (India) were tested for cytotoxicity and anticancer properties. The various solvent extract fractions obtained from the metabolites of the two bacteria were tested for their cytotoxicity against normal human liver cell lines and 2 cancer cell lines by (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole) assay. The two fractions obtained from B. cereus showed high cytotoxicity. These two fractions were further screened for anticancer activity by nuclear staining studies and DNA fragmentation analysis. Both the fractions demonstrated significant activity by membrane blebbing during nuclear staining and caused the damage the DNA patterns during DNA fragmentation analysis. On the other hand, the metabolites of B. pumilus revealed toxic effect against cancer cells as well as normal ones.     

Production of Cry11A and Cry11Ba Toxins in Bacillus sphaericus Confers Toxicity towards Aedes aegypti and Resistant Culex Populations

Cry11A from Bacillus thuringiensis subsp. israelensis and Cry11Ba from Bacillus thuringiensis subsp. jegathesan were introduced, separately and in combination, into the chromosome of Bacillus sphaericus 2297 by in vivo recombination. Two loci on the B. sphaericus chromosome were chosen as target sites for recombination: the binary toxin locus and the gene encoding the 36-kDa protease that may be responsible for the cleavage of the Mtx protein. Disruption of the protease gene did not increase the larvicidal activity of the recombinant strain against Aedes aegypti and Culex pipiens. Synthesis of the Cry11A and Cry11Ba toxins made the recombinant strains toxic to A. aegypti larvae to which the parental strain was not toxic. The strain containing Cry11Ba was more toxic than strains containing the added Cry11A or both Cry11A and Cry11Ba. The production of the two toxins together with the binary toxin did not significantly increase the toxicity of the recombinant strain to susceptible C. pipiens larvae. However, the production of Cry11A and/or Cry11Ba partially overcame the resistance of C. pipiens SPHAE and Culex quinquefasciatus GeoR to B. sphaericus strain 2297.     

Insecticidal Activity of Bacillus laterosporus

The Bacillus laterosporus strains 921 and 615 were shown to have toxicity for larvae of the mosquitoes Aedes aegypti, Anopheles stephensi, and Culex pipiens. The larvicidal activity of B. laterosporus was associated with spores and crystalline inclusions. Purified B. laterosporus 615 crystals were highly toxic for Aedes aegypti and Anopheles stephensi.     

Production of the bioinsecticide Bacillus thuringiensis subsp. israelensis with deltamethrin increases toxicity towards mosquito larvae

Bacillus thuringiensis subsp. israelensis is a bioinsecticide used for larval mosquito control and it represents a safe alternative to chemical insecticides. Despite its environmental safety, it is less efficient and persistent than chemical insecticides. To bypass these limitations, we propose to combine the advantages of chemical and biological insecticides by producing Bti in a medium supplemented with a chemical insecticide (DDT, deltamethrin, permethrin, propoxur or temephos). Among the investigated insecticides, the addition of deltamethrin in the medium induced a higher toxicity (over 6·72‐fold) of the composite deltamethrin‐Bti towards mosquito larvae as compared to Bti alone. This was mainly due to the insertion of deltamethrin into the membranes of Bti spores, as evidenced by a quantification of membrane‐extracted deltamethrin by HPLC. This composite larvicide is a promising tool to decrease the quantity of chemicals dispersed in the environment, to increase the efficacy of Bti and to facilitate its widespread use as a transition between chemical and biological insecticides. Further experiments are required to characterize the mechanisms that underline the incorporation of deltamethrin into Bti to optimize the production and the toxicity of this composite larvicide.

Significance and Impact of the Study

This study is the first report of an increased efficacy of the mosquitocidal bioinsecticide Bacillus thuringiensis subsp. israelensis (Bti) when produced with a chemical insecticide. The results clearly demonstrate that deltamethrin is able to synergize the insecticidal activity of Bti through inclusion into spore membranes, reducing off‐target and nonspecific toxicity occurring when the chemical is used alone as sprays. This new composite chemical–biological insecticide can become an invaluable tool as an intermediate between single chemical usage and the widespread use of Bti, notably in developing countries with limited financial resources for intensive mosquito control campaigns.     

Overexpression of polyphosphate kinase gene (ppk) increases bioinsecticide production by Bacillus thuringiensis

Polyphosphate (polyP), synthesized by polyP kinase (PPK) using the terminal phosphate of ATP as substrate, performs important functions in every living cell. The present work reports on the relationship between polyP metabolism and bioinsecticide production in Bacillus thuringiensis subsp. israelensis (Bti). The ppk gene of Bti was cloned into vector pHT315 and the effect of its overexpression on endotoxin production was determined. Endotoxin production by the recombinant strain was found to be consistently higher than that by the wild type strain and the strain that carried the empty plasmid. The toxicity of the recombinant mutant strain (LC₅₀ 5.8 ± 0.6 ng ml^?1) against late 2nd instar Culex quinquefasciatus was about 7.7 times higher than that of Bti (LC₅₀ 44.9 ± 7 ng ml^?1). To our knowledge this is the first reported study which relates polyP metabolism with bioinsecticide biosynthesis.     

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.     

Invasive Process and Repeated Cross-Sectional Surveys of the Mosquito Aedes japonicus japonicus Establishment in Belgium

When accidentally introduced in a new location, a species does not necessarily readily become invasive, but it usually needs several years to adapt to its new environment. In 2009, a national mosquito survey (MODIRISK) reported the introduction and possible establishment of an invasive mosquito species, Aedes j. japonicus, in Belgium. First collected in 2002 in the village of Natoye from a second-hand tire company, then sampled in 2003 and 2004, the presence of adults and larvae was confirmed in 2007 and 2008. A repeated cross-sectional survey of Ae. j. japonicus was then conducted in 2009 in Natoye to study the phenology of the species on two different sites using three kinds of traps: Mosquito Magnet Liberty Plus traps, BG sentinel traps and CDC Gravid traps. An analysis of the blood meals was done on females to assess the epidemiological risks. Five species of mosquitos were caught using the different kind of traps: Culex pipiens, Cx torrentium, Anopheles claviger, Aedes geniculatus and Ae. j. japonicus, Cx pipiens being the most abundant. The CDC gravid traps gave the best results. Surprisingly Ae. j. japonicus was only found on one site although both sites seem similar and are only distant of 2.5 km. Its population peak was reached in July. Most of the engorged mosquitoes tested acquired blood meals from humans (60%). No avian blood meals were unambiguously identified. Larvae were also collected, mostly from tires but also from buckets and from one tree hole. Only one larva was found in a puddle at 100 m of the tire storage. A first local treatment of Ae. j. japonicus larvae population was done in May 2012 using Bacillus thuringiensis subsp. israelensis (Bti) and was followed by preventive actions and public information. A monitoring is also presently implemented.     

Expression of the Bacillus thuringiensis Mosquitocidal Toxin Cry11Aa in the Aquatic Bacterium Asticcacaulis excentricus

A mosquitocidal aquatic bacterium has been developed by introducing an operon containing the cry11Aa, and p20 genes from Bacillus thuringiensis subsp. israelensis (Bti) into the gram-negative aquatic bacterium Asticcacaulis excentricus. After transformation, the cry11Aa gene was successfully expressed in recombinant A. excentricus under the tac promoter, at the level of 0.04 pg/cell. The recombinant bacteria were toxic to Aedes aegypti larvae with an LC₅₀ of 6.83 × 10⁵ cells/mL. We believe that these bacteria may have potential as genetically engineered microorganisms for the control of mosquito larvae.     

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.     

Identification and Characterization of Rhizosphere-Competent Bacteria of Wheat

To obtain rhizosphere-competent bacteria which could subsequently be modified for the development of biological control agents, bacteria were isolated from the rhizosphere and rhizoplane of wheat and barley plants by standard techniques. Of these isolates, 60 were selected for field testing as spring wheat seed inoculants in 1985. Isolates were marked genetically for resistance to antibiotics via selection of spontaneous mutants to detect and monitor isolates in the field. Forty-three days after planting, the average log₁₀ CFU/mg (dry weight) of roots and rhizosphere soil for the mutant isolates sampled ranged from 0 to 3.4. Twenty mutant isolates were retested in 1986. A total of 4 isolates were not detected, but the other 16 had an average root colonization value of log₁₀ 2.1 CFU and a range of log₁₀ 0.9 CFU to log₁₀ 3.2 CFU when sampled 32 days after planting. The average colonization value dropped to log₁₀ 1.1 CFU 51 days later. Some isolates detected previously were not detected in the second sampling; others had root colonization values similar to those obtained in the first sampling. Mutant isolates of rhizosphere bacteria included Bacillus pumilus, Bacillus subtilis, Pseudomonas fluorescens, Streptomyces spp., Xanthomonas maltophilia, and a saprophytic coryneform. Mixtures of isolates from different genera and species were compatible on seeds and roots.     

Transstadial Effects of Bti on Traits of Aedes aegypti and Infection with Dengue Virus

Most mosquito control efforts are primarily focused on reducing the adult population size mediated by reductions in the larval population, which should lower risk of disease transmission. Although the aim of larviciding is to reduce larval abundance and thus recruitment of adults, nonlethal effects on adults are possible, including transstadial effects on phenotypes of adults such as survival and pathogen infection and transmission. In addition, the mortality induced by control efforts may act in conjunction with other sources of mosquito mortality in nature. The consequences of these effects and interactions may alter the potential of the population to transmit pathogens. We tested experimentally the combined effects of a larvicide (Bacillus thuringiensis ssp. israelensis, Bti) and competition during the larval stages on subsequent Aedes aegypti (Linnaeus) traits, population performance, and susceptibility to dengue-1 virus infection. Ae. aegypti that survived exposure to Bti experienced accelerated development, were larger, and produced more eggs with increasing amounts of Bti, consistent with competitive release among surviving mosquitoes. Changing larval density had no significant interactive effect with Bti treatment on development and growth to adulthood. Larval density, but not Bti or treatment interaction, had a strong effect on survival of adult Ae. aegypti females. There were sharper declines in cumulative daily survival of adults from crowded than uncrowded larval conditions, suggesting that high competition conditions of larvae may be an impediment to transmission of dengue viruses. Rates of infection and dengue-1 virus disseminated infections were found to be 87±13% and 88±12%, respectively. There were no significant treatment effects on infection measurements. Our findings suggest that larvicide campaigns using Bti may reduce the number of emerged adults, but survivors will have a fitness advantage (growth, development, enhanced production of eggs) relative to conspecifics that are not under larvicide pressure. However, under most circumstances, these transstadial effects are unlikely to outweigh reductions in the adult population by Bti and altered risk of disease transmission.