Field of the invention.

The field relates generally to plants and disease resistance, and more specifically, to insecticidal treatments, insecticidal compositions or formulations, insecticidal proteins and transgenic plants producing insecticidal proteins.

Background of the invention.

Insects and pests cost farmers billions of dollars annually in crop losses and in the expense of keeping these pests under control. The losses caused by pests in agricultural production environments include decrease in crop yield, reduced crop quality, and increased harvesting costs.

The coffee berry borer (Hyphotenemus hampei Ferrari) is the most important economic plague in coffee plantations, and it is distributed in all coffee growing areas of the world. The insect attacks the berries during its entire life cycle, resulting in important quality and quantity loses of the crop. There are no known sources of H. hampei resistance in coffee germplasm (neither Coffea arabica nor Coffea canephora). The synthetic insecticide endosulphan can be used to control H. hamper, but it is banned in many countries because it is very toxic for humans and non-target animals including insects, birds, fish, and amphibians. Moreover, insects can develop resistance to the insecticide.

Cotton has huge economic interest worldwide. It is cultivated in over 60 countries worldwide. Five countries — China, India, Pakistan, United States and Uzbekistan — account for 75% of the production, 71% of the area and 70% of consumption. The cotton plant is attacked by several arthropods, including white fly, cotton borers, and mites. Among the most damaging insect pests for cotton are the boll weevil (Anthonomus grandis (Coleoptera: Curculionidae)) and the armyworm Spodoptera frugiperda (Lepidoptera: Noctuidae). The boll weevil is distributed throughout the United States, Mexico, Central America, Cuba, Haiti, Venezuela, Colombia, Argentina, Paraguay, Africa, China and Brazil. Control of this insect is typically achieved through the massive use of chemical pesticides. For many reasons, environmentally-sensitive methods for controlling or eradicating insect infestation are desirable. The most widely used environmentally-sensitive insecticidal formulations developed in recent years have been composed of microbial pesticides derived from the bacterium Bacillus thuringiensis. B. thuringiensis is a Gram-positive bacterium that produces crystal proteins or inclusion bodies that are specifically toxic to certain orders and species of insects. Many different strains of B. thuringiensis have been shown to produce insecticidal crystal proteins, which are quite toxic to the specific target insect, but harmless to plants and other non- targeted organisms. Primary sequence identity forms the basis of the groupings of crystal proteins and their nomenclature as well. Summary of the invention.

This disclosure provides a composition that may be used to control an insect infestation of plants, particularly in economically valuable crops such as coffee and cotton. The composition comprises at least two crystal polypeptides selected from SEQ ID NOs: 1 (cyt1 ), 3 (cry4A), 5 (cry4B), 7 (cry10), 9 (cry1 1 ), 1 1 (cyt2) and 13 (cry3), polypeptides having at least 90% identity to SEQ ID NOs: 1 (cyt1 ), 3 (cry4A), 5 (cry4B), 7 (cry 10), 9 (cry1 1 ), 1 1 (cyt2) and 13 (cry3), or processed crystal proteins. Particular compositions comprising SEQ ID NOs: 3 (cry 4A) and 5 (cry4B); SEQ ID NOs. 1 (cyt1 ) and 7 (cry 10); or SEQ ID NOs. 1 (cyt1 ), 3 (cry 4A) and 5 (cry4B) are also disclosed. In addition, the compositions may comprise polypeptides with at least 90% amino acid identity to SEQ ID NO. 3 (cry4A) and to SEQ ID NO. 5 (cry4B); or at least 90% amino acid identity to SEQ ID NO. 1 (cyt1 ) and to SEQ ID NO. 7 (cry10); or at least 90% amino acid identity to SEQ ID NO. 1 (cyt1 ), to SEQ ID NO. 3 (cry4A) and to SEQ ID NO. 5 (cry4B). The compositions may further comprise an insect attractant. The compositions can be used to kill insects, such as coleopterans or dipterans, and to control insect infestation of plants. Some plants of interest include Coffea arabica, Coffea robusta, Coffea canephora, Saccharum officinarum, Elaeis guineensis, Gossypium hirsutum, Gossypium barbadense, Gossypium arboretum and Gossypium herbaceum. Insects of interest include Hyphotenemus hampei (coffee berry borer), Metamasius hemipterus (rotten sugar cane borer), Aedes aegypti (mosquito), Rhynchophorus palmarum (American palm weevil), lps sexdentatus (six spined engraver beetle,), Tomicus piniperda (pine shoot beetle), Orthotomicus erosus.

This disclosure also details host cells comprising an expression vector comprising nucleic acid sequence encoding at least two of the crystal polypeptides discussed above (which includes the polypeptides with 90% sequence identity and processed polypeptides). The host cell may have two or more expression vectors, each comprising a sequence encoding a crystal polypeptide or a single expression vector that encodes two or more of the crystal polypeptides. Among suitable host cells are B. thuringiensis, Spodoptera frugiperda, or plant cells.

Also disclosed are transgenic plants comprising nucleotide sequences encoding at least two of the crystal polypeptides. Some types of transgenic plants include Coffea arabica, Coffea robusto, Coffea canephora, Saccharum officinarum, Elaeis guineensis, Gossypium hirsutum, Gossypium barbadense, Gossypium arboretum and Gossypium herbaceum.

Brief description of the drawings.

Figure 1 is a photograph of a polyacrylamide gel (10%) showing the proteins expressed by Bt- pSTAB-cry10, solubilized in NaOH 5OmM, DTT 25mM and CAPS 5OmM pH 1 1.5 (lane 1 ) and molecular weight marker (lane 2). Bands of 68 and 56 kDa (arrowheads) correspond to ORF 1 and ORF 2, respectively, from the cry10 operon, cloned in Bt-pSTAB-c/y^O. Figure 2 is a diagram that summarizes data presented in the Examples. Figure 3 depicts a strategy to obtain the recombinant virus that contains gene cry 10Aa (vSyncrylO). The plasmid pGemcry 10, was processed with the enzyme Eco Rl, the fragment of the gen cry 10 was cloned to the transfer vector pSynXIWI+X3, which was previously processed with Eco R1 generating plasmids pSyncry 10 (A). The plasmid pSyncry 10, was co-transfected (B) with the ADN virus vSynGalVI in insect cells 7n5B, and the recombinant virus vSyncry 10, were purified.

Figure 4 is a graph of mortality of A. grandis exposed to crystal proteins. The bars represent 95% confidence levels.

Detailed description of the invention.

The disclosure is directed to methods and compositions for treating plants to prevent, reduce, or get rid of coleopteran infestations. One family of coleopterans of interest is the Curculionidae family, which includes the coffee berry borer and boll weevil for cotton. Plants may be treated with crystal proteins from Bacillus thuringiensis, host cells such as B. thuringiensis or Spodoptera frugiperda that express crystal proteins, or transformed with nucleic acids encoding crystal proteins.

Nucleic acids encoding crystal proteins and crystal proteins.

As used herein, "crystal proteins" refers to any of the Cry and Cyt proteins, including pro-toxin and toxin forms of the proteins.

In some examples, nucleic acids encoding crystal proteins and crystal proteins are isolated from B. thuringiensis var. israelensis, a well known variety. B. thuringiensis serovar israelensis produces an arsenal of proteins active against insects. These crystal proteins are encoded on a single large plasmid, pBtoxis. The main components of the para-sporal crystal of this strain are: Cry4Aa, Cry4Ba, Cry1 1Aa and Cyt1 Aa (Crickmore, N, et. al. Revision of the nomenclature for the Bacillus thuringiensis pesticidal crystal proteins. Microbiology and Molecular Biology Review. 62:807-813, 1998), while small amounts of Cryi OAa (Guarduno et al., Applied and Environmental Microbiology, 277-279, 1988) and Cyt2Ba (Guerchicoff, A. et. al. Identification and characterization of a previously undescribed cyt gene in Bacillus thuringiensis subsp. israelensis. Applied and Environmental Microbiology 63(7): 2716-2721 , 1997) are also present. The production of proteins encoded by the gene cytiCa, has not yet been detected in the crystals, although this gene seems to be transcribed (Stein, C, et. al. Transcriptional Analysis of the Toxin-Coding Plasmid pBtoxis from Bacillus thuringiensis subsp. israelensis. Applied and Environmental Microbiology 72(3): 1771-1776, 2006). Other B. thuringiensis varieties which express israelensis-type toxins can also be used. Such toxins would have amino acid sequences and activities similar to toxins produced by B. thuringiensis var. israelensis. For example, B. thuringiensis isolates of the var. morrisoni serotype 8a, 8b have been reported to express B. thuringiensis var. israelensis-type toxins. As used herein, the term "Bacillus thuringiensis var. israelensis toxin" includes toxins that are similar or related to toxins expressed by B. thuringiensis var. israelensis, but which happen to be expressed by a different variety of Bacillus thuringiensis.

Equivalent crystal proteins and/or nucleic acid sequences encoding these proteins can be derived from other B. thuringiensis isolates and/or DNA libraries using the teachings provided herein. There are a number of methods for obtaining the pesticidal toxins of the instant invention. For example, antibodies to the pesticidal toxins can be used to identify and isolate other toxins from a mixture of proteins. Specifically, antibodies may be raised to the portions of the toxins which are most constant and most distinct from other B. thuringiensis toxins. These antibodies can then be used to specifically identify equivalent toxins with the characteristic activity by immunoprecipitation, enzyme linked immunosorbent assay (ELISA), or Western blotting. Antibodies to the toxins or to equivalent toxins, or fragments of these toxins, can readily be prepared using standard procedures in this art. The genes which encode these toxins can then be obtained from the microorganism. The complete sequence of pBtoxis is in GenBank (Accession No. AL731825). Amino acid and nucleotide sequences for crystal proteins and gene sequences from this plasmid can be found in the Sequence Listing: cyt1 (SEQ ID NOS: 1 and 2); cry4A (SEQ ID NOS: 3 and 4); cry4B (SEQ ID NOS: 5 and 6); cry10 (SEQ ID NOS: 7 and 8); and cry 1 1 (SEQ ID NOS: 9 and 10). Links to sequences of crystal proteins from other Bacillus thuringiensis strains are found in http://www.lifesci.sussex.ac.uk/home/Neil Crickmore/Bt/ (table 1 ).

Table 1

Briefly, nucleotide sequences include isolated genomic sequences encoding the protoxin form of a crystal protein, as well as sequences encoding the toxic form of a crystal protein. Furthermore, the sequences may be codon optimized for expression in a particular host cell. Amino acid and nucleic acid sequences may also include additional residues or nucleotides, such as additional N- or C-terminal amino acids or 5' or 3' sequences, so long as the protein maintains biological activity. The addition of terminal sequences particularly applies to nucleic acid sequences that may, for example, include various non-coding sequences flanking either of the 5' or 3' portions of the coding region or may include various internal sequences, i.e., introns. Nucleotide sequences encoding the crystal proteins can be obtained by a variety of methods. In one method, the nucleotide sequences presented in the Sequence Listing can be used to design amplification primers. To amplify the complete coding region, amplification primers will derive from flanking sequences. Sub-regions of the coding region, for example the region encoding the mature toxic form of a crystal protein, can also be amplified. DNA isolated from B. thuringiensis var. israelensis or other B. thuringiensis strain that encodes one or more of the same crystal proteins, is then amplified using the desired set of amplification primers. In another method, cDNA is generated by standard methodologies and then amplified using primer sets that hybridize to transcribed sequences. In yet another method, DNA from B. thuringiensis is digested with restriction enzyme(s) and fragments cloned into plasmid or phage vectors. Clones containing sequences encoding crystal proteins can be identified e.g., by nucleic acid hybridization with a probe or fragment having a complementary sequence. Such probes are easily synthesized on automatic synthesizers. Sequences of these probes can be determined from the Sequence Listing or GenBank sequences or the like. In yet another method, the nucleotide sequences encoding crystal proteins can be synthesized by e.g., an automated gene synthesizer. In addition to native nucleotide and protein sequences, variant sequences may be desirable in some circumstances. With regard to nucleotide sequences, it will be appreciated that a nucleotide sequence encoding a crystal protein may differ from the wild-type sequence due to codon degeneracies, nucleotide polymorphisms, or amino acid differences. For example, nucleic acid sequences may be prepared that encode the peptide sequence disclosed in SEQ ID NO. 1 (cyt1 ), SEQ ID NO. 3 (cry4A), SEQ ID NO. 5 (cry4B), SEQ ID NO. 7 (cry10), SEQ ID NO. 9 (cry1 1 ), SEQ ID NO. 1 1 (cyt2), SEQ ID NO. 13 (cry3). For use in plants, the codon usage can be optimized for expression. At times, nucleotide sequences should hybridize to a wild-type nucleotide sequence at conditions of normal stringency, which constitutes hybridization and wash conditions at approximately 25-3O ⁰ C below Tm of the native duplex (e.g., 1 M Na+ at 65 ⁰ C; 5X SSPE, 0.5% SDS, 5X Denhardt's solution, at 65 ⁰ C or equivalent conditions (see generally, Sambrook et al. Molecular Cloning: A laboratory Manual, 2nd ed., Cold Spring Harbor Press, 1987; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing, 1987). The Tm for other than short oligonucleotides can be calculated by the formula Tm = 81.5 + 0.41% (G+C) - log(Na+). Low stringency hybridizations are performed at conditions approximately 40 ⁰ C below Tm, and high stringency hybridizations are performed at conditions approximately 10 ⁰ C below Tm. At normal stringency conditions, hybridizing nucleic acids will have approximately at least 75% nucleotide identity to wild-type sequences. Accordingly, polynucleotide sequences that have at least about 75%, 80%, 85%, 90%, 95% or 99% identity to the polynucleotide sequence of SEQ ID NO. 2 (cyt1 ), SEQ ID NO. 4 (cry4A), SEQ ID NO. 6 (cry4B), SEQ ID NO. 8 (cry10), SEQ ID NO. 10 (cry11 ), SEQ ID NO. 12 (cyt2), SEQ ID NO. 14 (cry3) and encode a protein with similar functional activity are useful. For expression of a crystal protein, it may be desirable to have the nucleic acid sequence use codons that are preferred in the organism expressing the protein. For example, a nucleic acid sequence expressing Cryi OA in plants would have codons that are more likely used in plants. A Codon Usage Database (Nakamura et al., Nucl. Acids Res. 28:292, 2000) is available on-line at www.kazusa.or.ip/codon/. At the time of this application, its data source is NCBI-GenBank Flat File Release 160.0 (15 June 2007). Alterations of the nucleic acid sequence to achieve codon- optimization can be performed by any of a variety of methods, including site-directed mutagenesis, ligation of overlapping synthetic oligonucleotides and the like. With regard to amino acid sequences, variants can differ from native sequences by amino acid deletions (at the ends or internal to the sequence), additions (at the ends or internally), or alterations of one or more amino acids. Polypeptide sequences that are at least 80%, 85%, 90%, 95% or 99% identical over their matched length (using a tool such as BLAST) to SEQ ID NO. 1 (cyt1 ), SEQ ID NO. 3 (cry4A), SEQ ID NO. 5 (cry4B), SEQ ID NO. 7 (cry10), SEQ ID NO. 9 (cry1 1 ), SEQ ID NO. 1 1 (cyt2), SEQ ID NO. 13 (cry3) and have similar functional activity are useful. With regard to crystal protein sequences, the protein is initially produced as a protoxin, which is cleaved in vivo to yield an active fragment (i.e. a processed crystal protein, sometimes called delta-endotoxin). Activation involves proteolytic removal of an N-terminal peptide (approximately 25-30 residues for Cry1 proteins, approximately 49 residues for Cry2A, approximately 58 residues for Cry3A and CrylOA, approximately 43 for Cry4A and Cry4B, and approximately 28 for Cry11A) and approximately half of the remaining protein from the C-terminus in the case of the long Cry proteins (Cry1A, Cry4A, Cry4B). Moreover, internal cleavage can occur for some Cry proteins (e.g., Cry1A, Cry4A, Cry4B, and Cry11 ), but the fragments remain associated and retain toxicity. Cyt proteins undergo proteolytic cleavage of small portions of their N-terminus (about 30 amino acids) and C-terminus (about 15 amino acids) to activate the toxin (table 2). Active portions of crystal proteins and nucleic acids that encode them may be desirable.

The nucleic acid segments, regardless of the length of the coding sequence itself, may be combined with other nucleic acid sequences, such as promoters, Shine-Dalgarno sequences, initiation codons, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like. If desired, one may also prepare fusion proteins and peptides, e.g., where the peptide-coding regions are aligned within the same expression unit with other proteins or peptides having desired functions, such as for purification or immuno-detection purposes (e.g., proteins that may be purified by affinity chromatography and enzyme label coding regions, respectively) or for enhanced expression. A number of tag sequences are often used for these purposes; these sequences include His-tag (e.g., HiS ₆ ); T7-tag, S-tag, FLAG peptide, thioredoxin, lacZ, glutathione-S-transferase, and the like (R. C. Stevens. "Design of high-throughput methods of protein production for structural biology "Structure, 8, R177-R185 (2000), incorporated in its entirety). Table 2

Mature protein Amino acids

Protein Approximate important for

Cleavage sites size biological activity

Gly58-Lys695 60 KDa

Cry4Aa Two fragments: Tyr-202

Gly58-Gln236-

20 KDa and Lys695

45 KDa

Arg-158, Asn-166,Tyr-

Asn36 and 170, Pro389, Ser410,

Cry4Ba Gln677 Glu417, Tyr455,

Asn456

SeM O and

70 KDa Ser259 and Glu266, Lys643 important sites for

36 KDa (First

Cry 11Aa Ser10-Arg 360 synergism with fragment)

CytiAa.

32 KDa (Second

Asp361-Lys643 From Gly257 to Arg360 fragment)

Lys45- Lys643

CryiOAa 58 KDa (Prediction)

Variations of nucleotide sequences may be readily constructed using standard techniques, such as methods for making point mutations or by synthesis. Also, fragments of these genes can be made using commercially available exonucleases or endonucleases. For example, enzymes such as Bal31 can be used to systematically delete nucleotides from the ends of these nucleic acids. Also, sequences that encode active fragments may be obtained using a variety of restriction enzymes. Proteases may be used to directly obtain active fragments of these toxins. In addition to their use in directing the expression of crystal proteins or peptides, the nucleic acid sequences described herein also have a variety of other uses. For example, they may be used as probes or primers in nucleic acid hybridizations and amplifications. Oligonucleotide primers for amplification of a full-length cDNA are usually derived from sequences at the 5' and 3' ends of the coding region. Amplification of genomic sequences will use primers that span alternative intron/exon sequences and may use conditions that favor long amplification products (see Promega catalogue). Briefly, oligonucleotides used as amplification primers preferably do not have self-complementary sequences nor have complementary sequences at their 3' end (to prevent primer-dimer formation). Preferably, the primers have a GC content of about 50% and contain restriction sites to facilitate cloning. Generally, primers are between 15 and 50 nucleotides long and more usually between 20 and 35 nucleotides long. The primers are annealed to cDNA or genomic DNA and sufficient amplification cycles are performed to yield a detectable product, preferably one that is readily visualized by gel electrophoresis and staining. The amplified fragment is purified and inserted into a vector (e.g., a viral, phagemid or plasmid vector) and propagated.

Oligonucleotides for hybridization analysis (e.g., Southern, Northern, screening of libraries) may be designed based on the DNA sequence of crystal proteins disclosed herein. Oligonucleotides for screening are typically at least 11 bases long and more usually at least 20 or 25 bases long or 20-30 bases long. Such an oligonucleotide may be synthesized in an automated fashion. Alternatively, fragments of DNA encoding a crystal protein can be used. Fragments can be any size, but usually are a few hundred to one thousand base pairs. Fragments can be obtained by a variety of methods, including restriction enzyme digestion or amplification and isolation of an appropriate fragment. To facilitate detection, the oligonucleotide or fragment may be conveniently labeled, generally at the 5' end, with a reporter molecule, such as a radionuclide, (e.g., ³² P), enzymatic label, protein label, fluorescent label, or biotin. Hybridization conditions are tailored to the length and GC content of the oligonucleotide or fragment. Following denaturation, neutralization, and fixation of the DNA to the membrane, membranes are hybridized with labeled probe. Suitable hybridization conditions may be found in Sambrook et al., supra, Ausubel et al., supra, and furthermore hybridization solutions may contain additives such as tetramethylammonium chloride or other chaotropic reagents or hybotropic reagents to increase specificity of hybridization (see for example, PCT/US97/17413). Following hybridization, suitable detection methods reveal hybridizing DNA or colonies or phage that are then isolated and propagated. Candidate clones or amplified fragments may be verified as containing DNA encoding crystal protein by any of various means. For example, the candidate clones may be hybridized with a second, non-overlapping probe or subjected to DNA sequence analysis. In these ways, clones containing a crystal gene or gene fragment, which are suitable for use in the present invention, are isolated.

Recombinant expression of crystal proteins. Nucleotide sequences that encode crystal proteins, including protoxin forms, toxin forms and other variants, may be used to transform a suitable host. Recombinant expression of crystal proteins may be in plants, e.g., cotton or coffee, in bacteria, e.g., Bacillus, bacteria that associate with plants, bacteria used for high level expression, in insects, or in other organisms. The organisms may express an individual crystal protein or multiple crystal proteins. When an organism expresses multiple crystal proteins, the coding sequences may be located on a single vector or the coding sequences may be located on separate vectors. In some instances, more than one crystal protein encoding sequence will be incorporated into the genome of the transformed host cell. In certain situations, it may be desirable to have one, two, three, four, or even more B. thuringiensis crystal proteins expressed in the transformed transgenic organism. Of particular interest is expression of combinations of crystal proteins Cry4A plus Cry4B, Cyt1 plus Cry10, and Cyt1 plus Cry4A plus Cry4b. Optionally, other transgens may be introduced into the host cell to confer additional phenotypic traits to the host. For examples, but not limited to, such transgens may confer resistance to one or more insects, bacteria, fungi, viruses, nematodes, or other pathogens, may metabolize a chemical, or may increase yield.

Regardless of the organism, vector architecture is similar. A coding region for a crystal protein (either the protoxin or toxin form or other variant) is in operative linkage with a promoter. Depending on the desired expression profile, the promoter may be constitutively active, be inducible, be temporally active, or be active in specific cell types. Generally, the promoter will be heterologous, that is one not normally associated with a crystal protein gene. Promoters may include promoters normally associated with other genes, and they may be isolated from any bacterial, viral, eukaryotic, or plant cell. Naturally, it will be important to employ a promoter that effectively directs the expression of the DNA segment in the cell type, organism, or even animal, chosen for expression.

Other common elements that may be part of vector architecture include a transcription terminator region, selectable marker, origin of replication, enhancers, polylinkers, introns, or even gene sequences which have positive- or negative-regulating activity upon the cloned sequence encoding a crystal protein. The proteins thus produced may be used in situ, that is used by the host expressing the proteins, or may be isolated. Crystal proteins can be isolated by standard protein purification techniques such as size separation, by affinity chromatography, by other chromatography techniques, or by methods to enrich and isolate crystals, such as those described in the Examples.

Expression in plants.

Various methods of producing transgenic plants are well-known in the art. In common to these methods, a suitable plant host cell is transformed with a vector that comprises a promoter operatively linked to a coding region that encodes one or more crystal proteins. For agricultural applications, the vectors should be functional in plant cells. Suitable plants are those susceptible to coleopteran infestation and include, but are not limited to, coffee, cotton, wheat, rice, corn, soybeans, lupins, vegetables, potatoes, canola, nut trees, cassava, yam, alfalfa and other forage plants, cereals, legumes and the like. In some circumstances, the hosts are coffee and cotton, and the main coleopteran problems are coffee berry borer and boll weevil. Vectors that are functional in plants include binary plasmids derived from Agrobacterium plasmids, other plasmids, cosmids, phage, phagemids, baculovirus, viruses, virions, BACs (bacterial artificial chromosomes), and YACs (yeast artificial chromosomes). Such vectors are capable of transforming plant cells. Binary vectors contain left and right border sequences that are required for integration into the host (plant) chromosome. The vectors also typically contain a bacterial origin of replication for propagation in bacteria.

A nucleotide sequence encoding a crystal protein should be in operative linkage with a promoter that is functional in a plant cell. Typically, the promoter is derived from a host plant gene, but promoters from other plant species and other organisms, such as insects, fungi, viruses, mammals, and the like, may also be suitable, and at times preferred. The promoter may be constitutive or inducible, or may be active in a certain tissue or tissues (tissue type-specific promoter), in a certain cell or cells (cell-type specific promoter), or at a particular stage or stages of development (development-type specific promoter). The choice of a promoter depends at least in part upon the application. Many promoters have been identified and isolated. Examples of constitutive promoters include CaMV 35S promoter (US 5352605), opine promoters (e.g., US 5955646), plant ubiquitin promoters (e.g., US 5510474), rice actin 1 promoter (e.g., US 5641876). Examples of inducible promoters include: alcohol dehydrogenase promoter (e.g., US 6605754), tetracycline-regulated promoters (e.g., US 5851796), steroid-regulated promoters (e.g., mammalian glucocorticoid receptor promoter - US 5512483; ecdysone receptor promoters US 6379945), metal-regulated promoters (e.g., metallothionein promoter - US 4940661 ), pathogenesis-related protein promoters (e.g., US 5654414, US 5689044), temperature-regulated promoters (e.g., heat shock promoter - US 5447858, cold-inducible promoters - US 6479260), light-inducible promoter (US 5750385). A few examples of the many types of tissue-specific promoters include root promoters (e.g., US 2001/047525), fruit promoter (e.g., US 4943674), and seed-specific promoters (e.g., EP 255378 B2, US 5420034). Other promoters can be found in gene databases (see, generally, GenBank and EMBL databases) or may be isolated by well- known methods. For example, a genomic clone for a particular gene can be isolated by probe hybridization and its promoter region identified and isolated. For expression in plants or other eukaryotic cells, an intron sequence will improve expression. lntrons can be from genes of the host cell type or synthetic. Some introns enhance gene expression levels (e.g., WO 06/094976). Many different intron sequences are used in the art. Some of these include introns from castor bean catalase, maize tubA1, Adh1, Sh1, UbH, and petunia rbcS. Generally, the vector contains a selectable marker for identifying transformants. It is often desirable that the selectable marker confers a growth advantage under appropriate conditions. Often, selectable markers are drug resistance genes, such as neomycin phosphotransferase. Other drug resistance genes are known to those in the art and may be readily substituted. Selectable markers include ampicillin resistance, tetracycline resistance, kanamycin resistance, chloramphenicol resistance, and the like. The selectable marker also preferably has a linked constitutive or inducible promoter and a termination sequence, including a polyadenylation signal sequence. Other selection systems, such as positive selection can alternatively be used. A general vector suitable for use in the present invention is based on pCAMBIA 1305.2. Other vectors have been described (US 4536475, US 5733744, US 4940838, US 5464763, US 5501967, US 5731179) or may be constructed based on the guidelines presented herein. The plasmid contains a left and right border sequence for integration into a plant host chromosome and also contains a bacterial origin of replication and selectable marker. These border sequences flank two genes. One is a kanamycin resistance gene (neomycin phosphotransferase) driven by a nopaline synthase promoter and using a nopaline synthase polyadenylation site. The second is the E. coli gus gene (reporter gene) under control of the CaMV 35S promoter and polyadenlyated using a nopaline synthase polyadenylation site. The E. coli gus gene is replaced with a gene encoding a fungal gus gene, especially one that cleaves cellobiuronic acid. If appropriate, the CaMV 35S promoter is replaced by a different promoter. Either one of the expression units described above is additionally inserted or is inserted in place of the CaMV promoter and gus gene.

Plants may be transformed by any of several methods. For example, plasmid DNA may be introduced by Agrobacterium co-cultivation (e.g., US 5591616, US 440838) or bombardment (e.g., US 4945050, US 5036006, US 5100792, US 5371015). Other transformation methods include electroporation (US 5629183), CaPθ ₄ -mediated transfection, gene transfer to protoplasts (AU B 600221 ), microinjection, and the like (see, Gene Transfer to Plants, Ed. Potrykus and Spangenberg, Springer, 1995, for procedures). In part, the choice of transformation methods depends upon the plant to be transformed. Tissues can alternatively be efficiently infected by Agrobacterium utilizing a projectile or bombardment method. Bombardment is often used when naked DNA, typically Agrobacterium binary plasmids or pUC-based plasmids, is used for transformation or transient expression.

Other transformation methods are applicable such as pollinic tube transformation, the method of which is herein incorporated by reference (US 2006/0294619). For example, one may employ the methods of Sano et al., as taught in US 6392125, incorporated herein by reference, which teaches the method of producing stable transformants of coffee plants; transformant of coffee plants produced from embryogenic calli, using Agrobacterium method. Additionally, US 6392125 (Sano et al.), discloses the method of modifying a plant's traits, in particular disease tolerance or resistance. Further, the invention can apply to a variety of alternative hosts of H. hampei. The presence and expression of the crystal protein is conveniently assayed in whole plants or in selected tissues using a biochemical method such as amplification, Western blotting, microscopy identification of crystals, Northerns, Southerns, etc. Bioassays may alternatively or additionally be utilized. Furthermore, the transgenic plants may be propagated or used in introgression breeding. The progeny and seeds of the plants will have a crystal protein-encoding transgene stably incorporated into their genome, and such progeny plants will inherit the traits afforded by the introduction of a stable transgene.

Expression in bacteria.

The toxin-encoding genes harbored by the isolates disclosed herein can be introduced into a wide variety of microbial hosts. With suitable microbial hosts, e.g., Pseudomonas, the microbes can be applied to a niche of the pest, where the microbes will proliferate and be ingested by the pest, resulting in control of the pest. Alternatively, the microbe hosting the sequence encoding a crystal protein can be treated under conditions that prolong the activity of the toxin and stabilize the cell. The treated cell, which retains the toxic activity, then can be applied to the environment of the target pest. Where the B. thuringiensis toxin gene is introduced via a suitable vector into a microbial host, and said host is applied to the environment in a living state, it is advantageous to use certain host microbes. For example, microorganism hosts can be selected which are known to occupy the pest's habitat. Microorganism hosts may also live symbiotically with a specific species of coffee berry borers. These microorganisms are selected so as to be capable of successfully competing in the particular environment with the wild-type microorganisms, provide for stable maintenance and expression of the gene expressing the polypeptide pesticide, and, desirably, provide for improved protection of the pesticide from environmental degradation and inactivation. A wide variety of ways are available for introducing a B. thuringiensis gene encoding a toxin into a microorganism host under conditions which allow for stable maintenance and expression of the gene. These methods are well known to those skilled in the art and are described, for example, in US 5135867, which is incorporated herein by reference.

In one method, plasmids are used. For expression of a crystal protein, a promoter is used that is designed for expression of the proteins in a bacterial host. Suitable promoters are widely available and are well known in the art. Inducible or constitutive promoters are preferred. Such promoters for expression in bacteria include promoters from the T7 phage and other phages, such as T3, T5, and SP6, and the trp, Ipp, and lac operons. Hybrid promoters (see US 4551433), such as tac and trc, may also be used. For protein expression, a promoter is inserted in operative linkage with the coding region. Furthermore, the promoter may be controlled by a repressor. In some systems, the promoter can be de-repressed by altering the physiological conditions of the cell, for example, by the addition of a molecule that competitively binds the repressor, or by altering the temperature of the growth media. Preferred repressor proteins include, but are not limited to the E. coli LACI repressor responsive to IPTG induction, the temperature sensitive λ cl857 repressor, and the like. The E. coli LACI repressor is preferred. Other elements of vectors include a transcription terminator sequence and an origin of replication. Thus, for bacterial hosts, the vector usually contains a bacterial origin of replication. Such origins of replication include the f1-ori and col E1 origins of replication, especially the origin derived from pUC plasmids. The plasmids also preferably include at least one selectable gene that is functional in the host. A selectable gene includes any gene that confers a phenotype on the host that allows transformed cells to be identified and selectively grown. Suitable selectable marker genes for bacterial hosts include the ampicillin resistance gene (Ampr), tetracycline resistance gene (Tcr) and kanamycin resistance gene (Kanr). Suitable markers for eukaryotes usually complement a deficiency in the host (e.g., thymidine kinase (tk) in tk- hosts). However, drug markers are also available (e.g., G418 resistance and hygromycin resistance).

One skilled in the art appreciates that there are a wide variety of suitable vectors for expression in bacterial cells and which are readily obtainable. Vectors such as the pET series (Novagen, Madison, Wl) and the tac and trc series (Pharmacia, Uppsala, Sweden) are suitable for expression of a β-glucuronidase. A suitable plasmid is ampicillin resistant, has a colEI origin of replication, a laclq gene, a lac/trp hybrid promoter in front of the lac Shine-Dalgarno sequence, a hexa-his coding sequence that joins to the 3' end of the inserted gene, and an rrnB terminator sequence. The choice of a vector for expression is dictated in part by the bacteria. Commercially available vectors are paired with suitable hosts. The vector is introduced in bacterial cells by standard methodology. Typically, bacterial cells are treated to allow uptake of DNA (for protocols, see generally, Ausubel et al., supra; Sambrook et al., supra). Alternatively, the vector may be introduced by electroporation, phage infection, or another suitable method. B. thuringiensis cells can be cultured using standard art media and fermentation techniques. Upon completion of the fermentation cycle, the bacteria can be harvested by first separating the B. thuringiensis spores and crystals from the fermentation broth by means well known in the art. The recovered B. thuringiensis spores and crystals can be formulated into a wettable powder, liquid concentrate, granules or other formulations by the addition of surfactants, dispersants, inert carriers, and other components to facilitate handling and application for particular target pests. These formulations and application procedures are all well known in the art.

As mentioned above, B. thuringiensis or recombinant cells expressing a B. thuringiensis crystal protein can be treated to prolong the toxin activity and stabilize the cell by forming a cellular microcapsule. The pesticide microcapsule that is formed comprises the B. thuringiensis toxin within a cellular structure that has been stabilized and will protect the toxin when the microcapsule is applied to the environment of the target pest. Suitable host cells may include either prokaryotes or eukaryotes, normally being limited to those cells which do not produce substances toxic to higher organisms, such as mammals. However, organisms which produce substances toxic to higher organisms could be used, where the toxic substances are unstable or the level of application sufficiently low as to avoid any possibility of toxicity to a mammalian host. As hosts, of particular interest will be the prokaryotes and the lower eukaryotes, such as fungi. The cell will usually be intact and be substantially in the proliferative form when treated, rather than in a spore form, although in some instances spores may be employed.

Treatment of the microbial cell, e.g., a microbe containing the B. thuringiensis toxin gene, can be by chemical or physical means, or by a combination of chemical and/or physical means, so long as the technique does not deleteriously affect the properties of the toxin, nor diminish the cellular capability of protecting the toxin. Examples of chemical reagents are halogenating agents, particularly halogens of atomic no 17-80. More particularly, iodine can be used under mild conditions and for sufficient time to achieve the desired results. Other suitable techniques include treatment with aldehydes, such as glutaraldehyde; anti-infectives, such as zephiran chloride and cetylpyridinium chloride; alcohols, such as isopropyl and ethanol; various histologic fixatives, such as Lugol iodine, Bouin's fixative, various acids, and Helly's fixative or a combination of physical (heat) and chemical agents that preserve and prolong the activity of the toxin produced in the cell when the cell is administered to the host animal. Examples of physical means are short wavelength radiation such as gamma-radiation and X-radiation, freezing, UV irradiation, lyophilization, and the like. Methods for treatment of microbial cells are disclosed in US 4695455 and US 4695462, which are incorporated herein by reference.

Asporoqenous mutants.

Asporogenous mutants of Bacillus thuringiensis produce high yields of crystal proteins. Some B. thuringiensis asporogenous mutants have been generated (e.g., US 5827515; US 5279962, both incorporated in their entirety). Mutants of the isolates described herein can be made by procedures well known in the art. For example, an asporogenous mutant can be obtained through ethylmethane sulfonate (EMS) mutagenesis of a novel isolate. Alternatively, mutants can be made using ultraviolet light and nitrosoguanidine by procedures well known in the art. A smaller percentage of the asporogenous mutants will remain intact and not lyse for extended fermentation periods; these strains are designated lysis minus (-). Lysis minus strains can be identified by screening asporogenous mutants in shake flask media and selecting those mutants that are still intact and contain toxin crystals at the end of the fermentation. Lysis minus strains are suitable for a cell treatment process that will yield a protected, encapsulated toxin protein. To prepare a phage resistant variant of an asporogenous mutant, an aliquot of the phage lysate is spread onto nutrient agar and allowed to dry. An aliquot of the phage sensitive bacterial strain is then plated directly over the dried lysate and allowed to dry. The plates are incubated at 3O ⁰ C. The plates are incubated for 2 days and, at that time numerous colonies could be seen growing on the agar. Some of these colonies are picked and subcultured onto nutrient agar plates. These apparent resistant cultures are tested for resistance by cross streaking with the phage lysate. A line of the phage lysate is streaked on the plate and allowed to dry. The presumptive resistant cultures are then streaked across the phage line. Resistant bacterial cultures show no lysis anywhere in the streak across the phage line after overnight incubation at 3O ⁰ C. The resistance to phage is then reconfirmed by plating a lawn of the resistant culture onto a nutrient agar plate. The sensitive strain is also plated in the same manner to serve as the positive control. After drying, a drop of the phage lysate is plated in the center of the plate and allowed to dry. Resistant cultures show no lysis in the area where the phage lysate has been placed after incubation at 3O ⁰ C for 24 hours.

Expression in baculovirus.

Expression in insect cells can offer significant advantages, including high expression levels, ease of scale-up, production of proteins with posttranslational modifications, and simplified cell growth.

A variety of methods, vectors, and cell lines have been developed for expressing proteins in baculovirus. One such method is described in the Examples. Other methods and materials can be obtained from commercial vendors, including Invitrogen (CA), BD Biosciences (NJ), Clontech

(CA), and Protein Sciences (CT) (see also O'Reilly et al., 1992, Baculovirus expression vectors,

A laboratory manual, W. H. Freeman and Company, NY).

The principles that guide vector construction for bacteria and plants, as discussed herein, are applicable to baculovirus vectors. In general, vectors are well known and readily available. Briefly, the vector should have at least a promoter functional in the host in operative linkage with the transgene. Usually, the vector will also have one or more selectable markers, an origin of replication, a polyadenylation signal and a transcription terminator.

Expression in other organisms. A variety of other organisms are suitable for expressing crystal proteins. For example, various fungi, including yeasts, molds, and mushrooms, insects, especially vectors for diseases and pathogens, and other animals, such as cows, mice, goats, birds, aquatic animals (e.g., shrimp, turtles, fish, lobster and other crustaceans), amphibians and reptiles and the like, may be transformed with a sequence encoding and expressing crystal proteins. The principles that guide vector construction for bacteria and plants, as discussed above, are applicable to vectors for these organisms. In general, vectors are well known and readily available. Briefly, the vector should have at least a promoter functional in the host in operative linkage with the transgene. Usually, the vector will also have one or more selectable markers, an origin of replication, a polyadenylation signal and a transcription terminator. One of ordinary skill in the art will appreciate that a variety of techniques for producing transgenic animals exist. In this regard, the US patents nos. 5162215, 5545808, 5741957, 4873191 , 5780009, 4736866, 5567607 and 5633076 teach such methodologies and are thus incorporated herein by reference. Promoters for expression in eukaryotic cells include the P10 or polyhedrin gene promoter of baculovirus/insect cell expression systems (see, e.g., US 5243041 , US 5242687, US 5266317, US 4745051 and US 5169784), MMTV LTR, RSV LTR, SV40, metallothionein promoter (see, e.g., US 4870009) and other inducible promoters. Appropriate promoter systems contemplated for use in high-level expression include, but are not limited to the Pichia expression vector system (Pharmacia LKB Biotechnology).

lnsecticidal compositions and methods of use.

The polypeptide compositions disclosed herein have particular utility as insecticides for topical and/or systemic application to field crops, grasses, fruits and vegetables, lawns, trees, and/or ornamental plants. Alternatively, the polypeptides disclosed herein may be formulated as a spray, dust, powder, or other aqueous, atomized or aerosol for killing an insect, or controlling an insect population. The polypeptide compositions disclosed herein may be used prophylactically, or alternatively, may be administered to an environment once target insects, such as coffee berry borer has been identified in the particular environment to be treated.

Regardless of the method of application, the amount of the active polypeptide component(s) is applied at an insecticidal-effective amount, which will vary depending on such factors as, for example, the specific target insects to be controlled, the specific environment, location, plant, crop, or agricultural site to be treated, the environmental conditions, and the method, rate, concentration, stability, and quantity of application of the insecticidal-active polypeptide composition. The formulations may also vary with respect to climatic conditions, environmental considerations, and/or frequency of application and/or severity of insect infestation. The insecticide compositions described may be made by formulating the bacterial cell, crystal and/or spore suspension, or isolated protein component with the desired agriculturally-acceptable carrier. The compositions may be formulated prior to administration in an appropriate means such as lyophilized, freeze-dried, desiccated, or in an aqueous carrier, medium or suitable diluents, such as saline or other buffer. The formulated compositions may be in the form of a dust or granular material, or a suspension in oil (vegetable or mineral), or water or oil/water emulsions, or as a wettable powder, or in combination with any other carrier material suitable for agricultural application. Suitable agricultural carriers can be solid or liquid and are well known in the art. The term "agriculturally-acceptable carrier" covers all adjuvants, inert components, dispersants, surfactants, tackifiers, binders, etc. that are ordinarily used in insecticide formulation technology; these are well known to those skilled in insecticide formulation. The formulations may be mixed with one or more solid or liquid adjuvants and prepared by various means, e.g., by homogeneously mixing, blending and/or grinding the insecticidal composition with suitable adjuvants using conventional formulation techniques.

Formulated bait granules containing an attractant and spores and crystals of the B. thuringiensis isolates, or recombinant microbes comprising the genes obtainable from the B. thuringiensis isolates disclosed herein, can be applied to the environment of the insect, such as Coleopterans. The bait may be applied liberally since the toxin does not affect animals or humans. Product may also be formulated as a spray or powder. The B. thuringiensis isolate or recombinant host expressing the B. thuringiensis gene may also be incorporated into a bait or food source for the coffee berry borer or mosquito.

As would be appreciated by a person skilled in the art, the pesticidal concentration will vary widely depending upon the nature of the particular formulation, particularly whether it is a concentrate or to be used directly. The pesticide will be present in at least 1% by weight and may be 100% by weight. The dry formulations will have from about 1-95% by weight of the pesticide while the liquid formulations will generally be from about 1-60% by weight of the solids in the liquid phase. Formulations that contain cells will generally have from about 10 ² to about 10 ⁴ cells/mg. These formulations will be administered at about 50 mg (liquid or dry) to 1 Kg or more per hectare. The formulations can be applied to the environment of the insect, e.g., on plant foliage. The bioinsecticide composition of the invention may comprise an oil flowable suspension of bacterial cells which expresses one or more of the crystal proteins disclosed herein. Alternatively, the bioinsecticide composition comprises a water dispersible granule. This granule comprises bacterial cells which express one or more of the crystal proteins disclosed herein. In yet another alternative, the bioinsecticide composition comprises a wettable powder, dust, spore crystal formulation, cell pellet, or colloidal concentrate comprising transformed bacterial cells.

Dry forms of the insecticidal compositions may be formulated to dissolve immediately upon wetting, or alternatively, dissolve in a controlled-release, sustained-release, or other time- dependent manner. Such compositions may be applied to, or ingested by, the target insect, and as such, may be used to control the numbers of insects, or the spread of such insects in a given environment.

In another alternative, the bioinsecticide composition comprises an aqueous suspension of bacterial cells or an aqueous suspension of parasporal crystals, or an aqueous suspension of bacterial cell lysates or filtrates, etc., such as those described above that express the crystal protein. Such aqueous suspensions may be provided as a concentrated stock solution which is diluted prior to application, or alternatively, as a diluted solution ready-to-apply.

For these methods involving application of transformed bacterial cells, suitable bacterial cells include Bacillus thuringiensis serovar israelensis 4Q1 cells, other B. thuringiensis strains, B. megaterium, B. subtilis, B. cereus, E. coli, Salmonella spp., Agrobacterium spp., or Pseudomonas spp. The cellular host containing the crystal protein gene(s) may be grown in any convenient nutrient medium. These cells may then be harvested in accordance with conventional ways.

When the insecticidal compositions comprise intact B. thuringiensis cells expressing the protein(s) of interest, such bacteria may be formulated in a variety of ways. They may be employed as wettable powders, granules or dusts, by mixing with various inert materials, such as inorganic minerals (phyllosilicates, carbonates, sulfates, phosphates, and the like) or botanical materials (powdered corncobs, rice hulls, walnut shells, and the like). The formulations may include spreader-sticker adjuvants, stabilizing agents, other pesticidal additives, or surfactants. Liquid formulations may be aqueous-based or non-aqueous and employed as foams, suspensions, emulsifiable concentrates, or the like. The ingredients may include rheological agents, surfactants, emulsifiers, dispersants, or polymers.

Alternatively, the novel insecticidal polypeptides of the invention may be prepared by native or recombinant bacterial expression systems in vitro and isolated for subsequent field application. Such protein may be either in crude cell lysates, suspensions, colloids, etc., or alternatively may be purified, refined, buffered, and/or further processed, before formulating in an active biocidal formulation. Likewise, under certain circumstances, it may be desirable to isolate crystals and/or spores from bacterial cultures expressing the crystal protein and apply solutions, suspensions, or colloidal preparations of such crystals and/or spores as the active bioinsecticidal composition (e.g. with alcohol/methanol).

In certain circumstances, when the control of multiple insect species is desired, the insecticidal formulations described herein may also further comprise one or more chemical pesticides, (such as chemical pesticides, nematocides, fungicides, virucides, microbicides, amoebicides, insecticides, etc.), and/or one or more crystal proteins having the same, or different insecticidal activities or insecticidal specificities, as the insecticidal polypeptide. The insecticidal polypeptides of the invention may also be used in conjunction with other treatments such as fertilizers, weed killers, alcohol & methanol based attractants, cryoprotectants, surfactants, detergents, insecticidal soaps, dormant oils, polymers, and/or time-release or biodegradable carrier formulations that permit long-term dosing of a target area following a single application of the formulation. Likewise the formulations may be prepared into edible "baits" or fashioned into insect "traps" to permit feeding or ingestion by a target insect of the insecticidal formulation. The insecticidal compositions of the invention may also be used in consecutive or simultaneous application to an environmental site singly or in combination with one or more additional insecticides, pesticides, chemicals, fertilizers, or other compounds. In a specific example, one or more B. thuringiensis crystal proteins are administered to coffee berry borers to control this pest in coffee crops. The B. thuringiensis is administered in a manner wherein the coffee berry borer ingest the toxin. As would be appreciated by one skilled in the art, the exact method of administration is not critical. For example, the B. thuringiensis can be administered as a foliar spray onto coffee crops. This method of administration is effective in controlling adult coffee berry borers that feed on the berries of coffee plants. Another method of administration is accomplished by transforming a coffee plant to express one or more crystal proteins. Both adults and larvae grazing on the transgenic plant will thereby ingest the crystal protein. Advantageously, tissue-specific promoters can be employed to drive the expression of the B. thuringiensis gene so that the toxin is present in the tissue which is most likely to be eaten by the coffee berry borer. For example, root specific promoters can be employed to provide control of larvae and specific promoters can be employed to control adult coffee berry borer. The insecticidal compositions of the invention are applied to the environment of the target insect, typically onto the foliage of the plant or crop to be protected, by conventional methods, preferably by spraying. The strength and duration of insecticidal application will be set with regard to conditions specific to the particular pest(s), crop(s) to be treated and particular environmental conditions. The proportional ratio of active ingredient to carrier will naturally depend on the chemical nature, solubility, and stability of the insecticidal composition, as well as the particular formulation contemplated.

Other application techniques, including dusting, sprinkling, soil soaking, soil and plant injection, seed coating, seedling coating, foliar spraying, aerating, misting, atomizing, fumigating, aerosolizing, and the like, are also feasible and may be required under certain circumstances such as e.g., insects that cause root or stalk infestation, or for application to delicate vegetation or ornamental plants. These application procedures are also well-known to those of skill in the art.

The insecticidal compositions of the present invention may also be formulated for preventative or prophylactic application to an area, and may in certain circumstances be applied to pets, livestock, animal bedding, or in and around farm equipment, barns, domiciles, or agricultural or industrial facilities, and the like.

The concentration of insecticidal composition which is used for environmental, systemic, topical, or foliar application will vary widely depending upon the nature of the particular formulation, means of application, environmental conditions, and degree of biocidal activity. Typically, the bioinsecticidal composition will be present in the applied formulation at a concentration of at least about 1 % by weight and may be up to and including about 99% by weight. Dry formulations of the polypeptide compositions may be from about 1% to about 99% or more by weight of the protein composition, while liquid formulations may generally comprise from about 1 % to about 99% or more of the active ingredient by weight. As such, a variety of formulations are preparable, including those formulations that comprise from about 5% to about 95% or more by weight of the insecticidal polypeptide, including those formulations that comprise from about 10% to about 90% or more by weight of the insecticidal polypeptide. Naturally, compositions comprising from about 15% to about 85% or more by weight of the insecticidal polypeptide, and formulations comprising from about 20% to about 80% or more by weight of the insecticidal polypeptide are also considered to fall within the scope of the present disclosure. In the case of compositions in which intact bacterial cells that contain the insecticidal polypeptide are included, preparations will generally contain from about 10 ⁴ to about 10 ⁸ cells/mg, although in certain embodiments it may be desirable to utilize formulations comprising from about 10 ² to about 10 ⁴ cells/mg, or when more concentrated formulations are desired, compositions comprising from about 10 ⁸ to about 10 ¹⁰ or 10 ¹¹ cells/mg may also be formulated. Alternatively, cell pastes, spore concentrates, or crystal protein suspension concentrates may be prepared that contain the equivalent of from about 10 ¹² to 10 ¹³ cells/mg of the active polypeptide, and such concentrates may be diluted prior to application.

The insecticidal formulation described above may be administered to a particular plant or target area in one or more applications as needed, with a typical field application rate per hectare ranging on the order of from about 50 g/hectare to about 500 g/hectare of active ingredient, or alternatively, from about 500 g/hectare to about 1000 g/hectare may be utilized. In certain instances, it may even be desirable to apply the insecticidal formulation to a target area at an application rate of from about 1000 g/hectare to about 5000 g/hectare or more of active ingredient.

The following examples are offered by way of illustration, and not by way of limitation.

Example 1. Cloning of cry 10 gene.

The gene cryiOAa was cloned from plasmid DNA of B. thuringiensis serovar israelensis S-1806

(Genetic and Biotechnology Resources, EMBRAPA, Brazil).

This gene was amplified from 50 ng of total bacterial DNA in a reaction mix containing oligonucleotides (0.4 μM each) forward and reverse (table 3), 10 μM of each dNTP, 2.5μl_ of Taq DNA polymerase, 2 mM MgC^ and 1 U of Taq polymerase (Invitrogen) in a total volume of 25μl_. Amplification conditions were: one cycle of 94 ⁰ C for 5 min, then 35 cycles of 95 ⁰ C for 30 sec, 52 ⁰ C for 1.3 min, 72 ⁰ C for 4 min followed by an incubation at 72 ⁰ C for 8 min. The forward oligonucleotides contains a BamH I restriction site and the ATG initiation codon; the reverse oligonucleotide is complementary to nucleotides 2015-2042 and also contains a BamH I restriction site. The amplified fragment was cloned in plasmid pGEM® - T easy (Promega, Wl, U.S.) and named pGemcry 10Aa. The plasmid was introduced into competent E. coli DH5-α (Invitrogen, CA U.S.) cells. Plasmid DNA was purified using the DNA purification kit from Wizard®Plus SV Minipreps (Promega) and sequenced using an automatic sequencer MEGA BACE® 1000 (Amersham Bioscience, U.K.). In addition to oligonucleotides SP6 and T7 that flank the insert, oligonucleotides F-496 and R-1324, which are specific for the gene crylOAa, were also used to confirm the presence of the gene sequence (table 3). Sequences were analyzed by the programs Open Reading Frame (ORF) finder and BLAST, both available from National Center for Biotechnological Information (NCBI). BLAST analysis of the sequence revealed high identity with the gene cryWAa (GenBank accession number M12662; also SEQ ID NO. 8), but with differences in two nucleotide positions. These differences result in amino acid changes of T589A and T624S.

Table 3

Oligonucleotide Sequence of the oligonucleotide (5' -> 3') SEQ ID NO.

Forward (F) GGGATCCGGGAGGAATAGATΛ TGAATC 15

Reverse (R) ATAGTGAATGATTTATTTGTAAGGATCCTTTCC 16

F-496 GCACGTACACACGCTAATGC 17 R- 1324 GATATTCATCCAATTCAACAATA 18

Restriction sites for Bam HI (GGATCC) are highlighted in bold; the initiation codon (ATG) in Forward is underlined; Reverse is complementary to nucleotides 2015-2042.

Example 2. Cloning first open reading frame of cry 1OA gene in the expression vector pSTAB.

The first open reading frame of cryiOAa was cloned into the expression vector pSTAB (Park et al., 1999. FEMS Microbiology Letters 181 , 319-327, 1999). The gene was first inserted into pCR2.1 TOPO vector (Invitrogen, CA) and subsequently subcloned into pSTAB. The sequence of cry10 gene as reported from Bacillus thuringiensis subsp. israelensis plasmid pBtoxis (GenBank Accession No. AL731825) reveals two open reading frames and an intervening sequence. Primers designed to amplify the entire gene were developed using the Massachusetts Institute of Technology program Primer3. Restriction sites for Sal\ and Sph\, which are used for cloning into pSTAB, were added to the primer sequences. The forward (1Od) and reverse (1Or) primer sequences were:

1Od δ'-AATGTCGACTTGCAACAGAAAAGAGTTGTGTC-S' (SEQ ID NO. 19)

1Or δ'-CGAGCATGCACATTTCCCCACAATTTTCA-S' (SEQ ID NO. 20). The restriction sites for Sal\ and Sph\ are underlined. DNA was extracted from B. thuringiensis israelensis strain IPS 82 (Institut Pasteur) and 500 ng DNA was amplified in a reaction mix containing 2.5 μl buffer 10x, 2.5 μl of 225 mM MgCI ₂ , 0.5 μl dNTP, 20 μM each primer and 2.5 U Taq polymerase. The amplification conditions were: an initial step at 95 ⁰ C for 1 minute, and 30 cycles of one min at 95 ⁰ C, 1.5 min at 5O ⁰ C, and three min at 72 ⁰ C, followed by a final step of 10 min at 72 ⁰ C. Amplification reaction was electrophoresed in 1% agarose gel electrophoresis. The amplified product corresponding to the cry10 gene was cloned into pCR2.1TOPO vector (Invitrogen, CA), which was used to transform E. coli DH5α competent cells. Transformants were selected on medium containing carbenicillin, X-GaI and IPTG and subsequently grown. Plasmid DNA was digested with Sa/I and Sph\, electrophoresed in agarose gel, and the DNA fragment corresponding to the gene was purified with the QIAquick Gel Extraction Kit (Qiagen). Likewise, the B. thuringiensis expression vector pSTAB was digested with Sa/I and Sph\, and purified with the QIAquick Gel Extraction Kit. Purified cry10 gene fragment and vector were analyzed by agarose gel electrophoresis to determine the relative amount of each DNA. Ligation was carried out using a vector/insert ratio of 1 :3. Following incubation overnight at 16 ⁰ C, the reaction was used to transform competent E. coli DH5α cells. Verification of the plasmid was made by restriction enzyme analysis. Colonies having the expected restriction pattern were grown in LB liquid medium, and DNA was extracted by an alkaline maxiprep procedure (Sambrook and Rusell Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory. NY, 2001 ). Plasmid DNA was transfected into the acrystalliferous Bt strain 4Q7 (Lereclus et al. FEMS Microbiology Letters 60, 21 1-218, 1989) by electroporation. An expected Bt-cry10 recombinant transformant was obtained. Similarly, the first ORF of cryiOAa gene was also cloned into the pSTAB vector. Primers for amplifying the first ORF have the sequences AATGTCGACTTGCAACAGAAAAGAGTTGTGTC (1Od; SEQ ID NO. 19) and the reverse primer TCTAATAATGCATGAGTGATTGGAATAAATTCGA (SEQ ID NO. 21 ). The restriction sites for Sa/I and Λ/s/V, respectively, are underlined. An acrystalliferous Bt strain 4Q7 was also transformed with this construct.

Example 3. Construction of baculovirus expressing cry 1OA.

The scheme to obtain the recombinant virus that contains the gene cryiOAa (vSyncrylO) is illustrated in figure 3. Briefly, the DNA of plasmid pGemcry 10Aa was digested with Eco Rl, and the fragment containing the gene cry 10Aa was isolated following agarose gel electrophoresis. The fragment containing the gene cry 10Aa was purified from the gel using a GFX Kit® and ligated to an Eco Rl-digested transference vector pSynXIWI+X3 (Wang et al., Gene 100:131- 137, 1991 ). The ligated vector was used to transform competent E. coli DH5-α. Colonies were selected in selective medium. The presence and orientation of cryiOAa was verified by amplification reactions. Oligonucleotides for amplification of pSynXIVVI+X3 are: oligonucleotides ORF 603: 5'- ACAGCCATTGTAATGAGACG (SEQ ID NO. 22), which is derived from nucleotides +8 and -1 1 relative to the initiation codon of ORF 603, and polhR: δ'-CTAGATTCTGTGCGTTGTTG-S' (SEQ ID NO. 23), which is derived from nucleotides 34 to 54 after the termination codon of the polyedrine gene. These amplification primers enable verification of orientation of the insert. Cells of Trichoplusia ni (BTI-Tn5B1-4) were maintained at 27 ⁰ C in a medium of TC100 with 10 % of bovine fetal serum. These cells serve as host for the propagation in vitro of the baculovirus AcMNPV and its recombinants. The recombinant virus vSyngalVI-, that contains the gene of β- galactosidase in the locus of the gene of polyedrine (Wang et al., 1991 ), was used for the construction of AcMNPV recombinant, which contains the gene crylOAa.

One μg of the DNA of the recombinant plasmid (pSyncryl OAa) and 0.5 μg of the DNA of the virus vSyngalVI- previously linearized with the restriction enzyme Bsu 361, were used in co-transfection of BTI-TN5B1-4 cells (10 ⁶ cells), using liposomes and following the instructions of the manufacturer (Cellfectin ^® , Invitrogen). The plate was incubated for seven days at 27 ⁰ C, until viral occlusion bodies (OB) appeared; the floating coat of the plate was collected and used for the purification of the recombinant viruses in serial dilutions in plates of 96 wells. The only site of Bsu 36I in the virus vSynGalVI- is located in the gene β-galactosidase, and the linear virus is not infective, thus facilitating the purification of the recombinant virus. Additionally, plasmid pSyncryl OAa has, in addition to the gene cryWAa, the gene of polyedrine (absent in vSyngalVI-). After homologous recombination between the DNA of plasmid pSyncryl OAa, and the viral DNA, in which gene crylOAa replaced lac-Z, recombinant virus vSyncrylOAa was isolated. The virus was purified in three serial dilutions in plates with 96 wells (O'Reilly et al., Baculovirus expression vectors. A Laboratory Manual, Freeman. 1992. p. 345. 1992).

One hundred third stage larvae of S. frugiperda were infected by intra-hemocelic injection of 5 to 10 μl of a bank of BV (1.17 x 10 ⁷ pfu/mL) of the recombinant virus vSyncrylOAa. After 120 hours post infection (h.p.i.), the dead larvae were collected and the OBs with possible crystals of the recombinant proteins were purified (O ^' Reilly et al, 1992). After purification, the crystals of the recombinant protein and the viral polyhedra were analyzed via light microscopy (Axiophot 100, Zeiss), photographed and later stored at -8O ⁰ C.

CrylOA analysis. For analysis in SDS-PAGE, both the polyhedra of the wild virus and the crystals of recombinant protein were re-suspended in 100 μl_ of PBS (136 mM NaCI, 1.4 mM KH ₂ PO ₄ , 2.6 mM KCI, 8 mM Na2HPO ₄ .2H ₂ O, pH 7,4) and samples of 10 μL were analyzed in SDS-PAGE at 12% (Laemmli, 1970) using the device Mini-Protean II, according to the instructions of the manufacturer (Bio-Rad). The presence of a 74kDa polypeptide, corresponding to the recombinant protein Cryl OAa was detected in the extract of the cells infected with recombinant virus vSyncrylOAa. Confirmation of the 74kDa protein as Cryl OAa was made using a specific anti-serum against this protein. Furthermore, the crystals and polyhedra obtained from larvae of S. frugiperda infected with the virus vSyncrylOAa, were purified and processed to be then analyzed through electronic scanning microscopy, which revealed its cube shape. The crystals observed are formed by the recombinant protein CrylOAa. For transcription analysis of the recombinant virus vSyncrylOAa, 5 x 10 ⁶ cells (BTI-Tn5B1-4) were plated on two plates of 100 mm in diameter (TPP), and incubated for an hour at room temperature. The medium was removed, and the cells were infected with the recombinant virus at a multiplicity of infection (MOI) of 20. After one hour, the inoculums of the virus were removed and bovine fetal serum was added to 10%. After 96 h.p.i., the cells were collected and total RNA was extracted by reactive Trizol (Invitrogen).

The total RNA was used to obtain cDNA, using a specific oligonucleotide for the poly-A end of messenger RNA (T1 : 5'CCTGCAGGATCCTTAGGTTTTTTTTTTTTTTTTTT 3' (SEQ ID NO. 24) and the enzyme reverse transcriptase Mu-MLV (Invitrogen). cDNA was synthesized in the following manner. A mixture of 2 μl of total RNA, 9 μl of water MiIIi-Q "RNAse free", and 1 μl of T1 oligonucleotide was incubated at 65 ⁰ C for five minutes and then placed on ice. To this reaction mix was added 1 μl of dNTPs (10 mM each), 1 μl of DTT (0.1 M), 28 units of inhibitor RNAse (ARN guard ^® , Gibco), 5 μl of Taq 5X, 1 μl of the enzyme Reverse Transcriptase M-MLV RT (Gibco BRL), and Mili-Q ^® water to a total volume of 20 μl, and incubated at 37 ⁰ C for 50 min for synthesis of cDNA. The cDNA was amplified with oligonucleotides T2 (5 ^' CCTGCAGGATCCTTAGGTT 3 ^' (SEQ ID NO. 25) and oligo F-496, which is specific for the gene cryiOAa and maps to nucleotides +496 to +516. Presence of the transcribed gene cryiOAa was confirmed by detection of a 1600 bp fragment and by digestion of the fragment with Xho I, which cuts the gene in position +1064. Digestion produced the expected fragment of 1 100 bp and another one of about 548pb, thus confirming the specificity of the amplification.

Example 4. Preparation of solubilized and purified crystal proteins. The sources of cyt1 , cry3, cry4A, cry4B, cry10, and cry1 1 proteins were from recombinant or native strains of B. thuringiensis. The strains mentioned in the table 4 express individual B. thuringiensis subsp. israelensis toxins.

Table 4

Strain Expressed gene(s)

Sf-pSTAB- Cry10Aa ^* Cryi OAa pWF45 ^** CytiAa pHT4A+4B ^*** Cry4A and Cry4B pHT4B ^*** Cry4B

LBiT704 ^*** Cry11A

* Cloning and expression by Hernandez, 2006.

** Strain donated by B. Fedehci, University of California, Riverside.

*** Strains donated by A. Delecluse, lnstitut Pasteur, Paris.

The Cry3Aa toxin was isolated from the type strain of B. thuringiensis subsp. tenebrionis, obtained from the German Stock Culture Collection. All strains were grown in sporulation medium (8g/l nutrient broth; 1g/L yeast extract; 1g/L KH ₂ PO ₄ ; 1 mg/L CaCO ₃ ; 1 mg/L MgSO ₄ .7H ₂ O; 0.1 mg/L FeSO ₄ JH ₂ O; 0.1 mg/L MnSO ₄ JH ₂ O; 0.1 mg/L ZnSO ₄ JH ₂ O: pH adjusted to 7.0 by addition of NaOH), supplemented with 10 μg/ml erythromycin for maintenance of the plasmids in the recombinant strains, for 72 hours, 200 rpm, 30 ⁰ C. At this time, cultures were fully sporulated. Cultures (600 ml) were centrifuged at 12,800 x g for 30 minutes, at 4°C, the cell pellets were frozen for 16 h and lyophilized for 18 h in a Labconco model Lyphlock 18 freeze-dryer. Afterwards, the material was weighed for use in the bioassay. Alternatively, the supernatant from each culture was discarded, and the slurry of spores, cellular debris, and crystals was subjected to centrifugation through NaBr, sucrose or Renografin-60 gradients (Squibb Diagnostics, New Brunswick, NJ.) to produce purified crystals. Purified crystals of CryiOA were washed three times in deionized sterile water and collected by centrifugation (17,000 x g for 10 min.). Washed crystals were resuspended in 100 μl of deionized sterile water. 20 μl of the crystals are resuspended in equal volume of buffer (5OmM NaOH, 25 mM DTT, 50 mM CAPS pH 11.5) for 1 hr at 37 ⁰ C. Then, 10μl of a 4X buffer (2% SDS, 40% glycerol, 5% mercaptoethanol, 0.001% bromophenol blue, 0.0625M Tris-HCI, pH 8) was added and incubated for 3 min in a hot water bath. A total of 15 μl of the sample was analyzed in a 10% polyacrylamide gel; bands of 68 and 56 kDa, corresponding to ORF 1 and ORF 2, respectively, from the cry10 operon, cloned in Bt-pSTAB-cry^O, were observed (figure 1 ).

Example 5. Bioassay of crystal proteins on H. hampei and A. aegypti In this example, the activities of crystal proteins against two insects are determined.

H. hampei growth. For establishment and maintenance of a H. hampei colony, only females were inoculated. The insects were disinfected with 2.5% benzalkonium chloride, rinsed with sterilized distilled water and finally were sprayed with 0.1% benomyl (methyl [1-[(butylamino)carbonyl]-1 H- benzimidazol-2-yl]carbamate). They were then dried on a paper towel disinfected with 0.1% benomyl. Once the insects were dry, they were placed in a cylinder with a perforated top, which was used to distribute the H. hampei in 24-well plates containing diet. The inoculated plates were covered and placed in an incubator in total dark, at 27°C and a relative humidity of 85%. To prepare 1 L of diet, 150 g of unroasted coffee was pulverized to particles of approximately 2 mm diameter and sterilized for 15 minutes at 120 ⁰ C and 15 Ib of pressure. A sterile 2% agar solution (750 ml) was mixed with 150 g of the sterile coffee particles, 1.5 g of benomyl, 15 g of yeast, 15 g of casein, 2 g of benzoic acid, 0.5 g of Vanderzant vitamins, 0.8 g of Wesson salts, 10 ml of 95% alcohol and 1 ml of formaldehyde. The mixture is homogenized in a blender for approximately 2 minutes. Approximately 1 ml of this diet is dispersed per well in disposable 24- well plates. Demographic parameters. For evaluations of demographic parameters, 30 active H. hampei females were individually placed in a well of 24-well plates containing H. hampei diet. Each week, at random, 30 samples of diet were evaluated for the number of individuals present, per state within the life cycle, and the number of dead individuals.

The following formulas were used to measure the different demographic parameters:

Net maternity function (f _x ) : l _x m _x β Net reproductive rate (R ₀ ) : ∑l _x m _x x a β Intrinsic growth rate (r _m ) : ∑e ⁺⁰ l _x m _x = 1 x a

Where I _x is the probability that an individual will reach certain age x and m _x is the average number of states of a progeny produced by a female of a certain age, x. The m _x values were determined by multiplying the average number of eggs produced by a female of a certain age x. The number of eggs registered per female was calculated from the number of eggs obtained per sample. The number of eggs counted in the second sample was subtracted from the number of eggs in the previous sample, the difference was assumed as mx (Portilla, Revista Colombiana de Entomologfa 26(1-2): 31-37, 2000). Amount of humidity in the diet. The amount of humidity in the diet samples was determined by weight difference from data collected weekly for a period of 10 weeks from five samples of uninoculated diet.

Oviposition. Five samples of inoculated diet with active females were dissected every two days for a total of 10 days, and the presence of eggs was determined. Also, on a daily basis, the development of 20 fertile H. hampei eggs reaching adult phase was observed, as was the number of days in each prior phase.

Evaluation of natural mortality. Evaluations were carried out for (i) Eggs: 10 dozens of fertile eggs of H. hampei from colonies established in the diet; (ii). Larvae (L1 ): 70 H. hampei larvae that were recently hatched were selected from the diet, (iii) Pupae: 60 H. hampei pupae were selected from the diet and placed in groups of 15 individuals, (iv) Adults: 60 "light brown" H. hampei adults from a colony established in a diet were placed in groups of 15 individuals. All individuals evaluated (eggs, larvae, pupae and adults) were placed in pellets of H. hampei diet and covered. The mortality evaluation was carried out every two days for a period of 10 days. Bioassavs. Protocols were established for qualitative bioassays both for H. hampei adults and larvae. Larvae (L1 ): for bioassays with H. hampei larvae, the H. hampei diet was prepared at least a day prior and kept under refrigeration. The diet substrate was placed in a reading chamber two hours prior to the start of the bioassay at room temperature. Previously, 2.5 mg of the selected lyophilized strain of B. thuringiensis was weighed and reconstituted in 50 μl of 0.1% TWEEN 20 and 450 μl of distilled sterile water by 3 minutes of agitation in a Vortex and 5 minutes of sonication. Then, 20 μl of this solution was added to each well with diet and allowed to dry for 30 minutes. Once the suspension was dry, 10 H. hampei larvae from the first instar (larvae obtained 1 d post-hatching from eggs inoculated in diet) were placed in each well of the plates with the diet. The plates were sealed with auto adhesive plastic that was punctured (e.g., 3 times with a dissection needle) in order to facilitate gas exchange, and were placed in total darkness at 27° C and 85% relative humidity. The mortality of the larvae was evaluated 7 days later. Bioassays with adults were performed in the same fashion, except that five H. hampei adults were placed in each well with diet (adults obtained from pupae inoculated in H. hampei diet).

A fine bioassay (dose-dependent assay) to determine the LC ₅₀ was performed with serial 1 :1 dilutions from a 20 μg/ml spore and crystal solution. To determine synergistic effects of crystal proteins - e.g., CryiOA and Cyt1A proteins - a fine bioassay was performed with serial 1 :1 dilutions from a 100 ng/ml solution of each strain. The mortality was determined at 24 h and then subjected to Probit analysis. The synergistic potential was calculated using the formula of Tabashnik (Tabashnik, Appl and Environ Microbiol 58: 3343-3346, 1992). For bioassays with A. aegypti, fourth instar larvae were obtained from the Cinvestav lrapuato insectaria. The bioassay was performed in plastic cups containing 100 ml of water and 20 larvae (Mclaughlin et al., Bull. Ent. Soc. Amer. 30:26-29, 1983). Approximately 100 μg/ml of the spore and crystal solution of the Bt-pSTAB-cry10A strain was added to each container. A total of 200 larvae without crystal protein solution served as a negative control. The mortality was determined at 24h by Probit analysis. Mortality evaluation data. Mortality was determined in qualitative bioassays with larvae L1 and adults of Hypothenemus hampei exposed to a mix of spore and crystal complex of the strains pSTAB-Cry1 OAa and Sf-Cyt1Aa at doses of 5 μg/μl. Mortality was evaluated at day seven. The application of either cyt1 or cry10 alone resulted in a mortality rate of 50% and 20%, respectively. A combination of cyt1 and cry10 however resulted in a 100% mortality of H. hampei, demonstrating a synergistic effect (figure 2, table 5). Mortality was observed in larvae L1 of Hypothenemus hampei exposed to different combinations of the recombinant proteins present in the Bacillus thuringiensis israeliensis strain. The combination of the Cry10 and Cyt1 proteins killed 100% of the H. hampei insect larvae, while Cry10 killed 20% and Cyt1 protein alone resulted in 45% mortality of the larvae (figure 2). Additionally, the combination of cyt1 , cry 4A and cry4B killed 65% of insect larvae, while a combination of cry 4A and cry4B killed 53.75% of the larvae (table 6).

Qualitative bioassays were performed with -100 μg/ml of spore and crystal complex of the Bt- pSTAB-cry10A using A. aegypti fourth instar larvae. The qualitative bioassays with A. aegypti Table 5

Treated Dead % Mortality insects insects Control (-) Control (+) Strain Abbott

Larvae L1

Cry10Aa + CytiAa

Rep. 1 40 40 0 100 100 100

Rep. 2 40 40 0 100 100 100

Rep. 3 40 40 10 100 100 100

X ± DS 3.33 ± 5.77 100 ± 0 100 ± 0 100 ± 0

Adults

Cry10Aa + CytiAa

Rep. 1 40 4 0 65 10 10

Rep. 2 40 4 0 65 10 10

Rep. 3 40 4 0 65 10 10

X ± DS o ± o 65 ± 0 10 ± 0 10 ± 0

larvae demonstrated toxicity of the spore and crystal complex of the strain Bt-pSTAB-cry10A against this insect. The mortality was 70% at 24 hrs and 90% at 48 hrs at an approximate concentration of 100 μg/ml (table 7).

A bioassay of spore and crystal complex of the Bt-pSTAB-cry10A was performed using Aedes aegypti fourth instar larvae at 24h. This dose-dependent bioassay showed an LC50 of 2.061 μg/ml and a LC ₉₅ of 75.489 μg/ml (table 8). The dose-dependent bioassay of the spore-crystal complex of Bt-pSTAB-cry10 and Cyt1A crystals, on A. aegypti larvae at 24h is reported in table 9. In this dose-dependent bioassay, mixtures of the strains Cry10A-Cyt1Aa gave a LC ₅₀ of 40.909 ng each component/ml; while the LC95 was 121.2 ng/ml (table 9).

The LC ₅₀ of the spore and crystal complex of the strain CrylOA was 2 μg/ml, substantially better than reported by others researchers (Thorne, L et al. 1986. Journal of Bacteriology. 166: 801- 81 1 ; Delecluse, A et al. 1988. Molecular and General Genetics. 214: 42-47; Wirth, M et al. 2004. Journal of Medical Entomology. 41 : 935-941 ).

Example 6. Activity of other B. thuringiensis isolates.

Bacillus thuringiensis subsp. israelensis has an effective mortality rate of 100% against H. hampei. The strain B. thuringiensis subsp. israelensis LBIT315 lacks the Cry10 polypeptide (SEQ ID NO. 7), however, it maintains an effective mortality rate against H. hampei of 100%. To determine which other crystal proteins are responsible for the mortality, the effectiveness of cry4A and cry4B were tested independently and in combination with Cyt1 against H. hampei. Table 6

Treated Dead % Mortality insects insects Control (-) Control (+) Strain Abbott

Cry10Aa + Cry11A

Rep.1 40 16 0 100 40 40

Rep.2 40 15 0 100 38 38

Rep.3 40 16 5 100 40 37

X±DS 1.7±2.9 100±0 39.3±1.2 38±2

CryiOAaH ^■ Cry4A

Rep.1 40 10 0 100 25 25

Rep.2 40 9 5 100 23 19

Rep.3 40 10 0 100 25 25

X±DS 1.7±2.9 100±0 24.3±1.2 23±3

CryiOAaH ^■ Cry4B

Rep.1 40 7 0 97.5 17.5 17.5

Rep.2 40 7 0 100 17.5 17.5

Rep.3 40 8 0 100 20 20

X±DS O±O 99.2±1.44 18±0.87 18±0.87

CryiOAaH ^■ CytiAa

Rep.1 40 40 0 100 100 100

Rep.2 40 40 0 100 100 100

Rep.3 40 40 10 100 100 100

X±DS 3.33±5.77 100±0 100±0 100±0

Cry11A + CytiAa

Rep.1 40 14 0 100 35 35

Rep.2 40 15 10 100 37.5 30

Rep.3 40 12 0 100 30 30

X±DS 3.33±5.77 100±0 34.2±3.8 31.9±2.7

Cry 4A (pHT4A + AB) + CytiAa

Rep.1 40 26 0 100 65 65

Rep.2 40 26 0 100 65 65

Rep.3 40 27 0 100 67.5 67.5

X±DS O±O 100±0 65.8±1.44 65.8±1.44

Doses: 5μg/ul , the mortality was evaluated at seventh day. Table 7

24 hrs 48 hrs

Treatments

Treated Dead Mortality % Treated Dead Mortality %

Rep. 1 20 17 85 20 19 95

Rep. 2 20 14 70 20 17 85

Rep. 3 20 13 65 20 18 90

Rep. 4 20 14 70 20 18 90

Rep. 5 20 15 75 20 19 95

Rep. 6 20 13 65 20 18 90

Total 120 86 71.66±3.07 90.8±3.7

Negative control

Rep. 1 20 0 0 20 0 0

Rep. 2 20 0 0 20 0 0

Rep. 3 20 0 0 20 0 0

Total 60 0 0 60 0 0

Mortality Evaluation Data. Referring to figure 2, application of Cry4A to H. hampei resulted in a mortality rate of 47%, application of Cry1 1A resulted in 29.3% of mortality. The combination of Cyt1 , Cry4A, and Cry4B however, resulted in a mortality rate of 66.25%. The application of Cry4A and Cry4B, in the absence of Cyt1 , exhibited a 53.75% mortality rate against H. hampei. The use of cry4B alone resulted in a mortality rate of only 5%.

Example 7. Activity against A. grandis. In this example, activity of crystal proteins was assessed using CryiOA isolated from recombinant virus (example 3) or from recombinant plasmid (example 1 and 2). Third stage larvae of S. frugiperda were infected with 10 μL of the recombinant viruses (example 3), and after five days, the larvae were homogenized in 1 ml. of MiliQ® water for each insect cadaver. The homogenate was filtered in glass wool, and the suspension was centrifuged at 10,000 g for 10 min. The floating coat was discarded, and the sediments were re-suspended in a solution of 100 mM of EDTA, 40 mM of EGTA and 1.0 mM of PMSF. Cryi OAa was quantified in polyacrylamide gel, using a program of Image phoretix 2D® (Pharmacia). The program makes ratio calculations between the band of the recombinant protein and a band of known concentration of bovine serum albumin protein (100 mg). Bioassays with neonatal larvae of A. grandis were performed according to Martins (Martins, expressao e analise da patologia de proteinas Cry, derivadas de B. Thuringiensis, em insetos- praga. Brasilia UNB, 2005. 130 p. Dissertacao mestrado, 2005), which are briefly described here: K) K ⁾

Ol O O

Table 8

Reliability Reliability

Bioassay Dose Mortality Corrected LC 50 LC 95

Treated Dead Interval Interval (3 rep.) (μg/ml) % mortality (μg/ml) (μg/ml)

(μg /mL) (μg /mL)

1 20 60 50 83.3 83.0

2 10 60 47 78.3 77.9 w

3 5 58 36 62.1 61.4

4 2.5 60 37 61.6 61.0 2.061 1.451-2.927 75.489 30.248-188.393

5 1.25 56 25 44.6 43.7

6 0.625 56 14 25 23.7

Negative

0 60 0 0 0 Control

the artificial diet was poured into a Petri plate (15 mm x 20 mm), and after solidification, 25 holes were made. A neonatal larvae of A. grandis was placed in each hole, with a total of 25 larvae for each dose of Cry 10Aa protein. With a total of 75 larvae per dose, five doses of the recombinant protein Cryi OAa were evaluated (10.4; 8.32; 6.24; 4.16; 2.08 μg/ml), in addition to one control with the addition of the diet only. All the bioassays were performed in incubation chambers with photophase of 14:10 h (light and dark) at a temperature of 25 ⁰ C and relative air humidity of 75%. After seven days, the experimental reading was made, and the LC ₅₀ was determined by probit analysis (Finney, Probit analysis, Cambridge University Press. 1971 ).

The toxicity of the mix of OB and crystals of Cryi OAa purified from cadavers of Spodoptera frugiperda infected with recombinant baculovirus vSynCryi OAa was determined for neonatal larvae of Anthonomus grandis. The results in table 10 are the average of three repetitions. The

LC ₅₀ of the protein CryiOAa for the neonatal larvae of the boll weevil was 7.12 μg/mL (table 10).

The results demonstrate that the recombinant protein Cryi OAa was toxic for the insect analyzed, thus demonstrating the efficiency of the expression system based on baculovirus insect cells, for the production of Cry proteins that are biologically similar to the native proteins.

Table 10

Confidence limit Bibliographical

Sample n LC ₅₀ (μg /ml_)

(μg /mL) reference

CryiOAa 75 7.12 (5.27-9.8) ^a this document

Cryi la 75 21.5 (17.0-26.0) ^a Martins, 2005

S1806 75 300.0 (250-360) ^a Martins et al., 2006

S1989 75 740.0 (610-910) ³ Martins et at., 2006

LC _5O Lethal concentration for 50% of the individuals evaluated during one week. a samples with p >0.05 y G >0.04 n number of insects used per repetition.

In other assays, the required weight of powder for each dilution was taken up in 5 ml 0.01 % Tween 20 to achieve a more homogeneous suspension and this was added to 35ml of the artificial diet before it was poured out into Petri dishes and was punched with 48 holes. Each hole received a neonate larva. Five doses (from 0.10 to 1.5 mg/ml) were tested. Four replicates were prepared and one bacteria free control was included. The bioassay was kept in an incubator with photoperiod of 14/10 at 27 ⁰ C. A week later, the bioassay was evaluated (Praca et al, Coleoptera e Diptera Presq Ag Bras 39:11-16, 2004) and the LC ₅₀ was determined through Probit analysis

(Finney, 1971 ). The bioassay was repeated 3 times and the LC ₅₀ were compared by ANOVA through Sigmastat program. Bioassays with purified toxins were accomplished by adding dried toxin to a final concentration of 200 mg/ml of boll weevil artificial diet. Four replicates were prepared and one bacteria free control was included. The mortality rates were compared by ANOVA through Sigmastat program. When different toxin-expressing strains were assayed in combination, equal quantities of each powder were used and doses were adjusted so that the final total spore powder concentration remained at 200 mg/ml. Assays of B. thuringiensis against A. grandis and Lepidopteran targets. B. thuringiensis subsp. israelensis showed no toxicity against the Lepidopteran insects P. xylostella, A. gemmatalis or S. frugiperda in the single concentration assays at 200 mg/ml (results not shown), in contrast to results previously obtained with caterpillars from a different taxonomic family, Hylesia metabus (Lepidoptera: Saturniidae) (Vassal et al. FEMS Microbiol Lett 107: 199-204, 1993). However, close to 100% mortality of A. grandis was observed (figure 4). Therefore, the LC ₅₀ value at 7 days against A. grandis was determined alongside B. thuringiensis subsp. tenebrionis (T08017), a strain known to be active against Coleopteran insects (US 5382429) B. thuringiensis subsp. israelensis IPS82 showed an LC50 of 0.74 mg/ml (confidence limits: 0.61-0.91 mg/ml), comparable to that of B. thuringiensis subsp. tenebrionis (LC ₅₀ 0.32 mg/ml; confidence limits: 0.23-0.44 mg/ml) but produces a different profile of toxins. As a result, and since cloned genes were already available for B. thuringiensis subsp. israelensis, bioassays with strains expressing individual toxins were undertaken. The results of bioassays of individual toxins and their combinations are shown in figure 4. Of the individual toxins, the toxicity of the strain producing Cry11 Aa was the lowest. Cry4Aa and Cry4Ba producing strains were more active with the highest level of mortality produced by CytiAa. Relative levels of toxin production were assessed by densitometric scanning of SDS-PAGE gels of the different spore/crystal preparations. The levels of CytiAa and Cry1 1Aa were both approximately twice that of Cry4Aa, while the level of Cry4Ba was approximately 4 times that of Cry4Aa. As a result, Cry11Aa would appear to make little contribution to A. grandis toxicity. Cry4Aa, however, is present in the lowest amount but has one of the highest activities, although Cyt1 Aa may also contribute to toxicity to this insect.

Assays of combinations of toxins did not produce 100% mortality in our assays, even when all four toxin producing strains were used at high dose and toxicity was too low for LC50 to be determined. The low toxicity of the four toxins tested here, singly or in combination, suggests that these toxins are not the major determinants of activity against A. grandis and that other B. thuringiensis subsp. israelensis factors may be involved. While Cry4Aa, Cry4Ba, Cry1 1Aa and CytiAa are the major crystal proteins produced by this strain, the pBtoxis toxin-coding plasmid also bears the crylOAa, cyt2Ba and cytiCa genes all of which are expressed in the B. thuringiensis host. The Cyti Ca protein shows no mosquitocidal activity (Manasherob, et al., Cyti Ca from Bacillus thuringiensis subsp. israelensis: production in Escherichia coli and comparison of its biological activities with those of other Cyt-like proteins. Microbiol. 152: 2651- 2659, 2006) although its activity against other insects is unknown. It is possible that one or more of these minor crystal proteins is responsible for the toxicity against Coleoptera, either alone or in synergy with other toxins.