[go: up one dir, main page]

WO2013052536A2 - Exploitation de molécules hôtes pour augmenter la virulence de mycoinsecticides - Google Patents

Exploitation de molécules hôtes pour augmenter la virulence de mycoinsecticides Download PDF

Info

Publication number
WO2013052536A2
WO2013052536A2 PCT/US2012/058543 US2012058543W WO2013052536A2 WO 2013052536 A2 WO2013052536 A2 WO 2013052536A2 US 2012058543 W US2012058543 W US 2012058543W WO 2013052536 A2 WO2013052536 A2 WO 2013052536A2
Authority
WO
WIPO (PCT)
Prior art keywords
insect
entomopathogenic fungus
fungus
genetically modified
modified strain
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2012/058543
Other languages
English (en)
Other versions
WO2013052536A4 (fr
WO2013052536A3 (fr
Inventor
Nematollah KEYHANI
Yanhua FAN
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Florida
University of Florida Research Foundation Inc
Original Assignee
University of Florida
University of Florida Research Foundation Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University of Florida, University of Florida Research Foundation Inc filed Critical University of Florida
Publication of WO2013052536A2 publication Critical patent/WO2013052536A2/fr
Publication of WO2013052536A3 publication Critical patent/WO2013052536A3/fr
Publication of WO2013052536A4 publication Critical patent/WO2013052536A4/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/50—Isolated enzymes; Isolated proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects

Definitions

  • the present disclosure includes a sequence listing incorporated herein by reference in its entirety.
  • 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.
  • 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 has met with limited success because of the relatively long time (6-12 days) it takes for a fungus to kill target insects.
  • Entomopathogenic fungi are virulent to a wide range of Lepidopterans as well as mosquitoes and have been considered as possible candidates for reducing disease
  • 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).
  • 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
  • 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.
  • 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.
  • the gene controlling region has about
  • the B. bassiana chitinase gene signal peptide has the amino acid sequence according to SEQ ID No.: 2.
  • 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.
  • the insect-specific polypeptide can be selected from the group consisting of: ⁇ -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.
  • the entomopathogenic fungus can be Metarhizium anisopliae or Beauveria bassiana.
  • the target insect host can be the fire ant (Solenopsis invicta), a mosquito species, a Lepidopteran species, a Dipteran, or a Hemipteran species.
  • 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 10 7 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 10 6 conidia/ml of each strain over the indicated time course is presented. These data were used to calculate the LT 50 values, and a concentration curve was used to determine the LD 50 values.
  • 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
  • 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. 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.
  • P 0.86
  • 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-NH 2 ) (SEQ ID No.: 21 ), ⁇ - ⁇ (QPQFTPRL) (SEQ ID No.: 7), or ⁇ - ⁇ - ⁇ 2 and presented to untreated ants.
  • p-NP-NH 2 -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
  • Fig. 8 is a graph illustrating that Aea-JMOF reduces fecundity of Anopheles gambiae mosquitoes.
  • A. gambiae females sprayed with a suspension of 1x10 8 spores/ml of wild type or ⁇ -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.
  • 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.
  • compositions comprising, “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).
  • compositions or methods 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.
  • viral infection and viral strains 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.
  • entomopathogenic fungi 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 generally produce conidia.
  • Deuteromycete fungi include fungi of the genera Beauveria, Metarhizium, Paecilomyces, Tolypocladium, Aspergillus, Culicinonyces, Nomuraea, Sorosporella, and Hirsutella.
  • 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.
  • insect peptide and insect hormone 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.
  • insect hormones include allostatins, antidiuretic hormones (e.g.
  • Tenebrio molitor (beetle) ADH the Tenebrio molitor (beetle) ADH
  • juvenile hormone diapause hormone
  • proctolins the Tenebrio molitor (beetle) ADH
  • mykinins pyrokinins other than ⁇ - ⁇
  • diuretic hormones other than MSDH
  • tachykinins myosuppressins, and the like.
  • insect 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).
  • RNA nucleic acid molecule 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • coding sequence 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.
  • gene controlling regions 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
  • 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.
  • operably linked refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function.
  • 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.
  • 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.
  • 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.
  • recombinant as used with respect to a protein or polypeptide means a polypeptide produced by expression of a recombinant polynucleotide.
  • Recombinant host cells 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 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • MVA modified vaccinia Ankara
  • BEVS recombinant rhabdovirus vectors
  • alphavirus vectors recombinant alphavirus vectors
  • recombinant adenovirus expression systems recombinant DNA expression vectors, and combinations thereof.
  • 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
  • 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).
  • variant 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.
  • 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:
  • 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.
  • hydrophilicity 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).
  • 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.
  • 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.
  • 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
  • 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.
  • peptide refers 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.
  • polynucleotide generally refers to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.
  • 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.
  • polynucleotide 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.
  • polynucleotide includes DNAs or RNAs as described above that contain one or more modified bases.
  • DNAs or RNAs with backbones modified for stability or for other reasons are “polynucleotides” as that term is intended herein.
  • 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.
  • 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.
  • 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.
  • DNA may be obtained by any method.
  • 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.
  • cDNA complementary DNA
  • 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.
  • appropriate DNA fragments for example, linkers, restriction sites, and so on
  • transformants can be prepared by introducing the expression vector mentioned above into host cells.
  • 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).
  • bacteria Esscherichia and Bacillus
  • yeast Sacharomyces, Pichia, and such
  • animal cells insect cells, or plant cells.
  • NP neuropeptide
  • TMOF trypsin modulating oostatic factor
  • Aea-TMOF Aedes aegypti TMOF
  • MSDH Manduca sexta diuretic hormone
  • PBAN pyrokinin/pheromone biosynthesis activating neuropeptide
  • 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.
  • 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.
  • 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.
  • 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.
  • the method 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.
  • PBAN pyrokinin/pheromone biosynthesis activating neuropeptide
  • 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.
  • these peptides are C-terminal amidated, a modification often required for their activity.
  • the PBAN peptide is encoded on a translated ORF that is subsequently processed (cleaved) to yield diapause hormone (DH), and the ⁇ -, ⁇ -, and ⁇ -neuropeptides, along with the PBAN peptide itself (which is found between the ⁇ - and ⁇ -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 ⁇ - and ⁇ -neuropeptide homologs, but no a-neuropeptide.
  • DH diapause hormone
  • the impact of expressing the ⁇ - ⁇ peptide in the fungal insect pathogen B. bassiana The data shows a decrease in both the lethal dose (LD 50 ) and lethal time (LT 50 ) it takes to kill target fire ants in the ⁇ - ⁇ -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.
  • 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.
  • the virulence of a B. bassiana strain to fire ants by expressing a fire ant neuropeptide in the fungal pathogen Increased virulence in the ⁇ - ⁇ 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.
  • TMOFs Trypsin-modulating oostatic factors
  • 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
  • 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 ⁇ . 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.
  • TMOF-expressing B 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 (LD 50 ) 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 (LT 50 ) values were lower for blood-fed as compared to sugar-fed mosquitoes in infections with both wild type and TMOF-expressing strains.
  • infection using the latter resulted in 15% and 25% reduction in LT 50 values for sugar- and blood-fed mosquitoes, respectively, relative to the wild type parent.
  • 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.
  • /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.
  • 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 LC 50 and 38% reduction in survival times), indicating a robust increase in lethality without the concerns regarding expression of
  • 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
  • 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.
  • 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.
  • the entomopathogenic fungus can be of the class Hyphomycetes.
  • 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).
  • the entomopathogenic fungus can be virulent against a mosquito species.
  • the entomopathogenic fungus can be any suitable fungus.
  • the entomopathogenic fungus can be any suitable fungus.
  • 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.
  • the heterologous recombinant nucleotide sequence encodes an insect neurohormone.
  • the insect neurohormone can be a PBAN/pyrokinin or a bioactive fragment thereof.
  • the heterologous recombinant nucleotide sequence encodes an insect neurohormone, or a bioactive fragment thereof, specific to the fire ant Solenopsis invicta.
  • the insect neurohormone can be ⁇ - neuropeptide sequence specifically inducing a biochemical or physiological reaction in the fire ant Solenopsis invicta.
  • the heterologous recombinant nucleotide sequence can encode an insect diuretic hormone.
  • the heterologous recombinant nucleotide sequence can encode Manduca sexta diuretic hormone (MSDH).
  • MSDH Manduca sexta diuretic hormone
  • the heterologous recombinant nucleotide sequence can encode a trypsin modulating oostatic factor.
  • the trypsin modulating oostatic factor can be a trypsin modulating oostatic factor of Aedes aegyti or S. bullata.
  • 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.
  • 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.
  • the gene controlling region can have about 90% sequence similarity with a sequence encoding a B. bassiana chitinase gene signal peptide.
  • the B. bassiana chitinase gene signal peptide can have the amino acid sequence according to SEQ ID No.: 2.
  • 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.
  • the gene controlling region has about 90% sequence similarity with a sequence encoding a B. bassiana chitinase gene signal peptide
  • the B. bassiana chitinase gene signal peptide has the amino acid sequence according to SEQ ID No.: 2.
  • 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.
  • the insect-specific polypeptide can be selected from the group consisting of: ⁇ -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.
  • the entomopathogenic fungus can be Metarhizium anisopliae or Beauveria bassiana.
  • the target insect host can be the fire ant (Solenopsis invicta), a mosquito species, a Lepidopteran species, a Dipteran, or a Hemipteran species.
  • 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.
  • 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.
  • MAPFLQTSLALLPLLASTMVSASPLAPRAGRMPSLSIDLPMSVLRQKLSLEKERKVHALRAAAN RNFLNDIG 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)
  • 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 gpd A-Bb and pSP- MSDH.
  • the desired construct (P g p dA -Bb:SP-MSDH) was produced via primerless assembly in a reaction mixture containing: 5 ⁇ 5x Phusion Taq polymerase buffer, 2 ⁇ 2.5 mM dNTP, 30 ng PgpdA-Bb, 30 ng pSP-MSDH, 0.4U Phusion Taq DNA polymerase, total volume 25 ⁇ .
  • 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.
  • the S. invicta pyrokinin ⁇ -neuropeptide ( ⁇ - ⁇ having amino acid sequence QPQFTPRL
  • 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.
  • 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: 5 ⁇ 5 x Phusion Taq polymerase buffer, 2 ⁇ 2.5 mM dNTP, 30ng P gpd A-Bb, 30ng SP:p-NP, 0.4 U Phusion Taq DNA polymerase, total volume 25 ⁇ .
  • 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 .
  • PgpdA-Bb:SP-p-NP was subcloned from pDrive vector via EcoRI restriction sites into pUC-Bar, yielding pUC-Bar-P gpdA _ B b: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 .
  • 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 are 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.
  • 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.
  • 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:
  • the primer pairs were designed containing 20 bp overlap sequences, i.e.
  • P trp c bar and P gpdA -Bb, and (2) P gpdA -Bb and SP:AeTMOF, respectively.
  • the desired construct (P tr pc'.bar. Pgp dA . b:SP-AeTMOF) was produced via primerless assembly in a reaction mixture containing: 5 ⁇ 5x Phusion Taq polymerase buffer, 2 ⁇ 2.5 mM dNTP, 30ng P trp cBar, 30ng Pgp d A Bb, 30 ng SP:AeT OF, 0.4U phusion Taq DNA polymerase, total volume 25 ⁇ .
  • the eluted fraction (in acetonitrile) was dried by SpeedVac, resuspended in water-0.1 % TFA (0.6ml) and chromatographed on a C 18 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 N 2 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.
  • 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 (10 4 -10 8 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 (LT 50 ) was calculated by Probit analysis.
  • LT 50 median lethal mortality time
  • the conidial suspension was centrifuged at top speed in a tabletop microfuge (14,000xg) for 10 min, and resuspended in the final desired volume
  • grapeseed oil (depending upon the concentration wanted) 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.
  • 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 0.25 ⁇ of conidial suspensions (10 4 -10 8 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.
  • Trypsin activity measurement To measure trypsin activity, 5 larvae were manually
  • 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.
  • 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 LD 50 values (50% mortality) in both groups compared to the wild type control (Table 1 ). Table 1: LD 50 and LT 50 values of wild type and Aea-TMOF expressing Bb-Aa1 strain against
  • LT 50 values were calculated from bioassays in which mosquitoes were infected by spraying with a suspension of 1x10 8 conidia/ml. Statistical analysis was performed using the Student's T-test and values were considered significant if P ⁇ 0.05. LD 50 values were calculated from the 76-h time point using the Probit analysis.
  • LD 50 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 (LT 50 values) of sugar- and blood- fed mosquitoes, respectively, compared to the wild type strain. LT 50 values were also lower for blood-fed compared to sugar-fed mosquitoes infected with the same strain, regardless its type.
  • strain S Aa1 also has the potential of reducing the size of A. gambiae mosquito populations by severely compromising fecundity.
  • 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 3 ⁇ 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.
  • Table 2 Calculated LD 50 and LT 50 of WT, MSDH, and TMOF expressing B. bassiana strains against G. mellonella and Ae. Aegypti
  • a LD 50 calculated from 96 hr time point.
  • 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).
  • Bb::spAeaTMOF-infected females 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 LD 50 could not be calculated since a threshold inoculum appeared to be needed in order for effective control to occur.
  • TMOF peptides 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.
  • 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.
  • 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 ⁇ - ⁇ 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 N 2 and analyzed by LC-MS/MS. A standard curve using synthetic ⁇ - ⁇ was used to quantify the amount of peptide in the sample.
  • 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.
  • Ants 0.5 gm, approximately 2,000 individuals
  • 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 X 2 -test was first used to determine
  • 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.
  • the fire ant ⁇ - ⁇ 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 ⁇ - ⁇ 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 .
  • 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 LD 50 of 1.5 ⁇ 0.9 x 10 7 conidia/ml compared to an LD 50 of 1.0 ⁇ 0.7 x 10 8 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).
  • 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).
  • Ant corpses treated with the ⁇ - ⁇ - ⁇ 2 peptide were moved significantly faster than buffer treated ants, ⁇ - ⁇ -treated ants, or ants treated with a control eight-amino acid amidated peptide (P ⁇ 0.001 , Fig. 5).
  • ⁇ - ⁇ -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 ⁇ - ⁇ treatment than for any other treatment examined.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Plant Pathology (AREA)
  • Dentistry (AREA)
  • Biomedical Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Environmental Sciences (AREA)
  • Mycology (AREA)
  • Virology (AREA)
  • Agronomy & Crop Science (AREA)
  • Pest Control & Pesticides (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Physics & Mathematics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Insects & Arthropods (AREA)
  • Medicinal Chemistry (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne des procédés d'élimination d'insectes selon lesquels l'expression fongique de molécules dérivées d'insectes est exploitée pour une augmentation spécifique à la cible de la virulence de champignons entomopathogènes. Les avantages majeurs qui dépendent de la molécule hôte (peptide) choisie sont l'augmentation de virulence qui peut être adaptée pour être spécifique à l'hôte, et le développement d'une résistance qui est minimisé puisque les peptides hôtes et/ou les hormones régulent des procédés de développement qui sont spécifiques à l'espèce et au tissu. Toute mutation adaptative qui se produit dans l'insecte ciblé qui pourrait compenser le produit à expression fongique pendant l'infection conduira à des défauts de développement ou à des coûts en termes de condition physique bien supérieurs au développement d'une résistance à un pesticide. Il a été montré que les procédés, les souches fongiques et les constructions d'acides nucléiques de l'invention sont efficaces contre diverses espèces hôtes d'insectes.
PCT/US2012/058543 2011-10-05 2012-10-03 Exploitation de molécules hôtes pour augmenter la virulence de mycoinsecticides Ceased WO2013052536A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201161543345P 2011-10-05 2011-10-05
US61/543,345 2011-10-05

Publications (3)

Publication Number Publication Date
WO2013052536A2 true WO2013052536A2 (fr) 2013-04-11
WO2013052536A3 WO2013052536A3 (fr) 2013-06-06
WO2013052536A4 WO2013052536A4 (fr) 2013-07-25

Family

ID=48044380

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2012/058543 Ceased WO2013052536A2 (fr) 2011-10-05 2012-10-03 Exploitation de molécules hôtes pour augmenter la virulence de mycoinsecticides

Country Status (1)

Country Link
WO (1) WO2013052536A2 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111148434A (zh) * 2017-01-24 2020-05-12 旗舰创业创新五公司 用于制造食物和饲料的方法和相关组合物
CN114480300A (zh) * 2021-06-28 2022-05-13 吉林省农业科学院 一种提高球孢白僵菌毒力的真菌病毒及其传毒方法
CN115505538A (zh) * 2022-11-17 2022-12-23 中国农业科学院植物保护研究所 金龟子绿僵菌菌株CIPPMa0941、及防治红火蚁的应用和菌剂
CN115991759A (zh) * 2022-10-20 2023-04-21 曲靖师范学院 大蜡螟性信息素结合蛋白基因GmelPBPs及其应用
ES2978576A1 (es) * 2023-02-09 2024-09-16 Instituto Valenciano De Investig Agrarias Ivia Máquina para inoculación de conidios de hongos entomopatógenos en polvo sobre machos estériles de Ceratitis capitata

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5683689A (en) * 1991-04-18 1997-11-04 University Of Florida Research Foundation, Inc. Controlling cockroaches, carpenter ants, and pharaoh ants using strains of beauveria bassiana
US5516513A (en) * 1994-07-14 1996-05-14 Troy Biosciences, Inc. Biological ovicide for control of lepidopterous insects
EP1018887A1 (fr) * 1997-09-17 2000-07-19 The University Of Florida Procedes et materiaux de protection contre les termites

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111148434A (zh) * 2017-01-24 2020-05-12 旗舰创业创新五公司 用于制造食物和饲料的方法和相关组合物
EP3573465A4 (fr) * 2017-01-24 2020-11-04 Flagship Pioneering Innovations V, Inc. Procédés et compositions associées pour la fabrication d'aliments et d'aliments pour animaux
US11690387B2 (en) 2017-01-24 2023-07-04 Flagship Pioneering Innovations V, Inc. Methods and related compositions for manufacturing food and feed
CN111148434B (zh) * 2017-01-24 2023-11-17 旗舰创业创新五公司 用于制造食物和饲料的方法和相关组合物
CN114480300A (zh) * 2021-06-28 2022-05-13 吉林省农业科学院 一种提高球孢白僵菌毒力的真菌病毒及其传毒方法
CN115991759A (zh) * 2022-10-20 2023-04-21 曲靖师范学院 大蜡螟性信息素结合蛋白基因GmelPBPs及其应用
CN115505538A (zh) * 2022-11-17 2022-12-23 中国农业科学院植物保护研究所 金龟子绿僵菌菌株CIPPMa0941、及防治红火蚁的应用和菌剂
CN115505538B (zh) * 2022-11-17 2023-08-04 中国农业科学院植物保护研究所 金龟子绿僵菌菌株CIPPMa0941、及防治红火蚁的应用和菌剂
ES2978576A1 (es) * 2023-02-09 2024-09-16 Instituto Valenciano De Investig Agrarias Ivia Máquina para inoculación de conidios de hongos entomopatógenos en polvo sobre machos estériles de Ceratitis capitata

Also Published As

Publication number Publication date
WO2013052536A4 (fr) 2013-07-25
WO2013052536A3 (fr) 2013-06-06

Similar Documents

Publication Publication Date Title
US6797490B2 (en) Formicidae (ant) control using Bacillus thuringiensis toxin
US8362201B2 (en) Insecticidal compounds and methods for selection thereof
CN101755050A (zh) 蝎毒素基因转化的杀虫真菌工程菌株及应用
Fan et al. Pyrokinin β-neuropeptide affects necrophoretic behavior in fire ants (S. invicta), and expression of β-NP in a mycoinsecticide increases its virulence
WO2007142543A2 (fr) Nouvelle bactérie et ses utilisations
WO2013052536A2 (fr) Exploitation de molécules hôtes pour augmenter la virulence de mycoinsecticides
KR101644338B1 (ko) 살충 활성이 있는 바실러스 투린지엔시스 아종 쿠르스타키 cab565 균주 및 이의 용도
Ahmed et al. Entomopathogenic fungi as biopesticides against the Egyptian cotton leaf worm, Spodoptera littoralis: between biocontrol-promise and immune-limitation
US6583264B2 (en) Insecticidal compounds and methods for selection thereof
CN1952151A (zh) 苏云金芽胞杆菌的杀虫晶体蛋白基因cry7Bal
AU778146B2 (en) Biological control of nematodes
Leger Metarhizium anisopliae as a model for studying bioinsecticidal host pathogen interactions
CN116157018B (zh) 生物刺激素和生物保护肽及它们在农业中的用途
KR101649139B1 (ko) 살충 활성이 있는 바실러스 투린지엔시스 아종 아이자와이 cab566 균주 및 이의 용도
Narayanan Microbial control of insect pests: role of genetic engineering and tissue culture
CN108070534B (zh) 防治黄曲条跳甲的苏云金芽孢杆菌及制剂与应用
KR19990069306A (ko) 바실러스 투린지엔시스 엔티 0423 균주의 내독소 단백질 및 이를 이용한 미생물 살충제
KR100599414B1 (ko) 나비목 해충에 살충효과를 보이는 신규한 내독소 단백질유전자를 보유한 바실러스 투린지엔시스 케이-3 균주 및 이를 이용한 미생물 제제
Fan et al. Pyrokinin b-Neuropeptide Affects Necrophoretic Behavior in Fire Ants (S. invicta), and Expression
RU2804136C2 (ru) Композиции и соответствующие способы контроля заболеваний, передаваемых переносчиками
RU2777518C2 (ru) Композиции и соответствующие способы контроля заболеваний, передаваемых переносчиками
Bulla Jr et al. Formicidae (ant) control using Bacillus thuringiensis toxin
Narayanan Project Directorate of Biological Control, PB No. 2491, Hebbal, Bangalore 560 024, India
EP4271699A1 (fr) Toxines et cellules anti-nuisibles

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 12838527

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 12838527

Country of ref document: EP

Kind code of ref document: A2