WO2013052536A2

WO2013052536A2 - Exploiting host molecules to augment the virulence of mycoinsecticides

Info

Publication number: WO2013052536A2
Application number: PCT/US2012/058543
Authority: WO
Inventors: Nematollah KEYHANI; Yanhua FAN
Original assignee: University of Florida; University of Florida Research Foundation Inc
Current assignee: University of Florida; University of Florida Research Foundation Inc
Priority date: 2011-10-05
Filing date: 2012-10-03
Publication date: 2013-04-11
Anticipated expiration: 2014-04-05
Also published as: WO2013052536A4; WO2013052536A3

Abstract

The present disclosure provides methods of insect control in which fungal expression of insect-derived molecules is exploited for target-specific augmentation of entomopathogenic fungi virulence. The major advantages that depends upon the host molecule (peptide) chosen is the increase in virulence that can be tailored to be host specific, and the development of resistance that is be minimized since the host peptides and/or hormones regulate developmental processes that are species- and tissue-specific. Any adaptive mutations that arise in the targeted insect that could compensate for the fungal-expressed product during infection will lead to developmental defects or a fitness cost far greater than developing resistance to a pesticide. The methods, fungal strains, and nucleic acid constructs of the disclosure have been shown to be effective against a variety of insect host species.

Description

EXPLOITING HOST MOLECULES TO AUGMENT THE VIRULENCE OF

MYCOINSECTICIDES

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent Application Serial Number

61/543,345 entitled "EXPLOITING HOST MOLECULES TO AUGMENT THE VIRULENCE OF MYCOINSECTICIDES" and filed October 5, 201 1 , the entirety of which is hereby incorporated by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This disclosure was made with government support under USDA Grant No. 2010-34135-

21095 awarded by the U.S. Department of Agriculture and NSF Grant No. IOS-1 121392 of the National Science Foundation of the United States government. The government has certain rights in the disclosure.

SEQUENCE LISTING

The present disclosure includes a sequence listing incorporated herein by reference in its entirety.

TECHNICAL FIELD

The present disclosure is generally related to genetic modifications that expand the virulence of entomopathogenic fungi and the use of genetically modified entomopathogenic fungi for the control of a selected insect population.

BACKGROUND

There is a need for new tools in insect control particularly since few new chemical pesticides are being developed. Various Lepidopteran species cause billions of dollars of crop losses world-wide and are the most destructive pests of human agriculture. Overuse and reliance on chemical insecticides has resulted in significant ecosystem damage and the emergence of insecticide-resistant agricultural pests. Mosquitoes are vectors of many human and animal infectious diseases that cause death and economic hardship. World Health

Organization (WHO) recommendations suggest the use of different control strategies as part of integrated vector management (IVM) strategies to limit the emergence of insecticide-resistant mosquitoes. Effective strategies, however, for long-term reduction of mosquito populations remains elusive.

Entomopathogenic fungi such as Metarhizium anisopliae and Beauveria bassiana, both EPA approved biological control agents, offer environmentally friendly alternatives to chemical insecticides. The use of entomopathogenic fungi, however, has met with limited success because of the relatively long time (6-12 days) it takes for a fungus to kill target insects.

Expression of a scorpion toxin in M. anisopliae increased fungal toxicity about 9-fold against the yellow fever mosquito, Aedes aegypti. However, expression of non-species-specific toxins to control mosquitoes has the potential to promote the development of toxin-resistance. Ideally, a fungal strain with enhanced virulence towards target insects with minimal non-target effects, coupled to a decreased likelihood of the development of resistance to the agent, is most desired.

Entomopathogenic fungi are virulent to a wide range of Lepidopterans as well as mosquitoes and have been considered as possible candidates for reducing disease

transmission of vector-borne infectious agents. Methods have been developed for delivery of these agents in agricultural settings as well as to mosquito adults and larvae, and the fungi appear to be equally (or more) effective against insecticide resistant strains as compared to their insecticide susceptible parent strains.

The spread of fire ants is considered a classic example of world-wide biological invasions of a species into previously unoccupied habitats with the potential to result in significant ecosystem alterations. The red imported fire ant (Solenopsis invicta), native to South America, is considered by the World Conservation Unit as one of the top 100 worst invasive alien species, and its detrimental impact on humans, domestic and wild animals, agriculture, and ecosystems is well documented (Allen et al., (1994) Tex. J. Science 46: 51-59; Harris et al., (2003) Southwestern Entomologist 123-134; Jemal & Hughesjones (1993) Preventive Vet. Med. 17: 19-32). It is a major invasive pest insect to almost the entire Southeastern United States and continues to expand it range north and westwards causing agricultural and ecosystem disruptions that extend from crop losses to declines of native species (Callcott & Collins (1996) Florida Entomologist 79: 240-251 ). Fire ants have continued to spread despite the treatment of over 56 million hectares with Mirex bait alone and tons of other chemical insecticides (Williams et al., (2001 ) American Entomologist 47: 146-159), which themselves have significant damaging environmental consequences.

Biological control of fire ants using entomopathogenic fungi offers a more

environmentally friendly alternative to chemical pesticides (Williams et al., (2003) American Entomologist 49: 150-163; Oi et al., (2009) Biological Control 8: 310-315; Pereira R.M. (2003) J. Agricultural and Urban Entomol. 20: 123-130; Riggs et al. (2002) Southwestern Entomologist p31-41 ). The use of entomopathogenic fungi to control fire ant populations, however, has met with limited success partially due to the relatively long time (3-10 days) it can take for the fungus to kill target insects. Ants have posed a particular challenge due to communal behaviors such as grooming and nest cleaning that can decrease the efficacy of microbial agents (Oi &Pereira (1993) Florida Entomologist 76: 63-74). Previous work has shown that the potency of fungal insecticides can be improved (St Leger & Wang (2010) Applied Microbiol. & Biotechnol. 85: 901- 907).

SUMMARY

One aspect of the present disclosure, therefore, encompasses embodiments of a genetically modified strain of an entomopathogenic fungus comprising a heterologous recombinant nucleotide sequence encoding a peptide, polypeptide, or protein of a target insect host where, when the nucleotide sequence is expressed by the genetically modified

entomopathogenic fungus having infected the target insect host, the peptide, polypeptide, or protein can increase the virulence of the fungus in the target insect compared to the virulence of a non-genetically modified strain of the entomopathogenic fungus in the target insect host.

Another aspect of the present disclosure encompasses embodiments of an expression vector comprising a fungal gene expression controlling region nucleotide sequence operably linked to a nucleic acid encoding an insect-specific polypeptide wherein the gene expression controlling region directs production of a transcript that can be expressed by a

entomopathogenic fungus that has infected a target insect host, the insect specific polypeptide increasing the virulence of the fungus in the target insect compared to the virulence of a strain of the entomopathogenic fungus not expressing the insect specific polypeptide.

In embodiments of this aspect of the disclosure, the gene controlling region has about

90% sequence similarity with a sequence encoding a B. bassiana chitinase gene signal peptide.

In embodiments of this aspect of the disclosure, the B. bassiana chitinase gene signal peptide has the amino acid sequence according to SEQ ID No.: 2.

In some embodiments of this aspect of the disclosure, the insect-specific polypeptide can be selected from the group consisting of: an insect neurohormone, an insect diuretic hormone, a trypsin modulating oostatic factor, or any bioactive homolog or fragment thereof.

In embodiments of this aspect of the disclosure, the insect-specific polypeptide can be selected from the group consisting of: β-neuropeptide specific to the fire ant Solenopsis invicta, Manduca sexta diuretic hormone (MSDH), and a trypsin-modulating oostatic factor of Aedes aegyti or S. bullata.

Yet another aspect of the disclosure encompasses embodiments of a method of increasing the virulence of an entomopathogenic fungus in a target insect host, comprising the steps of: (a) obtaining a genetically-modified strain of a entomopathogenic fungus according to the disclosure; and (b) delivering the genetically modified strain of entomopathogenic fungus, or spores thereof, to a target insect host desired to be infected by the fungus, wherein the fungus delivers a target insect-specific polypeptide or peptide to the target insect host, thereby increasing the virulence of the entomopathogenic fungus in the target insect host.

In embodiments of this aspect of the disclosure, the entomopathogenic fungus can be Metarhizium anisopliae or Beauveria bassiana.

In embodiments of this aspect of the disclosure, the target insect host can be the fire ant (Solenopsis invicta), a mosquito species, a Lepidopteran species, a Dipteran, or a Hemipteran species.

BRIEF DESCRIPTION OF THE FIGURES

Further aspects of the present disclosure will be more readily appreciated upon review of the detailed description of its various embodiments, described below, when taken in conjunction with the accompanying drawings.

Figs. 1A-1 G illustrate insect bioassays and the effect of fungal infection on mosquito development.

Fig. 1A is a graph illustrating the mortality rate of Galleria melionella infected with conidial suspensions (5 x 10⁷ spores/ml) of wild type (top line) and a Bb::spMSDH, B. bassiana transformant expressing the Manduca sexta diuretic hormone (bottom line). These data combined with a dose-response curve were used to calculate the data presented in Table 1.

Fig. 1 B is a bar graph illustrating trypsin activity in uninfected and B. bass/ana-infected female mosquitoes.

Fig. 1 C is a bar graph illustrating the reduced fecundity in fungal-infected female mosquitoes.

Fig. 1 D is a bar graph illustrating mosquito larval length in control and fungal-infected insects.

Fig. 1 E is a digital image of a control (uninfected) larva.

Fig. 1 F is a digital image of a larva infected with wild type B. bassiana.

Fig. 1 G is a digital image of a larva infected with Aea-TMOF-expressing B. bassiana. Fig. 2 is a graph illustrating the results of bioassays of fire ant infected with either the wild type (WT) (closed circle, upper line) or Bb::spp-NP_gpd (closed circle, lower line) B. bassiana strains and buffer-treated controls (open circles, dashed line). The percent survival of S. invicta treated with 4 x 10⁶ conidia/ml of each strain over the indicated time course is presented. These data were used to calculate the LT₅₀ values, and a concentration curve was used to determine the LD₅₀ values. Fig. 3 is a series of digital images illustrating the distribution of dead ants after infection with wild type and Bb::spp-NP_gpd B. bassiana strains in mock mound assays. Top panels, ants in arenas with 10% sucrose, but no nest area; bottom panels, test arenas containing 10% sucrose and nest area.

Fig. 4 is a graph illustrating the responses of S. invicta to dead ants. Plots of the times between the introduction and removal of items in the test arena are shown. Boxes are bounded by the first quartile, median, and third quartile. Movement times of freeze-killed and fungal- killed, WT and Bb::spp-NP_gpd, ants presented to untreated ants. WT-killed ants were moved significantly more quickly than either freeze-killed or Bb::spp-NP_gpd-killed ants (P = 0.0014).

Fig. 5 is a graph illustrating the movement times of WT- or Bb::spp-NP_gpd-killed ants by untreated ants, WT-infected (2 days prior to assay) ants, or Bb::spp-NP_gpd-infected ants. No significant differences were noted between live ants treatments (P = 0.86), with WT-killed ants moved significantly faster than Bb::spp-NP_gpd-killed ants regardless of the tested infection state of the ants doing the moving (P < 0.001 ). This difference was consistent over the live ant treatments (interaction P-value = 0.53).

Fig. 6 is a graph illustrating the movement times of dead ants treated with synthetic peptides and presented to untreated ants. Freeze-killed ants were immersed in 100 nM solution of either a control amidated peptide (QAGVTGHA-NH₂) (SEQ ID No.: 21 ), β-ΝΡ (QPQFTPRL) (SEQ ID No.: 7), or β-ΝΡ-ΝΗ₂ and presented to untreated ants. p-NP-NH₂-treated ants were moved significantly faster than the control and other peptide treatments (P < 0.001 ).

Figs. 7A and 7B are graphs illustrating the expression of Aea-JMOF increases

Beauveria bassiana virulence to Anopheles gambiae mosquitoes. Sugar-fed (Fig. 7A) and blood-fed (Fig. 7B) adult female A. gambiae mosquitoes were challenged with Bb-ka strain expressing Aea-JMOF or the wild type parent by spraying with a suspension of 1x10⁸ spores/ml. Their survival was scored daily over the indicated period. Graphs represent percent survival as calculated by the Kaplan-Meier method for one representative experiment of each group. Statistical significance was calculated by the log rank test and survival curves were considered significantly different if P < 0.05.

Fig. 8 is a graph illustrating that Aea-JMOF reduces fecundity of Anopheles gambiae mosquitoes. A. gambiae females sprayed with a suspension of 1x10⁸ spores/ml of wild type or βώ-Aal strain were given a blood meal 24 h after fungal infection. Non-infected, blood-fed mosquitoes were used as controls. Each circle on the graph represents the number of eggs per mosquito. Medians are indicated by black lines and distributions were compared using the Kolmogorov-Smirnov test. N, number of mosquitoes in each group.

The drawings are described in greater detail in the description and examples below.

The details of some exemplary embodiments of the methods and systems of the present disclosure are set forth in the description below. Other features, objects, and advantages of the disclosure will be apparent to one of skill in the art upon examination of the following

description, drawings, examples and claims. It is intended that all such additional systems, methods, features, and advantages be included within this description, be within the scope of the present disclosure, and be protected by the accompanying claims.

DETAILED DESCRIPTION

Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed.

As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of medicine, organic chemistry, biochemistry, molecular biology, pharmacology, and the like, which are within the skill of the art. Such techniques are explained fully in the literature.

It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a support" includes a plurality of supports. In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings unless a contrary intention is apparent.

As used herein, the following terms have the meanings ascribed to them unless specified otherwise. In this disclosure, "comprises," "comprising," "containing" and "having" and the like can have the meaning ascribed to them in U.S. Patent law and can mean " includes," "including," and the like; "consisting essentially of or "consists essentially" or the like, when applied to methods and compositions encompassed by the present disclosure refers to compositions like those disclosed herein, but which may contain additional structural groups, composition components or method steps (or analogs or derivatives thereof as discussed above). Such additional structural groups, composition components or method steps, etc., however, do not materially affect the basic and novel characteristic(s) of the compositions or methods, compared to those of the corresponding compositions or methods disclosed herein. "Consisting essentially of or "consists essentially" or the like, when applied to methods and compositions encompassed by the present disclosure have the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.

Definitions

In describing and claiming the disclosed subject matter, the following terminology will be used in accordance with the definitions set forth below. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art of molecular biology.

Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described herein.

Further definitions are provided in context below. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art of molecular biology. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described herein.

Unless otherwise defined, all terms of art, notations and other scientific terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this disclosure pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. The techniques and procedures described or referenced herein are generally well understood and commonly employed using conventional methodology by those skilled in the art, such as, for example, the widely utilized molecular cloning methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd. edition (2001 ) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Current Protocols in Molecular Biology (Ausbel et al., eds., John Wiley & Sons, Inc. 2001 ). As appropriate, procedures involving the use of commercially available kits and reagents are generally carried out in accordance with manufacturer defined protocols and/or parameters unless otherwise noted.

The terms "virulence" and "virulent strains" as used herein refer to processes that are caused by entomopathogenic fungi and which lead to a reduction in some component of the host's fitness or mortality. Virulence and resistance are properties that emerge as a result of host-parasite interaction in a given environment.

The term "entomopathogenic fungi" as used herein refers to fungi capable of infecting and parasitizing and/or killing an insect. Such a fungus is considered a mycopesticide when directed for use in controlling a population of insects. Entomopathogenic fungi include those strains or isolates of fungal species in the class Hyphomycetes which possess characteristics allowing them to be virulent against insects. These characteristics include formation of stable infective conidia. An effective entomopathogenic fungus preferably is lethal for target insects but less harmful for non-target insects. Also, the entomopathogenic fungus preferably does not harm vegetation or animals that might come in contact with it. Such fungi include, but are not limited to, fungi of the class Deuteromycete. There are two classes of Deuteromycete:

Hyphomycetes and Coelomycetes. Hyphomycetes fungi generally produce conidia.

Deuteromycete fungi include fungi of the genera Beauveria, Metarhizium, Paecilomyces, Tolypocladium, Aspergillus, Culicinonyces, Nomuraea, Sorosporella, and Hirsutella. Examples of species of Deuteromycete fungi include Beauveria bassiana, Metarhizium flavoviride, Metarhizium anisopliae, Paecilomyces fumusoroseus, Paecilomyces farinosus, Nomuraea rileyi, and the like. The term "entomopathogenic fungi" further includes, but is not limited to, the Entomophthorales, an order of fungi of the subphylum, Entomophthoromycotina, and includes such species as Pandora neoaphidis, Entomophaga maimaiga (a biocontrol agent of gypsy moths), Entomophthora muscae (a pathogen of houseflies), Massospora spp., (pathogens of periodical cicadas), and the like.

The terms "insect peptide" and insect hormone" as used herein refer any peptide or polypeptide hormone recognized as having a physiological, biochemical or behavioral role in an insect. It is further understood that the peptide or polypeptide hormone of one species may have an analog (functionally similar but having a dissimilar amino acid sequence) or a homolog (having a sequence related but not identical) of another species. For example, but not intended to be limiting, such insect hormones include allostatins, antidiuretic hormones (e.g. the Tenebrio molitor (beetle) ADH), juvenile hormone, diapause hormone, proctolins, mykinins, pyrokinins other than β-ΝΡ, diuretic hormones other than MSDH, tachykinins, myosuppressins, and the like.

The term "insect" as used herein refers to any insect target desired to be controlled and may be of the orders Archaeognatha, Blattodea (Cockroaches), Coleoptera (Beetles),

Dermaptera (Earwigs), Diptera (Flies), Embioptera (Webspinners), Ephemeroptera (Mayflies), Hemiptera, Hymenoptera, Isoptera (Termites), Lepidoptera, Mantodea (Mantises), Mecoptera, Megaloptera, Neuroptera, Notoptera, Orthoptera, Phasmatodea, Phthiraptera (Lice), Plecoptera, Psocoptera, Raphidioptera (Snakeflies), Siphonaptera (Fleas), Strepsiptera, Thysanoptera (Thrips), Zoraptera, and Zygentoma (Thysanura).

The term "expressed" or "expression" as used herein refers to the transcription from a gene to give an RNA nucleic acid molecule at least complementary in part to a region of one of the two nucleic acid strands of the gene. The term "expressed" or "expression" as used herein also refers to the translation from said RNA nucleic acid molecule to give a protein, an amino acid sequence or a portion thereof. As used herein, the term "nucleic acid molecule" is intended to include DNA molecules (e.g. , cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNA generated using nucleotide analogs, and derivatives, fragments and homologs thereof. The nucleic acid molecule can be single-stranded or double-stranded, but advantageously is double- stranded DNA. An "isolated" nucleic acid molecule is one that is separated from other nucleic acid molecules that are present in the natural source of the nucleic acid. A "nucleoside" refers to a base linked to a sugar. The base may be adenine (A), guanine (G) (or its substitute, inosine (I)), cytosine (C), or thymine (T) (or its substitute, uracil (U)). The sugar may be ribose (the sugar of a natural nucleotide in RNA) or 2-deoxyribose (the sugar of a natural nucleotide in DNA). A "nucleotide" refers to a nucleoside linked to a single phosphate group.

As used herein, the term "oligonucleotide" refers to a series of linked nucleotide residues, which oligonucleotide has a sufficient number of nucleotide bases to be used in a PCR reaction. A short oligonucleotide sequence may be based on, or designed from, a genomic or cDNA sequence and is used to amplify, confirm, or reveal the presence of an identical, similar or complementary DNA or RNA in a particular cell or tissue. Oligonucleotides may be chemically synthesized and may be used as primers or probes. Oligonucleotide means any nucleotide of more than 3 bases in length used to facilitate detection or identification of a target nucleic acid, including probes and primers.

The term "transfection" refers to a process by which agents are introduced into a cell. The list of agents that can be transfected is large and includes, but is not limited to, siRNA, sense and/or anti-sense sequences, DNA encoding one or more genes and organized into an expression plasmid, proteins, protein fragments, and more. There are multiple methods for transfecting agents into a cell including, but not limited to, electroporation, calcium phosphate- based transfections, DEAE-dextran-based transfections, lipid-based transfections, molecular conjugate-based transfections (e.g. , polylysine-DNA conjugates), microinjection and others.

The term "coding sequence" as used herein refers to a sequence which "encodes" a selected polypeptide and is a nucleic acid molecule which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo when placed under the control of appropriate regulatory sequences (or "control elements"). The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. A coding sequence can include, but is not limited to, cDNA from viral, prokaryotic or eukaryotic mRNA, genomic DNA sequences from viral or prokaryotic DNA, and even synthetic DNA sequences. A transcription termination sequence may be located 3' to the coding sequence. The term "gene controlling regions " as used herein refers to, but is not limited to, transcription promoters, transcription enhancer elements, transcription termination signals, polyadenylation sequences (located 3' to the translation stop codon), sequences for

optimization of initiation of translation (located 5' to the coding sequence), and translation termination sequences, see e.g., McCaughan ef a/., (1995) Proc. Natl. Acad. Sci. U. S.A. 92: 5431 -5435; Kochetov ef a/., (1998) FEBS Letts. 440: 351-355.

A "nucleic acid" molecule can include, but is not limited to, prokaryotic sequences, eukaryotic mRNA, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. The term also captures sequences that include any of the known base analogs of DNA and RNA.

The term "operably linked" as used herein refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function. Thus, a given promoter operably linked to a coding sequence is capable of effecting the expression of the coding sequence when the proper enzymes are present. The promoter need not be contiguous with the coding sequence, so long as it functions to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between the promoter sequence and the coding sequence and the promoter sequence can still be considered "operably linked" to the coding sequence.

The term "recombinant" as used herein, and referring to a nucleic acid molecule, means a polynucleotide of genomic, cDNA, semisynthetic, or synthetic origin which, by virtue of its origin or manipulation: (1) is not associated with all or a portion of the polynucleotide with which it is associated in nature; and/or (2) is linked to a polynucleotide other than that to which it is linked in nature. The term "recombinant" as used with respect to a protein or polypeptide means a polypeptide produced by expression of a recombinant polynucleotide. "Recombinant host cells," "host cells," "cells," "cell lines," "cell cultures," and other such terms denoting prokaryotic microorganisms or eukaryotic cell lines cultured as unicellular entities, are used interchangeably, and refer to cells which can be, or have been, used as recipients for recombinant vectors or other transfer DNA, and include the progeny of the original cell which has been transfected. It is understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total DNA complement to the original parent, due to accidental or deliberate mutation. Progeny of the parental cell which are sufficiently similar to the parent to be characterized by the relevant property, such as the presence of a nucleotide sequence encoding a desired peptide, are included in the progeny intended by this definition, and are covered by the above terms. "Identity," as known in the art, is a relationship between two or more polypeptide sequences, as determined by comparing the sequences. In the art, "identity" also refers to the degree of sequence relatedness between polypeptides as determined by the match between strings of such sequences. "Identity" and "similarity" can be readily calculated by known methods, including, but not limited to, those described in Computational Molecular Biology, Lesk, A. M., Ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., Ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin & Griffin, Eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov & Devereux, Eds., M Stockton Press, New York, 1991 ; and Carillo & Lipman (1988) SI AM J. Applied Math., 48: 1073.

By way of example, a polypeptide sequence may be identical to the reference sequence, that is be 100% identical, or it may include up to a certain integer number of amino acid alterations as compared to the reference sequence such that the % identity is less than 100%. Such alterations are selected from at least one amino acid deletion, substitution (including conservative and non-conservative substitution), or insertion, and wherein said alterations may occur at the amino- or carboxy-terminus positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids in the reference sequence, or in one or more contiguous groups within the reference sequence. The number of amino acid alterations for a given % identity is determined by multiplying the total number of amino acids in the reference polypeptide by the numerical percent of the respective percent identity (divided by 100) and then subtracting that product from said total number of amino acids in the reference polypeptide.

Techniques for determining amino acid sequence "similarity" are well known in the art. In general, "similarity" means the exact amino acid to amino acid comparison of two or more polypeptides at the appropriate place, where amino acids are identical or possess similar chemical and/or physical properties such as charge or hydrophobicity. A so-termed "percent similarity" then can be determined between the compared polypeptide sequences. Techniques for determining nucleic acid and amino acid sequence identity also are well known in the art and include determining the nucleotide sequence of the mRNA for that gene (usually via a cDNA intermediate) and determining the amino acid sequence encoded thereby, and comparing this to a second amino acid sequence. In general, "identity" refers to an exact nucleotide to nucleotide or amino acid to amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Two or more polynucleotide sequences can be compared by determining their "percent identity." Two or more amino acid sequences likewise can be compared by determining their "percent identity." The percent identity of two sequences, whether nucleic acid or peptide sequences, is generally described as the number of exact matches between two aligned sequences divided by the length of the shorter sequence and multiplied by 100. An

approximate alignment for nucleic acid sequences is provided by the local homology algorithm of Smith & Waterman (1981 ) Adv. Applied Mathematics 2: 482-489. This algorithm can be extended to use with peptide sequences using the scoring matrix developed by Dayhoff, Atlas of Protein Sequences and Structure, M. O. Dayhoff ed. , 5 suppl. 3:353-358, National Biomedical Research Foundation, Washington, D.C., USA, and normalized by Gribskov (1986) Nucl. Acids Res. 14:6745-6763. Suitable programs for calculating the percent identity or similarity between sequences are generally known in the art.

A "vector" is a genetic unit (or replicon) to which or into which other DNA segments can be incorporated to effect replication, and optionally, expression of the attached segment.

Examples include, but are not limited to, plasmids, cosmids, viruses, chromosomes and minichromosomes. Exemplary expression vectors include, but are not limited to, baculovirus vectors, modified vaccinia Ankara (MVA) vectors, plasmid DNA vectors, recombinant poxvirus vectors, bacterial vectors, recombinant baculovirus expression systems (BEVS), recombinant rhabdovirus vectors, recombinant alphavirus vectors, recombinant adenovirus expression systems, recombinant DNA expression vectors, and combinations thereof.

The term "polypeptides" includes proteins and fragments thereof. Polypeptides are disclosed herein as amino acid residue sequences. Those sequences are written left to right in the direction from the amino to the carboxy terminus. In accordance with standard

nomenclature, amino acid residue sequences are denominated by either a three letter or a single letter code as indicated as follows: Alanine (Ala, A), Arginine (Arg, R), Asparagine (Asn, N), Aspartic Acid (Asp, D), Cysteine (Cys, C), Glutamine (Gin, Q), Glutamic Acid (Glu, E), Glycine (Gly, G), Histidine (His, H), Isoleucine (lie, I), Leucine (Leu, L), Lysine (Lys, K),

Methionine (Met, M), Phenylalanine (Phe, F), Proline (Pro, P), Serine (Ser, S), Threonine (Thr, T), Tryptophan (Trp, W), Tyrosine (Tyr, Y), and Valine (Val, V).

The term "variant" as used herein refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide, but retains essential properties. A typical variant of a polypeptide differs in amino acid sequence from another reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall (homologous) and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more modifications (e.g., substitutions, additions, and/or deletions). A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a polypeptide may be naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally.

Modifications and changes can be made in the structure of the polypeptides of this disclosure and still result in a molecule having similar characteristics to the polypeptide (e.g., a conservative amino acid substitution). For example, certain amino acids can be substituted for other amino acids in a sequence without appreciable loss of activity. Because it is the interactive capacity and nature of a polypeptide that defines that polypeptide's biological functional activity, certain amino acid sequence substitutions can be made in a polypeptide sequence and nevertheless obtain a polypeptide with like properties.

In making such changes, the hydropathic index of amino acids can be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a polypeptide is generally understood in the art. It is known that certain amino acids can be substituted for other amino acids having a similar hydropathic index or score and still result in a polypeptide with similar biological activity. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics. Those indices are:

isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cysteine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1 .6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

It is believed that the relative hydropathic character of the amino acid determines the secondary structure of the resultant polypeptide, which in turn defines the interaction of the polypeptide with other molecules, such as enzymes, substrates, receptors, antibodies, antigens, and the like. It is known in the art that an amino acid can be substituted by another amino acid having a similar hydropathic index and still obtain a functionally equivalent polypeptide. In such changes, the substitution of amino acids whose hydropathic indices are within ± 2 is preferred, those within ± 1 are particularly preferred, and those within ± 0.5 are even more particularly preferred.

Substitution of like amino acids can also be made on the basis of hydrophilicity, particularly where the biologically functional equivalent polypeptide or peptide thereby created is intended for use in immunological embodiments. The following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ± 1 ); glutamate (+3.0 ± 1 ); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); proline (-0.5 ± 1 ); threonine (-0.4); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1 .8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent polypeptide. In such changes, the substitution of amino acids whose hydrophilicity values are within ± 2 is preferred, those within ± 1 are particularly preferred, and those within ± 0.5 are even more particularly preferred.

As outlined above, amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take one or more of the foregoing characteristics into consideration are well known to those of skill in the art and include, but are not limited to (original residue: exemplary substitution): (Ala: Gly, Ser), (Arg: Lys), (Asn: Gin, His), (Asp: Glu, Cys, Ser), (Gin: Asn), (Glu: Asp), (Gly: Ala), (His: Asn, Gin), (lie: Leu, Val), (Leu: lie, Val), (Lys: Arg), (Met: Leu, Tyr), (Ser: Thr), (Thr: Ser), (Tip: Tyr), (Tyr: Trp, Phe), and (Val: lie, Leu). Embodiments of this disclosure thus contemplate functional or biological equivalents of a polypeptide as set forth above. In particular, embodiments of the polypeptides can include variants having about 50%, 60%, 70%, 80%, 90%, and 95% sequence identity to the

polypeptide of interest. The term "substantially homologous" is used herein to denote polypeptides of the present disclosure having about 50%, about 60%, about 70%, about 80%, about 90%, and preferably about 95% sequence identity to the reference sequence. Percent sequence identity is determined by conventional methods as discussed above. In general, homologous polypeptides of the present disclosure are characterized as having one or more amino acid substitutions, deletions, and/or additions.

Furthermore, unless the context demands otherwise, the terms "peptide", "polypeptide" and "protein" are herein used interchangeably to refer to amino acids in which the amino acid residues are linked by covalent peptide bonds or alternatively (where post-translational processing has removed an internal segment) by covalent disulfide bonds, etc. The amino acid chains can be of any length and comprise at least two amino acids. They can include domains of proteins or full-length proteins. Unless otherwise stated the terms peptide, polypeptide, and protein also encompass various modified forms thereof, including but not limited to glycosylated forms, phosphorylated forms, etc.

As used herein, the term "polynucleotide" generally refers to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. Thus, for instance, polynucleotides as used herein refers to, among others, single- and double- stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is a mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double- stranded or a mixture of single- and double-stranded regions. The terms "nucleic acid," "nucleic acid sequence," or "oligonucleotide" also encompass a "polynucleotide" as defined above.

In addition, "polynucleotide" as used herein refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The strands in such regions may be from the same molecule or from different molecules. The regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules. One of the molecules of a triple-helical region often is an oligonucleotide.

As used herein, the term polynucleotide includes DNAs or RNAs as described above that contain one or more modified bases. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleotides" as that term is intended herein. Moreover, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are polynucleotides as the term is used herein.

It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art. The term

polynucleotide as it is employed herein embraces such chemically, enzymatically, or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including simple and complex cells, inter alia.

By way of example, a polynucleotide sequence of the present disclosure may be identical to the reference sequence, that is be 100% identical, or it may include up to a certain integer number of nucleotide alterations as compared to the reference sequence. Such alterations are selected from the group including at least one nucleotide deletion, substitution, including transition and transversion, or insertion, and wherein said alterations may occur at the 5' or 3' terminus positions of the reference nucleotide sequence or anywhere between those terminus positions, interspersed either individually among the nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence. The number of nucleotide alterations is determined by multiplying the total number of nucleotides in the reference nucleotide by the numerical percent of the respective percent identity (divided by 100) and subtracting that product from said total number of nucleotides in the reference nucleotide. Alterations of a polynucleotide sequence encoding the polypeptide may alter the polypeptide encoded by the polynucleotide following such alterations. As used herein, DNA may be obtained by any method. For example, the DNA includes complementary DNA (cDNA) prepared from mRNA, DNA prepared from genomic DNA, DNA prepared by chemical synthesis, DNA obtained by PCR amplification with RNA or DNA as a template, and DNA constructed by appropriately combining these methods.

The DNA encoding the protein disclosed herein can be prepared by the usual methods: cloning cDNA from mRNA encoding the protein, isolating genomic DNA and splicing it, chemical synthesis, and so on.

An "expression vector" is useful for expressing the DNA encoding the protein used herein and for producing the protein. The expression vector is not limited as long as it expresses the gene encoding the protein in various prokaryotic and/or eukaryotic host cells and produces this protein. Examples thereof are pMAL C2, pEF-BOS (Nucleic Acids Res. 18:5322 (1990)), pME18S (Experimental Medicine: SUPPLEMENT, "Handbook of Genetic Engineering" (1992)), etc.

The expression vector used herein can be prepared by continuously and circularly linking at least the above-mentioned promoter, initiation codon, DNA encoding the protein, termination codon, and terminator region to an appropriate replicon. If desired, appropriate DNA fragments (for example, linkers, restriction sites, and so on), can be used by the usual method such as digestion with a restriction enzyme or ligation using T4 DNA ligase.

As used herein, "transformants" can be prepared by introducing the expression vector mentioned above into host cells.

As used herein, "host" cells are not limited as long as they are compatible with an expression vector mentioned above and can be transformed. Examples thereof are various cells such as wild-type cells or artificially established recombinant cells usually used in technical field (for example, bacteria (Escherichia and Bacillus), yeast (Saccharomyces, Pichia, and such), animal cells, insect cells, or plant cells).

Abbreviations

NP. neuropeptide; TMOF, trypsin modulating oostatic factor; Aea-TMOF, Aedes aegypti TMOF; MSDH, Manduca sexta diuretic hormone; PBAN, pyrokinin/pheromone biosynthesis activating neuropeptide;

Description

The present disclosure provides an approach to insect control in which expression of host, i.e. insect-derived, molecules in an insect pathogen is exploited for target-specific augmentation of virulence. The major advantages of such a strategy are: (1 ) that depending upon the host molecule (peptide) chosen, the increase in virulence can be tailored to be host specific, and (2) the development of resistance would be minimized, since the host peptides and/or hormones regulate developmental processes that are species and tissue specific. Unlike current approaches to insect control, any mutations that arise that could compensate for the fungal-expressed product during infection would be dependent upon the fungus for proper development, i.e. any potential resistance to the target molecule would lead to developmental defects or a fitness cost far greater than developing resistance to a pesticide. In contrast to methods which use an insecticidal compound, the development of resistance faces an additional burden. It is contemplated that multiple host molecules can be expressed in the same fungal strain to further augment the specificity and virulence of mycoinsecticides to produce more effective and safer insect biological control agents.

The present disclosure encompasses methods of increasing the virulence of

entomopathogenic fungi by genetically modifying such agents. The modifications allow the fungal agent to express a peptide, polypeptide or protein derived from the intended target insect host. It has been observed that by expressing such heterologous amino acid sequences that are native to the target insect, the virulence of the fungus to the insect is enhanced. Virulence is maintained since any resistance to the fungus that focuses on the expressed polypeptide will also target the native insect protein and result in further weakening of the host insect and a more rapid incapacity or death. The methods, fungal strains, and nucleic acid constructs of the disclosure have been shown to be effective against a variety of insect host species and that the genetic modifications of the attacking fungus can be effectively tailored to the host target without increasing fungal virulence against other species not desired to be attacked, and consequently are limited to the selected target host species. It is also contemplated that selection of the fungus and the insect-derived peptide or hormone expressed therein may be selected to provide virulence against a broader range of target insects than just a single species or closely related species. It is further contemplated that it is within the scope of the disclosure for the method to be adapted to target any insect species or group of species by identifying a peptide, polypeptide or protein variant specifically encoded by the target insect genome, and that it is possible to distinguish between closely related insect species as suitable targets of the methods herein disclosed. The methods, nucleic acid constructs, and fungal strains of the present disclosure have been found especially useful for inhibiting the growth, survival, and/or reproduction of significant pests such as the fire ant and mosquito and hence provides effective alternatives to chemical and biological insecticides that are ecologically disadvantageous and/or subject to the target insect developing resistance. The pyrokinin/pheromone biosynthesis activating neuropeptide (PBAN) family consists of insect neurohormones characterized by the presence of a C-terminal FXPRL amine sequence. First isolated from the cockroach, Leucophaea maderae, as a myotropic (visceral muscle contraction stimulatory) peptide, members of this peptide family are widely distributed within the Insecta, where depending upon the species, they function in a diverse range of physiological processes that includes stimulation of pheromone biosynthesis, melanization, acceleration of pupariation, and induction and/or termination of diapause. In the natural insect host, these peptides are C-terminal amidated, a modification often required for their activity.

In Lepidoptera, the PBAN peptide is encoded on a translated ORF that is subsequently processed (cleaved) to yield diapause hormone (DH), and the α-, β-, and γ-neuropeptides, along with the PBAN peptide itself (which is found between the β- and γ-neuropeptides). More recently, isolation of a cDNA sequence for the fire ant, S. invicta, led to the identification of PBAN and related peptide homologs. Analysis of the ORF revealed the presence of DH, as well as β- and γ-neuropeptide homologs, but no a-neuropeptide.

The impact of expressing the β-ΝΡ peptide in the fungal insect pathogen B. bassiana: The data shows a decrease in both the lethal dose (LD₅₀) and lethal time (LT₅₀) it takes to kill target fire ants in the β-ΝΡ-expressing strain as compared to its wild-type parent. The effect was host specific, and no increase in virulence was noted when the strain was tested against the greater wax moth, Galleria mellonella. By using a host molecule the chances of resistance are minimized due to the fungal-expressed peptide representing a host molecule that is regulated in both tissue specific and developmental patterns. Any mutations that could compensate for the increased dose given by the fungus during infection would be significantly compromised such that the host is now potentially dependent upon the fungus for proper development.

There has been much interest in the use of biological control strategies for fire ant control ranging from release of various parasites including mites and phorid flies, to the use of viruses, microsporidia, nematodes, and fungi. The use of entomopathogenic fungi, although promising in several studies, has thus far met with limited success. Although newer formulation technologies have increased their field efficacy, the relatively slow kill rate of these fungi coupled to ant behavioral responses such as grooming and corpse removal continue to pose significant obstacles to the use of entomopathogenic fungi.

The virulence of a B. bassiana strain to fire ants by expressing a fire ant neuropeptide in the fungal pathogen: Increased virulence in the β-ΝΡ expressing fungal strain was noted in both standard and mock mound assays. The increased virulence was specific and no effects were detected against Lepidopteran hosts (Galleria mellonella and Manduca sexta), indicating that target-specific virulence can be achieved. This has significant potential for fungal strain improvement regulatory agencies approval for insect control applications.

Unexpectedly, an altered behavioral pattern in ants infected with the β-ΝΡ expressing strain was seen. Rather than forming organized corpse piles as seen in uninfected and wild- type infected ant assays, the dead appeared to remain dispersed throughout the assay chambers. Removal of dead nestmates is thought to limit the potential spread of pathogens, particularly within a social society, and is a common behavior in many ant species.

This observation pointed to altered behavioral effects resulting from application of the β- NP-expressing strain on fire ants. These behavioral effects were further probed using the various fungal strains as well as synthetic peptides. Worker ants have chemosensory perception mechanisms that are able to discriminate between surface peptides, and the β-ΝΡ- NH₂ peptide, but not the non-amidated form, can act as a semiochemical specifically eliciting enhanced necrophoretic behavior. Furthermore, the observed effect on necrophoretic behavior after infection by the β-ΝΡ (non-amidated)-expressing B. bassiana may be due to agonistic interactions with the host's amidated peptide and/or receptor, likely in conjunction with microbial pathogen/infection detection mechanisms. This result links, for the first time, β-ΝΡ-ΝΗ₂ and necrophoretic behavior. Topical application of PBAN/pyrokinins are known to induce pheromotropic and myotropic activity in live insects: however, the full range of their

physiological activities remains obscure. Members of the PBAN/pyrokinin family can apparently act as necrophorectic-eliciting cues on dead insects, expanding the potential physiological and sanitary roles of these peptides.

To further test whether the host molecules could be exploited for increasing the virulence of entomopathogenic fungi, two insect peptides, the Manduca sexta diuretic hormone (MSDH) and the Ae. aegypti trypsin modulating oostatic factor (TMOF), were engineered in B. bassiana, to be expressed and secreted by the fungus as it infects its host. Accordingly, the exogenously- produced host molecule would disrupt the normal endocrine or neurological balance of the host, making it more susceptible to the invading fungus. A broad host range target (MSDH) was used as well as a more host specific peptide (TMOF) that participates in critical host physiological processes so as to minimize non-target effects.

Insect diuretic hormones participate in the regulation of water balance and the Manduca sexta diuretic hormone (MSDH) belongs to the corticotropin-releasing factor-related family of peptides. Synthetic MSDH has been shown to stimulate fluid excretion in vivo, resulting in pronounced loss of fluid through the gut and the epidermis, decreased feeding, and ultimately death of the insect. Trypsin-modulating oostatic factors (TMOFs) are unblocked deca and hexapeptides found in insects including mosquitoes and flies that terminate trypsin biosynthesis in the insect gut. Aedes aegypti TMOF (Aea-TMOF) circulates in the hemolymph, binds to gut receptors on the hemolymph side of the gut and inhibits trypsin biosynthesis by exerting a translational control on trypsin mRNA. Because TMOF resists proteolysis in the gut and easily traverses the gut epithelial cells into the hemolymph in adults and larvae, it was fed to different species of mosquito larvae causing inhibition of food digestion anorexia, ultimately leading to starvation and death. TMOF is currently under development as an insecticide and appears to be very specific against mosquitoes with minimal non-target effects. TMOFs from different insects have different peptide sequences, e.g. the Ae. aegypti Aea-TMOF sequence is

YDPAPPPPPP (SEQ ID No.: 12) whereas the grey flesh fly, S. bullata, Sb-TMOF sequence is NPTNLH (SEQ ID No.: 13).

The Manduca sexta diuretic hormone (MSDH-Gly, 42 amino acid) and the Ae. aegypti TMOF (YDPAPPPPPP (SEQ ID No.: 12)) peptides were expressed in B. bassiana via transformation of expression vectors containing a constitutive β. bassiana-derived gpd- promoter, and the nucleotide sequence corresponding to the MSDH or TMOF peptide fused to a 28-amino acid signal sequence derived from the B. bassiana chitinase (chitl) gene to produce strains Bb::spMSDH and Bb::spAeaTMOF Bb::spp-NP. Heterologous expression of the peptides was confirmed by partial purification and mass spectrometry analysis of culture supernatants. These data indicated the production of TMOF and MSDH peptides by respective transformants of the fungus at concentrations ranging from about 0.2 to about 1.0 μΜ.

The efficacy of wild type and transgenic Beauveria bassiana strain expressing Aedes aegypti TMOF was evaluated against sugar- and blood-fed adult mosquitoes of the major African malaria vector Anopheles gambiae using insect bioassays. TMOF-expressing B.

bassiana increased fungal toxicity towards sugar- and blood fed adult A. gambiae. Mean lethal dose (LD₅₀) values for both sugar and blood-fed mosquitoes were decreased by approximately 40% after application of the TMOF-expressing strain as compared to the wild type parent. Mean lethal time (LT₅₀) values were lower for blood-fed as compared to sugar-fed mosquitoes in infections with both wild type and TMOF-expressing strains. However, infection using the latter resulted in 15% and 25% reduction in LT₅₀ values for sugar- and blood-fed mosquitoes, respectively, relative to the wild type parent. In addition, infection with the TMOF-expressing strain resulted in a dramatic reduction in fecundity of the target mosquitoes. B. bassiana, therefore, expressing Ae. aegypti TMOF exhibited increased virulence against A. gambiae relative to the wild type strain. These data expand the range and utility of entomopathogenic fungi expressing mosquito-specific molecules to improve their biological control activities against mosquito vectors of disease.

The feasibility of expressing host peptide molecules (hormones) in a fungal

entomopathogen to increase its virulence has been shown. Three candidate host molecules representing a wide distribution of targets and effectors were examined. Disruption of insect water balance via exploitation of insect diuretic hormones and an effective means of delivering the peptide to insects has been demonstrated. The data show that fungal pathogens can serve as a vehicle for exploiting these compounds.

/Aea-TMOF does not have vertebrate toxicity and has passed EPA/FDA approval. The /Aea-TMOF-expressing B. bassiana strain was effective against adults and larvae, causing a decrease in fecundity and abnormal development, respectively. It is contemplated that the methods of the present disclosure are also useful for the expression of biopesticides. The increase in virulence using the various host molecules of the disclosure was similar to that reported for expression of a scorpion toxin in the entomopathogenic fungus M anisopliae when tested against Ae. aegypti (about a 9-fold lower LC₅₀ and 38% reduction in survival times), indicating a robust increase in lethality without the concerns regarding expression of

neurotoxins.

In embodiments of this aspect of the disclosure, the heterologous recombinant nucleotide sequence can be operably linked to a gene expression controlling region, where the gene expression controlling region directs production of a transcript from the heterologous recombinant nucleotide sequence in a recipient entomopathogenic fungus.

In some embodiments of this aspect of the disclosure, the entomopathogenic fungus can be of the class Hyphomycetes.

In embodiments of this aspect of the disclosure, the entomopathogenic fungus can be virulent against the fire ant (Solenopsis invicta), a mosquito species, a Lepidopteran species, a Dipteran, or a Hemipteran species. In some embodiments of this aspect of the disclosure, the entomopathogenic fungus can be virulent against the fire ant (Solenopsis invicta).

In some embodiments of this aspect of the disclosure, the entomopathogenic fungus can be virulent against a mosquito species.

In embodiments of this aspect of the disclosure, the entomopathogenic fungus can be

Metarhizium anisopliae or Beauveria bassiana.

In embodiments of this aspect of the disclosure, the heterologous recombinant nucleotide sequence encodes an amino acid sequence specifically inducing a biochemical or physiological reaction in a target insect, wherein the amino acid sequence can be an insect neurohormone, an insect diuretic hormone, or a trypsin modulating oostatic factor.

In some embodiments of this aspect of the disclosure, the heterologous recombinant nucleotide sequence encodes an insect neurohormone.

In some embodiments of this aspect of the disclosure, the insect neurohormone can be a PBAN/pyrokinin or a bioactive fragment thereof.

In embodiments of this aspect of the disclosure, the heterologous recombinant nucleotide sequence encodes an insect neurohormone, or a bioactive fragment thereof, specific to the fire ant Solenopsis invicta.

In embodiments of this aspect of the disclosure, the insect neurohormone can be β- neuropeptide sequence specifically inducing a biochemical or physiological reaction in the fire ant Solenopsis invicta.

In embodiments of this aspect of the disclosure, the heterologous recombinant nucleotide sequence can encode an insect diuretic hormone.

In embodiments of this aspect of the disclosure, the heterologous recombinant nucleotide sequence can encode Manduca sexta diuretic hormone (MSDH).

In embodiments of this aspect of the disclosure, the heterologous recombinant nucleotide sequence can encode a trypsin modulating oostatic factor.

In embodiments of this aspect of the disclosure, the trypsin modulating oostatic factor can be a trypsin modulating oostatic factor of Aedes aegyti or S. bullata.

In embodiments of this aspect of the disclosure, the heterologous recombinant nucleotide sequence can encode a polypeptide having about 90% sequence similarity with a sequence selected from the group consisting of: SEQ ID Nos. 1 , 22, 23, and 24.

In embodiments of this aspect of the disclosure, the heterologous recombinant nucleotide sequence can encode a polypeptide having about 95% sequence similarity with a sequence selected from the group consisting of: SEQ ID Nos. 1 , 22, 23, and 24. In embodiments of this aspect of the disclosure, the heterologous recombinant nucleotide sequence can encode a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID Nos. 1 , 22, 23, and 24.

In embodiments of this aspect of the disclosure, the gene controlling region can have about 90% sequence similarity with a sequence encoding a B. bassiana chitinase gene signal peptide.

In some embodiments of this aspect of the disclosure, the B. bassiana chitinase gene signal peptide can have the amino acid sequence according to SEQ ID No.: 2.

entomopathogenic fungus when said fungus has infected a target insect host, the insect-specific polypeptide increasing the virulence of the fungus in the target insect compared to the virulence of a strain of the entomopathogenic fungus not expressing the insect-specific polypeptide.

In embodiments of this aspect of the disclosure, the gene controlling region has about 90% sequence similarity with a sequence encoding a B. bassiana chitinase gene signal peptide

In embodiments of this aspect of the disclosure, the insect specific polypeptide can be selected from the group consisting of: an insect neurohormone, an insect diuretic hormone, and a trypsin modulating oostatic factor, or a bioactive homolog or fragment thereof.

In embodiments of this aspect of the disclosure, the insect-specific polypeptide can be selected from the group consisting of: β-neuropeptide specific to the fire ant Solenopsis invicta, Manduca sexta diuretic hormone (MSDH), and a trypsin modulating oostatic factor of Aedes aegyti or S. bullata.

Yet another aspect of the disclosure encompasses embodiments of a method of increasing the virulence of an entomopathogenic fungus in a target insect host, comprising the steps of: (a) obtaining a genetically-modified strain of a entomopathogenic fungus according to any of claims 1 -22; and (b) delivering the genetically modified strain of entomopathogenic fungus, or spores thereof, to a target insect host desired to be infected by the fungus, wherein the fungus delivers a target insect-specific polypeptide or peptide to the target insect host, thereby increasing the virulence of the entomopathogenic fungus in the target insect host. In embodiments of this aspect of the disclosure, the entomopathogenic fungus can be Metarhizium anisopliae or Beauveria bassiana.

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to perform the methods and use the compositions and compounds disclosed and claimed herein. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C, and pressure is at or near atmospheric. Standard temperature and pressure are defined as 20 °C and 1 atmosphere.

It should be noted that ratios, concentrations, amounts, and other numerical data may be expressed herein in a range format. It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a concentration range of "about 0.1 % to about 5%" should be interpreted to include not only the explicitly recited concentration of about 0.1 wt% to about 5 wt%, but also include individual concentrations (e.g., 1 %, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.5%, 1.1 %, 2.2%, 3.3%, and 4.4%) within the indicated range. The term "about" can include ±1 %, ±2%, ±3%, ±4%, ±5%, ±6%, ±7%, ±8%, ±9%, or ±10%, or more of the numerical value(s) being modified.

EXAMPLES

Example 1

Construction of expression vector and fungal transformation: The nucleotide sequence of a construct encoding the polypeptide

MAPFLQTSLALLPLLASTMVSASPLAPRAGRMPSLSIDLPMSVLRQKLSLEKERKVHALRAAAN RNFLNDIG (SEQ ID No.: 1 ) contained an ORF corresponding to the M sexta diuretic hormone fused to a 28 amino acid B. bassiana chitinase gene signal peptide

(MAPFLQTSLALLPLLASTMVSASPLAPRAG) (SEQ ID No.: 2) and was synthesized using a commercial DNA synthesis service (Bio Basic Inc, ON, Canada). The construct (pSP-MSDH) was then used as a template for subcloning into a B. bassiana expression vector. The primers PspMsDH-1 (5'-ATGGCTCCTTTTCTTCAAAC-3' (SEQ ID No.: 3)) and PspMsDH-2 (5'- TTAGCCAATGTCGTTGAGAAA-3' (SEQ ID No.: 4)) were used to subclone the gene fused to a B. bassiana glyceraldehyde phosphate dehydrogenase promoter (PgpctA-Bb) as amplified by primers PgpdA 1 (5'-GTTGGGTATGCTCCGGC-3' (SEQ ID No.; 5)) and PgpdA2 (5'- TGTTATTGATTAAAAGGGTGAGTTTGAAGAAAAGGAGCCAT-3' (SEQ ID No.: 6)). The latter primer pair was designed to contain a 20 bp overlap sequence between P_gpdA-Bb and pSP- MSDH.

The desired construct (P_gp_dA-Bb:SP-MSDH) was produced via primerless assembly in a reaction mixture containing: 5 μΙ 5x Phusion Taq polymerase buffer, 2μΙ 2.5 mM dNTP, 30 ng PgpdA-Bb, 30 ng pSP-MSDH, 0.4U Phusion Taq DNA polymerase, total volume 25μΙ. PCR reaction cycling conditions: 98 °C (2 min); followed by 25 cycles of: 98 °C (20 s), 56 °C (30sec), 72 °C (1 min); and 72 °C (5 min).

Primer pair PgpdAI (SEQ ID No.: 5) and PspMsDH-2 (SEQ ID No.: 4) were then used to obtain the PgpctA-Bb:SP-MSDH fragment using the assembled product as template. The assembled PCR product was cloned into pBlunt vector yielding pBP_gpd-SP- MSDH, and verified by sequencing. The fragment was then subcloned into a vector (pUC-bar) containing the bar gene encoding for phosphinothricin resistance as a selection marker for transformation into B. bassiana (Fan et al., (201 1 ) Invertebr. Pathol. 106: 274-279, incorporated herein by reference in its entirety) using EcoRI restriction sites to obtain pUC-foar-P_gpd-SP-MSDH. The vector was linearized with Xbal and transformed into B. bassiana competent cells as described.

The S. invicta pyrokinin β-neuropeptide (β-ΝΡ having amino acid sequence QPQFTPRL

(SEQ ID No.: 7)) was fused to the 28-amino acid signal peptide (SEQ ID No.: 2) derived from the B. bassiana chitinases-1 (chitl) gene (resulting in amino acid sequence

MAPFLQTSLALLPLLASTMVSASPLAPRAGQPQFTPRL (SEQ ID No.: 24)) and cloned under control of the B. bassiana glyceraldehyde phosphate dehydrogenase promoter (Pgpd-Bb). Primer pairs P1/P2 (5 -GTTGGGTATGCTCCGGCGCG (SEQ ID No.: 8), and 5'-

GGTTGTTATTGATTAAAAGG (SEQ ID No.: 9) were used to amplify P_gpdA-Bb using B. bassiana genomic DNA as templates. The B. bassiana chitinase (Bbchit1)-der\ved signal peptide (SEQ ID No.: 2) (SP) was obtained with the primer pair P3/P4 (P3: 5'- C CTTTTAATC AAT AAC AAC C ATG G CTC CTTTTCTTC AAAC (SEQ ID No.. 10); P4: 5'- TTAGAGGCGGGGGGTAAACTGGGGCTGTCGCGGCGCCAAGGGCGAGG (SEQ ID No.: 1 1 )) using B. bassiana genomic DNA as the template and with the β-ΝΡ coding sequence incorporated into primer P4 (SEQ ID No.: 1 1 ). These primers were designed containing a 20 bp overlap sequence between P_gpdA-Bb and SP:p-NP. The desired construct (PgpdA-Bb:p-NP) was produced via primer-less assembly in a reaction mixture containing: 5μΙ 5 x Phusion Taq polymerase buffer, 2μΙ 2.5 mM dNTP, 30ng P_gpdA-Bb, 30ng SP:p-NP, 0.4 U Phusion Taq DNA polymerase, total volume 25 μΙ. PCR reaction cycling conditions: 98 °C (2 min); followed by 25 cycles of: 98 °C (20 s), 56 °C (30sec), 72 °C (1 min); and 72 °C (5 min). Primer pair P1 & P4 were used to obtain the P_gpdA._Bb:SP-p-NP fragment using the assembled product as template. The obtained fragments were cloned into pDrive vector (Qiagen) and verified by sequencing. PgpdA-Bb:SP-p-NP was subcloned from pDrive vector via EcoRI restriction sites into pUC-Bar, yielding pUC-Bar-P_gpdA__Bb:SP-p-NP. This plasmid was linearized with Xbal and transformed into B. bassiana competent cells, as described in Zhang et al., (2010) Appl. Microbiol. Biotechnol. 87: 1 151-1 156, incorporated herein by reference in its entirety. The resultant strain was labeled Bb::spp-NP_gpd.

Example 2

Construction of expression vector and fungal transformation: Ae. aegypti and N. bullata TMOF peptides (YDPAPPPPPP (SEQ ID No.: 12) and NPTNLH (SEQ ID No.: 13), respectively) were fused to the 28 amino acid signal peptide (SEQ ID No.: 2) derived from the B. bassiana chitinase gene to secrete the peptides into the host. The peptide sequences

MAPFLQTSLALLPLLASTMVSASPLAPRAGYDPAPPPPPP (SEQ ID No.: 22) and

MAPFLQTSLALLPLLASTMVSASPLAPRAGNPTNLH (SEQ ID No.: 23) were cloned downstream of a B. bassiana glyceraldehyde phosphate dehydrogenase promoter (P_gpd.Bb) in the presence of the bar gene encoding for phosphinothricin resistance as a selection marker for transformation into B. bassiana. Primer pairs P1/P2 (5'-TCAGATCTCGGTGACGGGCAG (SEQ ID. No.: 14) and 5'-GTCGACAGAAGATGATATTG (SE ID No.: 15)) and P3/P4 (5'- CAATATCATCTTCTGTCGACCTCTAGAGAATTCGTTGGGT (SEQ ID No.: 16) and 5'- GGTTGTTATTGATTAAAAGGG (SEQ ID No.: 17)) were used to produce: (1 ) P_trpc:6ar, containing the bar gene cassette under control of the TrpC promoter, and (2) P_gpdA-_Bb, the B. bassiana gpd promoter, using the plasmid pBar-GPE1 and B. bassiana genomic DNA as templates, respectively.

The B. bassiana chitinase-derived signal peptide (SP) (SEQ ID No.: 2) and Ae. aegypti TMOF-coding sequences were incorporated into the primer pair P5/P6 (P5:

CCCTTTTAATCAATAACAACCATGGCTCCTTTTCTTCAAAC (SEQ ID No.: 18); P6:

TTAAGGAGGAGGAGGAGGAGGGGCGGGGTCGTATCGCGGCGCCAAGGGCGAGG (SEQ ID No.: 19)). The primer pairs were designed containing 20 bp overlap sequences, i.e.

between: (1 ) P_trpc:bar and P_gpdA-Bb, and (2) P_gpdA-Bb and SP:AeTMOF, respectively. The desired construct (P_trpc'.bar. Pgp_dA. b:SP-AeTMOF) was produced via primerless assembly in a reaction mixture containing: 5μΙ 5x Phusion Taq polymerase buffer, 2μΙ 2.5 mM dNTP, 30ng P_trpcBar, 30ng Pgp_dA Bb, 30 ng SP:AeT OF, 0.4U phusion Taq DNA polymerase, total volume 25μΙ. PCR reaction cycling conditions: 98 °C (2 min); followed by 25 cycles of: 98 °C (20 s), 56 °C (30 s), 72 (1 min); and 72°C (5 min). Primer pair P1 (SEQ ID No.: 14) and P6 (SEQ ID No.: 19) were used to obtain the P_Upc'.bar. Pgp_dA._Bb:SP-AeTMOF fragment using the assembled product as template. The N. bullata-JMOF construct P_lTpc.bar.P_gpdA._Bb:SP-NbTMOF) was produced by PCR using the primers P1 (SEQ ID No.: 14) and P7

(TTAGTGGAGGTTAGTGGGGTTTCGCGGCGCCAAGGGCGAGG (SEQ ID No.: 20)) using P_trpC: bar. Pg_pciA._Bb:SP-AeTMOF as the template. PCR products corresponding to P_tr_Pc: bar Ρ_9ρ£Μ. _Bb:SP-NbTMOF and P_Xrpc.bar.P_gp(j_A__Bb:SP-NbTMOF

cloned into pMD-T (Promega) and the integrity of the inserts verified by sequencing. The resultant strains were labeled Bb::spMSDH, Bb::spAeaTMOF, and Bb::spf3- NPgpct.

Example 3

Purification and identification of TMOF from fungi cultures using HPLC and MS/MS: To verify (extracellular) TMOF production in the recombinant B. bassiana strain fungal cultures

(Ptrpc'.bar.

and the WT parent) were grown first in SDBY (Sabouraud dextrose broth with 0.5% yeast extract) for 2 days, after which 2.5 g of washed cells were transferred to Czapek-dox broth (150 ml) for 3 days.

Fungal cells were removed by centrifugation, the resulting supernatant filtered through a 0.22μΐη filter, and the samples subsequently lyophilized and stored at -20 °C until required Lyophilized samples were rehydrated in 4.0 ml of water containing 0.1 % TFA, and applied onto a Ci₈ reverse phase SepPak column. The column was washed with 4.0ml 0.1 % TFA and peptides were eluted with 4.0ml acetonitrile-0.1 % TFA. The eluted fraction (in acetonitrile) was dried by SpeedVac, resuspended in water-0.1 % TFA (0.6ml) and chromatographed on a C₁₈ reversed phase HPLC column with a linear gradient of acetonitrile-water in the presence of 0.1 % TFA (0 to 100%) and eluted factions were monitored at 220nm. Fractions between 19 mins to 22 mins were collected, dried with a fine stream of nitrogen, rehydrated to 0.6ml with water-0.1 % TFA, and rechromatographed as above. Fractions 19-22 were collected dried under N₂ and analyzed by MS/MS. A standard curve using synthetic TMOF was made to quantify the amount of TMOF in the sample. The entire analysis was repeated twice.

Quantified samples represented a 200-fold dilution of the original sample, and the concentration of TMOF derived from each analysis was 4.97nM and 4.94nM, respectively, indicating an original concentration of TMOF in the culture supernatants of about 1 μΜ. Example 4

Insect Bioassays: Galleria mellonella larvae were treated via topical application of fungal conidia harvested in sterile distilled water. Individual insects were immersed in fungal solutions (10⁴-10⁸ conidia/ml) for 3-6 sec, and the excess liquid on the insect bodies removed by placement on dry paper towel. Controls were treated with sterile distilled water. Experimental and control larvae were placed in plastic chambers or large (150mm) Petri dishes and incubated at 26 °C. For each experimental condition, approximately 40 larvae were used, and all experiments were repeated three times. The number of dead insects was recorded daily and median lethal mortality time (LT₅₀) was calculated by Probit analysis.

Example 5

Mosquito larvae: Aedes aegypti larvae (in groups of 20, day 1 of hatching) were reared in glass jars (mouth diameter = 8 cm) containing 100 ml autoclaved tap water supplemented with 1 ml Brown food (3% bovine liver powder and 2% Brewer's yeast). Fungal cultures were grown on potato dextrose agar (PDA). Plates were incubated at 26 °C for 14-21 days, and aerial conidia (spores) were harvested by flooding or scraping the plates with sterile distilled water containing 0.05% Tween 80. Spore concentrations were determined by direct count using a

hemocytometer and adjusted to the desired concentration for use (typically between 10⁷-10⁸ conidia/ml). For larval assays, the conidial suspension was centrifuged at top speed in a tabletop microfuge (14,000xg) for 10 min, and resuspended in the final desired volume

(depending upon the concentration wanted) of grapeseed oil. The spore suspension (1 ml) was then applied onto the surface of water and briefly mixed in the glass test chamber. Experiments were performed at room temperature. All tests were done in triplicate using at least three different batches of conidia. Dead larvae were removed and mortality determined daily.

Example 6

Mosquito adults: Larvae of Ae. aegypti were reared at 27 °C on a diet of brewer's yeast and lactalbumin (1 : 1 ) with 16:8 light:dark cycle. Adults were fed on 10% sucrose or on chicken blood. Females were used 3-5 days after emergence. Bioassays were performed on blood fed females, 3 days after feeding. Mosquitoes were anesthetized using ether, and 0.25μΙ of conidial suspensions (10⁴-10⁸ conidia/ml in grapeseed oil) was placed on the abdomen of each individual insect. Experiments were performed at room temperature. All tests were done in triplicate using at least three different batches of conidia. Mortality was determined daily.

Example 7

Trypsin activity measurement: To measure trypsin activity, 5 larvae were manually

homogenized in Eppendorf tubes containing 500 ml of 50 mM Tris-HCI, pH 8.2, 20 mM CaCb. The homogenates were then centrifuged at 4 °C for 10 min at 10,000 x g and the supernatants were collected and aliquots assayed for trypsin activity. Assays were carried out in 96-well plates using 20 ml supernatant, 20 ml buffer and 160 ml of a substrate solution of 1 mM N-a- benzoyl-DL-arginine-p-nitroanilide (BapNA, final concentration 0.8mM) as described in Borovsky & Schlein (1988) Arch. Insect Biochem. Physiol. 8: 249-260, incorporated herein by reference in its entirety. The enzymatic reaction was continuously monitored at 405 nm over 20 min using the kinetic mode of BioTek μΩυβηί microplate reader (Winooski, Vermont).

Example 8

Anopheles gambiae G3 strain was reared as described by Danielli et a/., (2000) Proc. Natl. Acad. Sci. U.S.A. 97: 7136-7141 , incorporated herein by reference in its entirety. Mosquito eggs were treated routinely with 1 % VIRKON.RTM for 4-5 mins before floating them, to avoid spread of opportunistic infections in the colony.

Wild-type and SJb-Aa1 B. bassiana strains were cultured and spores collected and counted as described in Example 5.

Insect bioassays were performed by spraying batches of 45 mosquitoes each with spore suspensions of the indicated B. bassiana strains and mortality was scored on a daily basis. The

Kaplan-Meier survival test was used to calculate the percent mortality over the indicated time scale. Statistical significance of the observed differences was calculated by the Log-Rank test.

Differences were considered to be significant if P < 0.05. LT₅₀ values for both B. bassiana strains were calculated from survival curves of sugar- and blood fed mosquitoes infected with

1x10⁸ spores/ml using regression analysis. Statistical analysis of LT₅₀ values was performed using the Student's T-test. LD₅₀ values were determined using concentrations ranging from

1x10⁷ to 2x10⁸ spores/ml and calculated by Probit analysis.

To study the effect of fungus infection on mosquito fecundity, A. gambiae females sprayed with a suspension of 1x10⁸ spores/ml of wild type or S0-Aa1 strain, were given a blood meal 24 h after fungal infection. Blood-fed females were placed individually into paper cups and eggs were counted 48 h after blood feeding. Statistical analysis was performed using the Mann

Whitney test.

Example 9

A. gambiae: Sugar- and blood-fed adult female A. gambiae were exposed to spores (conidia) of strain B6-Aa1 or the wild type parent to determine the effect of \ea-TMOF expression on virulence. Bb-Aa1 was more potent than its wild type parent against both sugar and blood-fed adults causing 40% reduction in LD₅₀ values (50% mortality) in both groups compared to the wild type control (Table 1 ). Table 1: LD₅₀ and LT₅₀ values of wild type and Aea-TMOF expressing Bb-Aa1 strain against

Anopheles gambiae.

LT₅₀ values were calculated from bioassays in which mosquitoes were infected by spraying with a suspension of 1x10⁸ conidia/ml. Statistical analysis was performed using the Student's T-test and values were considered significant if P<0.05. LD₅₀ values were calculated from the 76-h time point using the Probit analysis.

LD₅₀ values were similar between sugar- and blood-fed mosquitoes infected with the same fungal strain, regardless its type (Bb- a^ or wild type). Infection with Bb-f a also induced a 15% and 25% reduction in the mean survival times (LT₅₀ values) of sugar- and blood- fed mosquitoes, respectively, compared to the wild type strain. LT₅₀ values were also lower for blood-fed compared to sugar-fed mosquitoes infected with the same strain, regardless its type. The fact that infections with the same strain, irrespective of its type, resulted in similar LD₅₀ values between sugar- and blood-fed mosquitoes, but lowered consistently the LT₅₀ values for the blood- compared to sugar fed group, indicates that the blood meal itself does not seem to alter the virulence of a particular B. bassiana strain but rather mosquito tolerance to infection.

Infection of A. gambiae mosquitoes with wild-type B. bassiana strain resulted in a significant reduction (approximately 16%) in fecundity compared to non-infected controls, as shown in Figs. 7A and 7B. Expression of /Aea-TMOF resulted in a significant reduction

(approximately 60%) in fecundity compared to controls, as shown in Fig. 8. These data suggest that strain S Aa1 also has the potential of reducing the size of A. gambiae mosquito populations by severely compromising fecundity.

Example 10

Assays for egg laying and egg development: Female mosquitoes (29 to 69 females) 72 h after treatment with wild type and TMOF-producing fungi were dissected under the microscope, oocytes removed and yolk length measured and expressed as μηη ± SEM (Borovsky, D. (1988) Arch. Insect Biochem. Physiol. 7: 187-329).

Groups of female Ae. aegypti (30 females per group) were fed a blood meal and immediately infected with fungi expressing TMOF and 5 days later the surviving adults were provided an oviposition site of wet paper towel and females were allowed to lay their eggs for 72 h. At that time the paper towels with eggs were removed, dried at room temperature for 24 h and counted under a dissecting microscope. Control groups included untreated and WT B. bassiana-treated insects. The number of eggs in the control and experimental groups were compared to determine if the genetically modified fungi expressing TMOF suppress egg development.

Example 11

N. bullata adult: For bioassay of adult flesh flies (N. bullata, Carolina Biologjcal Supply Co. , USA), the flies were briefly chilled (6 °C, 15 min) to reduce their movement, and 3μΙ of a conidial suspension (adjusted to desired spore concentration in grapeseed oil) were applied onto the abdominal region. Flies (batched of 25) were housed in a plastic container containing sugar cubes and water. Experiments were performed at room temperature. Test conditions were performed in triplicate and the entire experiment was performed with at least three different batches of conidia. Dead flies were removed and mortality determined daily.

Example 12

Expression of the Manduca sexta diuretic hormone by the entomopathogenic fungus B. bassiana resulted in greater than a 10-fold increase in virulence (LD₅₀) towards the

Lepidopteran host, the greater waxmoth, Galleria mellonella (Table 2).

Table 2: Calculated LD₅₀ and LT₅₀ of WT, MSDH, and TMOF expressing B. bassiana strains against G. mellonella and Ae. Aegypti

a LD₅₀ calculated from 96 hr time point.

b LD₅₀ calculated from 120 hr time point.

c Accurate LD₅₀ could not be determined in this case.

d Bioassay performed using spore concentration of 1 x 10⁷ conidia/ml.

e Bioassay performed using 0.25 μΙ of a 1 x 10⁸ conidia/ml spore solution applied to mosquito abdomen.

f Bioassay performed using spore concentration of 1 x 10⁸ conidia/ml. The MSDH-expressing strain also showed a 36% reduction in the survival time (LT₅₀) of the target (Table 1 , Fig. 1A). Ae. aegypti adults and larvae were tested with recombinant B. bassiana spores to determine the effect of /Aea-TMOF expression on virulence.

Bb::spAeaTMOF was more potent than its wild-type parent against blood-fed female adults, displaying the need for 7-fold fewer conidia of Bb::spAeaTMOF to obtain the same level of control as WT (Table 1 , P < 0.01 ). Expressing AeaTMOF also resulted in a 25% reduction in the survival time of the target mosquitoes (Table 1 , P < 0.01 ). Modulation of trypsin activity was confirmed after fungal infection, with Bb::spAeaTMOF-infected females showing a 50% reduction in trypsin activity (Fig. 1 B). In addition, a dramatic drop in fecundity was noted, with a 40% reduction in the number of eggs laid by TMOF-infected females as compared to WT- infected females (which were themselves 16% lower than uninfected controls, P < 0.01) (Fig. 1 C).

Microscopic examination of the ovaries of Bb::spAeaTMOF-infected females showed that many of the oocytes were smaller and underdeveloped as compared with WT and control, (that were essentially indistinguishable from each other). Bb::spAeaTMOF also displayed increased virulence towards Ae. aegypti larvae. In this instance, an accurate LD₅₀ could not be calculated since a threshold inoculum appeared to be needed in order for effective control to occur.

The LT₅₀ value, however, using spore concentrations high enough for the WT to kill most insects (1 x10⁸ conidia/ml), was dramatically reduced for Bb::spAeaTMOF as compared to the WT, dropping from 6.5 to 4.2 days (P < 0.01 ). In addition, Bb::spAeaTMOF treated larvae were significantly smaller that WT treated ones and their normal development was impaired (Figs. 1 D-1 F). Since the sequences of TMOF peptides do not occur in vertebrates and are different in mosquitoes and flesh flies, and host range of entomopathogenic fungi are in general determined by cuticular recognition cues, expression of TMOFs which would be active in post-penetration events is unlikely to compromise safety and selectivity. To investigate this and determine the potential level of specificity of Bb::spAeaTMOF, a B. bassiana strain expressing the S. bullata- TMOF (NPTNLH, Bb::spSbTMOF) was constructed. Bioassays using Bb::spAeaTMOF showed no increase in virulence when compared with the WT strain against S. bullata. Conversely, Bb::spSbTMOF was no better than the WT strain against Ae. aegypti.

Example 13

Purification and identification of β-ΝΡ from fungi cultures using HPLC and MS/MS: To verify (extracellular) β-ΝΡ production in the recombinant B. bassiana strain, fungal cultures (Bb::spp- NP_gpd and the WT parent) were grown first grown in SDBY (Sabouraud dextrose broth with yeast extract) for 2 days, after which 1.5 g of washes cells were transferred to Czapek-dox broth (50-100 ml) for 3 days. Fungal cells were removed by centrifugation, the resultant supernatant filtered through a 0.22μηη filter, and the supernatant samples subsequently lyophilized and stored at -20 °C until used. Lyophilized samples were rehydrated in 3.0ml of water containing 0.1 % TFA, and applied onto a C18 reverse phase SepPak column. The column was washed with 0.1 % TFA and peptides were eluted with 80% acetonitrile-0.1 % TFA. The eluted fraction (in acetonitrile) was dried in a SpeedVac, resuspended in water-0.1 % TFA (0.5ml) and chromatographed on a C18 reversed phase HPLC column with eluting factions monitored via absorbance at 214nm. Fractions eluting at the same retention time as an initial run using synthetic β-ΝΡ used as a standard were collected, dried with a fine stream of nitrogen, rehydrated to 0.2ml with water-0.1 % TFA, and rechromatographed as above. Fractions were collected as above, dried under N₂ and analyzed by LC-MS/MS. A standard curve using synthetic β-ΝΡ was used to quantify the amount of peptide in the sample.

Example 14

Insect Bioassays: S. invicta colonies were collected from the field, separated from the soil by drip flotation and maintained in Fluon-coated trays with a diet consisting of 10% sucrose solution, a variety of freeze-killed insects, fruits and vegetables, and chicken eggs.

Fungal cultures were grown on potato dextrose agar (PDA). Plates were incubated at 26 °C for 14-21 days, and aerial conidia (spores) were harvested by flooding or scraping the plates with sterile distilled water containing 0.05% Tween 80. Spore concentrations were determined by direct count using a hemocytometer and adjusted to the desired concentration for use (typically between 10⁶-10⁸ conidia/ml).

Two types of bioassays were used to assess the virulence of the fungal strains. A "classical bioassay" using S. invicta workers. Test groups of ants (25/chamber) were inoculated with fungal suspensions (concentrations ranging from 10⁶-10⁸ conidia/ml) using a spray tower as described in Pereira et a/., (1993) J. Invert. Pathol. 61 : 156-161. The ants were housed in plastic cups (0 = 6 cm) whose sides had been coated with Fluon and topped with a perforated lid. Ants were given 10% sucrose solutions in 1.5ml Eppendorf tubes with a cotton plug.

Experiments were performed at 26 °C and mortality was recorded daily. Controls were treated with Tween-80 and the mortality assays were repeated at least three times

In "mock mini-mound" assays, larger scale bioassays were performed using larger test chambers (0=19cm). Test chambers contained a small Petri dish (0 = 3cm) containing moist dental plaster that served as the nest for the mini-mound. Ants (0.5 gm, approximately 2,000 individuals) including 3-4 dealate reproductive females were placed in the test chamber that included 10% sucrose solution in an Eppendorf tube placed in the test chamber. Treatments and assay conditions were identical to the classical bioassay. Duplicate samples were performed for each experiment and the entire assay repeated three times with independent batches of fungal spores. For all experiments, a X²-test was first used to determine

homogeneity among variance of the repeats (p<0.05). Further statistical analysis of the mortality was performed using SPSS which was used to estimate the median lethal time (LT₅₀), the median lethal concentration (LC₅₀), fiducial limits and other regression parameters.

Example 15

Necrophoretic behavior assays: Assay chambers and methods were based upon a previously described protocol Pereira et a/., (1993) J. Invert. Pathol. 61 : 156-161 ). Briefly, the conical end of a 15 ml polypropylene tube (nest) was cut off and connected via a short tubing (ø = 8mm, 10cm long) to a round plastic container (ø = 19cm, foraging arena) into which a hole had been punched out in the bottom at the middle of the container. Test ants (0.1 gm, about 500 ants with at least one dealate) were placed in the assay chamber and allowed to equilibrate for 1-2 hr before the experiment was initiated.

Three separate experimental protocols were employed: (1 ) Freeze killed, ants killed by the WT B. bassiana strain, and ants killed by the Bb::spp-NP_gpd strain were presented to untreated ants. For the freeze-killed ants, ants were placed at -80 °C for 15 min, and then placed at room temperature for 24 hr before use. For fungal-killed ants, infections were performed as described above and the dead ants removed daily. Test ants were derived from those that died on day 4 post-infection. To measure necrophoretic behavior, the test items (5- 10 dead ants) were placed in a ring around (1 cm from) the nest entrance. The time interval between introduction and removal of each item was recorded up to a time limit of 600-800 minutes. The number of test objects that were not moved within this interval was also recorded. (2) The effect of infection on necrophoretic behavior was probed by presenting WT- or Bb::sp - NPgpd-killed ants to (a) uninfected ants; (b) WT-infected ants; or (c) Bb::sp -NP_gpd-infected ants. Ants were infected with the fungal strains (5 x 10⁷ conidia/ml) 2 days prior to testing. Test objects (dead ants) were prepared and tested as described above. (3) The effect of synthetic peptides on ant cadaver removal was evaluated by having three peptides; (a) β-ΝΡ

(QPQFTPRL (SEQ ID No.: 7, no C-terminal amidation), (b) β-ΝΡ-ΝΗ₂, and (c) QAGVTGHA (SEQ ID No.: 21 ) carboxy-terminus amidated (control amino acid amidated peptide) were synthesized (GenScript, Piscataway, NJ). Freeze-killed ants (15 min at -80 °C, allowed to thaw for 15 min at room temperature), were immersed in 100nM solutions (resuspended in sterile distilled water) of the test peptide or water alone for 30 sees and then allowed to air-dry for 30 mins on a Kim-wipe towel. Necrophoretic behavior to the ants was measured as described above. P-values were obtained from an analysis of variance (1 or 2 way-ANOVA) for each data set, using a permutation test to guard against possible non-normality. 10,000 permutations were used for each test statistic. The unknown (i.e. never moved test objects) data had no effect on the analysis.

Example 16

Construction and bioassay of β-ΝΡ expressing B. bassiana: The fire ant β-ΝΡ, comprised of the eight-amino acid sequence, QPQFTPRL (SEQ ID No.: 7) was expressed in B. bassiana via transformation of an expression vector containing a constitutive B. bass/ana-derived gpd- promoter, and the nucleotide sequence corresponding to the β-ΗΡ peptide fused to a 28-amino acid signal sequence derived from the B. bassiana chitinase (chitl) gene (SEQ ID No.: 21 ) to produce strain Bb::sp -NP_gpd. Heterologous expression of the peptide was confirmed by partial purification and mass spectrometry analysis of culture supernatants. These data indicated the production of a non-amidated ?-NP peptide by the fungus at a concentration of about 0.2-0.4 μΜ. Both classical worker group and mock mound assays were used to assess the virulence of WT and β- P expressing B. bassiana strains. Bb::spp-NP_gpd was much more potent (P < 0.001 ) than WT, causing 50% mortality against fire ants after 5 days post-infection with an LD₅₀ of 1.5 ± 0.9 x 10⁷ conidia/ml compared to an LD₅₀ of 1.0 ± 0.7 x 10⁸ conidia/ml for the WT parent. Thus, it takes 6-7-fold fewer conidia to provide the same level of mortality. Expressing /?-NP also significantly reduced survival times (Fig. 2). At a concentration of 2 x 10⁷ conidia/ml, the mean lethal time to achieve 50% mortality (LT₅₀) was reduced from 177 ± 1 1 hr for the WT to 122 ± 5 hr for the β- P expressing strain, representing approximately 30% reduction in the mean survival time. At lower spore concentrations (4 x 10⁶ conidia/ml) the effect was even more dramatic, with the WT LT₅₀ reaching 21 1 ± 23 hr and the β-ΝΡ expressing strain 135 ± 7 hr (P < 0.001 ). To determine whether expression of the fire ant β-ΝΡ would affect virulence towards other insect, bioassays were performed with several other insects. No significant difference was noted between the virulence of the WT and Β^:5ρβ-ΝΡ_9ρϋ strains towards the lepidopteran host, Galleria melloneila, in which the LT50 values were 158 ± 5 hr and 166 ± 8 hr, for the WT and β-ΝΡ expressing strains, respectively (P > 0.05). Similarly, no difference was noted between the WT and β-ΝΡ expressing strains when tested against the tobacco hornworm, Manduca sexta. Example 17

Alterations in ant social behavior mediated by β-ΝΡ: In the course of performing mock fire ant mound experiments we noted that ants infected with the Bb::spp-NP_gpd strain appeared altered in their necrophoretic, or disposal of the dead, behavior (Fig.3). Whereas mock-treated and WT B. Jbass/ana-infected ants disposed of their dead in well defined "bone piles", Bb::spp-NP_gpd- infected ants appeared to have randomly scattered piles of dead throughout the assay chamber, although typically at the periphery. To further probe this observation, the responses of workers to estimate corpses was examined by placing corpses near the nest entrance in an experimental arena and monitoring the time taken to remove the corpses. Workers moved ants killed by the WT B. bassiana strain faster than freeze killed ants (about 24 hr old, P = 0.0014), but not those killed by the Bb::spp-NP_gpd strain, which showed a wider variation, but was not significantly different from the response to the freeze killed ants (Fig. 4). Thus, expression of the β-ΝΡ peptide appeared to delay removal of corpses. The large variation in removal time observed with Bb::spp-NP_gpd -infected ants may be due to differences in levels of β-ΝΡ expression in infected ants resulting from differential fungal growth within individual ant hosts. The infection state of the ants themselves did not appear to make a significant difference (P = 0.86). When WT or Bb::spp-NP_gpd -infected ants were presented with either WT- or Bb::spp- NP_gpd-killed ants, they moved the Bb::spp-NP_gpd -killed ants more slowly than WT-killed ones (P < 0.001 , Fig. 5). These experiments confirmed that ants killed by Bb::spP-NP_gpd were treated differently than WT-killed ants, which were more rapidly removed regardless of the infection state of the ants themselves. This finding has potentially important application consequences since it may increase the lethality of the fungus in field applications due to reduced removal of cadavers which would increase the contact time and possible dispersal of the fungal agent within mounds.

To further probe the effects of β-ΝΡ, a series of synthetic peptides were examined.

Since both pheromonotropic and myotropic activity of pyrokinin/PBAN peptides have been demonstrated via topical application of the peptides onto insects to determine the effects of β- NP-NH₂ (C-terminal amidated), β-ΝΡ (non-amidated peptide), and a control amidated peptide (QAGVTGHA (SEQ ID No.: 21 ) carboxy-terminus amidated) on the necrophoretic behavior of the fire ants. Freeze-killed ants were immersed in a 100 nM solution of the tested synthetic peptides and presented to untreated ants.

Ant corpses treated with the β-ΝΡ-ΝΗ₂ peptide were moved significantly faster than buffer treated ants, β-ΝΡ-treated ants, or ants treated with a control eight-amino acid amidated peptide (P < 0.001 , Fig. 5). β-ΝΡ-treated ants were not moved any slower than control or buffer treated ants, although their distribution and the number of ants that were never removed within our assay conditions was larger for the β-ΝΡ treatment than for any other treatment examined.

Claims

CLAIMS We claim:

1 . A genetically modified strain of an entomopathogenic fungus comprising a heterologous recombinant nucleotide sequence encoding a peptide, polypeptide, or protein of a target insect host wherein, when said nucleotide sequence is expressed by the genetically modified entomopathogenic fungus when said fungus has infected the target insect host, the peptide, polypeptide, or protein increases the virulence of the fungus in the target insect compared to the virulence of a non-genetically modified strain of the entomopathogenic fungus in the target insect host.

2. The genetically modified strain of an entomopathogenic fungus of claim 1 , wherein the heterologous recombinant nucleotide sequence is operably linked to a gene expression controlling region, wherein the gene expression controlling region directs production of a transcript from the heterologous recombinant nucleotide sequence in a recipient

entomopathogenic fungus.

3. The genetically modified strain of an entomopathogenic fungus of claim 1 , wherein the entomopathogenic fungus is of the class Hyphomycetes.

4. The genetically modified strain of an entomopathogenic fungus of claim 1 , wherein the entomopathogenic fungus is virulent against the fire ant (Solenopsis invicta), a mosquito species, a Lepidopteran species, a Dipteran, or a Hemipteran species.

5. The genetically modified strain of an entomopathogenic fungus of claim 1 , wherein the entomopathogenic fungus is virulent against the fire ant (Solenopsis invicta).

6. The genetically modified strain of an entomopathogenic fungus of claim 1 , wherein the entomopathogenic fungus is virulent against a mosquito species.

7. The genetically modified strain of an entomopathogenic fungus of claim 1 , wherein the entomopathogenic fungus is a strain of Metarhizium anisopliae or Beauveria bassiana.

8. The genetically modified strain of an entomopathogenic fungus of claim 1 , wherein the heterologous recombinant nucleotide sequence encodes an amino acid sequence specifically inducing a biochemical or physiological reaction in a target insect, wherein the amino acid sequence is an insect neurohormone, an insect diuretic hormone, or a trypsin modulating oostatic factor.

9. The genetically modified strain of an entomopathogenic fungus of claim 1 , wherein the heterologous recombinant nucleotide sequence encodes an insect neurohormone, or a bioactive fragment thereof.

10. The genetically modified strain of an entomopathogenic fungus of claim 1 , wherein the insect neurohormone is a PBAN/pyrokinin or a bioactive fragment thereof.

1 1. The genetically modified strain of an entomopathogenic fungus of claim 9, wherein the heterologous recombinant nucleotide sequence encodes an insect neurohormone, or a bioactive fragment thereof, specifically inducing a biochemical or physiological reaction in the fire ant Solenopsis invicta.

12. The genetically modified strain of an entomopathogenic fungus of claim 1 1 , wherein the insect neurohormone is β-neuropeptide.

13. The genetically modified strain of an entomopathogenic fungus of claim 1 , wherein the heterologous recombinant nucleotide sequence encodes an insect diuretic hormone.

14. The genetically modified strain of an entomopathogenic fungus of claim 1 , wherein the heterologous recombinant nucleotide sequence encodes Manduca sexta diuretic hormone (MSDH).

15. The genetically modified strain of an entomopathogenic fungus of claim 1 , wherein the heterologous recombinant nucleotide sequence encodes a trypsin modulating oostatic factor.

16. The genetically modified strain of an entomopathogenic fungus of claim 1 , wherein the trypsin modulating oostatic factor is a trypsin modulating oostatic factor of Aedes aegyti or S. bullata.

17. The genetically modified strain of an entomopathogenic fungus of claim 1 , wherein the heterologous recombinant nucleotide sequence encodes a polypeptide having about 90% sequence similarity with a sequence selected from the group consisting of: SEQ ID Nos. 1 , 22, 23, and 24.

18. The genetically modified strain of an entomopathogenic fungus of claim 17, wherein the heterologous recombinant nucleotide sequence encodes a polypeptide having about 95% sequence similarity with a sequence selected from the group consisting of: SEQ ID Nos. 1 , 22, 23, and 24.

19. The genetically modified strain of an entomopathogenic fungus of claim 16, wherein the heterologous recombinant nucleotide sequence encodes a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID Nos. 1 , 22, 23, and 24.

20. The genetically modified strain of an entomopathogenic fungus of claim 2, wherein the gene controlling region has about 90% sequence similarity with a sequence encoding a B. bassiana chitinase gene signal peptide.

21. The genetically modified strain of an entomopathogenic fungus of claim 20, wherein the B. bassiana chitinase gene signal peptide has the amino acid sequence according to SEQ ID No.: 2.

22. An expression vector comprising a fungal gene expression controlling region nucleotide sequence operably linked to a nucleic acid encoding an insect-specific polypeptide wherein the gene expression controlling region directs production of a transcript, wherein when said transcript is expressed by a entomopathogenic fungus when said fungus has infected a target insect host, the insect-specific polypeptide increases the virulence of the fungus in the target insect compared to the virulence of a strain of the entomopathogenic fungus not expressing the insect specific polypeptide.

23. The expression vector of claim 22, wherein the gene controlling region has about 90% sequence similarity with a sequence encoding a B. bassiana chitinase gene signal peptide.

24. The expression vector of claim 22, wherein the B. bassiana chitinase gene signal peptide has the amino acid sequence according to SEQ ID No.: 2.

25. The expression vector of claim 22, wherein the insect-specific polypeptide is selected from the group consisting of: an insect neurohormone, an insect diuretic hormone, and a trypsin modulating oostatic factor, or a bioactive homolog or fragment thereof.

26. The expression vector of claim 25, wherein the insect-specific polypeptide is selected from the group consisting of: β-neuropeptide specific to the fire ant Solenopsis invicta, Manduca sexta diuretic hormone (MSDH), and a trypsin modulating oostatic factor of Aedes aegyti or S. bullata.

27. A method of increasing the virulence of an entomopathogenic fungus in a target insect host, comprising the steps of:

(a) obtaining a genetically modified strain of a entomopathogenic fungus according to claims 1 ; and

(b) delivering the genetically modified strain of entomopathogenic fungus, or spores thereof, to a target insect host desired to be infected by the fungus, wherein the fungus delivers a target insect-specific polypeptide or peptide to the target insect host, thereby increasing the virulence of the entomopathogenic fungus in the target insect host.

28. The method of claim 27, wherein the wherein the entomopathogenic fungus is Metarhizium anisopliae or Beauveria bassiana.

29. The method of claim 27, wherein the target insect host is the fire ant (Solenopsis invicta), a mosquito species, a Lepidopteran species, a Dipteran, or a Hemipteran species.