NOVEL PEPTIDES AND THE USE THEREOF TO CONTROL PESTS

DESCRIPTION

NOVEL PEPTIDES AND THE USE THEREOF TO CONTROL PESTS

Background of the Invention

Many blood-ingesting pests are known to feed on humans and animals, and many pests are vectors for pathogenic microorganisms which threaten human and animal health, including commercially important livestock, pets and other animals. Various species of mosquitoes, for example, transmit diseases caused by viruses, and many are vectors for disease-causing nematodes and protozoa. Mosquitoes of the genus Anopheles transmit Plasmodiitm, the protozoan which causes malaria, a devastating disease which results in approximately 1 million deaths annually. The mosquito species Aedes aegypti transmits an arbovirus that causes yellow fever in humans. Other arboviruses transmitted by Aedes species include the causative agents of dengue fever, eastern and western encephalitis, Venezuelan equine encephalitis, St. Louis encephalitis, chikungunya, oroponehe and bunyamidera. The genus Culex, which includes the common house mosquito C. pipiens, is implicated in the transmission of various forms of encephalitis and fϊlarial worms. The common house mosquito also transmits Wuchereria bancrofti and Brugia malayi, which cause various forms of lymphatic fϊlariasis, including elephantiasis. Trypanasoma cruzi, the causative agent of Chagas' disease, is transmitted by various species of blood-ingesting Triatominae bugs. The tsetse fly (Glossina spp.) transmits African trypanosomal diseases of humans and cattle. Many other diseases are transmitted by various blood-ingesting pest species. The order Diptera contains a large number of blood- ingesting and disease-bearing pests, including, for example, mosquitoes, black flies, no-see-ums (punkies), horse flies, deer flies and tsetse flies. Various pesticides have been employed in efforts to control or eradicate populations of disease-bearing pests, such as disease-bearing blood-ingesting pests. For example, DDT, a chlorinated hydrocarbon, has been used in attempts to eradicate malaria-bearing mosquitoes throughout the world. Other examples of chlorinated hydrocarbons are BHC, lindane, chlorobenzilate, methoxychlor, and the cyclodienes (e.g., aldrin, dieldrin, chlordane, heptachlor, and endrin). The long-term stability of many of these pesticides and their tendency to bioaccumulate render them particularly dangerous to many non-pest organisms.

Another common class of pesticides is the organophosphates, which is perhaps the largest and most versatile class of pesticides. Organophosphates include, for example, parathion, Malathion™, diazinon, naled, methyl parathion, and dichlorvos. Organophosphates are generally much more toxic than the chlorinated hydrocarbons. Their pesticidal effect results from their ability to inhibit the enzyme cholmesterase, an essential enzyme in the functioning of the insect nervous system. However, they also have toxic effects on many animals, including humans.

The carbamates, a relatively new group of pesticides, include such compounds as carbamyl, methomyl, and carbofuran. These compounds are rapidly detoxified and eliminated from animal tissues. Their toxicity is thought to involve a mechanism similar to the mechanism of the organophosphates; consequently, they exhibit similar shortcomings, including animal toxicity.

A major problem in pest control results from the capability of many species to develop pesticide resistance. Resistance results from the selection of naturally-occurring mutants possessing biochemical, physiological or behavioπstic factors that enable the pests to tolerate the pesticide. Species of Anopheles mosquitoes, for example, have been known to develop resistance to DDT and dieldrm. DDT substitutes, such as Malathion™, propoxur and fe trothion are available; however, the cost of these substitutes is much greater than the cost of DDT.

There is clearly a longstanding need in the art for pesticidal compounds that are pest- specifϊc, that reduce or eliminate direct and/or indirect threats to human health posed by presently available pesticides, that are environmentally compatible in the sense that they are biodegradable, and are not toxic to non-pest organisms, and have reduced or no tendency to bioaccummulate.

Many pests, including for example blood-mbibmg pests, must consume and digest a protemaceous meal to acquire sufficient essential ammo acids for growth, development and the production of mature eggs. Adult pests, such as adult mosquitoes, need these essential ammo acids for the production of vitellogemns by the fat body. These vitellogemns are precursors to yolk protems which are cπtical components of oogenesis. Many pests, such as house flies and mosquitoes, produce oostatic hormones that inhibit egg development by inhibiting digestion of the protein meal, and thereby limiting the availability of the essential ammo acids necessary for egg development.

Seπne esterases such as trypsin and trypsm-like enzymes (collectively referred to herein as "TTLE") are important components of the digestion of proteins by insects. In the mosquito, Aedes aegypti, an early trypsm that is found in the midgut of newly emerged females is replaced, following the blood meal, by a late trypsin . A female mosquito typically weighs about 2 mg and produces 4 to 6 μg of trypsin withm several hours after a ingesting blood meal. Continuous boisynthesis at this rate would exhaust the available metabolic energy of a female mosquito; as a result, the mosquito would be unable to produce mature eggs, or even to find an oviposition site. To conserve metabolic energy, the mosquito regulates TTLE biosynthesis with a peptide hormone named Trypsm Modulating Oostatic Factor (TMOF) TMOF mosquitoes produce in the fol cular epithelium of the ovary 12-35 hours after a blood meal, TMOF is then released into the hemolymph where it binds to a specific receptor on the midgut epithelial cells, signaling the termination of TTLE biosynthesis.

This regulatory mechanism is not unique for mosquitoes; flesh flies, fleas, sand flies, house flies, dog flies and other insect pests which need protein as part of their diet have similar regulatory mechanisms.

In 1985, Borovsky purified an oostatic hormone 7,000-fold and disclosed that injection of a hormone preparation into the body cavity of blood imbibed mosquitoes caused inhibition of egg development and sterility (Borovsky, D. [1985] Arch Insect Biochem Physiol 2:333- 349). Following these observations, Borovsky (Borovsky, D [1988] Arch. Ins Biochem Physiol. 7:187-210) reported that injection or passage of a peptide hormone preparation into mosquitoes inhibited the TTLE biosynthesis m the epithelial cells of the gut. This inhibition caused inefficient digestion of the blood meal and a reduction m the availability of essential ammo acids translocated by the hemolymph, resulting in arrested egg development in the treated insect. Borovsky observed that this inhibition of egg development does not occur when the oostatic hormone peptides are inside the lumen of the gut or other parts of the digestive system (Borovsky, D. [1988], supra). Following the 1985 report, the isolated hormone, (a ten amino acid peptide) and two

TMOF analogues were disclosed in U.S. Patent Nos. 5,011,909 and 5,130,253, and in a 1990 publication (Borovsky et al [1990] FASEB J 4:3015-3020). Additionally, U.S. Patent No. 5,358,934 discloses truncated forms of the full length TMOF which have prolmes removed from the carboxy terminus, including the peptides YDPAP, YDPAPP, YDPAPPP, and YDPAPPPP. Neuropeptides Y (NPY) are an abundant family of peptides that are widely distributed in the central nervous system of vertebrates. NPY peptides have also been recently isolated and identified in a cestode, a turbellaπan, and in terrestπal and maπne molluscs (Maule et al, 1991 "Neuropeptide F: A Novel Parasitic Flatworm Regulatory Peptide from Moniezia expansa (Cestoda: Cyclophyhdea)" Parasitology 102:309-316; Curry et al , 1992 "Neuropeptide F: Primary Structure from the Turbellaπan, Arthioposthia triangulata " Comp. Biochem. Physiol. 101C:269-274; Leung et al, 1992 "The Pπmary Structure of Neuropeptide F (NPF) from the Garden Snail, Helix aspersa " Regul. Pep. 41 :71-81; Rajpara et al , 1992 "Identification and Molecular Cloning of Neuropeptide Y Homolog that Produces Prolonged Inhibition in Aplysia Neurons" Neuron. 9:505-513). Invertebrate NPYs are highly homologous to vertebrate NPYs The major difference between vertebrate and invertebrate NPYs occurs at the C-termmus where the vertebrate NPY has an amidated tyrosine (Y) whereas invertebrates have an amidated phenylalanme (F). Because of this difference, the invertebrate peptides are referred to as NPF peptides. Cytoimmunochemical analyses of NPY peptides suggest that they are concentrated m the bram of various insects, including the Colorado potato beetle Leptinotarsa decemhneata (Verhaert et al , 1985 "Distinct Localization of FMRFamide- and Bovme Pancreatic Polypeptide-Like Mateπal m the Bram, Retrocerebal Complex and Subesophageal Ganglion of the Cockroach Perψlaneta americana " L Bram Res. 348:331-338; Veenstra et al , 1985 "Immunocytochemical Localization of Peptidergic Neurons and Neurosecretory Cells in the Neuro-Endocπne System of the Colorado Potato Beetle with Antisera to Vertebrate Regulatory Peptides" Histochemistry 82:9-18). Partial purification of NPY peptides m insects suggests that both NPY and NPF are synthesized in insects (Duve et al , 1981 "Isolation and Partial Charactenzation of Pancreatic Polypeptide-like Material in the Brain of the Blowfly alliphora vomitoria " Biochem. J. 197, 767-770).

Researchers have recently isolated two neuropeptides with NPF- ke immunoreactivity from brain extracts of the Colorado potato beetle. The researchers punfied the peptides using C,_g reversed phase high pressure liquid chromatography (HPLC), and determined their structure using mass spectrometry. The deduced structures of these peptides are: Ala-Arg-Gly-Pro-Gln- Leu-Arg-Leu-Arg-Phe-amide (SEQ LD NO. 1) and Ala-Pro-Ser-Arg-Leu-Arg-Phe-amide (SEQ ID NO. 2) designated NPF I and NPF II, respectively (Spittaels, Kurt, Peter, Verhaert, Chris Shaw, Richard N. Johnston et al [1996] Insect Biochem Molec. Biol 26(4):375-382).

Bnef Summary of the Invention The subject invention concerns novel pesticidal polypeptides and other compounds. In a preferred embodiment, the pesticidal agents of the subject invention (collectively referred to herem as "pesticidal compounds") inhibit digestion in pests by terminating or otherwise blocking synthesis of digestive enzymes by activating a TMOF receptor. The pesticidal polypeptides and other compounds of the present invention are usefully employed in the control of pests, such as mosquitoes, which mgest blood.

In one aspect, the pesticidal compounds of the present mvention comprise novel polypeptides compπsmg an ammo acid sequence having the formula:

A'A²A³A⁴A⁵F (Formula I) wherein: A¹ is selected from the group consisting of Y, A, D, F, G, M, P, S, and Y; A² is selected from the group consisting of A, D, E, F, G, N, P, S, and Y; A³ is optionally present and is selected from the group consisting of A, D, F, G, L, P, S, and Y;

A⁴ is optionally present when A³ is present and is selected from the group consisting of A, F, G, L, and Y,

A⁵ is optionally present when A⁴ is present and is selected from the group consisting of A, F, L, and P;

F is a flanking region which is optionally present and is selected from the group consisting of P, PP, PPP, PPPP, and PPPPP. The pesticidal compound preferably does not consist of YDPAP₆, DYPAP₆, PAP₆,

YDPAP, YDPAP₂, YDPAP₃, YDPAP₄, NPTNLH, or DF-OMe.

In a narrower aspect, the pesticidal compound compπses a polypeptide having an ammo acid sequence which consists essentially of the ammo acid sequence of Formula I. In a preferred aspect, where the ammo acid sequence is a TMOF fragment, the pesticidal compound lacks TMOF ammo acids adjacent to the TMOF fragment . In still another aspect, the peptide consists of the amino acid sequence of Formula I.

In another aspect, only A¹, A², A³, A⁴, and F of Formula I are present and F is attached to A⁴. In yet another aspect, only A¹, A², A³, and A⁴ of Formula I are present. In still a further aspect, only A¹, A², A³, and F of Formula I are present and F is attached to A⁴. In an additional aspect, only A¹, A², and A³ of Formula I are present. In another aspect, only A¹, A², and F of Formula I are present and F is attached to A², and in a further aspect, only A¹ and A² of Formula I are present. In a preferred mode, A and D are present m the amino acid sequence and, more preferably, A, D, and Y are present.

One embodiment of the subject invention concerns a peptide compπsmg an amino acid sequence having the formula A'A² (Formula II), wherein A¹ is an ammo acid residue selected from the group consisting of A, D, F, M, and Y, and A² is an ammo acid residue selected from the group consisting of A, D, E, P, and Y. In a preferred embodiment, the subject mvention is directed to peptides which comprise the ammo acids A, D, and Y.

The pesticidal compounds of the present invention have advantageous biological activity against pests. The novel polypeptides and other compounds of the invention are particularly active against blood-suckmg insects, particularly against species of mosquitoes such as Aedes aegypti that are common vectors of arthropod-borne viral diseases, such as arboviruses. Other bitmg pests such as flies, fleas, ticks, and lice can also be controlled using peptides and methods of the subject invention. These pests utilize as their pπmary blood-digestmg enzymes. The subject peptides can also be used to control pests of agπcultural crops These pests include, for example, coleopterans (beetles), lepidopterans (caterpillars), and mites The compounds of the subject invention can also be used to control household pests including, but not limited to, ants and cockroaches A further aspect of the subject invention are addition salts, complexes, or prodrugs such as esters of the peptides described herein, especially the nontoxic pharmaceutically or agriculturally acceptable acid addition salts The acid addition salts can be prepared using standard procedures m a suitable solvent from the parent compound and an excess of an acid, such as hydrochloric, hydrobromic, sulfuric, phosphoric, acetic, maleic, succimc, ethanedisulfomc or methanesulfonic acids Esteπfϊcation to form deπvatives such as the methyl or ethyl esters, can also be performed using standard procedures

The N-termmus and C-termmus of the pesticidal polypeptides can be blocked to further inhibit proteolysis by metabolic enzymes Deπvation of peptides to block the N-termmus or C- terminus is known in the art. For example, the N-termmus can be acetylated by methods known to those of ordinary skill in the art, and/or the C-termmus can be amidated as is well known in the art.

Peptides containing the above sequences in which only conservative substitutions have been made are also provided by the present mvention Analogues of the above-mentioned proteins and peptides which have one or more amino acid substitutions forming a branched peptide (e g , by substitution with an amino acid or ammo acid analogue having a free ammo- or carboxy-side chain that forms a peptide bond with a sequence of one or more amino acids, including but not limited to prolmes) or allowing circulaπzation of the peptide (eg , by substitution with a cysteme, or insertion of a cysteine at the ammo- or carboxy-termmus or internally, to provide a sulfhydryl group for disulfide bond formation), are also provided. The pesticidal polypeptides and other compounds of the present invention may also compπse D-conformation ammo acids which can inhibit the ability of proteases to degrade the polypeptιdes.Also, deπvation of the pesticidal compounds with long chain hydrocarbons will facilitate passage through the cuticle into the pest body cavity Therefore, m a further embodiment, the subject invention provides compositions compπsmg the pesticidal polypeptides bound to lipids or other earners

Yet another aspect of the subject invention pertains to DNA sequences encoding the pesticidal polypeptides disclosed herein These DNA sequences can readily be synthesized by a person skilled in the art, and can be used to transform an appropπate prokaryotic or eukaryotic host to enable the host to express the novel peptides Hosts of particular interest include bacteria, yeasts, viruses, and plants Furthermore, viruses may also be modified to transmit polynucleotides encoding the pesticidal polypeptides of the present invention For each of these hosts, the DNA sequences may be specifically designed by a person skilled in the art to utilize codons known to be optimally expressed in the particular hosts. Advantageous promoters can also easily be employed in the polynucleotide sequences Bacteria, yeasts, plants, and viruses each may be used in the production of pesticidal polypeptides for further use, or these hosts can be used as vehicles for direct application of the pesticidal polypeptides to the target pest. Plants can be transformed to render them toxic to a target pest species which feeds on the transformed plant. Methods for transforming plant cells utilizing, for example, Agrobacteria, are well known to those skilled in the art. Another aspect of the subject invention pertains to a method for controlling pests compnsmg admmisteπng to said pest an effective amount of a pesticidal polypeptide compound of the subject invention.

The subject invention provides pest control compositions compnsmg pesticidal compounds and a suitable pesticidal earner The pest control compositions are formulated for application to the target pests or their situs. In a specific embodiment, the present invention provides recombinant hosts transformed to express a pesticidal polypeptide. The recombinant host may be, for example, prokaryotic or eukaryotic cells such as yeast or algae which have been transformed to express a pesticidal compound of the subject invention. The transformed hosts can be applied to pest habitats, such as bodies of water inhibited by mosquito larvae. Ingestion of the transformed host by a pest species m control of the pest by the pesticidal polypeptide. In a preferred embodiment for the control of agricultural pests, the subject mvention provides transformed plants which express a pesticidal polypeptide. Pest control is achieved when the pest ingests the transformed plant mateπal.

The methods and materials of the subject invention provide a novel approach to controlling msects and insect-transmitted diseases. The peptides of the subject invention have advantageous activity and increased resistance to proteolysis over previously disclosed compounds.

As used herein, the term "pesticidally effective" is used to indicate an amount or concentration of a pesticidal compound which is sufficient to reduce the number of pests m a geographical locus as compared to a corresponding geographical locus in the absence of the amount or concentration of the pesticidal compound.

The term "pesticidal" is not intended to refer only to the ability to kill pests, but also includes the ability to interfere with a pest's life cycle m any way that results in an overall reduction in the pest population. For example, the term "pesticidal" includes inhibition of a pest from progressing from one form to a more mature form, e g., transition between vaπous larval mstars or transition from larva to pupa or pupa to adult Further, the term "pesticidal" is intended to encompass anti-pest activity duπng all phases of a pest's life cycle; thus, for example, the term mcludes larvacidal, ovicidal , and adulticidal activity.

The word "transform" is broadly used herein to refer to introduction of an exogenous polynucleotide sequence into a prokaryotic or eukaryotic cell by any means known in the art (including for example, direct transmission of a polynucleotide sequence from a cell or virus particle, transmission by infective virus particles and transmission by any other nucleotide- beaπng construct) resulting in a permanent or temporary alteration of genotype and in an immortal or non-immortal cell. The terms "polypeptide," "peptide," and "protein" as used herein are intended to refer to ammo acid sequences of any length.

Bnef Descnption of the Sequences SEQ ID NO. 1 is the amino acid sequence for TMOF. SEQ ID NOS. 2-42 are TMOF peptide analogs according to the subject invention.

SEQ ID NOS. 43-44 are TMOF receptors according to the subject invention.

Detailed Disclosure of the Invention The subject mvention concerns novel pest control compounds and methods for using such compounds. Specifically exemplified are novel pesticidal polypeptides, compositions compnsmg said pesticidal polypeptides and the use of such pesticidal polypeptides and compositions in controlling pests, such as mosquitoes.

In one aspect, the biological control agents comprise novel polypeptides, compnsmg an amino acid sequence of the formula: A'A²A³A⁴A⁵F (Formula I) wherein:

A¹ is selected from the group consisting of Y, A, D, F, G, M, P, S, and Y; A² is selected from the group consisting of A, D, E, F, G, N, P, S, and Y; A³ is optionally present and is selected from the group consisting of A, D, F, G, L, P, S, and Y;

A⁴ is optionally present when A³ is present and is selected from the group consisting of A, F, G, L, and Y;

A⁵ is optionally present when A⁴ is present and is selected from the group consisting of A, F, L, and P; F is a flanking region which is optionally present and is selected from the group consisting of P, PP, PPP, PPPP, and PPPPP

The peptide preferably does not consist of YDPAP₆, DYPAP₆, PAP₆, YDPAP, YDPAP₂, YDPAP₃, YDPAP₄, NPTNLH, or DF-OMe In a narrower aspect the polypeptide compπses an ammo acid sequence which consists essentially of the ammo acid sequence of Formula I. In a preferred aspect, where the ammo acid sequence is a TMOF fragment, the peptide or protein lacks TMOF amino acids adjacent to the TMOF fragment . In still another aspect, the pesticidal polypeptide consists of the ammo acid sequence of Formula I. In another aspect, only A¹, A², A³, A⁴, and F of Formula I are present and F is attached to A⁴. In yet another aspect, only A¹, A², A³, and A⁴ of Formula I are present. In an additional aspect, only A¹, A², A³ and F of Formula I are present and F is attached to A³. In another aspect, only A¹, A², and A³ of Formula I are present In another aspect, only A¹ , A², and F of Formula I are present, . and F is attached to A². In a further aspect, only A¹ and A² of Formula I are present. In a preferred mode, A and D are present in the amino acid sequence and, more preferably, A, D, and Y are present.

One embodiment of the subject mvention concerns a peptide having the formula A'A² (Formula II) wherein A¹ is an ammo acid selected from the group consisting of A, D, F, M, and Y, and A² is an ammo acid selected from the group consisting of A, D, E, P, and Y. In a preferred embodiment, the subject invention is directed to peptides which compnse the amino acids A, D, and Y.

In a preferred mode, the pesticidal polypeptides of the present invention compnse truncated forms of TMOF having 50% or fewer ammo acids than the corresponding TMOF. Preferably the truncated forms have 2-10 ammo acid residues. More preferably the truncated forms have 2-8 ammo acid residues. Most preferably, the truncated forms have 2-5 amino acid residues. Despite this substantial truncation, the pesticidal polypeptides retain some or all of the biological activity of the full-length peptide. Further, the pesticidal polypeptides of the subject invention are particularly advantageous because of more rapid and efficient penetration into the idgut, and are less expensive to produce by conventional chemical methods. Preferably, the subject peptides have an LD₅₀ against mosquito larvae of less than 3.0 μmole/ml. More preferably, the peptides have an LD₅₀ of less than 2.0 μmole/ml, and, most preferably, the peptides have an LD₅₀ of less than 1.0 μmole/ml. As used herein, "LD₅₀" refers to a lethal dose of a peptide able to cause 50% mortality of larvae maintained on a diet of 1 mg/ml autoclaved yeast supplemented with the pesticidal polypeptide (Borovsky, Dov, and Fanda Mahmood [1995] Regulatory Peptides 57:273-281). The one-letter symbol for the am o acids used herein is well known m the art. For convenience, the relationship of the three-letter abbreviation and the one-letter symbol for ammo acids is as follows:

Ala A Leu L

Arg R Lys K

Asn N Met M

Asp D Phe F

Cys C Pro P

Gin Q Ser S

Glu E Thr T

Gly G Trp w

His H Tyr Y

He I Val V

The pesticidal polypeptides of the present mvention may be provided as a fusion polypeptide, the ammo acid sequence of which mcludes one or more pesticidal polypeptides of the present invention, and optionally compnses one ore more heterologous polypeptides. In vaπous specific embodiments, two or more of the pesticidal polypeptides are linked, for example, by peptide bonds between the N-termmus of one portion and the C-termmus of another portion. In other aspects, one or more of the pesticidal polypeptides can be linked to one or more heterologous peptides or proteins to form pesticidal fusion polypeptides. Molecules compnsmg such portions linked by hydrocarbon linkages are also provided. Denvatives of the foregoing fusion protems are also provided (e g , branched, cychzed, or C-termmal chemically modified, etc.). Further, two or more peptides of the general Formulas I and/or II can be linked to each other.

Pesticidal polypeptides compnsmg the sequences of Formulas I and/or II m which only conservative substitutions have been made are also provided by the present invention. Analogues which have one or more ammo acid substitutions forming a branched peptide (e.g , by substitution with an ammo acid or ammo acid analogue having a free ammo- or carboxy-side cham that forms a peptide bond with a sequence of one or more ammo acids, including but not limited to prolmes) or allowing circulaπzation of the peptide (e.g., by substitution with a cysteme, or insertion of a cyste e at the ammo- or carboxy-terminus or internally, to provide a sulfhydryl group for disulfide bond formation), are also provided.

Nonclassical ammo acids and/or chemical ammo acid analogues can be introduced as a substitution for an existing ammo acid residue, or as an insertion into the amino acid sequence of the pesticidal polypeptides of the present invention, or as an addition to a terminus of the pesticidal polypeptides Non-classical ammo acids include but are not limited to the D-isomers of the common ammo acids, 2,4-dιammobutyπc acid, α-ammo lsobutyπc acid, 4-ammobutyπc acid, Abu, 2-ammo butyπc acid, γ-Abu, ε-Ahx, 6-ammo hexanoic acid, Aib, 2-amιno lsobutyπc acid, 3-ammo propionic acid, ornith e, norleuc e, norvalme, hydroxyprolme, sarcosme, citruhne, homocitrulline, cysteic acid, t-butylglycme, t-butylalanme, phenylglycme, cyclohexylalanme, β-alamne, fluoro-am o acids, designer ammo acids such as β-methyl ammo acids, C-methyl ammo acids, N-methyl ammo acids, and ammo acid analogues in general. Furthermore, the ammo acid can be D (dextrorotary) or L (levorotary). Dextrorotary ammo acids are indicated herein by a parenthetical D, i e , "(D)", immediately preceding the dextrorotary amino acid.

Thus, the pesticidal polypeptide deπvatives include peptides containing as a primary ammo acid sequence all or part of the peptide sequence of Formulas I and/or II, including altered sequences in which functionally equivalent ammo acid residues are substituted for residues withm the sequence, resulting m a peptide which is functionally active. For example, one or more ammo acid residues withm the sequence can be substituted by another amino acid of a similar polanty, which acts as a functional equivalent, resulting in a silent alteration. Conservative substitutions for an ammo acid within the sequence may be selected from other members of the class to which the ammo acid belongs (see Table 1) Such pesticidal polypeptide deπvatives can be made either by chemical peptide synthesis or by recombinant production from polynucleotide sequences encoding the pesticidal polypeptide. The subject invention further includes other pesticidal compounds which bind to a TMOF receptor. As used herein, "TMOF compounds" refers to pesticidal polypeptides of Formulas I and II as well as other polypeptides and compounds which bind to a TMOF receptor as described herein. TMOF receptors and polvnucleotides. In one embodiment, the subject invention is directed to the control of pests using a pesticidal compound which binds to or otherwise associates with a TMOF receptor. Specifically exemplified herein is a TMOF receptor compnsmg the ammo acid sequence shown in SEQ ID NO. 44. Preferably, the polypeptide is encoded by a complete polynucleotide nucleotide sequence of a TMOF receptor gene, or a fragment analogue, deπvative or other functional equivalent thereof which encodes polypeptides having TMOF receptor activity. In a specific embodiment, the TMOF receptor is encoded by a polynucleotide sequence compnsmg the coding sequence (nucleotides 1-186) shown in SEQ ID NO.43 or other polynucleotide sequence with codons encoding the ammo acid sequence of SEQ ID NO. 44. Isolated TMOF receptors can be used to produce antibodies according to known techniques These antibodies may be monoclonal or polyclonal These antibodies can be used to screen an expression library to identify other clones expressing polypeptides having TMOF receptor activity Alternatively, these antibodies may be used to identify TMOF receptors from their natural material such as mosquito gut material

A specific TMOF receptor sequence is exemplified herein. This sequence is merely exemplary of TMOF receptors Vanant or equivalent receptors (and nucleotide sequences coding for equivalent receptors) having the same or similar TMOF receptor activity can also be utilized Equivalent receptors will typically have amino acid homology with the exemplified receptor. This ammo acid identity will typically be greater than 60%, preferably be greater than 75%, more preferably greater than 80%, more preferably greater than 90%, and can be greater than 95%. These identities are as determined using standard alignment techniques. The ammo acid homology will be highest m cntical regions of the receptor that account for biological activity or are involved m the determination of three-dimensional configuration, which ultimately is responsible for the biological activity. In this regard, certain amino acid substitutions are acceptable and can be expected if these substitutions are in regions that are not cπtical to biological activity or are conservative amino acid substitutions that do not affect the three-dimensional configuration of the molecule. For example, ammo acids may be placed in the following classes: non-polar, uncharged polar, basic, and acidic. Conservative substitutions whereby an amino acid of one class is replaced with another ammo acid of the same type fall withm the scope of the subject invention so long as the substitution does not completely diminish the biological activity of the compound. Table 1 provides a listing of examples of amino acids belonging to each class.

Table 1.

Class of Ammo Acid Examples of Amino Acids

Nonpolar Ala, Val, Leu, He, Pro, Met, Phe, Tip

Uncharged Polar Gly, Ser, Thr, Cys, Tyr, Asn, Gin

Acidic Asp, Glu

Basic Lys, Arg, His

In some instances, non-conservative substitutions can also be made. The cπtical factor is that these substitutions must not completely dimmish the biological activity of the receptor. The use of polynucleotide probes is well known to those skilled in the art. In one specific example, a cDNA library for mosquito gut cells can be created by routine means, and DNA of interest can be isolated therefrom Polynucleotides of the subject mvention can be used to hybridize with DNA fragments of the constructed cDNA- brary, allowing identification of and selection (or "probing out") of the genes of interest, i e , those nucleotide sequences which hybridize with the probes of the subject mvention and encode polypeptides having TMOF receptor activity. The isolation of these genes can be performed by a person skilled in the art, having the benefit of the instant disclosure, using techniques which are well-known in the molecular biology art. Thus, it is possible, without the aid of biological analysis, to identify polynucleotide sequences encoding TMOF receptors Such a probe analysis provides a rapid method for identifying genes encoding TMOF receptors from a wide variety of hosts. The isolated genes can be inserted into appropriate vehicles which can then be used to transform a suitable host.

Vaπous degrees of stπngency of hybndization can be employed. The more severe the conditions, the greater the complementanty that is required for duplex formation. Seventy of conditions can be controlled by temperature, probe concentration, probe length, ionic strength, time, and the like. Preferably, hybndization is conducted under moderate to high stringency conditions by techniques well known in the art, as descnbed, for example, m Keller, G.H., M.M. Manak (1987) DNA Probes, Stockton Press, New York, NY., pp. 169-170. Examples of various stnngency conditions are provided herein. Hybndization of immobilized DNA on Southern blots with 32P-labeled gene-specific probes can be performed by standard methods ( Mamatis et al (1982) Molecular Cloning. A Laboratory Manual, Cold Spnng Harbor Laboratory, New York.). In general, hybndization and subsequent washes can be carried out under moderate to high stnngency conditions that allow for detection of target sequences with homology to the exemplified polynucleotide sequence. For double-stranded DNA gene probes, hybridization can be earned out overnight at 20-25° C below the melting temperature (Tm) of the DNA hybnd in 6X SSPE, 5X Denhardt's solution, 0.1% SDS, 0.1 mg/ml denatured DNA. The melting temperature is described by the following formula (Beltz et al. et al. [1983] Methods of Enzymology , R. Wu, L. Grossman and K. Moldave [eds.] Academic Press, New York 100:266-285).

Tm=81.5°C+16.6 Log[Na+]+0.41(%G+C)-0.61(%formamιde)-600/length of duplex in base pairs.

Washes are typically earned out as follows:

(1) twice at room temperature for 15 minutes m IX SSPE, 0.1% SDS (low stnngency wash); (2) once at Tm-20°C for 15 minutes in 0.2X SSPE, 0.1% SDS (moderate stringency wash).

For oligonucleotide probes, hybridization can be carried out overnight at 10-20°C below the melting temperature (Tm) of the hybrid m 6X SSPE, 5X Denhardt's solution, 0.1% SDS, 0.1 mg/ml denatured DNA. Tm for oligonucleotide probes can be determined by the following formula:

Tm (°C)=2(number T/A base pairs) +4(number G/C base pairs) (Suggs et al [1981] ICN-UCLA Symp Dev Biol Using Purified Genes, D.D. Brown [ed.], Academic Press, New York, 23:683-693). Washes can be carried out as follows:

(1) twice at room temperature for 15 minutes IX SSPE, 0.1% SDS (low stnngency wash;

(2) once at the hybridization temperature for 15 minutes m IX SSPE, 0.1 % SDS (moderate stnngency wash). In general, salt and/or temperature can be altered to change stnngency. With a labeled

DNA fragment >70 or so bases m length, the following conditions can be used: Low: 1 or 2X SSPE, room temperature

Low: l or 2X SSPE, 42°C

Moderate: 0.2X or IX SSPE, 65 °C High: 0.1X SSPE, 65°C.

Duplex formation and stability depend on substantial complementanty between the two strands of a hybrid and, as noted above, a certain degree of mismatch can be tolerated. Therefore, the probe sequences of the subject mvention include mutations (both single and multiple), deletions, insertions of the descπbed sequences, and combinations thereof, wherein said mutations, insertions and deletions permit formation of stable hybrids with the target polynucleotide of interest. Mutations, insertions and deletions can be produced in a given polynucleotide sequence m many ways, and these methods are known to an ordinanly skilled artisan. Other methods may become known in the future.

Identification of pest control compounds. The TMOF receptors can advantageously be used to identify pesticidal compounds and/or confirm or characteπze the activity of the pesticidal polypeptides of the subject invention. The pesticidal compounds of the subject invention are preferably those which bind to, or otherwise associate with, the TMOF receptor m a way which activates the TMOF receptor, thereby inhibiting biosynthesis of TTLE, resulting m control of the pest population. A person skilled in the art, having the benefit of the instant disclosure, can utilize the TMOF receptors described herein to identify and charactenze pesticidal compounds of the subject invention In one embodiment, the TMOF receptor can be punfied from its natural sources using, for example, antibodies to the TMOF receptor to obtain the punfied protein This punfied protein can then be used to identify compounds which bind to the receptor Compounds thus identified can then be further evaluated using, for example, appropnate bioassays to confirm and/or characterize the pest control activity of the compound

As an alternative to punfying TMOF receptors from their natural mateπal, recombinant TMOF receptor protein can be expressed in an appropnate recombinant host that has been transformed with a polynucleotide sequence encoding the TMOF receptor The polynucleotide sequence used to transform the appropnate host may compnse, for example, the polynucleotide coding sequence disclosed in SEQ ID NO 43 The host may be transformed so as to express the TMOF receptor at the cell surface or, alternatively, the TMOF receptor may be retained intracellularly or secreted into the surrounding media In any case, the expressed TMOF receptor may be isolated from the recombinant host using techniques known to those skilled in the art The recombinant punfied protein can then be used as descπbed above to identify compounds which bind to the receptor As an alternative embodiment, the receptor expressed at the surface of the recombinant cell can be used m conjunction with the whole cell to identify compounds which bind to the receptor.

In another embodiment, TMOF receptors of the subject invention can be applied to a chip or other suitable substrate to facilitate high throughput screening of potential pest control compounds.

Once compounds are identified which bind to the TMOF receptor, their pesticidal activity can be confirmed and/or characteπzed using bioassays known to those skilled m the art The pesticide compounds of the subject invention can have activity against a vaπety of pests These pests include agncultural pests which attack plants as well as pests of animals which attack humans, agncultural animals, domestic animals, and wild animals.

Preparation of novel pest control compounds The novel pesticidal compounds of the invention can be prepared by well-known synthetic procedures. For example, the pesticidal polypeptides of the present invention can be prepared by the well-known Mernfϊeld solid support method See Mernfield (1963) J Amer Chem Soc 85.2149-2154 and Mernfield (1965) Science 150:178-185. This procedure, using many of the same chemical reactions and blocking groups of classical peptide synthesis, provides a growing peptide cham anchored by its carboxyl terminus to a solid support, usually cross-linked polystyrene or styrenedivinylbenzene copolymer. This method conveniently simplifies the number of procedural manipulations, since removal of the excess reagents at each step is effected simply by washing the polymer Alternatively, these peptides can be prepared by use of well-known molecular biology procedures. Polynucleotide sequences encoding the pesticidal polypeptides of the invention can be readily synthesized . These polynucleotide sequences are a further aspect of the subject invention. These polynucleotides can be used to transform prokaryotic and eukaryotic cells, such as bactena, insect, plant, or fungi cells for synthesis of the peptides of the invention. These polynucleotides may also be employed to modify viruses such as bacteπophages for use as cloning and/or expression vectors. One example of a cell line useful in accordance with the present mvention includes, the insect cell line Sf9 (Spodoptera frugiperda), deposit number ATCC CRL 1711, which is available from the American Type Culture Collection, 12301 Parklawn Dnve, Rockville, MD 20852 USA. An example of a useful virus mcludes Baculovirus Autographa Californica Nuclear Polyhedrosis Virus (AcNPV) which is available from Texas A&M University, Texas Agricultural Experiment Station, College Station, TX 77843, and has been described in Smith, G., and M.D. Summers (1978) Virology 89:517-527; and (1979) J. Virology 30:828-838. Other nuclear polyhedrosis viruses (See World Health Organization Technical Report No. 531) such as Spodoptera frugiperda (Sf MNPV), Choristoneura fumiferana (Cf MNPV) (Smith, G., and M.D. Summers [1981] J. Virol. 39:125-137), or Spodoptera littoralis (SI NPV) (Harrap et al. [1977] Virology 79:14-31) can be used instead of Autographa californica NPV. Other insect cell lines can also be substituted for Spodoptera frugiperda (Sf9), for example, Trichoplusia ni (Volkman, L.E., and M.D. Summers [1975] J. Virol. 16:1630-1637), Spodoptera exigua, Choristoneura fumiferana (Smith, G., and M.D. Summers [1981] J Virol. 39:125-137) and Spodoptera littoralis (Harrap, K.A. et al. [1977] Virology 79:14-31).

In yet another embodiment, the subject invention is directed to polynucleotides which encode the subject pest control peptides. Polynucleotides can be produced by routine methods known in the art. See S.L. Beaucage and M.H. Caruthers (1981), Tetrahedron Lett. 22:1859. The polynucleotides may usefully be presented as expression vectors or as expression cassettes for insertion into expression vectors.

If desired, the polynucleotides of the present invention can be amplified using PCR. Polymerase Chain Reaction (PCR) is a repetitive, enzymatic, pnmed synthesis of a nucleic acid sequence. This procedure is well known and commonly used by those skilled m this art (see Mullis, U.S. Patent Nos. 4,683,195, 4,683,202, and 4,800,159; Saiki et al. et al. (1985) "Enzymatic Amplification of β-Globm Genomic Sequences and Restriction Site Analysis for Diagnosis of Sickle Cell Anemia," Science 230: 1350-1354.). PCR is based on the enzymatic amplification of a DNA fragment of interest that is flanked by two oligonucleotide pnmers that hybπdize to opposite strands of the target sequence. The pnmers are oriented with the 3 ' ends pointing towards each other. Repeated cycles of heat denaturation of the template, annealing of the primers to their complementary sequences, and extension of the annealed primers with a DNA polymerase results m the amplification of the segment defined by the 5 ' ends of the PCR pnmers. Since the extension product of each pπmer can serve as a template for the other pnmer, each cycle essentially doubles the amount of DNA fragment produced m the previous cycle. This results in the exponential accumulation of the specific target fragment, up to several million-fold m a few hours. By using a thermostable DNA polymerase such as Taq polymerase, which is isolated from the thermophilic bacteπum Thermus aquaticus, the amplification process can be completely automated. Other enzymes which can be used are known to those skilled in the art.

PCR pnmers can be designed from the DNA sequences of the subject mvention. In performing PCR amplification, a certain degree of mismatch can be tolerated between pπmer and template. Therefore, mutations, deletions and insertions (especially additions of nucleotides to the 5' end) of the exemplified sequences fall withm the scope of the subject invention. These PCR pnmers can be used to amplify genes of interest from a sample. Thus, this is another method by which polynucleotide sequences encoding the subject peptides can be identified and charactenzed.

The vanous methods employed in the preparation of plasmids compnsmg the pesticidal polypeptide encoding polynucleotides of the present invention, and transformation of host organisms are well known in the art and are descπbed, for example, in U.S. Patent Nos. 5,011,909 and 5,130,253. These patents are incorporated herein by reference. These procedures are also descnbed in Maniatis et al (1982) Molecular Cloning A Laboratory Manual, Cold Spnng Harbor Laboratory, New York. Thus, it is within the skill of those in the genetic engineenng art to extract DNA from microbial cells, perform restrictions enzyme digestions, electrophorese DNA fragments, tail and anneal plasmid and insert DNA, gate DNA, transform cells, e.g., E. coh or plant cells, prepare plasmid DNA, electrophorese protems, and sequence DNA.

Production of Recombinant Hosts. In another embodiment, the subject mvention is directed to a prokaryotic or eukaryotic cell transformed with a polynucleotide encoding a polypeptide of Formula I or II. Hosts which may be employed according to techniques well known in the art for the production of the polypeptides of the present invention include unicellular microorganisms, such as prokaryotes, e , bactena; and eukaryotes, such as fungi, including yeasts, algae, protozoa, molds, and the like, as well as plant cells, both m culture or inplanta, and animal cells Furthermore, virsus may also be modified to comprise a polynucleotide encoding a pesticidal polypeptide according to the present invention Specific bactena which are susceptible to transformation include members of the Enterobacteπaceae, such as strains of Eschenchia coll, Salmonella, Bacillaceae, such as Bacillus subtihs; Pseudomonas; Pneumococcus, Streptococcus; Haemophilus influenzae, and yeasts such as Saccharomyces, among others. In one embodiment of the present mvention, the transformed host is Bacillus sphaericus, which is known to be highly specific for control of mosquito larvae In a preferred embodiment, the host is Bacillus sphaericus, serotype H5a5b, available from Abbott Laboratoπes as VectoLex CG Biological Larvicide (EPA Reg No. 275-77). The polynucleotide sequences of the subject invention can be used to transform a host cell, for example, the polynucleotide sequences can be introduced directly mto the genome of the transformable host cell or can first be incorporated into a vector which is then introduced into the host. Exemplary methods of incorporation include transduction by recombinant phage or cosmids, transfection where specially treated host bacteπal cells can be caused to take up naked phage chromosomes, and transformation by calcium precipitation. These methods are well known m the art. Exemplary vectors include plasmids, cosmids, and phages.

It is well known m the art that when synthesizing a gene for improved expression in a host cell it is desirable to design the gene such that its frequency of codon usage approaches the frequency of preferred codon usage of the host cell. For purposes of the subject mvention, "frequency of preferred codon usage" refers to the preference exhibited by a specific host cell in usage of nucleotide codons to specify a given ammo acid. To determine the frequency of usage of a particular codon m a gene, the number of occurrences of that codon in the gene is divided by the total number of occurrences of all codons specifying the same amino acid in the gene. Similarly, the frequency of preferred codon usage exhibited by a host cell can be calculated by averaging frequency of preferred codon usage m a large number of genes expressed by the host cell. It is preferable to limit this analysis to genes that are highly expressed by the host cell.

Thus, m one embodiment of the subject invention, prokaryotic or eukaryotic cells such as bactena, algae, fungi, plant, or other cells can be genetically engineered, i e , transformed with polynucleotides encoding the subject peptides to attain desired expression levels of the subject peptides. To provide genes having enhanced expression, the DNA sequence of the gene can be modified to compnse codons preferred by highly expressed genes to attain an A+T content in nucleotide base composition which is substantially that found m the transformed host cell. It is also preferable to form an initiation sequence optimal for the host cell, and to eliminate sequences that cause destabihzation, inappropπate polyadenylation, degradation and termination of RNA and to avoid sequences that constitute secondary structure hairpins and RNA splice sites. For example, in synthetic genes the codons used to specify a given amino acid can be selected with regard to the distribution frequency of codon usage employed in highly expressed genes in the host cell to specify that amino acid. As is appreciated by those skilled in the art, the distribution frequency of codon usage utilized in the synthetic gene is a determinant of the level of expression.

Assembly of the polynucleotide sequences of this mvention can be performed using standard technology known in the art. For example, a structural gene designed for enhanced expression in a host cell can be assembled within a DNA vector from chemically synthesized oligonucleotide duplex segments. Preferably the DNA vector or construct has a suitable origin of replication; a selectable marker, such as antibiotic resistance or fluorescence; appropriate termination sequences; and an operable promoter.

Furthermore, chimeric toxins may be used according to the subject invention. Methods have been developed for making useful chimeric toxins by combining portions of proteins. The individual polypeptides which are combined to form the pesticidal chimeras need not be pesticidal, so long as the combination of portions creates a chimeric protein which is pesticidal. The chimeric toxins may include portions from toxins which do not necessarily act upon the TMOF receptor, for example, toxins from Bacillus thuringiensis (B.t.). e.g., B.t. israelensis, B.t. tenebrionis, B.t. san diego, B.t. aizawa, B.t. subtoxicus, B.t. alesti, B.t. gallaeriae, B.t. sotto, B.t. kurstaki, B.t. berliner, B.t. tolworthi, B.t. dendrolimus, and B.t. thuringiensis, and delta endotoxins as described in U.S. Patent No. 5,686,069.

With the teachings provided herein, one skilled in the art can readily produce and use the various toxins and polynucleotide sequences described herein.

The polynucleotide sequences and polypeptides useful according to the subject invention include not only the exemplified sequences but also fragments analogues, derivatives and variants thereof which retain some or all of the characteristic pesticidal activity of the TMOF peptides, or which exhibit improved activity as compared to the activity of the TMOF peptides. As used herein, the terms "variants" or "variations" of genes in reference to nucleotide sequences means nucleotide sequences encoding the same peptides or encoding equivalent peptides having pesticidal activity. As used herein, the term "equivalent peptides" refers to peptides having some or all of the same biological activity of the pesticidal peptides.

Variations of genes may be readily constructed using standard techniques for making point mutations. Also, fragments of these genes can be made using commercially available exonucleases or endonucleases according to standard procedures. For example, enzymes such as BAL31 or site-directed mutagenesis can be used to systematically excise nucleotides from the ends of these genes Also, genes which encode active fragments may be obtained using a vaπety of restriction enzymes Proteases may be used to directly obtain active fragments of these peptides.

Polynucleotide sequences encoding the pesticidal polypeptides of the present invention can be introduced mto a wide variety of microbial or plant hosts In the case of toxms, expression of the gene results, directly or indirectly, in the production and maintenance of the pesticide. With suitable microbial hosts, e g , yeast or Chlorella, the microbes can be applied to the situs of the pest where they will proliferate and be mgested,resultmg in control of the pest. Alternatively, the microbe hosting the gene can be killed and optionally treated under conditions that retain and/or 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 In one embodiment, the host is transformed such that the gene encoding the pesticidal polypeptide is only expressed or maintained for a relatively short period of time, such as days or months, so that the matenal does not persist m the environment. A wide vanety of means are available for introducing a polynucleotide sequence encoding a pesticidal polypeptide into a 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 descπbed, for example, m United States Patent No. 5,135,867, which is incorporated herein by reference. Synthetic genes that encode peptides that are functionally equivalent to the toxms of the subject invention can also be used to transform hosts. Methods for the production of synthetic genes can be found in, for example, U.S. Patent No. 5,380,831.

Recombinant cells expressing a pest control compound can be treated to prolong the toxin activity and stabilize the cell. For example, the pesticidal polypeptides can be formulated as pesticide microcapsules compnsmg the pesticidal polypeptide within a cellular structure that has been stabilized and protects the pesticidal polypeptide when the microcapsule is applied to the environment of the target pest. Suitable host cells include either prokaryotes or eukaryotes. As hosts, of particular interest are the prokaryotes and the lower eukaryotes, such as algae and fungi. The cell is preferably intact and substantially m the proliferative form when treated, rather than in a spore form.

Treatment of the microbial cell, e g , a microbe containing the polynucleotide sequence encoding the pesticidal polypeptide, can be by chemical or physical means, or by a combination of chemical and/or physical means, so long as the technique does not eliminate the pesticidal properties of the pesticidal polypeptide, nor eliminate the cellular capability of protecting the toxin Methods for treatment of microbial cells are disclosed in United States Patent Nos 4,695,455 and 4,695,462

Methods and Formulations for Control of Pests. Control of pests using the pest control compounds of the subject invention can be accomplished by a vaπety of methods known to those skilled in the art These methods include, for example, the application of recombinant microbes to the pests (or their habitats, food sources, etc.), and the transformation of plants with genes (polynucleotide sequences) encoding the pesticidal polypeptides of the subject invention Transformations can be accomplished by those skilled m the art using standard techniques. Materials necessary for these transformations are disclosed herein or are otherwise readily available to the skilled artisan.

The plant pests that can be controlled by the compounds of the subject invention include pests belonging to the orders Coleoptera, Lepidopterans, Hemiptera and Thysanoptera These pests all belong to the phylumArthropod . Other pests that can be controlled according to the subject invention include members of the orders Diptera, Siphonaptera, Hymenoptera and Phthiraptera. Other pests that can be controlled by the compounds of the subject invention include those in the family Arachnιda,such as ticks, mites and spiders.

The use of the compounds of the subject mvention to control pests can be accomplished readily by those skilled m the art having the benefit of the instant disclosure. For example, the compounds may be encapsulated, incorporated in a granular form, solubihzed m water or other appropnate solvent, powdered, and included mto any appropriate formulation for direct application to the pest or to a pest-mhabited locus. In a preferred embodiment for the control of plant pests, plants may be genetically transformed to express a pesticidal polypeptide, such that a pest feeding upon the plant will mgest pesticidal polypeptide and thereby be controlled.

Where the polynucleotide sequence is introduced via a suitable vector mto a microbial host, and said host is applied to the environment in a living state, it is preferred that certain host microbes be used. In one aspect, preferred microbes are known to occupy the "phytosphere" (phylloplane, phyllosphere, rhizosphere, and/or rhizoplane) of one or more crops of interest or the situs where the pest proliferates. These microorganisms are selected so as to be capable of successfully competing in the particular environment (crop and other insect habitats) with the wild-type organisms, provide for stable maintenance and expression of the gene expressing the pesticidal polypeptide and, desirably, provide for improved protection of the pesticide from environmental degradation and inactivation.

A large number of microorganisms are known to inhabit the phylloplane (the surface of the plant leaves) and/or the rhizosphere (the soil surrounding plant roots) of a wide vanety of important crops. These microorganisms include bactena, algae, and fungi. Of particular interest are microorganisms, such as bacteria, e.g., genera Pseudomonas, Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizobium, Rhodopseudomonas, Methylophilius, Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes; fungi, particularly yeast, e.g. , genera Saccharomyces, Cryptococcus, Kluyveromyces, Sporobolomyces, Rhodotorula, and Aureobasidium; and algae, e.g., Chlorella. Of particular interest are such phytosphere bacterial species as Pseudomonas syringae, Pseudomonas fluorescens, Serratia marcescens, Acetobacter xylinum, Agrobacterium tumefaciens, Rhodopseudomonas spheroides, Xanthomonas campestris, Rhizobium melioti, Alcaligenes entrophus, and Azotobacter vinlandii; and phytosphere yeast species such as Rhodotorula rubra, R. glutinis, R. marina, R. aurantiaca, Cryptococcus albidus, C diffluens, C. laurentii, Saccharomyces rosei, S. pretoriensis, S. cerevisiae, Sporobolomyces roseus, S. odorus, Kluyveromyces veronae, and Aureobasidium pollulans. The pigmented microorganisms are preferred.

Formulated bait granules containing an attractant and the pesticidal polypeptides of the present invention, or recombinant microbes comprising toxin-encoding polynucleotide sequences, can be applied to a pest-inhabited locus, such as to the soil. Formulated product can also be applied as a seed-coating or root treatment or total plant treatment at later stages of the crop cycle. Plant and soil treatments 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, gels, suspensions, emulsifiable concentrates or the like. The ingredients may include rheological agents, surfactants, emulsifiers, dispersants or polymers. 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 about 0.0001% by weight and may be 100% by weight. The dry formulations will have from about 0.0001-95% by weight of the pesticide while the liquid formulations will generally be from about 0.0001- 60% by weight of the solids in the liquid phase. The formulations that contain cells will generally have from about 1 to about 10¹⁰ 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 pest, e.g., soil and foliage, by spraying, dusting, sprinkling or the like. In applications to the environment of the target pest, the transformant strain can be applied to the natural habitat of the pest In some cases,the transformant strain will grow m the pest upon mgestion, while continuing to produce the pesticidal polypιptιde(s) The organism may be applied by a wide variety of methods known in the art, including pouring, spraying, soaking, injection mto the soil, seed coating, seedling coating or spraying or the like.

In aquatic environments, pest control may be attained at or below the surface by adjusting the specific gravity of the microbe This can be accomplished by, for example, varying the lipid content of the transformant microorganism strain. It is known that many indigenous aquatic algae float due to their lipid content. A variation m lipid content will allow the transformant strain to be distributed at desired depths below the water surface.

The pest control compounds may also be provided m tablets, pellets, bπquettes, bncks, blocks and the like which are formulated to float, maintain a specified depth or sink as desired. In one embodiment the formulations, according to the present mvention, are formulated to float on the surface of an aqueous medium; m another embodiment they are formulated to maintain a depth of 0 to 2 feet in an aqueous medium; m yet another embodiment the formulations are formulated to sink m an aqueous environment.

For commercial formulations, the organisms may be maintained in a nutrient medium which maintains selectivity and results m a low rate of proliferation. Various media may be used, such as yeast extract or L-broth. Once the organism is to be used in the field, the non- proliferating concentrate may be introduced into an appropriate selective nutnent medium, grown to high concentration, generally from about 10⁵ to 10⁹ cells/ml and may then be employed for introduction mto the environment of the pest.

All of the U.S. patents and other references cited herein are hereby incorporated by reference, as are co-filed U.S. Patent Application S.N. 09/295,846, (UF-223) Transformed Cells Useful for the Control of Pests; U.S. Patent Application S.N. 09/295,849, (UF-216) Neuropephdes and their use for Pest Control; U.S. Patent Application S.N. 09/296,113, (UF- 224) Materials and Methods Useful for the Control of Insect Larvae; and U.S. Patent Application S.N. 09/295,924, (IPTL Docket No. 4137-120) Compositions and Methods for Controlling Pests.

The following examples are simply illustrative of the practice of the present invention and should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted. Example 1 - Effect of TMOF Analogs on Mosquito Larvae

TMOF can traverse the gut epithelium, enter the hemolymph and bind a gut receptor (Borovsky, D. and F. Mahmood (1995) "Feeding the mosquito Aedes aegypti with TMOF and its analogs; effect on trypsin biosynthesis and egg development," Regulatory Peptides 57:273- 281.; Borovsky et al (1994) "Characterization and localization of mosquito-gut receptors for trypsin modulating oostatic factor using complementary peptide lmmunochemistry" FASEB J 8:350-355.). This charactenstic permits the testing of TMOF and its analogues by feeding them to mosquito and other pest larvae. To find out if truncated TMOF peptides have an effect on larval growth and development, a seπes of peptides were synthesized and tested by feeding them to mosquito larvae at concentrations of 0 to 5.0 mg/ml (Table 2). Individual, newly hatched Aedes aegypti larvae were maintained in separate microtiter plate wells on a diet of autoclaved yeast (1 mg/ml). The diet was supplemented with TMOF peptides (Table 2). An identical number of larvae maintained on yeast served as a control. Larvae fed on different concentrations of TMOF peptides (0 mg/ml to 5.0 mg/ml) were monitored for eight (8) days for survival and larval growth and development. All control groups survived and larval growth and development was normal. Since larvae swallow only a small portion of the yeast particles adsorbed the peptides, it is assumed that approximately 1 to 20 ng are taken orally at the high concentrations. The results are displayed m Table 2 as the Lethal Dose at 50% mortality (LD₅₀; ) of the TMOF peptides.

Table 2. The Effect of TMOF and its analogue peptides on mosquito larvae

Compound N LD₅₀ Compound N LD₅₀ mM±S.E.M. Mm±S.E.M.

1. YDPAP₆ 3 0.2 ± 0.02 23.DPA 3 0.4 ± 0.03

2. MPDYP₅ 3 >3.0 24. (D)YDP 3 0.51 ±0.05

3. YDPAF 3 0.33 ± 0.2 25.DAA 3 0.91 ±0.06

4. YEPAP 3 0.35 ±0.02 26.YDG 3 0.95 ±0.11

5. FDPAP 3 0.37 ±0.15 27.YDF 3 0.97 ±0.11

6. YDPLP 3 1.5 ±0.04 28. APA 3 1.0 ±0.07

7. YDPAL 3 0.52 ±0.03 29.AAP 3 1.08 ±0.07

8. YAPAP 3 0.54 ±0.13 30. YSF 3 1.08 ±0.12

9. YNPAP 3 0.55 ± 0.03 31.DYP 4 1.27 ±0.17

10. 3 0.56 ± 0.03 32.YDA 3 1.6±0.13

(D)YDPAP ll.YFPAP 3 0.64±0.03 33. FDP 3 1.98 ±0.6

12. YDPAP 3 1.64 ±0.03 34.YDP 5 2.3 ± 0.4

13. YDLAP 3 0.6 ±0.05 35. FSP 3 2.3 ±0.13

14. YDFAP 3 0.74 ±0.13 36. YAP 3 2.3 ± 0.5

15.YDAAP 3 1.0±0.18 37.PAA 3 2.4 ±0.34

16. YDPGP 5 1.1 ±0.18 38. PAP 3 3.17±0.14

17. 3 1.2 ±0.3 39.FAP 3 3.8 ±0.23

Y(D)DPAP

18.YSPAP 3 1.4 ±0.03 40.ADP 3 >6.6

19. YDPAA 3 1.6±0.13 41. YD 3 1.24 ±0.06

20. YDPFP 4 1.7 ±0.4 42. DY 3 3.0 ±0.8

21.ADPAP 4 2.0 ±0.36

22. YΓDDP 3 0.28 ±0.01

Groups of 12 to 24 mosquito larvae were incubated with different concentrations of TMOF and its analog peptides in 100 μ\ microtiter plates for 7 days. Results are expressed as LD₅₀±S.E.M.

Example 2 — Effect of TMOF Analogue Peptides on Heliothis virescens

Several analogues were chosen and were fed to fourth instar Heliothis virescens for seven (7) days and to first instars for fourteen (14) days (Tables 3 and 4). In both cases a reduction in weight gain and trypsin inhibition was noted (Tables 3 and 4).

Individual first instar and fourth instar larvae of H. virescens were maintained in separate plastic cups and were fed on artificial diet blocks on which different concentrations of

TMOF (0 to 1.6 mg) were adsorbed. Larvae were fed for 5 to 14 days and larval weight and trypsin activity were measured at the end of the experimental periods. A reduction in larval weight and trypsin biosynthesis was observed m fourth mstar larvae that were fed TMOF analogue peptides for 5 days (see Table 3 analogues 15, 14, and 18) When first instar larvae were fed for 14 days, an 18% and 26% reduction in weight was observed when analogues 15 and 16 were used (Table 4). These results indicate that the TMOF peptides of the subject mvention control trypsin biosynthesis in H virescens as was shown in mosquitoes and that these analogues can be used to control these agricultural pest msects.

These results indicate that short TMOF peptides can be used efficiently to block larval growth in mosquitos and other pests An advantage of using short analogs is that they can penetrate the midgut much faster than longer peptides and are less expensive to synthesize by conventional chemical methods. Synthetic organic mimics of these peptides can also be prepared. These organic compounds can penetrate the larval skin and thus, can be used to spray plants for pest control.

Table 3. 1 Effect of TMOF analogues on growth and trypsin biosynthesis on fourth mstar H virescens

TMOF Weight Weight Trypsin Inhibition analog mg±S.E.M. Gam (mg) g±S.E Vl. (%±S.E.M. peptide )

Start End

Control 35.63±1.54 219±8.2 183.5 2.5±0.15 0

DYP(31) 36.2±2.4 216.7±13 180.5 2.2±0.3 14±1.8

YDPGP(16) 31.7±1.6 199.8±11 163.1 2.1±0.1 17±1

YDP(34) 37±1.5 223.4±16 186.3 2.1±0.3 19±3.2

ADAAP(21) 35.7±1.5 209.7±12 174.1 2.4±0.3 5±0.6

YDAAP(25) 38.2±1.3 217±9.5 179 2.1±0.2 17±1.6

YDFAP(14) 37±1.3 201±12 164 2.1±0.2 19±1.5

YSPAP(16) 30.6±1.2 188±10.6 151 2.0±0.2 19±2

Y(D)DPAP(1 34.6±2 188±12 153 2.1±0.2 15±1.3 7)

Fourth instar larvae were weighed and fed on synthetic food and 0.8 μg of TMOF analogs for 5 days. After feeding, larvae were weighed and guts were removed and groups of 3 to 4 guts were incubated with [³H]DFP and analyzed for trypsm biosynthesis. Results are average of 3 to 10 expenments ±S.E.M. Table 4. Feeding ofH virescens on TMOF analogs for 14 days

TMOF analog N Number of Dead Weight (mg)±S.E.M. Weight Reduction

Larvae (%)±S.E.M.

Control 8 2 163±12 0

DYP(31) 9 1 149±9 9±0.5

YDPGP(16) 8 2 153±10 6±0.4

YDP(34) 9 0 157±10 4±0.2

ADAAP(21) 10 0 141±9 7±0.4

YDAAP(15) 10 0 133±7 18±1

YDFAP(14) 9 1 121±7 26±1.5

YSPAP(17) 10 0 168±11 0

Y(D)DPAP(2 9 1 152±27 7±1 0)

First mstar larvae were fed individually 1.6 μg of TMOF analogs for 14 days. After feeding, the weight of each larvae was determined and expressed as an average of 9 to 10 determinations ± S.E.M.

Example 3 — Biological Activity of Compounds Which Bind to TMOF Receptors

Control agents which bind with TMOF receptors can be tested to confirm and charactenze pest control activity. Many bioassays are known to those skilled m the art for the purpose of evaluating pesticidal activity. Assays for evaluating mosquito control activity are known to those skilled m the art and are described m, for example, U.S. Patent No. 5,436,002. Bioassays for evaluating the pest control activity against other targets are also known to those skilled in the art and are descπbed in, for example, U.S. Patent Nos. 5,596,071; 5,188,960; and 5,366,892.

Example 4 — Bioassays for Activity Against Lepidopterons and Coleopterans

Biological activity of the pesticidal compounds of the subject invention can be confirmed using standard bioassay procedures. One such assay is the budworm-bollworm (Heliothis virescens [Fabπcius] and Helicoverpa zea [Boddie]) assay. Lepidoptera bioassays can be conducted with either surface application to artificial insect diet or diet incorporation of samples. All Lepidopteran insects can be tested from the neonate stage to the final mstar. All assays can be conducted with artificial diet, such as toasted soy flour artificial diet or black cutworm artificial diet (BioServ, Frenchtown, NJ). Diet incorporation can be conducted by mixing the samples with artificial diet at a rate of 6 mL suspension plus 54 mL diet. After vortexmg, this mixture is poured mto plastic trays with compartmentalized 3 -ml wells (Nutrend Container Corporation, Jacksonville, FL). A water blank containing no pesticidal polypeptιde(s) serve as the control. First instar larvae (USDA- ARS, Stoneville, MS) can be placed onto the diet mixture. Wells can then be sealed with Mylar sheeting (ClearLam Packaging, IL) using a tackmg iron, and several pmholes can be made m each well to provide gas exchange. Larvae can be maintained at 25°C for 6 days m a 14: 10 (hghtdark) holding room. Mortality and stunting can be recorded after six days.

Bioassay by the top load method can utilize the same sample and diet preparations as listed above. The samples can be applied to the surface of the insect diet. In a specific embodiment, surface area can range from 0.3 to approximately 0.8 cm² depending on the tray size; 96 well tissue culture plates can be used in addition to the format described above Following application, samples can be allowed to air dry before insect infestation. A water blank containing no pesticidal polypeptιde(s) serve as the control. Eggs can be applied to each treated well and then can be sealed with Mylar sheetmg (ClearLam Packaging, IL) using a tacking iron, and pmholes can be made in each well to provide gas exchange. Bioassays can be maintained at 25°C for 7 days in a 14:10 (hghtdark) or 28°C for 4 days in a 14:10 (hghtdark) holding room. Mortality and insect stunting can be recorded at the end of each bioassay.

Another assay useful accordmg to the subject mvention is the Western Corn Rootworm assay. Compounds can be bioassayed for pesticidal activity against neonate Western Com Rootworm larvae (Diabrotica virgifera virgifera) via top-loading of sample onto an agar-based artificial diet. Artificial diet can be dispensed mto 0.78 cm² wells m 48-well tissue culture or similar plates and allowed to harden. After the diet solidifies, samples are dispensed by pipette onto the diet surface. Excess liquid is then evaporated from the surface pπor to transferπng approximately three neonate larvae per well onto the diet surface by camel's hair brush. To prevent insect escape while allowing gas exchange, wells are heat-sealed with 2-mιl punched polyester film with 27HT adhesive (Oliver Products Company, Grand Rapids, Michigan).

Bioassays can be maintained held in darkness at 25°C, and mortality scored after four days.

Analogous bioassays can be performed by those skilled in the art to assess activity against other pests, such as the black cutworm (Agrotis ipsilon).

Example 5 — Target Pests

Pesticidal compounds of the subject invention can be used, alone or m combination with other pesticides, to control one or more non-mammalian pests. These pests may be, for example, those listed in Table 5. Activity can readily be confirmed using the bioassays provided herein, adaptations of these bioassays, and/or other bioassays well known to those skilled in the art.

Table 5. Examples of Target pest species

ORDER/Common Name Latin Name

LEPIDOPTERA

European Corn Borer Ostrinia nubilalis

European Corn Borer resistant to Cryl A Ostrinia nubilalis

Black Cutworm Agrotis ipsilon

Fall Armyworm Spodoptera frugiperda

Southwestern Com Borer Diatraea grandiosella

Com Earworm Bollwoπn Helicoverpa zea

Tobacco Budworm Heliothis virescens

Tobacco Budworm Rs Heliothis virescens

Sunflower Head Moth Homeosoma ellectellum

Banded Sunflower Moth Cochylis hospes

Argentine Looper Rachiplusia nu

Cabbage Looper Trichopluia nil

Spilosoma Spilosoma virginica

Bertha Armyworm Mamestra configurata

Diamondback Moth Plutella xylostells

COLEOPTERA

Red Sunflower Seed Weevil Smicronyx fulvus Sunflower Stem Weevil Cylindrocopturus adspersus Sunflower Beetle Zygoramma exclamationis Canola Flea Beetle Phyllotreta cruciferae Western Com Rootworm Diabrotica virgifera virgifera

DIPTERA

Hessian Fly Mayetiola destructor

HOMOPTERA

Greenbug Schizaphis graminum

HEMΓPTERA

Lygus Bug Lygus lineolaris

NEMATODA Heterodera glycines Example 6 — Insertion of Toxin Genes Into Plants

One aspect of the subject invention is the transformation of plants with genes encoding the msecticidal toxin of the present mvention The transformed plants are resistant to attack by the target pest

Genes encoding pesticidal toxins, as disclosed herein, can be inserted mto plant cells using a vanety of techniques which are well known m the art. For example, a large number of cloning vectors comprising a replication system in E coh and a marker that permits selection of the transformed cells are available for transforming higher plants, e,g„ pBR322, pUC series, Ml 3mp seπes, pACYC 184, etc Accordingly, the sequence encoding the pesticidal peptide can be inserted into the vector at a suitable restnction site. The resulting plasmid can be used for transformation mto E coh The E coh cells can be cultivated m a suitable nutrient medium, then harvested and lysed The plasmid can be recovered. Sequence analysis, restnction analysis, electrophoresis, and other biochemical and/or molecular biological methods can be generally earned out as methods of analysis. After each manipulation, the DNA sequence used can be cleaved and joined to the next DNA sequence Each plasmid sequence can be cloned in the same or other plasmids. Depending on the method of inserting desired genes mto the plant, other DNA sequences may be necessary. If, for example, the Ti plasmid (the tumor-mducmg plasmid of the plant-pathogenic bactenum Agrobacterium tumefaciens) or Ri plasmid (the root- inducing plasmid of Agrobacterium rhizogenes) can be used for the transformation of the plant cell, then at least the πght border, but often the nght and the left border of the Ti or Ri plasmid

T-DNA ("Transferred DNA"), must be joined as the flanking region of the genes to be inserted.

A large number of techniques are available for inserting DNA mto a plant host cell.

These techniques include transformation with T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes as transformation agent, fusion, injection, biolistics (microparticle bombardment), or electroporation as well as other possible methods.

One of the most widely used approaches for the introduction of DNA into plant cells exploits the natural DNA-transferπng properties of Agrobacterium tumefaciens and Agrobacterium rhizogenes, the two species which cause crown gall and hairy root. Their ability to cause disease depends on the presence of large plasmids, in excess of 100 kb, which are referred to as the Ti and Ri plasmids, respectively.

A region referred to as the T-DNA ("Transferred DNA") is transferred from an infecting Agrobacterium cell mto the nucleus of the plant cell, where it is integrated mto the plant genome. Transfer of the T-DNA depends on a set of genes called vir if they are on the Ti plasmid, or chv if they are on the chromosome These genes are induced m response to vanous compounds in exudates from wounded plants The T-DNA itself is flanked by repeated sequences of around 25 base pairs, called border repeats (or left and nght borders) The T-DNA contains a group of genes referred to as the one genes, which are responsible for the oncogemcity of the T-DNA. The use of Agrobacterium in the genetic manipulation of plants involves the insertion of foreign DNA mto the T-DNA of a bacterial cell and subsequent transfer of the DNA by the transformed bacterium mto the plant As long as the necessary protems are provided by the bacterium, any sequences flanked by the T-DNA border repeats can be transferred mto the recipient plant cell genome. The Ti plasmids are too large to manipulate directly, but this problem can be circumvented by using comtegrative and binary systems.

The two ma components of a comtegrative system are a Ti plasmid that has typically been modified by the replacement of matenal between the border repeats (including the one sequences) by pBR322; and an intermediate vector, which is a modified pBR322 containing an extra marker, such as kanamycm resistance. The gene to be introduced into the target plant is first cloned into the intermediate vector, and this construct is then introduced mto Agrobacterium containing the Ti vector The pBR322-based plasmid cannot replicate efficiently inside Agrobacterium, so selection for kanamycm resistance identifies those Agrobacterium cells where the pBR322-based intermediate plasmid has been integrated by homologous recombination mto the Ti plasmid. Because the recombination is homologous, it will take place across the pBR322 sequences and therefore result m integration between the border repeats.

The need for cointegration of the plasmids can be circumvented by use of a binary vector, such as pBml9, a small plasmid containing a pair of left and πght borders The lacL region, located withm the borders, facilitates insertion and detection of DNA A neomycin phosphotransferase gene, typically modified for expression in plants by addition of nopalme synthase expression sequences, is also present withm the borders. Outside the left and πght borders, there is typically a kanamycm resistance gene that will function in prokaryotes and a broad host-range oπgm denved from the plasmid pRK252. The protems that catalyze transfer of the T-DNA mto the host plant do not have to be czs-encoded (i.e., do not have to be encoded by the same molecule). Therefore, if the binary vector is introduced mto Agrobacterium that already contams a resident Ti plasmid, the resident plasmid can provide all the functions needed to transfer into a plant nucleus the DNA between the borders of the binary vector. Other, more sophisticated binary vectors, are also known in the art, for example pROKl. These vectors typically have plant promoters incorporated to dnve expression. Others have cos sites to allow packaging mto lambda phage heads. When the correct sequences have been incorporated mto a vector (whether binary or comtegrative), the vector must then be transferred to an Agrobacterium strain carrying an appropnate Ti plasmid. This is usually accomplished either by electroporation with naked DNA or by a tπparental mating involving the Agrobacterium strain, an E coh strain containing the vector to be transferred, and an E coh strain with a plasmid capable of mobilizing the binary or intermediate vector mto Agrobacterium.

Once the binary vector of the comtegrative vector has been introduced mto a suitable Agrobacterium strain (and comtegration has occmred), the next stage is to permit the Agrobacterium to mfect plant cells. Vaπous methods exist, including inoculation of mtact plants with Agrobacterium cultures by injection, but the most widely used is to incubate discs cut from leaves of the target plant with an Agrobacterium culture. The bactenum will attack cells around the edge of the wounded leaf disc and transfer its T-DNA back mto them. The leaf discs are then transferred to a suitable medium to select for transformation. The neomycm phosphotransferase gene is widely used, confernng resistance to aminoglycoside antibiotics, such as neomycm, kanamycm, and G518. On a suitable selective medium, shoots form around the edges of the treated leaf discs. The shoots can then be regenerated into intact plants. See Howe, Gene Cloning and Manipulation (1995).

The transformed cells are regenerated mto morphologically normal plants in the usual manner. If a transformation event involves a germ line cell, then the inserted DNA and corresponding phenotypic traιt(s) will be transmitted to progeny plants. Such plants can be grown in the normal manner and crossed with plants that have the same transformed hereditary factors or other hereditary factors. The resulting hybrid individuals have the corresponding phenotypic properties.

In a preferred embodiment of the subject mvention, plants will be transformed with genes wherem the codon usage has been optimized for plants. See, for example, U.S. Patent No.

5,380,831. Also, advantageously, plants encoding a pesticidal polypeptide will be used. The pesticidal polypeptide typically will encode less than about 50% of the full length toxm but may be longer or shorter than TMOF.

It should be understood that the examples and embodiments descnbed herein are for illustrative purposes only and that vaπous modifications or changes m light thereof will be suggested to persons skilled in the art and are to be included withm the spmt and purview of this application and the scope of the appended claims.