MXPA97008495A - Insect control with multip toxins - Google Patents

Abstract

The present invention provides a method that accelerates the rate of extermination rate of pests such as those of the order Lepidoptera. The method comprises treating the pest or its habitat with at least two different insect toxins, which are expressed from at least one recombinant microbe. It has been found that pairs of toxins that do not compete with each other for the same binding site and that differ in their pharmacology provide synergistic control. The preferred insecticidal microbes are baculovir

Description

INSECT CONTROL WITH MULTIPLE TOXINS FIELD OF THE INVENTION The present invention relates generally to the use of insect toxins to control insects, and more particularly to insecticidal recombinant microbes that express insect-selective toxins in synergistic combinations to increase the insect mortality rate. This invention was made with government support under Contract No. 92-37302-6185, granted by the United States Department of Agriculture. The Government of the United States has certain rights over the invention.

BACKGROUND OF THE INVENTION The family of Lepidoptera Noctuidae includes some of the most destructive agricultural pests, such as the genera Heliothis, Helicoverpa, Spodoptera, and Tri chop usia. For example, included within this family are the tobacco budworm. { Heliothis virescens), the worm of the cotton capsule. { Helicoverpa zea), the worm of cotton leaves. { Alabama REF: 025963 argillacea), the spotted cutworm. { Ama thes niarum), the brilliant cutworm. { Devastating Crymodes), the tanned cutworm. { Nephel odes emmedoma), the worm of the autumn branches. { Laphygma frugiperda), the worm of the beet branches. { Spodoptera exigua), and the variegated cutworm. { Peridroma saucia). The resistance of agricultural pests, such as Noctuidae (and others), to pesticides leads to environmental and human health risks. This problem of resistance to insecticides leads to the use of non-selective and toxic compounds, to overcome the resistance of pests. This creates a destructive and viscous cycle. Selective natural toxins have been suggested for use in insect control. These toxins include substances which are produced in specialized glandular tissues in the body of a poisonous animal. The poison can be introduced into the body of its prey or opponent, such as by the help of a stinging-penetrating device, to paralyze and / or kill it, although other means of releasing poison are known. Scorpions, for example, contain in their venom numerous proteins, or neurothioxins, which are toxic and act on excitable systems. Among the insect-specific toxins suggested for use in insect control are the toxins of Bacillus thuringiensis, of the scorpions Buthus eupeus and Androctonus australis, Lei urus qumqustpatus hebraeus, Lei urus qumqustriatus quinquustriatus, and the termite Pyemotes tp tici. Poisons derived from scorpions that belong to the Buthmae subfamily have four major groups of polypeptide neurotoxins, which modify the conductance of axonal sodium. A group of scorpion neurotoxins are the a-toxins, which selectively affect mammals through an extreme prolongation of the action potentials due to the lethargy of the blockade of the sodium channel inactivation (Catterall, Science, 223: 653- 661 (1984); Rochat et al., Advances in Cytopharmacology, pp. 325-334 (1979)). The second group of toxins, the ß-toxins, affect the activation of the sodium channel (Couraud and Jover m Handbook of Natural Toxins (Tu, A. Ed.) Vol. 2, pp. 659-678 (1984) New York: Marcel Dekker). The third group of neurotoxins are the selective toxins of depressive insects which induce flaccid paralysis that develops progressively from the insects due to the blockage of the action potentials substantially due to the suppression of the sodium current (Lester et al., Biochim, Biophys, Acta, 701: 310-381 (1982); Zlotkm et al., Arch. Biochem. Biophys. , 2.0: 877-887 (1985)). The fourth group of neurotoxins are the excitatory insect selective toxins, which cause an immediate spastic paralysis (knock down) of the insects due to the induction of repetitive tables in their motor nerves due to an increase in the peak sodium current and the lethargy dependent on the voltage of its inactivation (Walther et al., J. Insect Physiol., 22: 1181-1194 (1976); Pelhate et al., J. Physi ol. , 30: 318-319 (1981)). In addition to the scorpion and termite toxins, other insect-selective toxins have been identified in the venoms of snails, spiders, and numerous other arthropods. See the review of Zlotkín, Comprehensi ve Insect Physiology, Biochemistry and Pharmacology, Vol. 10, Chapter 5, p. 499-541 (1985)]. The wasp poisons brancoides are highly toxic to lepidopteran larvae. The Bracon hebetor brancoid venom causes flaccid paralysis in lepidopteran larvae by inducing the presympathetic interruption of the excitatory glutaminergic transmission in the neuromuscular joint of the insect (Piek et al., Comp.Chemchem. Physiol., 72C: 303- 309 (1982)). The poisons of the solitary wasps are toxic to a large number of insects and spiders of different orders (Rathmeyer, Z. Vergl. Physi ol., .5: 453-462 (1962)). An example of such poisons is the poison of Philan thus tpangulum, which induces in the insects a flaccidity paralysis substantially due to the presynaptic block of the neuromuscular transmission; this poison affects both the excitatory and inhibitory transmission (May et al., Insect. Physiol., 25: 285-691 (1979)). The venom of the black widow spider, Lan trodectus mactans, contains components which are neurotoxic to insects, but not to mas, and other components with the opposite selectivity (Fritz et al., Nature, 238: 486-487 (1980 ), Ornberg et al., Toxicon, 14: 329-333 (1976)). More recently, a toxin was designated as LqhalT, which strongly resembles toxins in its primary structure and electrophysiological effects of L venom. quinquestriatus hebraeus, and showed to mainly affect insects (Eitan et al., Biochemistry, 29 (1990), pp. 5941-5947). Poisonous animal venom is composed of a variety of toxins that affect different target sites in the excitable systems of the prey. On the basis of the data comparing the activity of the toxins and their respective crude venom to lepidopteran larvae it is clear that the potency of the crude venom can not be explained by the activity of the toxin alone. The greater potency of crude venom could be related to the cooperation of the different toxins in the venom that affect different target sites on the same ion channels (Table 3, Trainer et al., JBC, 268, 17114-17119 (1993)), in different ion channels on the same excitable cells (Olivera et al., Sci ence, 249, 257-263 (1990)), and / or different binding sites on adjacent excitable cells (nerves and / or muscles) (Olivera et al. ., Science, 249, 257-263 (1990)). Repressive and excitatory insect selective toxins do not compete with insect toxin A for their binding site (Gordon and Zlotkin, FEBS Lett., 315 (1993), pp. 125-128). In contrast to the lobster or cockroach neuronal membranes, the excitatory toxins do not displace depressive toxins from their binding site on neuronal membranes of lepidopteran larvae (Gordon et al., Biochemistry, 31 (1992), pp. 76-22 -7628; Moskowitz et al., Insect Biochem, Molec. Biol., 24 (1994), pp. 13-19). Recently, Autographa californica nuclear polyhedrosis virus (AcNPV), of the Baculoviridae family, has been genetically modified to increase the rate of extermination by the expression of insect-selective toxins. The introduction of an insect-selective toxin into an insect pathogenic virus has resulted in a reduction in the extermination time of insect hosts, as described in US Patent Application No. 08 / 229,417, filed on April 15. of 1994, which is a continuation request in part of the North American Patent Application No. 07 / 629,603, filed December 19, 1990, which has (in part) the common allocation with the present. Tomalski et al., US Patent Number 5,266,317, issued November 30, 1993, discusses the use of recombinant baculoviruses expressing an insect-specific paralytic neurotoxin from an insect predator acaricide. Barton et al., US Patent No. 5,177,308, issued January 5, 1993, takes a different method to create transgenic plants that express a specific insect toxin derived from scorpion and / or a soil-dwelling microorganism toxin. In a co-pending application, from the common assignment hereof, Hammock and McCutchen, Application Serial No. 08 / 279,956, filed July 5, 1994, discuss insect control with a combination of a recombinant virus and an organic insecticide. These new emerging tools that use recombinant strategies to control insect pest populations are particularly promising because the large-scale presence of pest resistance to organic insecticides, such as pyrethroids, has begun to result in substantial crop losses. . In cotton alone, the presence of Heliothis pyr-R species has begun to result in millions of dollars in losses annually. In fact, several cases of pyrethroid insecticides have completely failed to control infestations of Heliothis larvae in cotton, which has resulted in complete destruction of the crop.

BRIEF DESCRIPTION OF THE INVENTION In one aspect of the present invention, a method for controlling a variety of pests is provided through the use of genetically engineered insecticidal microbes. Controlled pests controlled in accordance with the invention are, for example, from the group of insects, acarids and nematodes. In this way, the invention is applicable to Lepidoptera as well as to other orders, and to Noctuidae as well as to other families. Such pests are treated (or their habitats are treated) with a kinetic combination of toxins expressed by one or more recombinant microbes. For example, the method can use a combination of a first recombinant pathogen that expresses a first neurotoxin and a second recombinant pathogen that expresses a second neurotoxin, or can use a single recombinant virus expressing a plurality (such as the first and second) of neurotoxins. The method of the invention accelerates the rate of extermination of the pests by the virus.

BRIEF DESCRIPTION OF THE DRAWING In the drawing, Figure 1 illustrates the nucleotide sequence of a synthetic LqhlV gene, SEQ ID NO: 1, which is one of the preferred portions for practicing the invention.

DETAILED DESCRIPTION OF THE PREFERRED MODALITIES The present invention is the use of insecticidal microbes, genetically engineered in combinations for treating pests, such as insects. Although recombinant baculoviruses will be used herein as an illustration of the preferred microbes, this invention can be practiced with a variety of microbes as recombinant delivery systems. Thus, the microbes useful in the present invention include DNA and RNA viruses, such as baculoviruses, fungi and bacteria. Forty viruses of the nuclear polyhedrosis of insect species have been isolated. (See, for example, Atlas od Invertebra te Virures Adams and Bonami, editors, CRC Press, Inc., 1991). Several baculoviruses, including those that infect the cotton capsule worm, Helicoverpa zea, tobacco budworm, Heli othis virescens, Douglas fir herb butterfly, Orgia pseudotsuga ta, lizard, Lymantria di spar, caterpillar of the alfalfa, Autographa calif ornica, European pine fly, Neodi iprion sertifer, and small butterfly, Laspeyresia pomonella, have been registered as pesticides and all those baculoviruses of insect species are suitable for practicing the invention. Numerous fungi are capable of infecting insects. The introduction of insect-selective toxin into the genome of such fungi could increase their potency as pesticides. For example, Beauvaria bassania and Beauvaria brongniartii have a wide range of hosts and have been suggested as candidates for microbial pesticides (see review by Miller et al., Science, 219: 115-121, 1983). Bacteria (other than Bacillus thuringiensis) that have been consid as agents to control insects include Bacill us popilliae, B. l entimorbus, and B. sphaericus. Its potential as a pesticide can be increased by improving its potency through the incorporation of a selective insect toxin in its genome. The practice of the invention involves the combined use of two toxins that act synergistically in the control of insects. These two can be expressed- by means of a single recombinant microbe in which both genes of the toxin have been introduced, or it can be practiced by preparing two recombinant microbes, each of which has been constructed by cloning a gene that codes for the respective insect toxins in the genome. The combinations of pairs of selected toxins are determined in several ways. As will be described below (as described in the selection techniques of Example 6), toxins are preferably selected that act on the same cellular channels (typically the sodium channels) but at overlapping sites, as will be better described later herein. As mentioned at the beginning, the preferred insecticidal microbes for practicing the invention are baculoviruses. "Baculovirus" means any baculovirus of the Baculoviridae family, such as a nuclear polyhedrosis virus (NPV). Baculoviruses are a large group of evolutionarily related viruses, which infect only arthropods; in fact, some baculoviruses only infect insects that are pests of commercially important agricultural and forestry crops, while others are known to specifically infect other insect pests. Because baculoviruses infect only arthropods, they have little or no risk to humans, plants or the environment. The DNA of suitable viruses, in addition to the Baculoviridae, are the entomo-viral viruses (EPV), such as the EPV Melolontha melonotha, EPV Amsacta moorei, EPV Locusta migratoria, EPV Melanoplus. sanguinipes, EPV Schistocerca gregaria, EPV Aedes aogypti, and EPV Chirono us luridus. Other suitable DNA viruses are viruses of the grabulosis (GV). Suitable RNA viruses include toga viruses, flaviviruses, picornaviruses, cytoplasmic polyhedrosis viruses (CPV), and the like. The double-stranded DNA virus subfamily Eubaculovirinae includes two genera, NPV and GV, which are particularly useful for biological control because they produce body occlusion in their life cycle. Examples of GV include GV Cidia pomonella (small butterfly GV), GV Pieris brassicae, GV Trichoplusia ni, GV Artogenia rapae, and GV Plodia interpunctella (Indian flour butterfly). Baculoviruses suitable for practicing this invention may be occluded or non-occluded. Nuclear polyhedrosis viruses ("NPV") are a subgroup of baculoviruses, which are "occluded". That is, a distinctive feature of the NPV group is that many virions are included in a crystal protein matrix referred to as an "occlusion body". Examples of NPV include NPV Lymantria dispar (Lizard NPV), MNPV Autographa californica, NPV Anagrapha falcifera (NPV from the celery caterpillar), NPV Spodoptera li tturalis, NPV Spodoptera frugiperda.

NPV Heliothis armígera, NPV Mamestra brassicae, NPV Choristoneura fumiferana, NPV Trichopl usia ni, NPV Hel i coverda zea, and NPV Rachipl usia ou. For use in the field, occluded viruses are often preferred because of their high stability since the viral polyhedrin coating provides protection to the enclosed infectious nucleocapsids. Among illustrative baculoviruses useful in the practice of this invention are Anagrapha falcifera, Anticarsia gemma talis, Buzura supressuria, Cydia pomonella, Helicoverpa zea, Heliothis armigera, Manes tia brassicae, Pl utella xylostella, Spodoptera exigua, Spodoptera li t torali s, and Spodoptera li tura. A baculovirus "NPV" particularly useful for practicing this invention is AcNPV, which is a nuclear polyhedrosis virus of Autographa cali fornica. Autographa californica is of particular interest because several of the major pest species within the genera Spodoptera, Trichoplusia, and Heliothis are susceptible to this virus. The expressed insecticidal toxins are particularly a neurotoxin derived or similar to an arthropod toxin or other invertebrate, such as a scorpion toxin, a wasp toxin, a snail toxin, a mite toxin, or a spider toxin. A useful scorpion toxin is, for example, AaIT from Androctonus australis. Zlotkin et al., Bi ochemie, 53, 1073-1078 (1971). A useful snail venom is that which comes from the conus Conus querciones, which the animal releases by mouth and some individual toxins from which you appear to be selective for arthropods including insects. See, for example, Olivera et al., "Diversity of Conus Neuropeptides," Science, 249: 251-263 (1990). Even the peptides that normally appear in the life of the insect development can operate as an insecticidal toxin, and can be used in accordance with this invention. For example, the early appearance of juvenile hormone esterase ("JHE") will reduce the juvenile hormone titers in a host insect, which typically results in an irreversible termination of the feeding stage, pupation attempt, and death of the insect. of the plague. The amino acid sequence of the JHE is known, and the gene has been cloned. Preferred embodiments of the present invention include recombinant microbes expressing juvenile hormone esterase (JHE) mutations, and exemplary methods for preparing such mutations or deletions of JHE, various useful JHE mutations, and recombinant expression vectors for use in the control of insects (having JHE or sequences encoding mutant JHE) according to that described in WO 94/03588, published on February 17, 1994, invented by Hammock et al., incorporated herein by reference. Two mutants described in WO 94/03588 of Hammock et al., Are a double -Utiline utante (K29R, K522R), wherein the normal JHE-Usinas at position 29 and position 522 were changed to arginine by site-directed mutagenesis. site. Another mutant described was where serine 201 was changed to glycine and the mutant was designated "S201G". The insecticidal activity of the catalytically deficient mutant S201G of the JHE provides a similar time for death in 50% of the test insects to the scorpion toxins (when the A.cNPV was designed). Thus, the JHE insect protein found in nature, which is normally non-toxic, can be modified by means such as site-directed mutagenesis (or on the other hand) to a toxic agent. In addition to changes in the residual amino acids, other JHE mutants could be prepared such as by the deletion of 19 N-teral amino acids, which are a signal sequence for the newly produced protein to enter the secretory pathway, be glycosylated, and get out of the cell. As with JHE, the amino acid sequence of the excitatory toxin of Androctonus australis (AsIT) has also been determined, the sequence has been published (Darbon 1982), and the AaiT gene has been cloned and inserted into expression vectors for insect control. (See WO 92/11363, published July 9, 1992, inventors Belangaje et al). The toxin AaIT exhibits toxicity to insects, although it is not toxic to isopods and mammals. Another toxin suitable still for practicing the invention affects the sodium channels of the insect in a manner very similar to the effect of the a-toxins of the sodium channels of mammals. This neurotoxin was derived from a yellow scorpion Leuirus quinquestriatus hebraeus, and was named here LqhalT. The identification and purification of this toxin was described in "A Scorpion Venom Neurotoxin Paralytic to Insects that Affects Sodium Current Inactivation: Purification, Primary Structure, and Mode of Action", published by Eitan et al., Biochemistry, 25: 5941-5947 ( 1990) . Two preferred toxins for practicing the invention are novel in isolated and purified form, and will be described more fully hereinafter. Briefly, those two were designated as "LqhlV" and "LqhVI". Those two toxins are found in the venom of L. quinqestriatus hebraeus, which contains numerous individual toxins in mixture in the native form. The LqhlV toxin is an extremely potent lepidopteran toxin, shows positive cooperativity with scorpion toxins when injected into lepidopteran larvae, and has no or low toxicity in weak mammals. A synthetic gene for this LqhlV toxin is illustrated by Figure 1, SEQ ID NO: l. In this way, the genes for these two preterm toxins can be synthesized (since the sizes of peptide sequences are small enough to make DNA synthesis possible). Alternatively, the genes can be cloned. The coding sequences can then be cloned into a transfer vector, as will be more fully exemplified hereinafter. The aspects of the invention have been demonstrated with the synergistic combination of the AaIT and LqhalT toxins in bluebottle or bluefly larvae of the meat as larvae Heliothis larvae where the insecticidal activity of those selective insect neurotoxins was increased five to ten times when it was used in combination. Other combinations illustrating the invention and experimental details will be discussed more fully hereinafter. Various other scorpion toxins (for example, buckwheat) can be used for synergistic combinations, such as LqqIT2, which is a depressive insect toxin from Lei urus quinquestria tus quinquestria tus. The purification method used to obtain this neurotoxin was published by Zlotkin et al., Archi ves of Biochem. Bi ophys. , 240: 811-881 (1985).BjlT2 is another depressive insect toxin and is from Bothotus judai cos. The purification has been published in Lester et al., Biochim. Bi ophys. Acta, 701: 310-381 (1982). BjIT2 exists in two isoforms which differ in amino acid sequence in position 15. Form 1 has isoleucine in this position while form 2 has valine. LqhIT2 is another depressive insect toxin of Lei urus quinquestriatus hebraeus which was purified using inverted phase CLAP. Other more toxins, purified from the cactoid scorpion venom, Scorpio maurus palmatus, can also be used. For example, SmpIT2, from the cactus-like scorpion, Scorpio maurus palma tus, is a depressive insect toxin. This purification is described in Lazarovici et al., J. Biol. Chem., 257: 8397-844 (1982). Other toxins purified from the venom of the cactoid scorpion, Scorpio maurus palmatus, are SmpCT2 and SmpCT3, and toxins from crustaceans, whose purification has been described in Lazarovici, pH. D. Thesis (1980), Hebrew University, Jerusalem, "Studies on the Compositiion and Action of the Venom of the Scorpion Scorpio maurus palma tus (Scorpionidae)".

The Table lists some preferred toxins for practicing this invention together with citations for its purification and characterization.

TABLE 1 TOXINS ILLUSTRATIVE REFERENCES AaIT Zlotkin et al., Biochi, 53, 1075-1078 (1971). AaI! Loret et al., Biochem, 29, 1492-1501 (1990). AaIT2 Loret et al., Biochem, 29, 1492-1501 (1990). LqqlT! Zlotkin et al., Arch. F Biochem, & Biophys. , 240, 877-887 (1985). BjIT! Lester et al., Biochem. Biophys. Acta, 701, 370-387 (1982). LqhIT2 Zlotkin et al., Biochem. , 30, 4814-4821 (1991). LqqlT; Zlotkin et al., Arch. F Biochem, & Biophys. , 240, 877-887 (1985).

BjIT2 Lester et al., Biochem. Biophys. Acta, 701, 370-387 (1982). LqhalT Eitan et al., Biochem. , 29, 5941-5947 (1990). TSvn Bechis et al., Biochem. Biophys. Res. Com. , 122, 1146-1153 (1984). Acarid toxin Tomalski et al., Toxicon, 27, 1151-1167 (1989). a-conotoxins Gray et al., JBC, 256, 4734-4740 (1981); Gray et al., Biochem. , 23, 2796-2802 (1984). μ-conotoxins Cruz et al., JBC, 260, 9280-9288 (1989); Crus et al., Biochem. , 28, 3437-3442 (1989). Chlorotoxin Debin et al., Am. J. Physiol. , 264, 361-369 (1993). ? -conotoxins Olivera et al., Biochem, 23, 5087-5090 (1984); Rivier et al., JBC. , 262, 1194-1198 (1987). PLTX1 Branton et al., Soc. Neurosci. Abs. , 12, 176 (1986). PLTX2 Branton et al., Soc. Neurosci. Abs. , 12, 176 (1986).

PLTX3 Branton et al., Soc. Neurosci. Abs. , 12, 176 (1986). Agí Kerr et al., Soc. Neurosci. Abs. , 13, 182 (1987); Sugi ori et al., Soc. Neurosci. Abs. , 13, 228 (1987). Ag2 Kerr et al., Soc. Neurosci. Abs. , 13, 182 (1987); Jackson et al., Soc. Neurosci. Abs. , 13, 1078 (1987). -Agatoxin Adams et al., JBC. , 265, 861-867 (1990). μ-Adatoxin Adams et al., JBC. , 265, 861-867 (1990).

Hol Bowers et al., PNAS. , 84, 3506-3510 (1987). a-Laterotoxin Grasso, m Neurotoxins m Neurochemistry, ed. Dolly, 67-79 (1988). Toxin Steatoda Cavalieri et al., Toxicon, 25, 965-974 (1987). Bom III Vargas et al., Eur. J. Biochem. , 162, 589-599 (1987).

The cDNA libraries for many of the organisms of which the illustrative toxins of Table 1 can be purified are available as described by: Zilbergerg et al., (1992), Insect Biochem. Molec. Biol, 22 (2), 199-203. { Leiurus quinquestriatus hebraeus); Gurevitz et al. (1990) Febs Lett. , 259 (1), 229-332 (Buthus judaicus); Bougis et al. (1989), JBC, 264 (32), 19259-19256 [Androctonus australi s); Martin-Euclaire et al. (1992) Febs. Let t. , 302. { 3), 220-222. { Ti tyus serrula tus); Woodward et al. (1990) EMBO J., 9 (4), 1015-1020. { Conus textile); and Colledge et al. (1992) Toxicon, 30. { 9), 1111-11116. { Conus geographus). For others, synthetic genes that code for toxins can be constructed, in a manner analogous to that exemplified by Example 7. As mentioned at the outset, two toxins suitable for use in the practice of the present invention are novel in their isolated and purified form. One of those was designated as "LqhlV," and has the amino acid sequence shown as SEQ. ID. NO: 2: GVRDAYIADD KNCVYTCGAN SYCNTECTKN GAESGYCQWF GKYGNACWCI KLPDKVPIRI PGKCR. The sixty-five amino acid peptide of SEQ. ID. NO: 2 is best described in Example 5. Another novel toxin, designated "LghVI", has the amino acid sequence given by SEQ. ID. NO: 3: GVRDGYIAQP ENCVYHCFPG SPGCDTLCKG DGASSGHCGF KEGHGLACWC NDLPDKVGII VEGEKCH. This peptide of sixty-seven amino acids is also better described in Example 5. Toxins, such as the preferred toxins listed in Table 1, or as SEQ. ID. NOS: 2 and 3, can be selected to form synergistic combinations more easily by first making experimental combinations of toxins that have different pharmacologies. For example, the AaIT is an excitatory insect toxin while the LqhlT. It is a depressive toxin. For routine binding protocols (see, for example Gordon et al., Bi ochim Biophys. Acta, 778, 349-358 (1984) for AaIT, BjiT and Bj? T2 with locusts membrane vesicles Locus ta migratory) , the activity of the same channel is selected but in non-superimposed sites for the particular insect of interest. This is because, as is known in the art, there is variability between several insect neuronal membranes. For example, several recent articles have reported that unlike the lobster or cockroach neuronal membranes, the neuronal membranes of Lepidoptera larvae can bind to insect depressant and excitatory toxins at the same time. In the example noted at the beginning of a cmérgica combination of AaIT and LqhalT, there was twice the synergistic power for the combination of blowfly larvae or blue fly of the meat, than for the larvae of Heli othis. In contrast, with the combination of AaIT and LqhIT2, there was a synergistic combination (power of five times) when applied to Heliothis larvae, but no increased potency with respect to each of the toxins themselves when applied to blowfly larvae . These combinations of toxins can be used to increase selectivity within groups of insects. To produce recombinant microbes, such as baculoviruses, for the purposes of controlling insects, a secretory signal sequence is preferably included. The secretion signal sequences can be derived from bacterial, yeast, fungal, or higher eukaryote proteins, including both animals and plants (for examples, see Watson, Nuci, Ac. Res., 12: 5145-5164 (1984). more preferred are secretion signal sequences from proteins of insect origin, for example those of cecropin B from Hialophora cecropia (van Hofsten et al., PNAS, 82-2240-2243 (1985)), and the hormone from the ecclusion of Manduca sixth (Horodyski et al., PNAS, 86: 8123-8121 (1989)). Secretion signal sequences naturally associated with scorpion toxins are also preferred, which can be determined by the analysis of mRNA, cDNA, or Genomic DNA The most preferred is the natural secretion signal sequence of AaIT (Bougis et al., J. Biol. Chem., 264: 19259-19265 (1989)). Toxins from recombinant microbes can be expressed as derivatives of the toxin, a "functional derivative" of the toxin is a compound that possesses a biological activity (either functional or structural) that is substantially similar to a biological activity of the toxin. The term "functional derivative" is intended to include "fragments," "variants," "analogues," or "chemical derivatives" of a molecule. By "fragment" of a molecule such as a toxin refers to any polypeptide group in the molecule. A "variant" of a molecule such as a toxin refers to a molecule substantially similar in structure and function to any complete molecule, or a fragment of it. A molecule is said to be "substantially similar" to another molecule if both molecules have substantially similar structures or if both molecules possess similar biological activity. Thus, as long as the two molecules have a similar activity, they are considered variants according to the term used here even if the structure of one of the molecules is not found in the other, or if the residual amino acid sequence is not identical. "Analogous" of a molecule such as a toxin refers to a molecule substantially similar in function to either the entire molecule or a fragment thereof. As used herein, a molecule is said to be a "chemical derivative" of another molecule when it contains additional chemical moieties that are not normally part of the molecule. Such portions can improve the solubility, absorption, biological half-life, etc., of the molecule. Portions capable of mediating such effects are described in Remington's Pharmaceutical Sciences (1980). Methods for coupling such portions to a molecule are well known in the art. The expression of the toxin (or toxins), in general, will include a sufficient promoter region to direct the initiation of RNA synthesis. A baculovirus promoter gene is one that codes for polyhedrin, since the polyhedrin protein is one of the most highly expressed eukaryotic genes known, although other promoters and hybrid promoter sequences can be used, for example, such as plO. Recombinant baculoviruses expressing a toxin can be prepared by protocols now known in the art (for example Tomalski et al., US Patent No. 5,266,317, which exemplifies insect parasitic termite neurotoxins.; McCutchen et al., Bio / Technology, 9, 848-852 (1992) and Maeda et al., "Insecticidal Effects of an Insect-Specific Neurotoxin Expressed by a Recombinant Baculovirus", Virology, 184, 111-780 (1991), illustrating the construction of a recombinant baculovirus expressing AaIT). The preparation of a single baculovirus capable of expressing two different toxins can be by protocols analogous to: Belayev and Roy, Nucleic Acid Research, 21: 5, 1219-1223 (1993)); Wang et al., Gene, 100, 131-137 (1991), with appropriate modifications. Example 1 illustrates such analogous protocols.

EXAMPLE 1 Two insect toxin genes can be cloned into a transfer vector such as PacUW51P2 using standard molecular cloning techniques. This transfer vector is a vector based on Autographa californica polyhedral lobster (AcNPV) containing a copy of the AcNPV plO promoter and the SV40 transcription termination signals inserted in cascade, upstream of the polyhedral promoter, but in opposite orientation. This facilitates the assertion of an external gene coding for the region in a Ba Hl site under the control of the polyhedral promoter, and a second external gene coding for the region in the BglII cloning site, under the control of the plO promoter. In this way, the resulting recombinant viruses express two external proteins. The recombinant AcNPV thus prepared can be isolated by propagating Spodoptera frugiperda (sf21) cells, which are cotransfected by calcium precipitation with the recombinant plasmid. Cells infected with polyhedra can be identified and harvested after infection and purified recombinant virus plate by selection. The purification of the recombinant virus can be by standard protocols, with the resulting pure recombinant propagated and stored, such as at 4 ° C and 80 ° C. Standard protocols are described, for example, in O'Reilly, Miller and Luckow, Baculovirus Expression Vector, A Labora tory Manual.

EXAMPLE 2 The activities of four different insect toxins were determined towards two different insects and towards mice (since it is preferred to use insect toxins that have little or no effect on mammals). Those were purified by the established methods from the respective crude poisons. Its toxicity to flies and larvae of blowfly and lepidoptera was determined according to the method of Reed and Muench (1938). Table 2 shows the activity of the toxins towards insects and mice in terms of fifty percent endpoints (paralytic or lethal dose PU5o, LD50 respectively). The PU.o values of toxins for blowfly larvae were in agreement with previously published results (Zlotkin et al., Biochim, 53, 1075-1078 (1971); and Eitan et al., Bi ochem., 29 , 5941-5947 (1990)). The toxicity of those toxins to the larvae of Heliothis virescens lepidoptera is comparable to their toxicity to larvae of Spodoptera li ttoralis. The LqhalT showed greater toxicity towards mice (Swiss Webster), but the other toxins showed no toxicity towards mammals (3 μg / g bw injected subcutaneously had no effect, in contrast to the LD50 of a mammalian toxin AaHn - 0.018 μg / 20 g bw (DeLima et al., 1986) The LqhlV and LqhVI toxins are of considerable interest since the LqhlV is the most potent lepidopteran toxin isolated from scorpion venom to date, while the LqVI toxin has a toxicity towards mammals of a month.

TABLE 2 Three replicates of 25-40 blowfly larvae were injected each / with each of the toxins and the PU? 0 were determined. The P 50 of the excitatory toxins AaIT, LghVI, and LqhIT3 was determined as a contraction paralysis immediately after the injection. The PU50 of the depressive toxin LqhIT2 was determined as paralysis due to flaccidity 5 minutes after the injection. The PU50 of the insect toxins to LqhalT and LqhlV was determined as a delayed contraction paralysis and sustained 5 minutes after the injection. u Three replicates of 25-40 lepidopteran larvae were injected each, with each of the toxins and the PU50 was determined as the inability to move or rotate when inverted on their back 24 hours after injection. Two replicates of eight mice were injected subcutaneously and the DL ^ 0 for the mice was determined 24 hours after the injection.

EXAMPLE 3 Combinations of toxins were injected simultaneously, and toxicity was measured as summarized in Table 3. Toxin combinations included amounts corresponding to 1 PU50 unit of each toxin and its dilutions. The pairs of toxins that did not compete with each other in the same binding site and differ in their pharmacology were synergistic. As shown in Table 3, the degree of competitiveness does not depend on the toxin combinations but on the test animal.

TABLE 3 Three replicates of 25-40 blowfly larvae were injected each, with a combination of toxins and the PU50 was determined as the rapid contraction of the larva within one minute after injection. '"Three replicates of 25-40 lepidopteran larvae were injected each with a combination of toxins and the PU0 was determined as the inability to move or rotate when reversed on their back.The PU50 was determined 24 hours after injection . "Two replicates of eight mice were injected subcutaneously and the DL5o for mice was determined 24 hours after injection. Potency was estimated as the amount of toxin protein (a 1: 1 ratio of toxins was used in several dilutions) which caused an effect compared to PU < , 0 of each toxin alone.

As illustrated in Table 3, combinations with a potency greater than one had responses at higher doses than potentiation. Thus, such combinations are synergistic for the extermination rate rate as illustrated in the preferred embodiments of the invention.

EXAMPLE 4 In the practice of the invention, the pests are controlled and treated (and / or treated in their habitat) with recombinant baculoviruses expressing such combinations. In this example, the combined application of two viruses that express two different toxins showed to reduce the extermination time of a host insect when compared with the application of each of the respective viruses itself. Thus, as shown in Table 4, the combined application of the recombinant AcAalT with a recombinant AcLqhalT resulted in substantially reduced kill time.

TABLE 4 Lethal times (LT) were derived based on the response of third instar larvae of H. virescens to AcAalT (10000 PIB), AcLqhalT (lOOOü GDP) and a combined application of AcAalT (5000 PIB) and AcLqhalT (5000 PIB ). A small diet plug was placed in individual wells of microtiter plates and inoculated with any of the respective viruses. The third instar larvae of H. virescens were then added to the plates and maintained at 27 ° C. Mortality was recorded at intervals of 5-50. The LTs were analyzed with a Probit analysis program. Thus, the data in Table 4 are from an extermination rate study expressed as lethal times (LT) and analogous methods can be used to determine lethal doses, which are probably of greater economic importance. Taking, for example, the lethal time at which 50% of the larvae died, it is observed that the combination of toxins in the practice of the method of the invention provided a reduction of approximately 12. to 18% of the time required to exterminate the larvae host with respect to the applications of individual recombinants. When considering that the treatment with recombinant AcAalT represents an approximate reduction of 40% in the time required to exterminate the host larvae when compared to the natural AcNPV, a substantial decrease in insect feeding damage is observed and significantly less damaged plants as a result of the practice of the invention. In addition, larvae infected with recombinant microbes typically begin to show symptoms of paralysis and stop feeding a number of hours before death, further increasing the practical insecticidal effects of the method of the invention.

EXAMPLE 5 Purification of LqhlV and LqhVT The scorpion venom L. quinqestríatus hebraeus was obtained from Sigma (USA). The lyophilized poison of L. quinqestriatus hebraeus (50 mg) was suspended and homogenized in 2 ml of 10 mM ammonium acetate pH = 6.4. The insoluble material dried was removed by centrifugation at 2700% for 20 minutes. The supernatant was collected and the pellet was resuspended in an additional 2ml of 10mM ammonium acetate pH = 6.4, homogenized and centrifuged again. This extraction was done 4 times to increase the yield of protein extracted from the poison. The supernatant of all centrifugations was pooled, loaded onto a cation exchange column (10 ml of CM-52) and eluted with a linear gradient of ammonium acetate 0.01-0.5 MICROBIOS pH = 6.4 at a flow rate of 10 ml / hr. The absorbance was determined at 280 nm and consequently the peaks were collected. Fraction CM-III and CM-VI of cation exchange chromatography were further purified on RO-CLAP on a Vydac C4 Column. LqhlV was purified from CM-VI as follows: buffer A was 5. ACN with 0.1% TFA. and buffer B was 95% ACN with 0.1. of TFA. The column was equilibrated in buffer A and eluted with a linear B gradient of 0-60. in 70 min, the flow rate was 0.6 ml / min. The absorbance was determined at 214 nm and the peaks of LqhVI collected accordingly were purified from CM-III as follows: buffer A was 5. ACN with 0.1. of HFBA and buffer B was 95. of ACN with 0.1. of HFBA. The column was equilibrated in buffer A and eluted with a linear B gradient of 0-90% in 105 min, the flow rate was 0.6 ml / min. The absorbance was determined at 214 nm and the peaks were collected accordingly. The eluted fractions were collected and tested to determine their activity (Table 2) and purity.

Purity of toxins The homogeneity and purity of LqhlV and LqhVI was tested by Free Capillary Lectrotoresis of Solution (Applied Biosystems Model 270A). The capillary was equilibrated with 20 mM sodium citrate pH = 2.9 and the samples (0.02 mg / ml protein) were loaded using vacuum for two seconds. The test buffer was 20 mM sodium citrate pH = 2.9, the electric force was 20KV. í? Determination of the sequence μg of each toxin were reduced and carboxymethylated using the established method (Fernandez et al. al., "Techmques in Protein Chemistry", Vol. 5, page 215). The N-thermal sequence was determined using an HP sequence analyzer by automated Edman degradation. The Reduced and Carboxymethylated LqhlV was digested using Endoprotemase Asp-N and peptides were generated. The separation of the peptides digests were performed on microboron CLAP (Ultrafast Microprotem analyzer-Michrom BioResources Inc) using a polymer column. Buffer A was 5. ACN with 0.1% TFA and Buffer B was 95% ACN with 0.1% TFA. The column was equilibrated in buffer A and eluted with a linear gradient of B of 0-50% in 50 mm, the flow velocity was 0.05 ml / min. The absorbance was determined at 214 nm and consequently the peaks were collected. Peptide P2 was sequenced to determine the complete amino acid sequence of the toxin.

EXAMPLE 6 Union Protocol 0 Preparation of Membranes Insect Neuronella All dissections and preparations of neuronal insect tissues were made in a fluid buffer of the following composition: 0.25 M mannitol, 5 mM EDTA pH = 7.4, 5 mM HEPES (adjusted to pH 7.4 with Tris), 50 μg / ml of phenylmethylsulfonyl fluoride, 1 μM of peptatin A, 1 mM iodoacetamide and 1 mM 1, 10-phenanthroline. The nerve tissues of the insect were dissected and homogenized in an ice-cooled buffer, the remains were removed by centrifugation at 1,000 g for 10 minutes. The supernatant was centrifuged at 27,000g for 45 minutes and the membranes (P2) were collected. The P2 were suspended in the buffer and adjusted to 10% Ficoll (in the buffer) and centrifuged at 10,000 g for 75 minutes. The resulting floating film b representing the enriched synaptosomal fraction was collected. After treatment with hypertonic media (5 nM Tris-HCl pH = 7.4, 1 mM EDTA, 50 μg / ml phenylmethylsulfonyl fluoride, 1 μM pepstatin A, 1 mM iodoacetamide and 1 mM 1, 10-phenanthroline) vesicles were formed of membrane. The membrane vesicles were harvested in a small volume of dissecting buffer after centrifugation at 27,000 g for 45 minutes and stored at -80 ° C until used.

Radioiodination of Toxins Toxins were iodinated by iodogen (Pierce Chemical Co., Rockville, MD) using 0.5 mCi of carrier-free NaL5I (~ 0.3 nmol) (Amersham) and 5 mg (~ 0.7 nmol of toxin. by CLAP using an Ultrapore C3 RPSC column from Beckman (4.6x75 mm) the fractions were eluted in a gradient of solvent B of 10-80% solvent A = 0.1% TFA, solvent B = 50% ACN, 50% 2 -propánol and 0.1% TFA) at a flow rate of 0.5 ml / min. The monoiodinated toxin was eluted as the first peak of the radioactive protein (approximately 30% of solvent B) after the peak of the native toxins (approximately 28% of solvent B). The concentration of the radioactively labeled toxin was estimated according to the specific radioactivity of 1% and corresponds to 2424 dp / fmol of monoiodinated toxin.

Union tests Competitive binding assays were performed under equilibrium conditions using increasing concentrations of an unlabelled toxin in the presence of a constant concentration of a labeled toxin. Analyzes of all binding assays were performed using the interactive computer program LIGAND (P. J. Munson and D. Rodbard, modified by McPherson 1985). Insect membrane vesicles were suspended in binding medium containing choline chloride 0.13 M, EDTA lmM pH = 7.4, HPES / Tris 20 mM pH = 7.4 and 5 mg / ml of BSA. After 1 hour of incubation with the toxins, the reaction mixture was diluted with 2 ml of ice-cooled wash buffer (150 mM choline chloride, 5 mM HEPES / Tris pH-7.4, lmM EDTA pH «7.4, and mg / ml BSA) and filtered over GF / F filters (What an, RU) under vacuum, followed by two more washes of the filters with 2 ml of wash buffer each time. The non-specific toxin binding was not determined in the presence of 1 μM of unlabeled toxin.

EXAMPLE 7 Synthetic Gene Construction (Fiqura 1, SEQ ID NO: 1) The protein sequence of a toxin was converted to a nucleotide sequence using the preferred codon of use of a baculovirus. The toxin gene together with the nucleotide sequence of a leader sequence (bo bixin, native leader or other) and the appropriate restriction enzyme sites were used to design and synthesize 5 complementary pairs of oligonucleotides. The oligonucleotides were phosphorylated, annealed, ligated and amplified by PCR using the external oligonucleotides as primers. The PCR product was ligated at the cut end into a PCRscript plasmid and the correct sequence was confirmed by sequencing. A BamHI restriction fragment of this cloned plasmid was rescued in a baculovirus transfer vector under a baculovirus promoter (plO, polyhedrin, Basic, IE1, etc.). The plasmid containing the correct sequence of the gene and the leader sequence was confirmed by sequencing. A recombinant virus expressing the toxin was constructed using the resulting transfer vector and standard procedures.

Construction of a Virus that Expresses AaIT and LqhlV The leader sequence of the bombixin and the gene coding for the LqhlV toxin were designed and synthesized as described above. The correct sequence was confirmed and the gene was cloned into a double expression transfer vector that already contained the gene for AalT. The transfer vector PacUW51P2 is a positive vector to the polyhedrin with two cloning sites, a BglII site and the LqhlV gene with the leader sequence of bombixin were cloned in the BamHI site. Sf21 cells were cotransfected with the resulting transfer vector and infectious virus particles using the lipofectin procedure. Recombinant viruses were selected as a polyhedrin positive phenotype in a standard plaque assay. The sf21 cells were inoculated with the recombinant AcAaLq virus according to standard procedures. Protein extracts from cells infected with the virus were separated on PAGE gels with 15% SDS and then electroeluted to nitrocellulose membranes. Membranes were probed with antibodies to AaIT and LqhlV, the bound antibodies were detected using conjugates of rabbit HRP IgG.

In conclusion, the genetically engineered insecticidal microbes were produced according to the invention and were then used to control a variety of pests. To be able to produce this, a single recombinant virus expressing a plurality of neurotoxins can be used. The combination of the toxins is determined by the selection of the toxins that act in the same cellular channels (typically sodium channels) but not in overlapping sites. Alternatively, two (or more) recombinant insecticidal microbes each may be used to express a different toxin. Again, the different expressed toxins are selected as already described. These combinations of expressed toxins accelerate the rate or rate of extermination of pests by the virus or viruses beyond simply an "additive" function. For example, the lethality of AaIT and LqhalT toxins in blowfly larvae and Heliothis larvae increased 5-10 fold when used in combination. In addition, combinations of toxins can be used to increase selectivity within groups of insects. The conventional application means of the recombinant microbes (spraying, atomizing, dusting, scattering, or pouring) of formulations such as powders, microgranules, granules, as well as encapsulations such as in polymeric substances can be used.

. The compositions will typically include aarea carrier, such as clay, lactose, defatted soybeans, and the like to aid in applications, to apply the recombinant microoils that express synergistic combinations of insecticidal toxins. It should be understood that although the invention has been described above in conjunction with the preferred specific embodiments, the description and examples are intended to illustrate and not limit the scope of the invention, which is defined in scope by the appended claims.

LIST OF SEQUENCES (1. GENERAL INFORMATION: (1) APPLICANT: The Regents of the University of California (ii) TITLE OF THE INVENTION: Control of Insects with Multiple Toxins (iii) SEQUENCE NUMBER: 3 (iv) ADDRESS FOR CORRESPONDENCE: (A) ADDRESS: Majestic, Parsons, Siebert & Hsue (B) STREET: Four Embarcadero Center, Suite 1100 (C) CITY: San Francisco (D) STATE: California (E) COUNTRY: E.U.A. (F) CP: 94111-4106 (V) COMPUTER LEGIBLE FORM: (A) TYPE OF MEDIA: Flexible Disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) PROGRAM: Patentln Relay # 1.0, Version # 1.25 (vi) DATA OF THE CURRENT APPLICATION: (A) APPLICATION NUMBER: PCT / U596 / 06075 (B) DATE OF SUBMISSION: APRIL 30, 1996 (C) CLASSIFICATION: (vii) DATA FROM THE PREVIOUS APPLICATION: (A) APPLICATION NUMBER: US 08 / 435.040 (B) DATE OF SUBMISSION: 08-MAY-1995 (viii) MANDATORY / INFORMATION AGENT: (A) NAME: Siebert, J. Suzanne (B) REGISTRATION NUMBER: 28,758 (C) REFERENCE NUMBER / FILE: 2500.078WO0 (ix) INFORMATION FOR TELECOMMUNICATION: (A) TELEPHONE: (415) 248-5500 (B) TELEFAX: (415) 362-5418 (C) TELEX: 278638 MGPS (2) INFORMATION FOR SEQ ID NO: l: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 285 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: double (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETICAL : NO (ív) ANTICIPATION: NO (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: l: AGATCTGGAT CCATGAAGAT CCTCCTTGCT ATTGCCCTTA TGCTTAGCAC CGTGATGTGG 60 GTGAGCACCG GCGTGCGCGA CGCCTACATC GCCGACGACA AGAACTGCGT GTACACCTGC 120 GGCGCCAACT CTTACTGCAA CACCGACTGC ACCAAGAACG GCGCCGACTC TGGCTACTGC 180 CAATGGTTCG GCAAATACGG CAACGCATGC TGGTGCATCA AACTTCCCGA CAAAGTGCCC 24U ATCCGCATTC CCGGCAAATG CCGCTAAGGA TCCAGATCTG AGCTC 285 (2) INFORMATION FOR SEQ ID NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 65 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (iii) HYPOTHETIC: NO (v) ANTI-SENSE: NO (v) TYPE OF FRAGMENT: N-terminal (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 2: Gly Val Arg Asp Ala Tyr He Ala Asp Asp Lys Asn Cys Val Tyr Thr 1 5 10 15 Cys Gly Wing Asn Ser Tyr Cys Asn Thr Glu Cys Thr Lys Asn Gly Wing 20 25 30 Glu Ser Gly Tyr Cys Gín Trp Phe Gly Lys Tyr Gly Asn Ala Cys Trp 40 45 Cys He Lys Leu Pro Asp Lys Val Pro He Arg He Pro Gly Lys Cys 50 55 60 Arg 65 (2) INFORMATION FOR SEQ ID NO: 3: (I) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 67 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (II) TYPE OF MOLECULE: peptide (iii) HYPOTHETIC: NO (iv) ANTI-SENSE: NO (v) TYPE OF FRAGMENT: N-terminal (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 3: Gly Val Arg Asp Gly Tyr He Wing Gin Pro Glu Asn Cys Val Tyr His 1 5 10 15 Cys Phe Pro Gly Pro Pro Gly Cys Asp Thr Leu Cys Lys Gly Asp Gly 20 25 30 Ala Ser Ser Gly His Cys Gly Phe Lys Glu Gly His Gly Leu Ala Cys 35 40 45 Trp Cys Asn Asp Leu Pro Asp Lys Val Gly He He Val Glu Gly Glu Lys Cys His 65 It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention. Having described the above invention, the property contained in the following is claimed as property:

Claims (20)

1. A method for controlling pests of insect groups, acarids, and nematodes, characterized in that it comprises: treating the pests or their habitats with at least two different insect toxins, the source of the toxin is at least one recombinant microbe or is a plurality of recombinant microbes, the toxin has non-overlapping binding sites in a channel of the insect's cell membrane.

2. The method according to claim 1, characterized in that the source of the toxins are recombinant insect viruses.

3. The method according to claim 1, characterized in that the source of insect viruses, recombinants, are baculoviruses.

4. The method in accordance with the claim 3, characterized in that the baculoviruses are nuclear polyhedrosis viruses.

5. The method according to claim 3, characterized in that the baculoviruses are from Autographa califomica, Anagrapha falcifera, Anticarsia gemma talis, Buzura suppressuria, Cydia pomonella, Helicoverpa zea, Heliothis arpgera, Mariestia brassicae, Pl utella xylostella, Spodoptera exigua, Spodoptera li ttoralis , or Spodoptera li tura.

6. The method according to claim 1, characterized in that the toxins are combinations of neurotoxins of AaIT, LqhIT2, LqhalT, and LqhIT3.

7. The method according to claim 1, characterized in that the toxins include a mutant JHE.

8. The recombinant baculovirus according to claim 5, characterized in that the toxins include the AaIT and any of LqhIT2, LqhalT, or LqhIV.

9. The recombinant baculovirus according to claim 1, characterized in that at least one toxin is a component of the scorpion venom, wasp, snail, acarid or spider.

10. An insecticidal composition, characterized in that it comprises a recombinant baculovirus according to any of claims 1-9.

11. An insecticidal composition, characterized in that it comprises: a first recombinant baculovirus expressing a first toxin; and a second recombinant baculovirus expressing a second toxin, wherein both the first and second toxins bind to the same ion channel, but at non-superimposed sites, the first and second toxins together have an increased insecticidal potency.

12. A substantially pure insect toxin, useful in insect control, characterized in that it has the amino acid sequence of SEQ. ID. NO: 2 or SEQ. ID. NO: 3

13. A method for controlling pests of the group of insects, acarids, and nematodes, characterized in that it comprises: treating the pests or their habitats with one or a plurality of recombinant baculoviruses, the one or the plurality of baculoviruses expressing at least two different toxins in the pests infected with them, each toxin has an insecticidal potency and binding to an ion channel of the membrane at a non-superimposed binding site with respect to the other toxin or toxins, where the toxins together have an increased insecticidal potency.

14. The method according to claim 13, characterized in that the baculoviruses are nuclear polyhedrosis viruses.

15. The method according to claim 13, characterized in that the baculoviruses are from Autographa californica, Anagrapha falcifera, Anticarsia gemmatalis, Buzura suppressuria, Cydia pomonella, Helicoverpa zea, Heliothis arrigera, Mariestia brassicae, Plutella xylostella, Spodoptera exigua, Spodoptera littoralis, or Spodoptera. li ture.

16. The method according to claim 13, characterized in that the ion channel of the membrane is a sodium ion channel.

17. The method in accordance with the claim 16, characterized in that the toxin is AalT.

18. The method according to claim 16, characterized in that it is LqhlV or LqhVI.

19. The method according to claim 13, characterized in that the pest is Heliothis virescens or blowfly.

20. The method according to claim 19, characterized in that the toxins include AaIT and any of LqhIT2, LqhalT, or LqhIV.