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Background: Conventional methods often fail to control the flatheaded borers Capnodis spp., major pests of stone fruit trees; the larvae are protected from insecticides and predation because they feed deep in the roots. A potential solution is transgenic trees producing in their roots toxic compounds such as Cry proteins of Bacillus thuringiensis (Bt)., Results: Toxicities against Capnodis larvae were demonstrated by exploiting a recently designed artificial larval diet and an available collection of field isolated Bt. An isolate of Bt tenebrionis (Btt) from commercial bioinsecticide (Novodor) displayed LC50 and LC95 values of 3.2 and 164 mg g(-1) , respectively, against neonates of Capnodis tenebrionis, whereas values of the most toxic field isolate K-7 were 1.9 and 25.6 mg g(-1) respectively. Weights of surviving larvae after 1 month on diets containing low concentrations of K-7 (0.1-1.0 mg g(-1) ) were lower than on Btt or untreated larvae. K-7 was also toxic against larvae of C. cariosa and C. miliaris and found to harbour genes encoding Cry9Ea-like and Cry23Aa/Cry37Aa binary toxins., Conclusion: Larvae of Capnodis spp. are susceptible to Bt Cry toxins. Expressing cry genes active against these pests thus seems a feasible solution towards production of transgenic rootstock trees resilient to the pest., (© 2013 Society of Chemical Industry.)

Bacillus thuringiensis subsp. israelensis (Bti) is the first Bacillus thuringiensis to be found and used as an effective biological control agent against larvae of many mosquito and black fly species around the world. Its larvicidal activity resides in four major (of 134, 128, 72 and 27 kDa) and at least two minor (of 78 and 29 kDa) polypeptides encoded respectively by cry4Aa, cry4Ba, cry11Aa, cyt1Aa, cry10Aa and cyt2Ba, all mapped on the 128 kb plasmid known as pBtoxis. These six δ-endotoxins form a complex parasporal crystalline body with remarkably high, specific and different toxicities to Aedes, Culex and Anopheles larvae. Cry toxins are composed of three domains (perforating domain I and receptor binding II and III) and create cation-selective channels, whereas Cyts are composed of one domain that acts as well as a detergent-like membrane perforator. Despite the low toxicities of Cyt1Aa and Cyt2Ba alone against exposed larvae, they are highly synergistic with the Cry toxins and hence their combinations prevent emergence of resistance in the targets. The lack of significant levels of resistance in field mosquito populations treated for decades with Bti-bioinsecticide suggests that this bacterium will be an effective biocontrol agent for years to come.

During sporulation, Bacillus thuringiensis subsp. israelensis produces a mosquito larvicidal protein complex containing several crystalline and cytolytic (Cyt) toxins. Here, the activated monomeric form of Cyt1Aa, the most toxic Cyt family member, was isolated and crystallized, and its structure was determined for the first time at 2.2 Å resolution. Cyt1Aa adopts a typical cytolysin fold containing a β-sheet held by two surrounding α-helical layers. The absence of a β-strand (between residues V26 and I37) in the dimeric structure of Cyt2Aa led us to deduce that this is the only essential segment for dimer formation and that activation of the toxin occurs by proteolytic processing of its N-terminus. Based on the Cyt1Aa structure, we suggest that the toxicity of Cyt1Aa and other nonrelated proteins, all sharing a cytolysin fold, is correlated with their ability to undergo conformational changes that are necessary prior to their membrane insertion and perforation. This fold allows the α-helical layers to swing away, exposing the β-sheet to insert into the membrane. The identification of a putative lipid binding pocket between the β-sheet and the helical layer of Cyt1Aa supports this mechanism. Sequence-based structural analysis of Cyt1Aa revealed that the lack of activity of Cyt1Ca may be related to the latter's inability to undergo this conformational change due to its lack of flexibility. The pattern of the hemolytic activity of Cyt1Aa presented here (resembling that of pore-forming agents), while differing from that imposed by ionic and nonionic detergents, further supports the pore-forming model by which conformational changes occur prior to membrane insertion and perforation., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Modular transparent column system was designed to study depth-dependence behavior of mosquito larvae. The system was used in preliminary experiments to evaluate the effect of water depth on the larvicidal activity of Bacillus thuringiensis subsp. israelensis de Barjac against bottom feeder larvae of Aedes aegypti (Linn.) (Diptera: Culicidae), and suggestions for increasing the efficiency of the device are discussed., (Copyright © 2010 Elsevier B.V. All rights reserved.)

A new gene, cry11Bb2 from a field isolate of Bacillus thuringiensis, was cloned for expression in Escherichia coli. The encoded protein, with a deduced molecular mass of 89.5 kDa, exhibits 97 and 79% identities with the overlap regions of Cry11Bb1 from B. thuringiensis ssp. medellin and Cry11Ba1 from ssp. jegathesan, respectively. It is however longer than Cry11Bb1 by 42 amino acids in its carboxy-terminus, of which 32 comprise 2 tandem repeats additional to the 5 existing in the latter polypeptide. Possible roles for this recurrent motif among Cry toxins and their accessory proteins, and for their encoding genes are proposed., (Copyright © 2011 S. Karger AG, Basel.)

Various subspecies (ssp.) of Bacillus thuringiensis (Bt) are considered the best agents known so far to control insects, being highly specific and safe, easily mass produced and with long shelf life.1 The para-crystalline body that is produced during sporulation in the exosporium includes polypeptides named δ-endotoxins, each killing a specific set of insects. The different entomopathogenic toxins of various Bt ssp. can be manipulated genetically in an educated way to construct more efficient transgenic bacteria or plants that express combinations of toxin genes to control pests.2 Joint research projects in our respective laboratories during the last decade demonstrate what can be done by implementing certain ideas using molecular biology with Bt ssp. israelensis (Bti) as a model system. Here, we describe our progress achieved with Gram-negative bacterial species, including cyanobacteria, and some preliminary experiments to form transgenic plants, mainly to control mosquitoes (Diptera), but also a particular Lepidopteran and Coleopteran pest species. In addition, a system is described by which environment-damaging genes can be removed from the recombinants thus alleviating procedures for obtaining permits to release them in nature., (© 2010 Landes Bioscience)

Comparing activities of purified toxins from Bacillus thuringiensis ssp. israelensis against larvae of seven mosquito species (vectors of tropical diseases) that belong to three genera, gleaned from the literature, disclosed highly significant variations in the levels of LC(50) as well as in the hierarchy of susceptibilities. Similar toxicity comparisons were performed between nine transgenic Gram-negative species, four of which are cyanobacterial, expressing various combinations of cry genes, cyt1Aa and p20, against larvae of four mosquito species as potential agents for biological control. Reasons for inconsistencies are listed and discussed. Standard conditions for toxin isolation and presentation to larvae are sought. A set of lyophilized powders prepared identically from six Escherichia coli clones expressing combinations of four genes displayed toxicities against larvae of three mosquito species, with levels that differed between them but with identical hierarchy.

The Cyt family of proteins consists of delta-endotoxins expressed during sporulation of several subspecies of Bacillus thuringiensis. Its members possess insecticidal, hemolytic, and cytolytic activities through pore formation and attract attention due to their potential use as vehicles for targeted membrane destruction. The delta-endotoxins of subsp. israelensis include three Cyt species: a major Cyt1Aa and two minor proteins, Cyt2Ba and Cyt1Ca. A cleaved Cyt protein that lacks the N- and C-terminal segments forms a toxic monomer. Here, we describe the crystal structure of Cyt2Ba, cleaved at its amino and carboxy termini by bacterial endogenous protease(s). Overall, its fold resembles that of the previously described volvatoxin A2 and the nontoxic form of Cyt2Aa. The structural similarity between these three proteins may provide information regarding the mechanism(s) of membrane-perforating toxins.

The genes cry1Ac and cry1Ca from Bacillus thuringiensis subsps. kurstaki HD-73 and aizawai 4J4, respectively, encoding delta-endotoxins against lepidopteran larvae were isolated, cloned and expressed in Escherichia coli, with and without cyt1Aa (encoding cytolytic protein) and p20 (accessory protein) from subsp. israelensis. Nine combinations of the genes under control of an early T7, P A1 inducible promoter, produced the encoding proteins. Toxicities were examined against larvae of three major agricultural pests: Pectinophora gossypiella, Helicoverpa armigera and Spodoptera littoralis. The clones expressing cyt1Aa, with or without p20, were not toxic. The clone expressing cry1Ac (pBt-1A) was the most toxic to P. gossypiella (LC50 of 0.27 x 10(8) cells g(-1)). Clone pBt-1CA expressing cry1Ca and cry1Ac displayed the highest toxicity (LC50 of 0.12 x 10(8) cells ml(-1)) against S. littoralis. Clone pBt-1CARCy expressing all four genes (cry1Ca, cry1Ac, p20, cyt1Aa) in tandem exhibited the highest toxicity to H. armigera (LC50 of 0.16 x 10(8) cells ml(-1)). Cyt1Aa failed to raise the toxicity of these Cry toxins against P. gossypiella and S. littoralis but significantly enhanced toxicity against H. armigera. Two additional clones expressing either cry1Ac or cry1Ca under tandem promoters, P A1 and P psbA (constitutive), displayed significantly higher toxicities (7.5- to 140-fold) than their counterparts with P A1 alone, reducing the LC50 values to below 10(7) cells ml(-1).

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

The larvicidal activity of Bacillus thuringiensis subsp. israelensis against dipteran larvae is determined by four major polypeptides of the parasporal crystalline body produced during sporulation. Cyt1Aa shows the lowest toxicity when used alone but is the most synergistic with any of the other proteins. The sequence of the plasmid pBtoxis, which contains all the toxin genes in this subspecies, revealed a new cyt-like coding sequence named cyt1Ca. In addition to the Cyt-like region, the predicted Cyt1Ca contained an extra domain at the C terminus, which appeared to be a beta-trefoil carbohydrate-binding motif, as found in several ricin-like toxins. The gene was PCR-amplified from pBtoxis and cloned in several vectors, allowing high-level expression in Escherichia coli. Cyt1Ca was purified by nickel-nitrilotriacetic acid affinity chromatography, characterized, and its biological activity was determined. Toxicity against larvae of Aedes aegypti of Cyt1Ca in recombinant E. coli cells was compared with that of Cyt1Aa and Cyt2Ba, and the ability of these proteins to enhance the activity of Cry4Aa was assessed. Although Cyt2Ba appeared able to interact with Cry4Aa, no activity for Cyt1Ca was observed, even when produced in truncated form. Furthermore, in contrast to Cyt1Aa, Cyt1Ca did not lyse sheep erythrocytes, and it was not bactericidal to the host cell.

The gene cyt2Ba of Bacillus thuringiensis subsp. israelensis was cloned for expression, together with p20, in an acrystalliferous strain. The large hexagonal crystals formed were composed of Cyt2Ba, which facilitated its purification. Crystal solubilization in the presence of endogenous proteases (with spores and cell debris) enabled quick and simple procedure to obtain rather pure and active toxin species by cleavage between amino acid residues 34 and 35, most likely by a camelysin-like protease that was discovered in association with activated Cyt2Ba. The product of this cleavage displayed haemolytic activity comparable to that of exogenously activated Cyt2Ba. The sequence of this putative protease shares high homology with the cell envelope-bound metalloprotease (camelysin) of the closely related species Bacillus cereus.

Compaction of the Escherichia coli nucleoid in the cell's centre was associated with the loss of colony-forming ability; these effects were caused by induction of Cyt1Aa, the cytotoxic 27 kDa protein from Bacillus thuringiensis subsp. israelensis. Cyt1Aa-affected compaction of the nucleoids was delayed but eventually more intense than compaction caused by chloramphenicol. The possibility that small, compact nucleoids in Cyt1Aa-expressing cells resulted in DNA replication run-out and segregation following cell division was ruled out by measuring relative nucleoid length. Treatments with membrane-perforating substances other than Cyt1Aa did not cause such compaction of the nucleoids, but rather the nucleoids overexpanded to occupy nearly all of the cell volume. These findings support the suggestion that, in addition to its perforating ability, Cyt1Aa causes specific disruption of nucleoid associations with the cytoplasmic membrane. In situ immunofluorescence labelling with Alexa did not demonstrate a great amount of Cyt1Aa associated with the membrane. Clear separation between Alexa-labelled Cyt1Aa and 4',6-diamidino-2-phenylindole (DAPI)-stained DNA indicates that the nucleoid does not bind Cyt1Aa. Around 2 h after induction, nucleoids in Cyt1Aa-expressing cells started to decompact and expanded to fill the whole cell volume, most likely due to partial cell lysis without massive peptidoglycan destruction.

Genes encoding the mosquito larvicidal toxins Cry4Aa, Cry11Aa, Cyt1Aa and the regulatory P20 from Bacillus thuringiensis subsp. israelensis were introduced into the nitrogen-fixing, filamentous cyanobacterium Anabaena PCC 7120 for expression under control of two strong promoters P(psbA) and P(A1). The clone pRVE4-ADRC displayed toxicity against fourth-instar larvae of Aedes aegypti, the highest ever achieved in cyanobacteria. It was about 2.5-fold more toxic than the respective clone without cyt1Aa [Wu et al., Appl. Environ. Microbiol. 63 (1997) 4971-4975]. Cyt1Aa synergized the combination of Crys by about five-fold. Consistently, the lethal times exerted by pRVE4-ADRC were also reduced (it killed exposed larvae more quickly). This clone may become a useful biological control agent which reduces the probability of resistance development in the target organisms [Wirth et al., Proc. Natl. Acad. Sci. USA 94 (1997) 10536-10540].

A transgenic strain of the nitrogen-fixing filamentous cyanobacterium Anabaena PCC 7120 protected expressed delta-endotoxin proteins of Bacillus thuringiensis subsp. israelensis from damage inflicted by UV-B, a sunlight component that penetrates Earth's ozone layer. This organism, which serves as a food source to mosquito larvae and could multiply in their breeding sites, may solve the environment-imposed limitations of B. thuringiensis subsp. israelensis as a mosquito biological control agent.

The genes cyt1Aa and p20, encoding, respectively, cytolytic and accessory proteins of Bacillus thuringiensis subsp. israelensis, were introduced into previously constructed clones expressing cry4Aa and cry11Aa in Escherichia coli (Ben-Dov et al., 1995). Fifteen clones with all possible combinations of the four genes were obtained and found to express the genes included. Two new combinations, pVE4-ADRC and pVE4-ARC, expressing cyt1Aa, p20 and cry4Aa, with or without cry11Aa, respectively, were more toxic than their counterparts without cyt1Aa. They displayed the highest toxicity against Aedes aegypti larvae ever reached in transgenic bacteria. Five out of the six clones (except pVE4-DC) containing cry4Aa or cry11Aa (with or without p20) displayed varying levels of synergism with cyt1Aa: they are 1.5-to 34-fold more toxic than the respective clones without cyt1Aa against exposed larvae. Their lethal times also decreased (they kill larvae quicker), more so at higher cell concentrations. These clones are anticipated to dramatically reduce the likelihood of resistant development in the target organisms (Wirth et al., 1997).

The gene coding for the accessory protein P19 of Bacillus thuringiensis subsp. israelensis was expressed in Escherichia coli and its product was characterized. To investigate its putative role in delta-endotoxin crystallization as a P20-like polypeptide, each of the two encoding genes, p20 and p19, was cloned for inducible expression coordinatively with cyt1Aa. The latter is known to kill its transgenic host. P20 but not P19 stabilized Cyt1Aa and protected the host cells from its lethal effect. Neither GroEL nor GroES, expressed in trans, affected Cyt1Aa as did P20. The function of P20 is thus more specific than that of the chaperones, but that of P19 remains enigmatic. The correct sequence of p19, confirmed in all five isolates of B. thuringiensis subsp. israelensis, does not explain the slow electrophoretic mobility of its 179 amino acids product.

An alternative PCR analysis to screen for cry7 genes is proposed, based on the five conserved blocks of amino acids of Bacillus thuringiensis toxins and their encoding DNA sequences. A complete set of five primers was constructed, four direct and one reverse, yielding four specific amplicons. Modified profiles can identify new cry genes.

An extended PCR method was established to rapidly identify and classify Bacillus thuringiensis strains containing cry (crystal protein) genes toxic to lepidopteran, coleopteran, and dipteran pests (Ben-Dov et al., Appl. Environ. Microbiol. 63:4883-4890, 1997). To optimize identification of all reported cry genes, this methodology needs a complete PCR set of primers. In the study reported here, a set of universal (Un9) and specific primers for multiplex rapid screening for all four known genes from the cry9 group was designed. PCR analyses were performed for cry9 genes on 16 standard strains and 215 field isolates of B. thuringiensis. Among the standard strains, only B. thuringiensis subsp. aizawai HD-133, which harbors cry1 and cry2 genes, was positive with Un9 but negative to all four specific primers for cry9 genes. DNA of 22 field-collected isolates was also found to be positive with Un9. These isolates were classified into three cry9 profiles using specific primers; all of them harbor cry1 and cry2. This newly designed set of primers complements the existing PCR methodology for most currently known cry genes.

The P19 gene and cyt1A gene were obtained by PCR with 9.7 kb HindIII fragment containing P19 gene and cyt1A gene from 72 MD plasmid of Bt as a template. Digested PCR products were ligated to expression vector pUHE24 and transformed into E. coli XL-1. Three clones, LZ19 harboring P19 gene, LZcyt1A harboring cyt1A gene, LZ19A harboring P19 gene and cyt1A gene, were screened. The growth cure of cloned strains were determined under IPTG induction. The result showed that pLZ19 did not affect the growth of E. coli, pLZcyt1A is typically Lethal for E. coli, Lethal initial efficiency of pLZ19A is much higher than that of pLZcyt1A. Probably, this was a result that P19 gene enhanced initial expression of cyt1A gene in E. coli.

In order to understand the influence of the 20 kDa protein on the cytolytic activity of CytA protein, a set of PCR primers were designed to amplify 20 kDa protein and CytA protein genes. The genes were ligated to E. coli expression vector pUHE24 and transformed into E. coli XL1 and DH5 alpha. The clones, LZ20, LZcytA and LZ20A containing 20 kDa protein gene, cytA protein gene and 20 kDa-cytA genes were obtained respectively. The influence of the clones on the growth of E. coli cells was determined under induction of IPTG. The results showed that the growth of LZ20 cells were not affected, LZcytA cells were killed, and the growth of LZ20A cells were not affected. It was suggested that expression product of 20 kDa protein gene protected the host cells from the cytolytic effect of CytA protein. This was supported by using different host strains.

An extended multiplex PCR method was established to rapidly identify and classify Bacillus thuringiensis strains containing cry (crystal protein) genes toxic to species of Lepidoptera, Coleoptera, and Diptera. The technique enriches current strategies and simplifies the initial stages of large-scale screening of cry genes by pinpointing isolates that contain specific genes or unique combinations of interest with potential insecticidal activities, thus facilitating subsequent toxicity assays. Five pairs of universal primers were designed to probe the highly conserved sequences and classify most (34 of about 60) genes known in the following groups: 20 cry1, 3 cry2, 4 cry3, 2 cry4, 2 cry7, and 3 cry8 genes. The DNA of each positive strain was probed with a set of specific primers designed for 20 of these genes and for cry11A. Twenty-two distinct cry-type profiles were identified from 126 field-collected B. thuringiensis strains. Several of them were found to be different from all published profiles. Some of the field-collected strains, but none of the 16 standard strains, were positive for cry2Ac. Three standard and 38 field-collected strains were positive by universal primers but negative by specific primers for all five known genes of cry7 and cry8. These field-collected strains seem to contain a new gene or genes that seem promising for biological control of insects and management of resistance.

Various combinations of the genes cryIVA (cry4A), cryIVD (cry11A), and p20 from Bacillus thuringiensis subsp. israelensis were introduced into the nitrogen-fixing cyanobacterium Anabaena sp. strain PCC 7120 by means of Escherichia coli-Anabaena shuttle vector pRL488p and were expressed under control of two tandem strong promoters, a cyanobacterial promoter (PpsbA) and an E. coli T7 promoter (PA1). Two of the clones carrying cryIVA plus cryIVD, one with p20 and one without p20, displayed toxicity against third-instar larvae of Aedes aegypti at levels greater than any level previously reported for transgenic cyanobacteria.

A large plasmid containing all delta-endotoxin genes was isolated from Bacillus thuringiensis subsp. israelensis; restricted by BamHI, EcoRI, HindIII, KpnI, PstI, SacI, and SalI; and cloned as appropriate libraries in Escherichia coli. The libraries were screened for inserts containing recognition sites for BamHI, SacI, and SalI. Each was labeled with 32P and hybridized to Southern blots of gels with fragments generated by cleaving the plasmid with several restriction endonucleases, to align at least two fragments of the relevant enzymes. All nine BamHI fragments and all eight SacI fragments were mapped in two overlapping linkage groups (with total sizes of about 76 and 56 kb, respectively). The homology observed between some fragments is apparently a consequence of the presence of transposons and repeated insertion sequences. Four delta-endotoxin genes (cryIVB-D and cytA) and two genes for regulatory polypeptides (of 19 and 20 kDa) were localized on a 21-kb stretch of the plasmid; without cytA, they are placed on a single BamHI fragment. This convergence enables subcloning of delta-endotoxin genes (excluding cryIVA, localized on the other linkage group) as an intact natural fragment.

The genes cryIVA and cryIVD, encoding 134- and 72-kDa proteins, respectively, and the gene for a regulatory 20-kDa polypeptide of Bacillus thuringiensis subsp. israelensis (serovar H14) were cloned in all seven possible combinations by the Escherichia coli expression vectors pT7 and pUHE. The four combinations containing cryIVA (cryIVA alone, with cryIVD, with the 20-kDa-protein gene, and with both) displayed high levels of mosquito larvicidal activity in pUHE. The toxicity of the combination of cryIVA and cryIVD, with or without the 20-kDa-protein gene, was higher than has ever been achieved with delta-endotoxin genes in recombinant E. coli. Fifty percent lethal concentrations against third-instar Aedes aegypti larvae for these clones decreased (i.e., toxicity increased) continuously to about 3 x 10(5) cells ml-1 after 4 h of induction. Larvicidal activities, obtained after 30 min of induction, were lower for clones in pT7 and decreased for an additional 3.5 h. Induction of either cryIVD or the 20-kDa-protein gene alone resulted in no larvicidal activity in either pT7 or pUHE20. Cloned together, these genes were slightly toxic in pT7 but not in pUHE20. Five minutes of induction of this combination (cryIVD with the 20-kDa-protein gene) in pT7 yielded a maximal mortality of about 40%, which decreased rapidly and disappeared completely after 50 min. CryIVD is thus apparently degraded in E. coli and partially stabilized by the 20-kDa regulatory protein. Larvicidal activity of the combination of cryIVA and cryIVD was sevenfold higher than that of cryIVA alone, probably because of the cross-stabilization of the polypeptides or the synergism between their activities.

The toxicity of Bacillus thuringiensis var. israelensis (Bti) in mosquito larvae was enhanced by encapsulation in the protozoan Tetrahymena pyriformis. Aedes aegypti larvae which fed on T. pyriformis loaded with Bti died about three times faster than when fed on the same concentrations of Bti alone due to ingestion of higher toxin concentrations, reflected by shorter death times of exposed populations. The best larvicidal activities were achieved at ratios of cell/spore numbers in the range of 1:200 to 1:500. This enhancement of mortality by preincubation with T. pyriformis was higher at low Bti concentrations or in late third-instar larvae. Ninety minutes of preincubation yielded the best enhancement effect. Toxicity enhancement is very likely a consequence of concentrating large quantities of Bti spores and crystals (containing delta-endotoxin) by T. pyriformis cells and delivering them to the larvae. Shortening larval mortality time by encapsulation in T. pyriformis should reduce the exposure time of Bti to unfavorable field conditions that inactivate its larvicidal activity. Whether this method will indeed improve Bti efficacy is still to be determined.