Mosquitocidal Bacillus Thuringiensis Research Articles

De novo determination of mosquitocidal Cry11Aa and Cry11Ba structures from naturally-occurring nanocrystals

Cry11Aa and Cry11Ba are the two most potent toxins produced by mosquitocidal Bacillus thuringiensis subsp. israelensis and jegathesan, respectively. The toxins naturally crystallize within the host; however, the crystals are too small for structure determination at synchrotron sources. Therefore, we applied serial femtosecond crystallography at X-ray free electron lasers to in vivo-grown nanocrystals of these toxins. The structure of Cry11Aa was determined de novo using the single-wavelength anomalous dispersion method, which in turn enabled the determination of the Cry11Ba structure by molecular replacement. The two structures reveal a new pattern for in vivo crystallization of Cry toxins, whereby each of their three domains packs with a symmetrically identical domain, and a cleavable crystal packing motif is located within the protoxin rather than at the termini. The diversity of in vivo crystallization patterns suggests explanations for their varied levels of toxicity and rational approaches to improve these toxins for mosquito control.

Larvicidal activities of local Bacillus thuringiensis isolates and toxins from nematode bacterial symbionts against the Rift Valley fever vector, Aedes caspius (Diptera: Culicidae)

Mosquito resistance to insecticides has recently increased the burden of the majority of mosquito-borne diseases, and controlling the spread of these diseases should mainly rely on vector control measures. The present study aimed to investigate the toxicity of locally isolated mosquitocidal Bacillus thuringiensis (Bt) isolates and toxin complexes (TCs) from the two nematode bacterial symbionts Photorhabdus luminescens akhurstii (HRM1) and Photorhabdus luminescens akhurstii (HS1) on Aedes caspius. Out of sixty-eight local Bt isolates, four showed LC50 values ranging from 13.8–15.6 ppm, similar to LC50 recorded against the reference Bacillus thuringiensis israelensis (Bti-H14) (13.0 ppm). Seventeen isolates were more toxic than the Bti-H14 by 1.3–11.1 fold (LC50 values 1.17–9.9 ppm vs 13.0 ppm). Two isolates were less toxic than Bti-H14, showing LC50 values of 39.4 and 24.1 ppm, respectively. Additionally, the TCs from HRM1 and HS1 showed promising larvicidal toxicity, with HS1 being 2.74 fold more toxic than HRM1. Larval morphological features and body size were altered upon HS1 toxin-treatment. This study suggests that there is a new effective local Bt strain, and TCs toxin complexes could be used as eco-friendly biocontrol agents that might contribute to controlling mosquito-borne diseases. The synergistic effect of the Bt-TCs combination on mosquito larvae should be further investigated.

Computational identification and evolutionary analysis of toxins in Mosquitocidal Bacillus thuringiensis strain S2160-1.

Mosquitocidal Bacillus thuringiensis (Bt) strain S2160-1 was proposed to be an alternative to Bacillus thuringiensis subsp. israelensis (Bti). Discovering and validating a toxic gene by experimentation was a complex and time-consuming task, which can benefit from high-throughput sequencing analysis. In this research, we predicted and identified toxic proteins in the strain S2160-1 based on the draft whole genome sequence data. Through a local BLASP, 46 putative toxins were identified in S2160-1 genome, by searching against a customized B. thuringiensis toxin proteins database containing 653 protein or peptide sequences retrieved from public accessible resources and PCR/clone results in our laboratory (e value = 1e - 5). These putative toxins consist of 42 to 1216 amino acids. The molecular weights are ranged from 4.86 to 137.28kDa. The isoelectric point of these candidate toxins varied from 4.3 to 10.06, and 16 out of which had a pH greater than 7.0. The analysis of tertiary structure and PFAM domain showed that 12 potential plasmid toxins may share higher similarity (9/12 QMEAN4 score > 0.3) with known Bt toxins. In addition, functional annotation indicated that these 12 potential toxins were involved in "sporulation resulting in formation of a cellular spore" and "toxin activity". Moreover, multiple alignment and phylogenetic analysis were carried out to elucidate the evolutionary relationship among 101 known crystal or toxin proteins from public database and them with MEGA 6.0. It indicated that PS2160P2_1 and PS2160P2_153 may be potential Cry4-like toxins in Bt S2160-1. This research may lay the foundation for future functional analysis of Bt S2160-1 toxin proteins to reveal their biological roles.

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.

Mosquito Resistance to Bacterial Larvicidal Toxins

Insecticide resistance to the microbial insecticides Bacillus thuringiensis subsp. israelensis (Bti) and Bacillus sphaericus (Bs) represents a serious threat to their success. Available evidence indicates that the risk for resistance to Bti is low due to the makeup of its parasporal crystal, which contains Cyt1A, Cry4A, Cry4B, and Cry11A toxic proteins. Disrupting the toxin complex in Bti enables resistance to evolve, especially in the absence of the key factor, the cytolytic toxin, Cyt1A. Cross-resistance is widespread among mosquitocidal Bacillus thuringiensis Cry toxins and the mechanisms of Cry resistance in mosquitoes are not known. Bacillus sphaericus (Bs) is at higher risk for resistance due to its single- site action and field cases have been reported from a number of locations worldwide. Cross-resistance is reported among the various Bs isolates, although some isolates produce additional toxic proteins that can reduce cross-resistance and slow resistance evolution. Field and lab evolved resistant populations consistently show recessive and monofactorial inheritance of resistance. Resistant populations, however, have evolved a variety of molecular mechanisms causing that resistance. Traditional resistance management strategies with promise include rotations and mixtures of Bti and Bs, as well as untreated areas that provide natural refuges for susceptible alleles. Promising new strategies include genetic engineering to increase the toxin complexity targeted toward mosquito larvae, to enhance the host range of the mosquito control product, and to avoid the evolution of insecticide resistance. Regardless of the control strategy, a resistance- monitoring program alongside an integrative pest management approach is the best strategy to delay insecticide resistance.

The potential of the novel mosquitocidal Bacillus thuringiensis strain LLP29 for use in practice

1Key Laboratory of Biopesticide and Chemical Biology, Ministry of Education, Fujian Agriculture and Forestry University, 350002 Fuzhou, Fujian, People’s Republic of China 2Fujian International Travel Health Care Center, 350001 Fuzhou, Fujian, People’s Republic of China 3Biological Centre of the Academy of Sciences of the Czech Republic, Institute of Entomology, Branisovska 31, 37005 Ceske Budějovice, Czech Republic, gelbic@entu.cas.cz

A novel mosquitocidal Bacillus thuringiensis strain LLP29 isolated from the phylloplane of Magnolia denudata

Eleven Bacillus thuringiensis isolates were recovered from phylloplanes of Magnolia denudata, a specific source of new strains of B. thuringiensis. Among these, a new strain, LLP29, was found to be most toxic to mosquitoes based on the results of preliminary toxicity analysis. Phase contrast microscopy, mosquitocidal activity, polymerase chain reaction (PCR) analysis and parasporal inclusion were performed to learn more about the characteristics of this novel mosquitocidal isolate. The LC(50) values of LLP29 against Aedes albopictus and Culex quinquefasciatus were 0.33 and 0.04 ng of protein/ml, respectively. The cyt1 gene, which encodes the Cyt protein that is toxic to mosquitoes, was subsequently detected, cloned, sequenced and expressed in acrystalliferous Bt HD73 Cry(-). The results indicated that it might be a member of the cyt1Aa gene group. The novel strain LLP29 appears to be a new subspecies of B. thuringiensis and should prove useful in the control of mosquitoes and mosquito-borne diseases.

Distribution of mosquitocidal Bacillus thuringiensis and Bacillus sphaericus from sediment samples in Florida

A spore-forming soil bacterium, Bacillus thuringiensis subsp. israelensis has been used most widely and extensively in many regions of the world to control mosquito and blackfly larvae. It contains four major proteins in three different inclusion types assembled into a spherical parasporal body. Although no resistance to B. thuringiensis subsp. israelensis has been reported in the field yet, laboratory selection of mosquitoes with B. thuringiensis subsp. israelensis Cry proteins results in high levels of resistance. Furthermore, mosquito resistance to any of these proteins results in significant cross-resistance to the others. Another spore-forming bacterium, B. sphaericus also has been used to control mosquito larvae. However, as this bacterium contains only a single binary toxin, high levels of resistance to B. sphaericus have already been reported in the field in several countries. Consequently, there is an urgent need to search for and isolate new indigenous and more effective mosquitocidal bacterial strains. Therefore, in this study, we have screened sediment samples from various mosquito-breeding sites in Florida to isolate novel mosquitocidal bacilli with high toxicity. The results showed that mosquitocidal B. thuringiensis and B. sphaericus can be found almost everywhere.

Characterization of three mosquitocidal Bacillus thuringiensis strains isolated from urban soils of Hanoi, Vietnam

Characterization of three mosquitocidal Bacillus thuringiensis strains isolated from urban soils of Hanoi, Vietnam

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.

Cloning and Characterization of Two Novel Genes, cry24B and s1orf2, from a Mosquitocidal Strain of Bacillus thuringiensis serovar sotto

Two new crystal protein genes, cry24B and s1orf2, were cloned from a mosquitocidal Bacillus thuringiensis serovar sotto strain. The cry24B and s1orf2 genes encoded a 76-kDa and 62-kDa protein, respectively. The Cry24B protein retained five conserved regions commonly found in the existing Cry proteins. The amino acid sequence of the S1ORF2 had a high homology to that of the ORF2 protein of B. thuringiensis serovar jegathesan. Southern hybridization experiments with a cry24B gene-specific probe revealed that these genes are located on two large plasmids of > 100 kb. When the two genes, cry24B and s1orf2, were expressed in an acrystalliferous B. thuringiensis host, the proteins were synthesized and accumulated as inclusions. These inclusions exhibited no larvicidal activities against three mosquito species: Aedes aegypti, Anopheles stephensi, and Culex pipiens molestus. Likewise, the inclusions contained no cytocidal activity against HeLa cells.

Characterization of a mosquitocidal Bacillus thuringiensis serovar sotto strain isolated from Okinawa, Japan.

To characterize the mosquitocidal activity of parasporal inclusions of the Bacillus thuringiensis serovar sotto strain 96-OK-85-24, for comparison with two well-characterized mosquitocidal strains. The strain 96-OK-85-24 significantly differed from the existing mosquitocidal B. thuringiensis strains in: (1) lacking the larvicidal activity against Culex pipiens molestus and haemolytic activity, and (2) SDS-PAGE profiles, immunological properties and N-terminal amino acid sequences of parasporal inclusion proteins. It is clear from the results that the strain 96-OK-85-24 synthesizes a novel mosquitocidal Cry protein with a unique toxicity spectrum. This is the first report of the occurrence of a mosquitocidal B. thuringiensis strain with an unusual toxicity spectrum, lacking the activity against the culicine mosquito.

Cry29A and Cry30A: Two Novel δ-endotoxins Isolated from Bacillus thuringiensis serovar medellin

Two novel crystal protein genes from a highly mosquitocidal Bacillus thuringiensis serovar medellin strain were cloned and sequenced. The corresponding proteins, Cry29A and Cry30A, were nontoxic when tested individually against the mosquito species bioassayed (Aedes aegypti, Culex pipiens and Anopheles stephensi). However, Cry29A synergized the toxicity of Cry11Bb against Aedes aegypti by a four-fold factor.

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.

Identification and characterization of a previously undescribed cyt gene in Bacillus thuringiensis subsp. israelensis.

Mosquitocidal Bacillus thuringiensis strains show as a common feature the presence of toxic proteins with cytolytic and hemolytic activities, Cyt1Aa1 being the characteristic cytolytic toxin of Bacillus thuringiensis subsp. israelensis. We have detected the presence of another cyt gene in this subspecies, highly homologous to cyt2An1, coding for the 29-kDa cytolytic toxin from B. thuringiensis subsp. kyushuensis. This gene, designated cyt2Ba1, maps upstream of cry4B coding for the 130-kDa crystal toxin, on the 72-MDa plasmid of strain 4Q2-72. Sequence analysis revealed, as a remarkable feature, a 5' mRNA stabilizing region similar to those described for some cry genes. PCR amplification and Southern analysis confirmed the presence of this gene in other mosquitocidal subspecies. Interestingly, anticoleopteran B. thuringiensis subsp. tenebrionis belonging to the morrisoni serovar also showed this gene. On the other hand, negative results were obtained with the anti-lepidopteran strains B. thuringiensis subsp. kurstaki HD-1 and subsp. aizawai HD-137. Western analysis failed to reveal Cyt2A-related polypeptides in B. thuringiensis subsp. israelensis 4Q2-72. However, B. thuringiensis subsp. israelensis 1884 and B. thuringiensis subsp. tenebrionis did show cross-reactive products, although in very small amounts.

Characterization of six highly mosquitocidal Bacillus thuringiensis strains that do not belong to H-14 serotype.

Four strains belonging to Bacillus thuringiensis serovars thompsoni, malaysiensis, canadensis, jegathesan and two auto-agglutinating B.t. strains were identified as being highly toxic to the mosquito larvae of the species Aedes aegypti, Anopheles stephensi, and Culex pipiens. Their larvicidal and hemolytic activities were determined and compared with those of strains known to be highly mosquitocidal and/or cytolytic from serovars of B.t. israelensis, morrisoni, darmstadiensis, medellin, kyushuensis, and fukuokaensis. The electrophoretic protein profiles of purified crystals and immunological relationships with B.t.i. polypeptides were studied. Five out of the six new strains showed the same larvicidal and hemolytic activities and the same crystal proteins and toxin genes as B.t.i. One strain, B.t. jegathesan 367, presented a novel pattern of larvicidal activity and a protein profile different from those of other strains.

Isolation and Identification of novel toxins from a new mosquitocidal isolate from Malaysia, Bacillus thuringiensis subsp. jegathesan.

A new mosquitocidal Bacillus thuringiensis subsp., jegathesan, has recently been isolated from Malaysia. Parasporal crystal inclusions were purified from this strain and bioassayed against fourth-instar larvae of Culex quinquefasciatus, Aedes aegypti, Aedes togoi, Aedes albopictus, Anopheles maculatus, and Mansonia uniformis. The 50% lethal concentration of crystal inclusions for each species was 0.34, 8.08, 0.34, 17.59, 3.91, and 120 ng/ml, respectively. These values show that parasporal inclusions from this new subspecies have mosquitocidal toxicity comparable to that of inclusions isolated from B. thuringiensis subsp. israelensis. Solubilized and chymotrypsin-activated parasporal inclusions possessed low-level hemolytic activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the crystals were composed of polypeptides of 77, 74, 72, 68, 55, 38, 35, 27, and 23 kDa. Analysis by Western blotting (immunoblotting) with polyclonal antisera raised against toxins purified from B. thuringiensis subsp. israelensis reveals that proteins in parasporal inclusions of subsp. jegathesan are distinct, because little cross-reactivity was shown. Analysis of the plasmid content of B. thuringiensis subsp. jegathesan indicates that the genes for toxin production may be located on 105- to 120-kb plasmids. Cry- clones that have been cured of these plasmids are nontoxic. Southern blot analysis of plasmid and chromosomal DNA from subsp. jegathesan showed little or low homology to the genes coding for CryIVA, CryIVB, and CryIVD from B. thuringiensis subsp. israelensis.

Evaluation of Bacillus thuringiensis H-14 isolates from Nigerian soils for use in mosquito control

Eleven mosquitocidal Bacillus thuringiensis isolates from Nigerian soils were screened for the degree of toxicity against Aedes aegypti larvae. The six most larvicidal isolates, code-named OBGI, OBG8, BUS4, BAR3, GSC3 and GNA13, were identified as subsp. israelensis (B.t.i). pores of the six isolates of a B. t. i. from a commercial powder (R-153-78, Roger Bellon Laboratory, Belgium) and of IPS 82 (Institut Pasteur (Standards)) were subjected to ultraviolet irradiation (280-350 nm) for up to 75 min. After irradiation, the toxicity of OBG8, OBGI, BUS4, and BAR3 was higher than that of R-153-78 but lower than that of IPS 82.

Effect of promoter modification on mosquitocidal cryIVB gene expression in Synechococcus sp. strain PCC 7942.

The impact of promoter modification on the expression of the mosquitocidal Bacillus thuringiensis subsp. israelensis cryIVB gene when used to transform the cyanobacterium Synechococcus sp. strain PCC 7942 has been examined. Maximal transcript and protein abundances were achieved by the addition of the lacZ promoter upstream of the cryIVB sequence. Replacement of the endogenous corresponding Bacillus sequences with the Synechococcus petF1 promoter, ribosome binding site, and initiation codon also resulted in increased expression of the cryIVB gene relative to the expression obtained with the Bacillus promoter alone but decreased expression relative to the expression achieved with the tandem array of the Bacillus and lacZ promoters. Synechococcus cells carrying plasmids in which the expression of the cryIVB gene was regulated by either the lacZ or the petF1 promoter were readily consumed by first-instar Culex restuans larvae and proved to be toxic for these organisms.

Investigation of possible homologies between crystal proteins of three mosquitocidal strains ofBacillus thuringiensis

The possible presence of protein δ-endotoxins with homology to a cloned mosquitocidal Bacillus thuringiensis var. israelensis δ-endotoxin was investigated in two other mosquitocidal B. thuringiensis strains. Antiserum raised against the var. israelensis 27-kDa toxic polypeptide cross-reacted strongly with a 27-kDa crystal polypeptide of var. PG14 (serotype 8a:8b, a serotype more usually associated only with lepidopteran toxicity). In addition hybridisation studies using a DNA probe derived from the cloned var. israelensis 27-kDa δ-endotoxin gene located a homologous region on a 72–74-MDa plasmid of PG14. A less toxic strain, var. Kyushuensis , showed neither immunological cross-reaction with the antiserum nor DNA hybridization with the probe.