US20190365853A1 - Compositions and related methods for controlling vector-borne diseases - Google Patents

Compositions and related methods for controlling vector-borne diseases Download PDF

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US20190365853A1
US20190365853A1 US16/480,053 US201816480053A US2019365853A1 US 20190365853 A1 US20190365853 A1 US 20190365853A1 US 201816480053 A US201816480053 A US 201816480053A US 2019365853 A1 US2019365853 A1 US 2019365853A1
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host
aphids
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treatment
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Ignacio Martinez
Zachary Garo Armen
Christine Cezar
Barry Andrew Martin
Maier Steve Avendano Amado
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Flagship Pioneering Innovations V Inc
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Flagship Pioneering Innovations V Inc
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Assigned to FLAGSHIP PIONEERING INNOVATIONS V, INC. reassignment FLAGSHIP PIONEERING INNOVATIONS V, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: FLAGSHIP PIONEERING, INC.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A61K38/1729—Cationic antimicrobial peptides, e.g. defensins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1767—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • 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
    • 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
    • C07K14/43572—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from bees
    • 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
    • C07K14/43577—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from flies
    • C07K14/43581—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from flies from Drosophila
    • 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
    • C07K14/43586—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from silkworms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • Insects function as vectors for pathogens causing severe human disease such as dengue, trypanosomiases, and malaria. With 174 million diagnoses and 655,000 million deaths in 2011, malaria is considered as one of the most significant diseases worldwide. Thus, there is need in the art for methods and compositions to control insects that carry vector-borne diseases.
  • compositions and methods for modulating the fitness of insects for controlling the spread of vector-borne diseases in humans includes an agent that alters a level, activity, or metabolism of one or more microorganisms resident in a host, the alteration resulting in a modulation in the host's fitness.
  • a method of decreasing fitness of a vector for a human pathogen, the method including delivering an antimicrobial peptide having at least 90% sequence identity (e.g., at least 90%, 92%, 94%, 96%, 98%, or 100% sequence identity) with one or more of the following: cecropin (SEQ ID NO: 82), melittin, copsin, drosomycin (SEQ ID NO: 93), dermcidin (SEQ ID NO: 81), andropin (SEQ ID NO: 83), moricin (SEQ ID NO: 84), ceratotoxin (SEQ ID NO: 85), abaecin (SEQ ID NO: 86), apidaecin (SEQ ID NO: 87), prophenin (SEQ ID NO: 88), indolicidin (SEQ ID NO: 89), protegrin (SEQ ID NO: 90), tachyplesin (SEQ ID NO: 91), or def
  • the delivery includes delivering the antimicrobial peptide to at least one habitat where the vector grows, lives, reproduces, feeds, or infests.
  • the antimicrobial peptide may be delivered in an insect comestible composition for ingestion by the vector.
  • the antimicrobial peptide may be formulated as a liquid, a solid, an aerosol, a paste, a gel, or a gas composition.
  • the insect may be at least one of a mosquito, midge, louse, sandfly, tick, triatomine bug, tsetse fly, or flea.
  • compositions including an antimicrobial peptide having at least 90% sequence identity (e.g., at least 90%, 92%, 94%, 96%, 98%, or 100% sequence identity) with one or more of the following: cecropin (SEQ ID NO: 82), melittin, copsin, drosomycin (SEQ ID NO: 93), dermcidin (SEQ ID NO: 81), andropin (SEQ ID NO: 83), moricin (SEQ ID NO: 84), ceratotoxin (SEQ ID NO: 85), abaecin (SEQ ID NO: 86), apidaecin (SEQ ID NO: 87), prophenin (SEQ ID NO: 88), indolicidin (SEQ ID NO: 89), protegrin (SEQ ID NO: 90), tachyplesin (SEQ ID NO: 91), or defensin (SEQ ID NO: 92) formulated for targeting a microorganism in a vector (
  • the antimicrobial peptide may be at a concentration of about 0.1 ng/g to about 100 mg/g (about 0.1 ng/g to about 1 ng/g, about 1 ng/g to about 10 ng/g, about 10 ng/g to about 100 ng/g, about 100 ng/g to about 1000 ng/g, about 1 mg/g to about 10 mg/g, about 10 mg/g to about 100 mg/g) or about 0.1 ng/mL to about 100 mg/mL (about 0.1 ng/mL to about 1 ng/mL, about 1 ng/mL to about 10 ng/mL, about 10 ng/mL to about 100 ng/mL, about 100 ng/mL to about 1000 ng/mL, about 1 mg/mL to about 10 mg/mL, about 10 mg/mL to about 100 mg/mL) in the composition.
  • the antimicrobial peptide may further include a targeting domain.
  • the antimicrobial peptide may further include a cell penetrating peptide.
  • the composition includes an agent that alters a level, activity, or metabolism of one or more microorganisms resident in an insect host, the alteration resulting in a decrease in the insect host's fitness.
  • the one or more microorganisms may be a bacterium or fungus resident in the host.
  • the bacterium resident in the host is at least one selected from the group consisting of Candidatus spp, Buchenera spp, Blattabacterium spp, Baumania spp, Wigglesworthia spp, Wolbachia spp, Rickettsia spp, Orientia spp, Sodalis spp, Burkholderia spp, Cupriavidus spp, Frankia spp, Snirhizobium spp, Streptococcus spp, Wolinella spp, Xylella spp, Erwinia spp, Agrobacterium spp, Bacillus spp, Paenibacillus spp, Streptomyces spp, Micrococcus spp, Corynebacterium s
  • the fungus resident in the host is at least one selected from the group consisting of Candida, Metschnikowia, Debaromyces, Starmerella, Pichia, Cryptococcus, Pseudozyma, Symbiotaphrina bucneri, Symbiotaphrina Scheffersomyces shehatae, Scheffersomyces stipites, Cryptococcus, Trichosporon, Amylostereum areolatum, Epichloe spp, Pichia pinus, Hansenula capsulate, Daldinia decipien, Ceratocytis spp, Ophiostoma spp, and Attamyces bromatificus .
  • the bacteria is a Wolbachia spp. (e.g., in a mosquito host). In certain embodiments, the bacteria is a Rickettsia spp. (e.g., in a tick host).
  • the agent which hereinafter may also be referred to as a modulating agent, may alter the growth, division, viability, metabolism, and/or longevity of the microorganism resident in the host.
  • the modulating agent may decrease the viability of the one or more microorganisms resident in the host.
  • the modulating agent increases growth or viability of the one or more microorganisms resident in the host.
  • the modulating agent is a phage, a polypeptide, a small molecule, an antibiotic, a bacterium, or any combination thereof.
  • the phage binds a cell surface protein on a bacterium resident in the host. In some embodiments, the phage is virulent to a bacterium resident in the host. In some embodiments, the phage is at least one selected from the group consisting of Myoviridae, Siphoviridae, Podoviridae, Lipothrixviridae, Rudiviridae, Ampullaviridae, Bicaudaviridae, Clavaviridae, Corticoviridae, Cystoviridae, Fuselloviridae, Gluboloviridae, Guttaviridae, Inoviridae, Leviviridae, Microviridae, Plasmaviridae, and Tectiviridae.
  • the polypeptide is at least one of a bacteriocin, R-type bacteriocin, nodule C-rich peptide, antimicrobial peptide, lysin, or bacteriocyte regulatory peptide.
  • the small molecule is a metabolite.
  • the antibiotic is a broad-spectrum antibiotic.
  • the modulating agent is a naturally occurring bacteria.
  • the bacteria is at least one selected from the group consisting of Bartonella apis, Parasaccharibacter apium, Frischella perrara, Snodgrassella alvi, Gilliamela apicola, Bifidobacterium spp, and Lactobacillus spp.
  • the bacterium is at least one selected from the group consisting of Candidatus spp, Buchenera spp, Blattabacterium spp, Baumania spp, Wigglesworthia spp, Wolbachia spp, Rickettsia spp, Orientia spp, Sodalis spp, Burkholderia spp, Cupriavidus spp, Frankia spp, Snirhizobium spp, Streptococcus spp, Wolinella spp, Xylella spp, Erwinia spp, Agrobacterium spp, Bacillus spp, Paenibacillus spp, Streptomyces spp, Micrococcus spp, Corynebacterium spp, Corynebacterium spp, Corynebacterium spp, Corynebacterium spp, Corynebacterium spp, Coryn
  • host fitness may be measured by survival, reproduction, or metabolism of the host.
  • the modulating agent may modulate the host's fitness by increasing pesticidal susceptibility of the host (e.g., susceptibility to a pesticide listed in Table 12).
  • the modulating agent modulates the host's fitness by increasing pesticidal susceptibility of the host.
  • the pesticidal susceptibility is bactericidal or fungicidal susceptibility.
  • the pesticidal susceptibility is insecticidal susceptibility.
  • the composition may include a plurality of different modulating agents.
  • the composition includes a modulating agent and a pesticidal agent (e.g., a pesticide listed in Table 12).
  • the pesticidal agent is a bactericidal or fungicidal agent.
  • the pesticidal agent is an insecticidal agent.
  • modulating agent may be linked to a second moiety.
  • the second moiety is a modulating agent.
  • the modulating agent may be linked to a targeting domain.
  • the targeting domain targets the modulating agent to a target site in the host.
  • the targeting domain targets the modulating agent to the one or more microorganisms resident in the host.
  • the modulating agent may include an inactivating pre- or pro-sequence, thereby forming a precursor modulating agent.
  • the precursor modulating agent is converted to an active form in the host.
  • the modulating agent may include a linker.
  • the linker is a cleavable linker.
  • the composition may further include a carrier.
  • the carrier may be an agriculturally acceptable carrier.
  • the composition may further include a host bait, a sticky agent, or a combination thereof.
  • the host bait is a comestible agent and/or a chemoattractant.
  • the composition may be at a dose effective to modulate host fitness.
  • the composition may be formulated for delivery to a microorganism inhabiting the gut of the host. In any of the above compositions, the composition may be formulated for delivery to a microorganism inhabiting a bacteriocyte of the host and/or the gut of the host.
  • the composition may be formulated for delivery to a plant. In some embodiments, the composition may be formulated for use in a host feeding station.
  • the composition may be formulated as a liquid, a powder, granules, or nanoparticles.
  • the composition is formulated as one selected from the group consisting of a liposome, polymer, bacteria secreting peptide, and synthetic nanocapsule.
  • the synthetic nanocapsule delivers the composition to a target site in the host.
  • the target site is the gut of the host.
  • the target site is a bacteriocyte in the host.
  • hosts that include any of the above compositions.
  • the host is an insect.
  • the insect is a mosquito, midge, louse, sandfly, tick, triatomine bug, tsetse fly, or flea.
  • the insect is a mosquito.
  • the insect is a tick.
  • the insect is a mite.
  • the insect is a louse.
  • a system for modulating a host's fitness comprising a modulating agent that targets a microorganism that is required for a host's fitness, wherein the system is effective to modulate the host's fitness, and wherein the host is an insect.
  • the modulating agent may include any of the compositions described herein.
  • the modulating agent is formulated as a powder.
  • the modulating agent is formulated as a solvent.
  • the modulating agent is formulated as a concentrate.
  • the modulating agent is formulated as a diluent.
  • the modulating agent is prepared for delivery by combining any of the previous compositions with a carrier.
  • the method of modulating the fitness of an insect host includes delivering the composition of any one of the previous claims to the host, wherein the modulating agent targets the one or more microorganisms resident in the host, and thereby modulates the host's fitness.
  • the method of modulating microbial diversity in an insect host includes delivering the composition of any one of the previous claims to the host, wherein the modulating agent targets the one or more microorganisms resident in the host, and thereby modulates microbial diversity in the host.
  • the modulating agent may alter the levels of the one or more microorganisms resident in the host. In some embodiments of any of the above methods, the modulating agent may alter the function of the one or more microorganisms resident in the host. In some embodiments, the one or more microorganisms may be a bacterium and/or fungus. In some embodiments, the one or more microorganisms are required for host fitness. In some embodiments, the one or more microorganisms are required for host survival.
  • the delivering step may include providing the modulating agent at a dose and time sufficient to effect the one or more microorganisms, thereby modulating microbial diversity in the host.
  • the delivering step includes topical application of any of the previous compositions to a plant.
  • the delivering step includes providing the modulating agent through a genetically engineered plant.
  • the delivering step includes providing the modulating agent to the host as a comestible.
  • the delivering step includes providing a host carrying the modulating agent.
  • the host carrying the modulating agent can transmit the modulating agent to one or more additional hosts.
  • the composition may be effective to increase the host's sensitivity to a pesticidal agent (e.g., a pesticide listed in Table 12).
  • the host is resistant to the pesticidal agent prior to delivery of the modulating agent.
  • the pesticidal agent is an allelochemical agent.
  • the allelochemical agent is caffeine, soyacystatin N, monoterpenes, diterpene acids, or phenolic compounds.
  • the composition is effective to selectively kill the host.
  • the composition is effective to decrease host fitness.
  • the composition is effective to decrease the production of essential amino acids and/or vitamins in the host.
  • the host is an insect.
  • the host is a vector for a human pathogen.
  • the vector is a. mosquito, midge, louse, sandfly, tick, triatomine bug, tsetse fly, or flea.
  • the vector is a mosquito.
  • the vector is a tick.
  • the vector is a mite.
  • the vector is a louse.
  • the human pathogen is a virus, a protozoan, a bacterium, a protist, or a nematoda.
  • the virus is one belonging to the group Togaviridae, Flaviviridae, Bunyaviridae, Rhabdoviridae, or Orbiviridae.
  • the bacterium is one belonging to the genus Yersinia, Francisella, Rickettsia, Orientia , or Borrelia .
  • the protozoan is one belonging to the genus Plasmodium, Trypanosoma, Leishmania , or Babesia .
  • the nematode is one belonging to the genus Brugia .
  • the composition is effective to prevent or decrease transmission of the pathogen to humans.
  • the composition is effective to prevent or decrease horizontal or vertical transmission of the pathogen between hosts.
  • the composition is effective to decrease host fitness, host development, or vectorial competence.
  • screening assays to identify modulating agent that modulate the fitness of a host includes the steps of (a) exposing a microorganism that can be resident in the host to one or more candidate modulating agents and (b) identifying a modulating agent that decreases the fitness of the host.
  • the modulating agent is a microorganism resident in the host.
  • the microorganism is a bacterium.
  • the bacterium when resident in the host, decreases host fitness.
  • the modulating agent affects an allelochemical-degrading microorganism.
  • the modulating agent is a phage, an antibiotic, or a test compound.
  • the antibiotic is timentin or azithromycin.
  • the host may be an invertebrate.
  • the invertebrate is an insect.
  • the insect is a mosquito.
  • the insect is a tick.
  • the insect is a mite.
  • the insect is a louse.
  • host fitness may be modulated by modulating the host microbiota.
  • bacteriocin refers to a peptide or polypeptide that possesses anti-microbial properties. Naturally occurring bacteriocins are produced by certain prokaryotes and act against organisms related to the producer strain, but not against the producer strain itself. Bacteriocins contemplated herein include, but are not limited to, naturally occurring bacteriocins, such as bacteriocins produced by bacteria, and derivatives thereof, such as engineered bacteriocins, recombinantly expressed bacteriocins, and chemically synthesized bacteriocins. In some instances, the bacteriocin is a functionally active variant of the bacteriocins described herein.
  • the variant of the bacteriocin has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, e.g., over a specified region or over the entire sequence, to a sequence of a bacteriocin described herein or a naturally occurring bacteriocin.
  • bacteriocyte refers to a specialized cell found in certain insects where intracellular bacteria reside with symbiotic bacterial properties.
  • the term “effective amount” refers to an amount of a modulating agent (e.g., a phage, lysin, bacteriocin, small molecule, or antibiotic) or composition including said agent sufficient to effect the recited result, e.g., to decrease or reduce the fitness of a host organism (e.g., insect, e.g., mosquito, tick, mite, louse); to reach a target level (e.g., a predetermined or threshold level) of a modulating agent concentration inside a target host; to reach a target level (e.g., a predetermined or threshold level) of a modulating agent concentration inside a target host gut; to reach a target level (e.g., a predetermined or threshold level) of a modulating agent concentration inside a target host bacteriocyte; to modulate the level, or an activity, of one or more microorganism (e.g., endosymbiont) in the target host.
  • a modulating agent e.g
  • fitness refers to the ability of a host organism to survive, and/or to produce surviving offspring.
  • Fitness of an organism may be measured by one or more parameters, including, but not limited to, life span, reproductive rate, mobility, body weight, and metabolic rate. Fitness may additionally be measured based on measures of activity (e.g., biting animals or humans) or disease transmission (e.g., vector-vector transmission or vector-human transmission).
  • gut refers to any portion of a host's gut, including, the foregut, midgut, or hindgut of the host.
  • the term “host” refers to an organism (e.g., insect, e.g., mosquito, louse, mite, or tick) carrying resident microorganisms (e.g., endogenous microorganisms, endosymbiotic microorganisms (e.g., primary or secondary endosymbionts), commensal organisms, and/or pathogenic microorganisms).
  • an organism e.g., insect, e.g., mosquito, louse, mite, or tick
  • resident microorganisms e.g., endogenous microorganisms, endosymbiotic microorganisms (e.g., primary or secondary endosymbionts), commensal organisms, and/or pathogenic microorganisms.
  • decreasing host fitness or “decreasing host fitness” refers to any disruption to host physiology, or any activity carried out by said host, as a consequence of administration of a modulating agent, including, but not limited to, any one or more of the following desired effects: (1) decreasing a population of a host by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (2) decreasing the reproductive rate of a host (e.g., insect, e.g., mosquito, tick, mite, louse) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (3) decreasing the mobility of a host (e.g., insect, e.g., mosquito, tick, mite, louse) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (4) decreasing the body weight of a host (e.
  • insects includes any organism belonging to the phylum Arthropoda and to the class Insecta or the class Arachnida, in any stage of development, i.e., immature and adult insects.
  • lysin also known as endolysin, autolysin, murein hydrolase, peptidoglycan hydrolase, or cell wall hydrolase refers to a hydrolytic enzyme that can lyse a bacterium by cleaving peptidoglycan in the cell wall of the bacterium.
  • Lysins contemplated herein include, but are not limited to, naturally occurring lysins, such as lysins produced by phages, lysins produced by bacteria, and derivatives thereof, such as engineered lysins, recombinantly expressed lysins, and chemically synthesized lysins.
  • a functionally active variant of the bacteriocin may have at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, e.g., over a specified region or over the entire sequence, to a sequence of a synthetic, recombinant, or naturally derived bacteriocin, including any described herein.
  • microorganism refers to bacteria or fungi. Microorganisms may refer to microorganisms resident in a host organism (e.g., endogenous microorganisms, endosymbiotic microorganisms (e.g., primary or secondary endosymbionts)) or microorganisms exogenous to the host, including those that may act as modulating agents.
  • target microorganism refers to a microorganism that is resident in the host and impacted by a modulating agent, either directly or indirectly.
  • agent refers to an agent that is capable of altering the levels and/or functioning of microorganisms resident in a host organism (e.g., insect, e.g., mosquito, tick, mite, louse), and thereby modulate (e.g., decrease) the fitness of the host organism (e.g., insect, e.g., mosquito, tick, mite, louse).
  • a host organism e.g., insect, e.g., mosquito, tick, mite, louse
  • pesticide or “pesticidal agent” refers to a substance that can be used in the control of agricultural, environmental, or domestic/household pests, such as insects, fungi, bacteria, or viruses.
  • pesticide is understood to encompass naturally occurring or synthetic insecticides (larvicides, and adulticides), insect growth regulators, acaricides (miticides), nematicides, ectoparasiticides, bactericides, fungicides, or herbicides (substance which can be used in agriculture to control or modify plant growth). Further examples of pesticides or pesticidal agents are listed in Table 12. In some instances, the pesticide is an allelochemical.
  • allelochemical or “allelochemical agent” is a substance produced by an organism that can effect a physiological function (e.g., the germination, growth, survival, or reproduction) of another organism (e.g., a host insect, e.g., mosquito).
  • a physiological function e.g., the germination, growth, survival, or reproduction
  • another organism e.g., a host insect, e.g., mosquito
  • peptide encompasses any chain of naturally or non-naturally occurring amino acids (either D- or L-amino acids), regardless of length (e.g., at least 2, 3, 4, 5, 6, 7, 10, 12, 14, 16, 18, 20, 25, 30, 40, 50, 100, or more amino acids), the presence or absence of post-translational modifications (e.g., glycosylation or phosphorylation), or the presence of, e.g., one or more non-amino acyl groups (for example, sugar, lipid, etc.) covalently linked to the peptide, and includes, for example, natural proteins, synthetic, or recombinant polypeptides and peptides, hybrid molecules, peptoids, or peptidomimetics.
  • amino acids either D- or L-amino acids
  • length e.g., at least 2, 3, 4, 5, 6, 7, 10, 12, 14, 16, 18, 20, 25, 30, 40, 50, 100, or more amino acids
  • post-translational modifications e.g., glycosylation or phosphorylation
  • percent identity between two sequences is determined by the BLAST 2.0 algorithm, which is described in Altschul et al., (1990) J. Mol. Biol. 215:403-410. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.
  • bacteriophage or “phage” refers to a virus that infects and replicates in bacteria. Bacteriophages replicate within bacteria following the injection of their genome into the cytoplasm and do so using either a lytic cycle, which results in bacterial cell lysis, or a lysogenic (non-lytic) cycle, which leaves the bacterial cell intact.
  • the phage may be a naturally occurring phage isolate, or an engineered phage, including vectors, or nucleic acids that encode either a partial phage genome (e.g., including at least all essential genes necessary to carry out the life cycle of the phage inside a host bacterium) or the full phage genome.
  • plant refers to whole plants, plant organs, plant tissues, seeds, plant cells, seeds, and progeny of the same.
  • Plant cells include, without limitation, cells from seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores.
  • Plant parts include differentiated and undifferentiated tissues including, but not limited to the following: roots, stems, shoots, leaves, pollen, seeds, tumor tissue, and various forms of cells and culture (e.g., single cells, protoplasts, embryos, and callus tissue).
  • the plant tissue may be in a plant or in a plant organ, tissue, or cell culture.
  • a plant may be genetically engineered to produce a heterologous protein or RNA, for example, of any of the modulating agents in the methods or compositions described herein.
  • vector refers to an insect that can carry or transmit a human pathogen from a reservoir to a human.
  • exemplary vectors include insects, such as those with piercing-sucking mouthparts, as found in Hemiptera and some Hymenoptera and Diptera such as mosquitoes, bees, wasps, midges, lice, tsetse fly, fleas and ants, as well as members of the Arachnidae such as ticks and mites.
  • FIG. 1A-1G show images of different antibiotic delivery systems.
  • First instar LSR-1 aphids were treated with different therapeutic solutions by delivery through plants ( FIG. 1A ), leaf coating ( FIG. 1B ), microinjection ( FIG. 1C ), topical delivery ( FIG. 1D ), leaf perfusion and cutting ( FIG. 1E ), leaf perfusion and through plant ( FIG. 1F ), and combination treatment of spraying both plant and aphid, and delivery though plant ( FIG. 1G ).
  • FIG. 2A-2C show the delay in aphid development during rifampicin treatment in first instar LSR-1 aphids treated by delivery through plants with three different conditions: artificial diet without essential amino acids (AD only), artificial diet without essential amino acids with 100 ⁇ g/ml rifampicin (AD+Rif), and artificial diet with 100 ⁇ g/ml rifampicin and essential amino acids (AD+Rif+EAA).
  • FIG. 2B shows representative images from each treatment taken at 12 days. Scale bars 2.5 mm.
  • FIG. 2C shows area measurements from aphid bodies showing the drastic effect of rifampicin treatment. Adding back essential amino acids partially rescues development defects.
  • FIG. 4 is a graph showing that rifampicin treatment resulted in loss of reproduction in aphids.
  • First instar LSR-1 aphids were treated by delivery through plants with artificial diet without essential amino acids (AD only), artificial diet without essential amino acids with 100 ug/ml rifampicin (AD+Rif), and artificial diet with 100 ug/ml rifampicin and (AD+Rif+EAA) and the number of offspring produced each day after aphid reached adulthood was measured. Shown is the mean number of offspring produced per day after aphid reached adulthood ⁇ S.D.
  • FIG. 5 is a graph showing that rifampicin treatment eliminated endosymbiotic Buchnera .
  • Symbiont titer was determined for the different conditions at 7 days post-treatment.
  • DNA from aphids was extracted and qPCR was performed to determine the ratio of Buchnera DNA to aphid DNA. Shown is the mean ratio of Buchnera DNA to aphid DNA ⁇ SD of 3 aphids per group.
  • Statistically significant differences were determined using a one-way-ANOVA followed by Tukey's Post-Test; *, p ⁇ 0.05.
  • FIGS. 6A and 6B show that rifampicin treatment delivered through leaf coating delayed aphid development.
  • First instar eNASCO aphids were treated by coating leaves with 100 ⁇ l of two different solutions: solvent control (0.025% Silwet L-77), and 50 ⁇ g/ml rifampicin.
  • FIG. 6B is a graph showing area measurements from aphid bodies showing the drastic effect of rifampicin coated leaves on aphid size. Statistically significant differences were determined using a one-way-ANOVA followed by Tukey's Post-Test; *, p ⁇ 0.05.
  • FIG. 7 shows that rifampicin treatment delivered through leaf coating resulted in aphid death. Survival was monitored daily for eNASCO aphids treated by coating leaves with 100 ⁇ l of two different solutions: solvent control (Silwet L-77), and 50 ⁇ g/ml rifampicin. Treatment affects survival rate of aphids.
  • FIG. 8 shows that rifampicin treatment delivered through leaf coating eliminated endosymbiotic Buchnera .
  • Symbiont titer was determined for the two conditions at 6 days post-treatment.
  • DNA from aphids was extracted and qPCR was performed to determine the ratio of Buchnera DNA to aphid DNA. Shown is the mean ratio of Buchnera DNA to aphid DNA ⁇ SD.
  • Statistically significant differences were determined using a one-way-ANOVA followed by Tukey's Post-Test; *, p ⁇ 0.05.
  • FIG. 9 is a graph showing rifampicin treatment by microinjection eliminated endosymbiotic Buchnera .
  • Symbiont titer was determined 4 days post-injection with the indicated conditions.
  • Control sample is the solvent, 0.025% Silwet L-77 described before.
  • DNA from aphids was extracted and qPCR was performed to determine the ratio of Buchnera DNA to aphid DNA. Shown is the mean ratio of Buchnera DNA to aphid DNA ⁇ SD.
  • Statistically significant differences were determined using a one-way-ANOVA followed by Tukey's Post-Test; *, p ⁇ 0.05.
  • FIG. 10 is a graph showing that rifampicin treatment delivered through topical treatment eliminated endosymbiotic Buchnera .
  • Symbiont titer was determined 3 days post-spraying with: solvent (silwet L-77) or the rifampicin solution diluted in solvent.
  • DNA from aphids was extracted and qPCR was performed to determine the ratio of Buchnera DNA to aphid DNA. Shown is the mean ratio of Buchnera DNA to aphid DNA ⁇ SD.
  • Statistically significant differences were determined using a one-way-ANOVA followed by Tukey's Post-Test; *, p ⁇ 0.05.
  • FIG. 12 shows a graph demonstrating survival of 1 st and 2 nd instar LSR-1 aphids placed on leaves perfused with water plus food coloring or 50 ⁇ g/ml rifampicin in water plus food coloring. Number in parentheses represents the number of aphids in each group. Statistical significance was determined by Log-Rank Test.
  • FIG. 13 shows a graph demonstrating symbiont titer determined 8 days post-treatment with leaves perfused with water and food coloring or rifampicin plus water and food coloring.
  • DNA from aphids was extracted and qPCR was performed to determine the ratio of Buchnera DNA to aphid DNA. Shown is the mean ratio of Buchnera DNA to aphid DNA ⁇ SD. Number in box indicates the median of the experimental group.
  • FIG. 15 is a graph demonstrating survival of 1 st and 2 nd instar LSR-1 aphids placed on leaves perfused and treated with water plus food coloring or 100 ⁇ g/ml rifampicin in water plus food coloring. Number in parentheses represents the number of aphids in each group. A Log-Rank Test was performed and determined that there were no statistically significant differences between groups.
  • FIGS. 16A and 16B are graphs showing symbiont titer determined 6 ( 16 A) and 8 ( 16 B) days post-treatment in aphids feeding on leaves perfused and treated with water and food coloring or rifampicin plus water and food coloring.
  • DNA was extracted from aphids and qPCR was performed to determine the ratio of Buchnera DNA to aphid DNA. Shown is the mean ratio of Buchnera DNA to aphid DNA ⁇ SD. Number in box indicates the median of the experimental group.
  • FIG. 18 is a graph showing 1st and 2nd instar LSR-1 aphids were treated with control solutions of a combination of treatments containing rifampicin. Number in parentheses represents the number of aphids in each group. A Log-Rank Test was performed and determined that there were no statistically significant differences between groups.
  • FIG. 19 is a graph showing symbiont titer determined at 7 days post-treatment with control or rifampicin solutions.
  • DNA from aphids was extracted and qPCR was performed to determine the ratio of Buchnera DNA to aphid DNA. Shown is the mean ratio of Buchnera DNA to aphid DNA ⁇ SD. Number in box indicates the median of the experimental group. Statistically significant differences were determined by t-test.
  • FIG. 20 is an image showing the chitosan delivery system. A. pisum aphids were treated with a therapeutic solution by delivery through leaf perfusion and through the plants as shown.
  • FIG. 21 is a panel of graphs showing that chitosan treatment resulted in delayed aphid development.
  • First and second instar A pisum aphids were treated by delivery through plants and leaf perfusion with the control solution (Water), and 300 ug/ml chitosan in water. Developmental stage was monitored throughout the experiment. Shown are the percent of aphids at each developmental stage (1st instar, 2nd instar, 3rd instar, 4th instar, 5th instar, or 5R which represents a reproducing 5th instar) per treatment group.
  • FIG. 22 is a graph showing there was a decrease in insect survival upon treatment with chitosan.
  • First and second instar A pisum aphids were treated by delivery through plants and leaf perfusion with just water or chitosan solution and survival was monitored daily over the course of the experiment.
  • Number in parentheses represents the total number of aphids in the treatment group.
  • FIG. 23 is a graph showing treatment with chitosan reduced endosymbiotic Buchnera .
  • First and second instar A pisum aphids were treated by delivery through plants and leaf perfusion with water or 300 ug/ml chitosan in water.
  • DNA from aphids was extracted and qPCR was performed to determine the ratio of Buchnera DNA to aphid DNA. Shown is the mean ratio of Buchnera DNA to aphid DNA ⁇ SD of 6 aphids/group. The median value for each group is shown in box.
  • FIG. 24 is a panel of graphs showing treatment with nisin resulted in delayed aphid development.
  • FIG. 25 is a graph showing there was a dose dependent decrease in insect survival upon treatment with nisin.
  • First and second instar LSR-1 A pisum aphids were treated with water (control) or 1.6 or 7 mg/ml nisin via delivery by leaf injection and through the plant and survival was monitored over time. Number in parentheses indicates the number of aphids/group. Statistically significant differences were determined by Log Rank (Mantel-Cox) test.
  • FIG. 26 is a graph showing treatment with nisin reduced endosymbiotic Buchnera .
  • First and second instar LSR-1 A pisum aphids were treated with water (control) or 1.6 mg/ml nisin via delivery by leaf injection and through the plant and DNA was extracted from select aphids at eight days post-treatment and used for qPCR to determine Buchnera copy numbers. Shown are the mean Buchnera /aphid ratios for each treatment+/ ⁇ SEM. Number in the box above each experimental group indicates the median value for that group. Each data point represents a single aphid.
  • FIG. 27 is a panel of graphs showing treatment with levulinic acid resulted in delayed aphid development.
  • FIG. 28 is a graph showing there was a decrease in insect survival upon treatment with levulinic acid.
  • FIG. 29 is a panel of graphs showing treatment with levulinic acid reduced endosymbiotic Buchnera .
  • First and second instar eNASCO A pisum aphids were treated with water (control) or 0.03 or 0.3% levulinic acid via delivery by leaf injection and through the plant and DNA was extracted from select aphids at seven and eleven days post-treatment and used for qPCR to determine Buchnera copy numbers. Shown are the mean Buchnera /aphid ratios for each treatment+/ ⁇ SEM. Statistically significant differences were determined by One-way ANOVA and Dunnett's Multiple Comparison Test; *, p ⁇ 0.05. Each data point represents a single aphid.
  • FIGS. 30A and 30B show graphs demonstrating that gossypol treatment resulted in delayed aphid development.
  • First and second instar A pisum aphids were treated by delivery through plants with artificial diet without essential amino acids (AD only), and artificial diet without essential amino acids with different concentrations of gossypol (0.05%, 0.25% and 0.5%). Developmental stage was monitored throughout the experiment.
  • FIG. 30A is a series of graphs showing the mean number of aphids at each developmental stage (1st instar, 2nd instar, 3rd instar, 4th instar, 5th instar, or 5R which represents a reproducing 5th instar) per treatment group. At the indicated time, aphids were imaged and their size was determined using Image J.
  • FIG. 30B is a graph showing the mean aphid area ⁇ SD of artificial diet treated (Control) or gossypol treated aphids. Statistical significance was determined using a One-Way ANOVA followed by Tukey's post-test. *, p ⁇ 0.05. **, p ⁇ 0.01.
  • FIG. 31 is a graph showing a dose-dependent decrease in survival of aphids upon treatment with the allelochemical gossypol.
  • FIGS. 32A and 32B are two graphs showing that treatment with 0.25% gossypol resulted in decreased fecundity.
  • First and second instar A pisum aphids were treated by delivery through plants with artificial diet without essential amino acids (AD5-2 no EAA), or artificial diet without essential amino acids with 0.25% gossypol acetic acid (AD5-2 no EAA+0.25% gossypol), and fecundity was determined throughout the time course of the experiment.
  • FIG. 32A shows the mean day ⁇ SD at which aphids began producing offspring was measured and gossypol treatment delayed production of offspring.
  • FIG. 32B shows the mean number of offspring produced after the aphid began a reproducing adult ⁇ SD was measured and gossypol treatment results in decreased number of offspring produced. Each data point represents one aphid.
  • FIG. 33 is a graph showing that treatment with different concentrations of gossypol reduced endosymbiotic Buchnera .
  • Control artificial diet without essential amino acids
  • 0.05% gossypol 0.5%, 0.25%, or 0.05% gossypol.
  • DNA from aphids was extracted and qPCR was performed to determine the ratio of Buchnera DNA to aphid DNA. Shown is the mean ratio of Buchnera DNA to aphid DNA ⁇ SD of 2-6 aphids/group. Statistically significant differences were determined by Unpaired T-test; *, p ⁇ 0.05.
  • FIG. 34 is a graph showing that microinjection of gossypol resulted in decreased Buchnera levels in aphids.
  • AD essential amino acids
  • gossypol 0.05%)
  • Three days after injection DNA was extracted from aphids and Buchnera levels were assessed by qPCR. Shown are the mean ratios of Buchnera /aphid DNA ⁇ SD. Each data point represents one aphid.
  • FIG. 35 is a panel of graphs showing Trans-cinnemaldehyde treatment resulted in delayed aphid development.
  • FIG. 36 is a graph showing there was a dose-dependent decrease in survival upon treatment the natural antimicrobial trans-cinnemaldehyde.
  • FIG. 37 is a graph showing treatment with different concentrations of trans-cinnemaldehyde reduced endosymbiotic Buchnera .
  • First and second instar A pisum aphids were treated by delivery through plants with water and water with different concentrations of trans-cinnemaldehyde (0.05%, 0.5%, and 5%).
  • DNA from aphids was extracted and qPCR was performed to determine the ratio of Buchnera DNA to aphid DNA. Shown is the mean ratio of Buchnera DNA to aphid DNA ⁇ SD of 2-11 aphids/group. The median of each treatment group is shown in the box above the data points. Statistically significant differences were determined by Unpaired T-test; *, p ⁇ 0.05. There was a statistically significant difference between the water control and the 0.5% trans-cinnemaldehyde group.
  • FIG. 38 is a panel of graphs showing treatment with scorpion peptide Uy192 resulted in delayed aphid development.
  • First and second instar A pisum aphids were treated by delivery through plants and leaf perfusion with the control solution (water), and 100 ug/ml Uy192 in water. a) developmental stage was monitored throughout the experiment. Shown are the percent of aphids at each developmental stage (1st instar, 2nd instar, 3rd instar, 4th instar, 5th instar, or 5R which represents a reproducing 5th instar) per treatment group.
  • FIG. 39 is a graph showing there was a decrease in insect survival upon treatment with the scorpion AMP Uy192.
  • First and second instar A pisum aphids were treated by delivery through plants and leaf perfusion with just water or Uy192 solution and survival was monitored daily over the course of the experiment. Number in parentheses represents the total number of aphids in the treatment group.
  • FIG. 40 is a graph showing treatment with Uy192 reduced endosymbiotic Buchnera .
  • First and second instar A pisum aphids were treated by delivery through plants and leaf perfusion with water or 100 ug/ml Uy192 in water, at 8 days post-treatment, DNA from aphids was extracted and qPCR was performed to determine the ratio of Buchnera DNA to aphid DNA. Shown is the mean ratio of Buchnera DNA to aphid DNA ⁇ SD of 2-6 aphids/group. The median value for each group is shown in box.
  • FIG. 41 is a graph showing a decrease in survival in aphids microinjected with scorpion peptides D10 and D3.
  • LSR-1 A pisum aphids were microinjected with water (control) or with 100 ng of either scorpion peptide D3 or D10. After injection, aphids were released to fava bean leaves and survival was monitored throughout the course of the experiment. The number in parentheses indicates the number of aphids in each experimental treatment group.
  • FIG. 42 is a graph showing a decrease in endosymbiont titers upon injection with scorpion peptides D3 and D10.
  • FIG. 43 is a graph showing a decrease in insect survival upon treatment with a cocktail of scorpion AMPs.
  • First and second instar eNASCO aphids were treated by delivery through leaf perfusion and through plants with a cocktail of scorpion peptides (40 ⁇ g/ml of each of Uy17, D3, UyCt3, and D10) and survival was monitored over the course of the experiment.
  • the number in parentheses represents the number of aphids in each treatment group.
  • FIG. 44 is a panel of graphs showing treatment with scorpion peptide fused to a cell penetrating peptide resulted in delayed aphid development.
  • FIG. 45 is a graph showing treatment of aphids with a scorpion peptide fused to a cell penetrating peptide increased mortality.
  • FIG. 46 is a graph showing treatment with Uy192+CPP+FAM reduced endosymbiotic Buchnera .
  • First instar LSR-1 A pisum aphids were treated with water or 100 ⁇ g/ml Uy192+CPP+FAM (peptide) in water delivered by leaf injection and through the plant. DNA was extracted from select aphids at five days post-treatment and used for qPCR to determine Buchnera copy numbers. Shown are the mean Buchnera /aphid ratios for each treatment+/ ⁇ SEM. Number in the box above each experimental group indicates the median value for that group. Each data point represents a single aphid. Statistically significant differences were determined by Student's T-test; ****, p ⁇ 0.0001.
  • FIG. 47 is a panel of images showing Uy192+CPP+FAM penetrated bacteriocyte membranes. Bacteriocytes were dissected from the aphids and incubated with 250 ⁇ g/ml of the Uy192+CPP+FAM peptide for 30 min. Upon washing and imaging, the Uy192+CPP+FAM can be seen at high quantities inside the bacteriocytes.
  • FIG. 48A and FIG. 48B are a panel of graphs showing pantothenol treatment delayed aphid development.
  • First instar and second eNASCO aphids were treated by delivery through plants with three different conditions: artificial diet without essential amino acids (AD no EAA), artificial diet without essential amino acids with 10 uM pantothenol (10 uM pantothenol), and artificial diet without essential amino acids with 100 uM pantothenol (100 uM pantothenol), artificial diet without essential amino acids with 100 uM pantothenol, and artificial diet without essential amino acids with 10 uM pantothenol.
  • FIG. 48A shows developmental stage monitored over time for each condition.
  • FIG. 48B shows relative area measurements from aphid bodies showing the drastic effect of pantothenol treatment.
  • FIG. 49 is a graph showing that treatment with pantothenol increased aphid mortality. Survival was monitored daily for eNASCO aphids treated by delivery through plants with artificial diet without essential amino acids, or artificial diet without essential amino acids containing 10 or 100 uM pantothenol. Number in parentheses represents number of aphids in each group.
  • FIGS. 50A, 50B, and 50C are a panel of graphs showing Pantothenol treatment resulted in loss of reproduction.
  • First and second instar eNASCO aphids were treated by delivery through plants with artificial diet without essential amino acids or with artificial diet without essential amino acids with 10 or 100 uM pantothenol.
  • FIG. 50A shows the fraction of aphids surviving to maturity and reproducing.
  • FIG. 50B shows the mean day aphids in each group began reproducing. Shown is the mean day an aphid began reproducing ⁇ SD.
  • FIG. 50C shows the mean number of offspring produced per day after an aphid began reproducing. Shown are the mean number of offspring/day ⁇ SD.
  • FIG. 51 is a graph showing Pantothenol treatment did not affect endosymbiotic Buchnera .
  • Symbiont titer was determined for the different conditions at 8 days post-treatment.
  • DNA from aphids was extracted and qPCR was performed to determine the ratio of Buchnera DNA to aphid DNA. Shown is the mean ratio of Buchnera DNA to aphid DNA ⁇ SD of 6 aphids per group.
  • FIG. 52 is a panel of graphs showing Pantothenol treatment delivered through plants did not affect aphid development.
  • FIGS. 54A and 54B are a panel of graphs showing treatment with a cocktail of amino acid analogs delayed aphid development.
  • First instar LSR-1 aphids were treated by delivery through leaf perfusion and through plants with water or a cocktail of amino acid analogs in water (AA cocktail).
  • FIG. 54A shows the developmental stage measured over time for each condition. Shown are the percentage of living aphids at each developmental stage.
  • FIG. 54B shows the area measurements from aphid bodies showing the drastic effect of treatment with an amino acid analog cocktail (AA cocktail). Statistically significant differences were determined using a Student's T-test; ****, p ⁇ 0.0001.
  • FIG. 55 is a graph showing treatment with a cocktail of amino acid analogs eliminated endosymbiotic Buchnera .
  • Symbiont titer was determined for the different conditions at 6 days post-treatment.
  • DNA from aphids was extracted and qPCR was performed to determine the ratio of Buchnera DNA to aphid DNA. Shown are the mean ratios of Buchnera DNA to aphid DNA ⁇ SD of 19-20 aphids per group. Each data point represents an individual aphid. Statistically significant differences were determined using a Student's T-test; *, p ⁇ 0.05.
  • FIGS. 56A and 56B is a panel of graphs showing treatment with a combination of three agents delayed aphid development.
  • First instar LSR-1 aphids were treated by delivery through leaf perfusion and through plants with water or a combination of three agents in water (Pep-Rif-Chitosan).
  • FIG. 56A shows the developmental stage measured over time for each condition. Shown are the percentage of living aphids at each developmental stage.
  • FIG. 56B shows the area measurements from aphid bodies showing the drastic effect of treatment with a combination of three treatments (Pep-Rif-Chitosan).
  • Statistically significant differences were determined using a Student's T-test; ****, p ⁇ 0.0001.
  • FIG. 57 is a graph showing treatment with a combination of a peptide, antibiotic, and natural antimicrobial agent increased aphid mortality.
  • LSR-1 aphids were treated with water or a combination of three treatments (Pep-Rif-Chitosan) and survival was monitored daily after treatment.
  • FIG. 58 is a graph showing treatment with a combination of a peptide, antibiotic, and natural antimicrobial agent eliminated endosymbiotic Buchnera .
  • Symbiont titer was determined for the different conditions at 6 days post-treatment.
  • DNA from aphids was extracted and qPCR was performed to determine the ratio of Buchnera DNA to aphid DNA. Shown are the mean ratios of Buchnera DNA to aphid DNA ⁇ SD of 20-21 aphids per group. Each data point represents an individual aphid.
  • FIGS. 59A and 59B are a panel of images showing ciprofloxacin coated and penetrated corn kernels. Corn kernels were soaked in water (no antibiotic) or the indicated concentration of ciprofloxacin in water and whole kernels or kernel were tested to see whether they can inhibit the growth of E. coli DH5a.
  • FIG. 59A shows bacterial growth in the presence of a corn kernel soaked in water without antibiotics and
  • FIG. 59B shows the inhibition of bacterial growth when whole or half corn kernels that have been soaked in antibiotics are placed on a plate spread with E. coli.
  • FIG. 60 is a graph showing that adult S. zeamais weevils were treated with ciprofloxacin (250 ug/ml or 2.5 mg/ml) or mock treated with water. After 18 days of treatment, genomic DNA was isolated from weevils and the amount of Sitophilus primary endosymbiont was determined by qPCR. Shown is the mean ⁇ SEM of each group. Each data point represents one weevil. The median of each group is listed above the dataset.
  • FIGS. 61A and 61B are graphs showing weevil development after treatment with ciprofloxacin.
  • FIG. 61A shows individual corn kernels cut open 25 days after adults were removed from one replicate each of the initial corn kernels soaked/coated with water (control) or ciprofloxacin (250 ug/ml or 2.5 mg/ml) and examined for the presence of larvae, pupae, or almost fully developed (adult) weevils. Shown is the percent of each life stage found in kernels from each treatment group. The total number of offspring found in the kernels from each treatment group is indicated above each dataset.
  • FIG. 61A shows individual corn kernels cut open 25 days after adults were removed from one replicate each of the initial corn kernels soaked/coated with water (control) or ciprofloxacin (250 ug/ml or 2.5 mg/ml) and examined for the presence of larvae, pupae, or almost fully developed (adult) weevils. Shown is the percent of each life stage found
  • 61B shows genomic DNA isolated from offspring dissected from corn kernels from the control (water) and 2.5 mg/ml ciprofloxacin treatment groups and qPCR was done to measure the amount of Sitophilus primary endosymbiont present. Shown are the mean ⁇ SD for each group. Statistically significant differences were determined by unpaired t-test; ***, p ⁇ 0.001.
  • FIGS. 62A and 62B are graphs showing the two remaining replicates of corn kernels mock treated (water) or treated with 250 ug/ml or 2.5 mg/ml ciprofloxacin monitored for the emergence of offspring after mating pairs were removed (at 7 days post-treatment).
  • FIG. 62A shows the mean number of newly emerged weevils over time ⁇ SD for each treatment group.
  • FIG. 62B shows the mean number ⁇ SEM of emerged weevils for each treatment group at 43 days after mating pairs were removed.
  • FIG. 63 is a graph showing rifampicin and doxycycline treatment resulted in mite mortality. Survival was monitored daily for untreated two-spotted spider mites and mites treated with 250 ⁇ g/ml rifampicin and 500 ⁇ g/ml doxycycline in 0.025% Silwet L-77.
  • FIG. 64 is a panel of graphs showing the results of a Seahorse flux assay for bacterial respiration.
  • Bacteria were grown to logarithmic phase and loaded into Seahorse XFe96 plates for temporal measurements of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) as described in methods. Treatments were injected into the wells after approximately 20 minutes and bacteria were monitored to detect changes in growth.
  • OCR oxygen consumption rate
  • ECAR extracellular acidification rate
  • Treatments were injected into the wells after approximately 20 minutes and bacteria were monitored to detect changes in growth.
  • Rifampicin 100 ⁇ g/mL
  • Chloramphenicol 25 ⁇ g/mL
  • Phages (T7 for E. coli and ⁇ SmVL-C1 for S. marcescens ) were lysates diluted either 1:2 or 1:100 in SM Buffer.
  • the markers on each line are solely provided as indicators of the condition to which each line corresponds, and are not indicative
  • FIG. 65 is a graph showing phage against S. marcescens reduced fly mortality. Flies that were pricked with S. marcescens were all dead within a day, whereas a sizeable portion of the flies that were pricked with both S. marcescens and the phage survived for five days after the treatment. Almost all of the control flies which were not treated in anyway survived till the end of the experiment. Log-rank test was used to compare the curves for statistical significance, asterisk denotes p ⁇ 0.0001.
  • compositions useful for human health e.g., for altering a level, activity, or metabolism of one or more microorganisms resident in a host insect (e.g., arthropod, e.g., insect, e.g., a human pathogen vector, e.g., mosquito, mite, louse, or tick), the alteration resulting in a decrease in the fitness of the host.
  • a host insect e.g., arthropod, e.g., insect, e.g., a human pathogen vector, e.g., mosquito, mite, louse, or tick
  • the invention features a composition that includes a modulating agent (e.g., phage, peptide, small molecule, antibiotic, or combinations thereof) that can alter the host's microbiota in a manner that is detrimental to the host.
  • a modulating agent e.g., phage, peptide, small molecule, antibiotic, or combinations thereof
  • compositions described herein are based in part on the examples provided herein, which illustrate how modulating agents, for example antibiotics (e.g., oxytetracycline, doxycycline, or a combination thereof) can be used to target symbiotic microorganisms in a host (e.g., endosymbionts e.g., endosymbiotic Wolbachia in mosquitos or Rickettsia in ticks) in insect vectors of human pathogens, to decrease the fitness of the host by altering the level, activity, or metabolism of the microorganisms within the hosts.
  • antibiotics e.g., oxytetracycline, doxycycline, or a combination thereof
  • symbiotic microorganisms in a host e.g., endosymbionts e.g., endosymbiotic Wolbachia in mosquitos or Rickettsia in ticks
  • the present disclosure describes a variety of different approaches for the use of agents that alter a level, activity, or metabolism of one or more microorganisms resident in a host (e.g., a vector of a human pathogen, e.g., a mosquito, mite, louse or a tick) the alteration resulting in a decrease in the host's fitness.
  • a host e.g., a vector of a human pathogen, e.g., a mosquito, mite, louse or a tick
  • compositions provided herein may be used with any insect host that is considered a vector for a pathogen that is capable of causing disease in humans
  • the insect host may include, but is not limited to those with piercing-sucking mouthparts, as found in Hemiptera and some Hymenoptera and Diptera such as mosquitoes, bees, wasps, midges, lice, tsetse fly, fleas and ants, as well as members of the Arachnidae such as ticks and mites; order, class or family of Acarina (ticks and mites) e.g.
  • the insect is a blood-sucking insect from the order Diptera (e.g., suborder Nematocera, e.g., family Colicidae).
  • the insect is from the subfamilies Culicinae, Corethrinae, Ceratopogonidae, or Simuliidae.
  • the insect is of a Culex spp., Theobaldia spp., Aedes spp., Anopheles spp., Aedes spp., Forciponiyia spp., Culicoides spp., or Helea spp.
  • the insect is a mosquito. In certain instances, the insect is a tick. In certain instances, the insect is a mite. In certain instances, the insect is a biting louse.
  • the methods and compositions provided herein may be used to decrease the fitness of any of the hosts described herein.
  • the decrease in fitness may arise from any alterations in microorganisms resident in the host, wherein the alterations are a consequence of administration of a modulating agent and have detrimental effects on the host.
  • the decrease in host fitness may manifest as a deterioration or decline in the physiology of the host (e.g., reduced health or survival) as a consequence of administration of a modulating agent.
  • the fitness of an organism may be measured by one or more parameters, including, but not limited to, reproductive rate, lifespan, mobility, fecundity, body weight, metabolic rate or activity, or survival in comparison to a host organism to which the modulating agent has not been administered.
  • the methods or compositions provided herein may be effective to decrease the overall health of the host or to decrease the overall survival of the host.
  • the decreased survival of the host is about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or greater than 100% greater relative to a reference level (e.g., a level found in a host that does not receive a modulating agent).
  • a reference level e.g., a level found in a host that does not receive a modulating agent.
  • the methods and compositions are effective to decrease host reproduction (e.g., reproductive rate) in comparison to a host organism to which the modulating agent has not been administered.
  • the methods and compositions are effective to decrease other physiological parameters, such as mobility, body weight, life span, fecundity, or metabolic rate, by about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or greater than 100% relative to a reference level (e.g., a level found in a host that does not receive a modulating agent).
  • a reference level e.g., a level found in a host that does not receive a modulating agent.
  • the decrease in host fitness may manifest as a decrease in the production of one or more nutrients in the host (e.g., vitamins, carbohydrates, amino acids, or polypeptides).
  • the methods or compositions provided herein may be effective to decrease the production of nutrients in the host (e.g., vitamins, carbohydrates, amino acids, or polypeptides) by about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or greater than 100% relative to a reference level (e.g., a level found in a host that does not receive a modulating agent).
  • the methods or compositions provided herein may decrease nutrients in the host by decreasing the production of nutrients by one or more microorganisms (e.g., endosymbiont) in the host in comparison to a host organism to which the modulating agent has not been administered.
  • microorganisms e.g., endosymbiont
  • the decrease in host fitness may manifest as an increase in the host's sensitivity to a pesticidal agent (e.g., a pesticide listed in Table 12) and/or a decrease in the host's resistance to a pesticidal agent (e.g., a pesticide listed in Table 12) in comparison to a host organism to which the modulating agent has not been administered.
  • a pesticidal agent e.g., a pesticide listed in Table 12
  • a decrease in the host's resistance to a pesticidal agent e.g., a pesticide listed in Table 12
  • the methods or compositions provided herein may be effective to increase the host's sensitivity to a pesticidal agent (e.g., a pesticide listed in Table 12) by about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or greater than 100% relative to a reference level (e.g., a level found in a host that does not receive a modulating agent).
  • a pesticidal agent e.g., a pesticide listed in Table 12
  • the pesticidal agent may be any pesticidal agent known in the art, including insecticidal agents.
  • the methods or compositions provided herein may increase the host's sensitivity to a pesticidal agent (e.g., a pesticide listed in Table 12) by decreasing the host's ability to metabolize or degrade the pesticidal agent into usable substrates.
  • a pesticidal agent e.g., a pesticide listed in Table 12
  • the decrease in host fitness may manifest as an increase in the host's sensitivity to an allelochemical agent and/or a decrease in the host's resistance to an allelochemical agent in comparison to a host organism to which the modulating agent has not been administered.
  • the methods or compositions provided herein may be effective to decrease the host's resistance to an allelochemical agent by about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or greater than 100% relative to a reference level (e.g., a level found in a host that does not receive a modulating agent).
  • the allelochemical agent is caffeine, soyacystatin N, monoterpenes, diterpene acids, or phenolic compounds.
  • the methods or compositions provided herein may increase the host's sensitivity to an allelochemical agent by decreasing the host's ability to metabolize or degrade the allelochemical agent into usable substrates in comparison to a host organism to which the modulating agent has not been administered.
  • the methods or compositions provided herein may be effective to decease the host's resistance to parasites or pathogens (e.g., fungal, bacterial, or viral pathogens or parasites) in comparison to a host organism to which the modulating agent has not been administered.
  • the methods or compositions provided herein may be effective to decrease the host's resistance to a pathogen or parasite (e.g., fungal, bacterial, or viral pathogens; or parasitic mites) by about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or greater than 100% relative to a reference level (e.g., a level found in a host that does not receive a modulating agent).
  • a reference level e.g., a level found in a host that does not receive a modulating agent.
  • the decrease in host fitness may manifest as other fitness disadvantages, such as decreased tolerance to certain environmental factors (e.g., a high or low temperature tolerance), decreased ability to survive in certain habitats, or a decreased ability to sustain a certain diet in comparison to a host organism to which the modulating agent has not been administered.
  • the methods or compositions provided herein may be effective to decrease host fitness in any plurality of ways described herein.
  • the modulating agent may decrease host fitness in any number of host classes, orders, families, genera, or species (e.g., 1 host species, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 200, 250, 500, or more host species).
  • the modulating agent acts on a single host class, order, family, genus, or species.
  • Host fitness may be evaluated using any standard methods in the art. In some instances, host fitness may be evaluated by assessing an individual host. Alternatively, host fitness may be evaluated by assessing a host population. For example, a decrease in host fitness may manifest as a decrease in successful competition against other insects, thereby leading to a decrease in the size of the host population.
  • the modulating agents provided herein are effective to reduce the spread of vector-borne diseases.
  • the modulating agent may be delivered to the insects using any of the formulations and delivery methods described herein, in an amount and for a duration effective to reduce transmission of the disease, e.g., reduce vertical or horizontal transmission between vectors and/or reduce transmission to humans.
  • the modulating agent described herein may reduce vertical or horizontal transmission of a vector-borne pathogen by about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more in comparison to a host organism to which the modulating agent has not been administered.
  • the modulating agent described herein may reduce vectorial competence of an insect vector by about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more in comparison to a host organism to which the modulating agent has not been administered.
  • Non-limiting examples of diseases that may be controlled by the compositions and methods provided herein include diseases caused by Togaviridae viruses (e.g., Chikungunya, Ross River fever, Mayaro, Onyon-nyong fever, Sindbis fever, Eastern equine enchephalomyeltis, Wesetern equine encephalomyelitis, deciualan equine encephalomyelitis, or Barmah forest); diseases caused by Flavivirdae viruses (e.g., Dengue fever, Yellow fever, Kyasanur Forest disease, Omsk haemorrhagic fever, Japaenese encephalitis, Murray Valley encephalitis, Rocio, St.
  • Togaviridae viruses e.g., Chikungunya, Ross River fever, Mayaro, Onyon-nyong fever, Sindbis fever, Eastern equine enchephalomyeltis, Wesetern equine ence
  • microorganisms targeted by the modulating agent described herein may include any microorganism resident in or on the host, including, but not limited to, any bacteria and/or fungi described herein.
  • Microorganisms resident in the host may include, for example, symbiotic (e.g., endosymbiotic microorganisms that provide beneficial nutrients or enzymes to the host), commensal, pathogenic, or parasitic microorganisms.
  • An endosymbiotic microorganism may be a primary endosymbiont or a secondary endosymbiont.
  • a symbiotic microorganism e.g., bacteria or fungi
  • Microorganisms resident in the host may be acquired by any mode of transmission, including vertical, horizontal, or multiple origins of transmission.
  • Exemplary bacteria that may be targeted in accordance with the methods and compositions provided herein, include, but are not limited to, Xenorhabdus spp, Photorhabdus spp, Candidatus spp, Buchnera spp, Blattabacterium spp, Baumania spp, Wigglesworthia spp, Wolbachia spp, Rickettsia spp, Orientia spp, Sodalis spp, Burkholderia spp, Cupriavidus spp, Frankia spp, Snirhizobium spp, Streptococcus spp, Wolinella spp, Xylella spp, Erwinia spp, Agrobacterium spp, Bacillus spp, Paenibacillus spp, Streptomyces spp, Micrococcus spp, Corynebacterium spp, Acetobacter spp, Cyanobacteri
  • Non-limiting examples of bacteria that may be targeted by the methods and compositions provided herein are shown in Table 1.
  • the 16S rRNA sequence of the bacteria targeted by the modulating agent has at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, 99.9%, or 100% identity with a sequence listed in Table 1.
  • CACATGCAAGTCGAACGGCAGC ACGGAGAGCTTGCTCTGGTG GCGAGTGGCGAACGGGTGAGTA ATGCATCGGAACGTACCGAGTA ATGGGGGATAACTGTCCGAAAG GATGGCTAATACCGCATACGCC CTGAGGGGGAAAGCGGGGGAT CGAAAGACCTCGCGTTATTTGAG CGGCCGATGTTGGATTAGCTAG TTGGTGGGGTAAAGGCCTACCA AGGCGACGATCCATAGCGGGTC TGAGAGGATGATCCGCCACATT GGGACTGAGACACGGCCCAAAC TCCTACGGGAGGCAGCAGTGGG GAATTTTGGACAATGGGGGGAA CCCTGATCCAGCCATGCCGCGT GTCTGAAGAAGGCCTTCGGGTT GTAAAGGACTTTTGTTAGGGAAG AAAAGCCGGGTGTTAATACCATC TGGTGCTGACGGTACCTAAAGA ATAAGCACCGGCTAACTACGTG CCAGCAGCCGCGGTAATACGTA GGGTGCGAGCGTTA
  • a mosquito e.g., Aedes spp. or Anopheles spp. harbors symbiotic bacteria that modulate the mosquito's immune response and influence vectorial competence to pathogens.
  • the modulating agent described herein may be useful in targeting bacteria resident in the mosquito, including, but not limited to, EspZ, Seratia spp (e.g., Serratia marcescens ), Enterbacterioaceae spp., Enterobacter spp. (e.g., Enterobacter cloacae, Enterobacter amnigenus, Enterobacter ludwigii ), .
  • Proteus spp. Acinetobacter spp., Wigglesworthia spp.
  • Xanthomonas spp. e.g., Xanthomonas maltophilia
  • Pseudomonas spp. e.g., Pseudomonas aeruginosa, Pseudomonas stutzeri, Pseudomonas rhodesiae
  • Escherichia spp. e.g., Escherchia coli
  • Cedecea spp. e.g., Cedecea lapagei
  • Ewingella spp. e.g., Ewingella americana
  • Wolbachia spp. e.g., Wolbachia -wMel, Wolbachia -wAlbB, Wolbachia -wMelPop, Wolbachia -wMelPop-CLA.
  • the modulating agent may target a single bacterial species.
  • the modulating agent may target at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 500, or more distinct bacterial species.
  • the modulating agent may target any one of about 1 to about 5, about 5 to about 10, about 10 to about 20, about 20 to about 50, about 50 to about 100, about 100 to about 200, about 200 to about 500, about 10 to about 50, about 5 to about 20, or about 10 to about 100 distinct bacterial species.
  • the modulating agent may target at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more phyla, classes, orders, families, or genera of bacteria.
  • the modulating agent may increase a population of one or more bacteria (e.g., pathogenic bacteria, toxin-producing bacteria) by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in the host in comparison to a host organism to which the modulating agent has not been administered.
  • bacteria e.g., pathogenic bacteria, toxin-producing bacteria
  • the modulating agent may reduce the population of one or more bacteria (e.g., symbiotic bacteria, a pesticide-degrading bacterium, e.g., a bacterium that degrades a pesticide listed in Table 12) by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more in the host in comparison to a host organism to which the modulating agent has not been administered.
  • the modulating agent may eradicate the population of a bacterium (e.g., symbiotic bacteria, a pesticide-degrading bacterium) in the host.
  • the modulating agent may increase the level of one or more pathogenic bacteria by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in the host and/or decreases the level of one or more symbiotic bacteria by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more in the host in comparison to a host organism to which the modulating agent has not been administered.
  • the modulating agent may alter the bacterial diversity and/or bacterial composition of the host. In some instances, the modulating agent may increase the bacterial diversity in the host relative to a starting diversity by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more in comparison to a host organism to which the modulating agent has not been administered. In some instances, the modulating agent may decrease the bacterial diversity in the host relative to a starting diversity by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more in comparison to a host organism to which the modulating agent has not been administered.
  • the modulating agent may alter the function, activity, growth, and/or division of one or more bacterial cells.
  • the modulating agent may alter the expression of one or genes in the bacteria.
  • the modulating agent may alter the function of one or more proteins in the bacteria.
  • the modulating agent may alter the function of one or more cellular structures (e.g., the cell wall, the outer or inner membrane) in the bacteria.
  • the modulating agent may kill (e.g., lyse) the bacteria.
  • the target bacterium may reside in one or more parts of the insect. Further, the target bacteria may be intracellular or extracellular. In some instances, the bacteria reside in or on one or more parts of the host gut, including, e.g., the foregut, midgut, and/or hindgut. In some instances, the bacteria reside as an intracellular bacteria within a cell of the host insect. In some instances, the bacteria reside in a bacteriocyte of the host insect.
  • Changes to the populations of bacteria in the host may be determined by any methods known in the art, such as microarray, polymerase chain reaction (PCR), real-time PCR, flow cytometry, fluorescence microscopy, transmission electron microscopy, fluorescence in situ hybridization (e.g., FISH), spectrophotometry, matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS), and DNA sequencing.
  • a sample of the host treated with a modulating agent is sequenced (e.g., by metagenomics sequencing of 16S rRNA or rDNA) to determine the microbiome of the host after delivery or administration of the modulating agent.
  • a sample of a host that did not receive the modulating agent is also sequenced to provide a reference.
  • Exemplary fungi that may be targeted in accordance with the methods and compositions provided herein, include, but are not limited to Amylostereum areolatum, Epichloe spp, Pichia pinus, Hansenula capsulate, Daldinia decipien, Ceratocytis spp, Ophiostoma spp, and Attamyces bromatificus .
  • Non-limiting examples of yeast and yeast-like symbionts found in insects include Candida, Metschnikowia, Debaromyces, Scheffersomyces shehatae and Scheffersomyces stipites, Starmerella, Pichia, Trichosporon, Cryptococcus, Pseudozyma , and yeast-like symbionts from the subphylum Pezizomycotina (e.g., Symbiotaphrina bucneri and Symbiotaphrina kochii ).
  • yeast that may be targeted by the methods and compositions herein are listed in Table 2.
  • Harpium inquisitor Coleoptera Cerambycidae Mycetomes ( Candida rhagii ) Harpium mordax Coleoptera: Cerambycidae Cecae around midgut ( Candida tenuis ) H.
  • cerambyciformis Leptura sanguinolenta Coleoptera Cerambycidae Cecae around midgut ( Candida sp.)
  • Rhagium bifasciatum Coleoptera Cerambycidae Cecae around midgut ( Candida tenuis )
  • Rhagium inquisitor Coleoptera Cerambycidae Cecae around midgut ( Candida guilliermondii )
  • Rhagium mordax Coleoptera Cerambycidae Cecae around midgut (Candida)
  • Carpophilus Coleoptera Nitidulidae Intestinal tract (10 yeast species) hemipterus
  • Odontotaenius Coleoptera Passalidae Hindgut ( Enteroramus dimorphus ) disjunctus
  • Odontotaenius Coleoptera Passalidae Gut ( Pichia stipitis, P.
  • Dendroctonus frontalis Coleoptera Scolytidae Midgut ( Candida sp.)
  • Ips sexdentatus Coleoptera Scolytidae Digestive tract ( Pichia bovis , P . rhodanensis ) Hansenula holstii ( Candida rhagii ) Digestive tract ( Candida pulcherina )
  • Ips typographus Coleoptera Scolytidae Alimentary canal Alimentary tracts ( Hansenula capsulata , Candida parapsilosis ) Guts and beetle homogenates ( Hansenula holstii , H . capsulata , Candida diddensii , C .
  • Homoptera Aphididae Tissue sections Hamiltonaphis styraci Glyphinaphis bambusae Cerataphis sp. Hamiltonaphis styraci Homoptera: Aphididae Abdominal hemocoel Cofana unimaculata Homoptera: Cicadellidae Fat body Leofa unicolor Homoptera: Cicadellidae Fat body Lecaniines , etc. Homoptera: Coccoidea d Hemolymph, fatty tissue, etc. Lecanium sp.
  • Homoptera Coccidae Hemolymph, adipose tissue Ceroplastes (4 sp.) Homoptera: Coccidae Blood smears Laodelphax striatellus Homoptera: Delphacidae Fat body Eggs Eggs ( Candida ) Nilaparvata lugens Homoptera: Delphacidae Fat body Eggs (2 unidentified yeast species) Eggs, nymphs ( Candida ) Eggs (7 unidentified yeast species) Eggs ( Candida ) Nisia nervosa Homoptera: Delphacidae Fat body Nisia grandiceps Perkinsiella spp.
  • Homoptera Delphacidae Fat body Amrasca devastans
  • Homoptera Jassidae Eggs, mycetomes, hemolymph Tachardina lobata
  • Homoptera Kerriidae Blood smears ( Torula variabilis ) sp.
  • Comperia merceti Hymenoptera Encyrtidae Hemolymph, gut, poison gland
  • Solenopsis invicta Hymenoptera Form icidae Hemolymph ( Myrmecomyces annellisae )
  • S. quinquecuspis Solenopsis invicta Hymenoptera: Form icidae Fourth instar larvae ( Candida parapsilosis , Yarrowia lipolytica ) Gut and hemolymph ( Candida parapsilosis , C. lipolytica , C . guillermondii , C.
  • the modulating agent may target a single fungal species.
  • the modulating agent may target at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 500, or more distinct fungal species.
  • the modulating agent may target any one of about 1 to about 5, about 5 to about 10, about 10 to about 20, about 20 to about 50, about 50 to about 100, about 100 to about 200, about 200 to about 500, about 10 to about 50, about 5 to about 20, or about 10 to about 100 distinct fungal species.
  • the modulating agent may target at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more phyla, classes, orders, families, or genera of fungi.
  • the modulating agent may increase a population of one or more fungi (e.g., pathogenic or parasitic fungi) by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in the host in comparison to a host organism to which the modulating agent has not been administered.
  • the modulating agent may reduce the population of one or more fungi (e.g., symbiotic fungi) by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in the host in comparison to a host organism to which the modulating agent has not been administered.
  • the modulating agent may eradicate the population of a fungi (e.g., symbiotic fungi) in the host.
  • the modulating agent may increase the level of one or more symbiotic fungi by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in the host and/or may decrease the level of one or more symbiotic fungi by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in the host in comparison to a host organism to which the modulating agent has not been administered.
  • the modulating agent may alter the fungal diversity and/or fungal composition of the host.
  • the modulating agent may increase the fungal diversity in the host relative to a starting diversity by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more in comparison to a host organism to which the modulating agent has not been administered.
  • the modulating agent may decrease the fungal diversity in the host relative to a starting diversity by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more in comparison to a host organism to which the modulating agent has not been administered.
  • the modulating agent may alter the function, activity, growth, and/or division of one or more fungi.
  • the modulating agent may alter the expression of one or more genes in the fungus.
  • the modulating agent may alter the function of one or more proteins in the fungus.
  • the modulating agent may alter the function of one or more cellular components in the fungus.
  • the modulating agent may kill the fungus.
  • the target fungus may reside in one or more parts of the insect.
  • the fungus resides in or on one or more parts of the insect gut, including, e.g., the foregut, midgut, and/or hindgut.
  • the fungus lives extracellularly in the hemolymph, fat bodies or in specialized structures in the host.
  • Changes to the population of fungi in the host may be determined by any methods known in the art, such as microarray, polymerase chain reaction (PCR), real-time PCR, flow cytometry, fluorescence microscopy, transmission electron microscopy, fluorescence in situ hybridization (e.g., FISH), spectrophotometry, matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS), and DNA sequencing.
  • PCR polymerase chain reaction
  • FISH fluorescence in situ hybridization
  • MALDI-MS matrix-assisted laser desorption ionization-mass spectrometry
  • DNA sequencing DNA sequencing.
  • a sample of the host treated with a modulating agent is sequenced (e.g., by metagenomics sequencing) to determine the microbiome of the host after delivery or administration of the modulating agent.
  • a sample of a host that did not receive the modulating agent is also sequenced to provide a reference.
  • the modulating agent of the methods and compositions provided herein may include a phage, a polypeptide, a small molecule, an antibiotic, a secondary metabolite, a bacterium, a fungus, or any combination thereof.
  • the modulating agent described herein may include a phage (e.g., a lytic phage or a non-lytic phage).
  • a phage e.g., a lytic phage or a non-lytic phage.
  • an effective concentration of any phage described herein may alter a level, activity, or metabolism of one or more microorganisms (as described herein) resident in a host described herein (e.g., a vector of a human pathogen, e.g., a mosquito, a mite, a biting louse, or a tick), the modulation resulting in a decrease in the host's fitness (e.g., as outlined herein).
  • the modulating agent includes at least one phage selected from the order Tectiviridae, Myoviridae, Siphoviridae, Podoviridae, Caudovirales, Lipothrixviridae, Rudiviridae, or Ligamenvirales.
  • the composition includes at least one phage selected from the family Myoviridae, Siphoviridae, Podoviridae, Lipothrixviridae, Rudiviridae, Ampullaviridae, Bicaudaviridae, Clavaviridae, Corticoviridae, Cystoviridae, Fuselloviridae, Gluboloviridae, Guttaviridae, Inoviridae, Leviviridae, Microviridae, Plasmaviridae, and Tectiviridae. Further non-limiting examples of phages useful in the methods and compositions are listed in Table 3.
  • Apidae family XP15 (NC_007024), phiL7 Apis mellifera ; (NC_012742) Drosphilidae family; and Chloropidae family PP1 (NC_019542), PM1 Pectobacterium Apidae family (NC_023865) carotovorum subsp.
  • NC_009382 carotovorum ⁇ RSA1 (NC_009382), Ralstonia Bombyx mori ⁇ RSB1 (NC_011201), ⁇ RSL1 solanacearum (NC_010811), RSM1 (NC_008574) SF1(NC_028807) Streptomyces Philantus sp.; scabies Trachypus sp ECML-4 (NC)_025446), Escherichia coli Apidae family; ECML-117 (NC_025441), Varroa destructor ECML-134 (NC_025449) SSP5(JX274646.1), SSP6 Salmonella sp.
  • Drosphilidae family (NC_004831), SFP10 (NC_016073), F18SE (NC_028698) y (NC_001416), Bcp1 Bacillus sp. Gypsy moth; (NC_024137) Lymantria dispar ; Varroa destructor Phil (NC_009821) Enterococcus Schistocerca gragaria sp. ⁇ KMV (NC 005045), Pseudomonas Lymantria dispar ; ⁇ EL(AJ697969.1), ⁇ KZ sp. Apidae family (NC 004629) A2 (NC_004112), phig1e Lactobacilli sp.
  • NC_004305) Drosophila family
  • Varroa destructor KLPN1 NC_028760
  • Klebsiella sp. C. capitata vB_AbaM_Acibe1004 Acinetobacter Schistocerca gragaria (NC_025462), vB_AbaP_Acibe1007 sp. (NC_025457)
  • a modulating agent includes a lytic phage.
  • the phage causes lysis in the target bacterial cell.
  • the lytic phage targets and kills a bacterium resident in a host insect to decrease the fitness of the host.
  • the phage of the modulating agent may be a non-lytic phage (also referred to as lysogenic or temperate phage).
  • the bacterial cell may remain viable and able to stably maintain expression of genes encoded in the phage genome.
  • a non-lytic phage is used to alter gene expression in a bacterium resident in a host insect to decrease the fitness of the host.
  • the modulating agent includes a mixture of lytic and non-lytic phage.
  • the phage is a naturally occurring phage.
  • a naturally occurring phage may be isolated from an environmental sample containing a mixture of different phages.
  • the naturally occurring phage may be isolated using methods known in the art to isolate, purify, and identify phage that target a particular microorganism (e.g., a bacterial endosymbiont in an insect host).
  • the phage may be engineered based on a naturally occurring phage.
  • the modulating agent described herein may include phage with either a narrow or broad bacterial target range.
  • the phage has a narrow bacterial target range.
  • the phage is a promiscuous phage with a large bacterial target range.
  • the promiscuous phage may target at least about any of 5, 10, 20, 30, 40, 50, or more bacterium resident in the host.
  • a phage with a narrow bacterial target range may target a specific bacterial strain in the host without affecting another, e.g., non-targeted, bacterium in the host.
  • the phage may target no more than about any of 50, 40, 30, 20, 10, 8, 6, 4, 2, or 1 bacterium resident in the host.
  • the phage described herein may be useful in targeting one or more bacteria resident in the mosquito, including, but not limited to, EspZ, Seratia spp (e.g., Serratia marcescens ), Enterbacterioaceae spp., Enterobacter spp. (e.g., Enterobacter cloacae, Enterobacter amnigenus, Enterobacter ludwigii ), . Proteus spp., Acinetobacter spp., Wigglesworthia spp. ( Wigglesworthia gloosinidia ), Xanthomonas spp.
  • EspZ Seratia spp
  • Seratia spp e.g., Serratia marcescens
  • Enterbacterioaceae spp. Enterobacter spp.
  • Enterobacter spp. e.g., Enterobacter cloacae, Enterobacter amnigenus, Enterobacter ludwigii
  • Pseudomonas spp. e.g., Pseudomonas aeruginosa, Pseudomonas stutzeri, Pseudomonas rhodesiae
  • Escherichia spp. e.g., Escherchia coli
  • Cedecea spp. e.g., Cedecea lapagel
  • Ewingella spp. e.g., Ewingella americana
  • Bacillus spp. e.g., Bacillus pumilus
  • Comamonas spp. Comamonas spp., or Vagococcus spp.
  • Wolbachia spp. e.g., Wolbachia -wMel, Wolbachia -wAlbB, Wolbachia -wMelPop, Wolbachia -wMelPop-CLA.
  • compositions described herein may include any number of phage, such as at least about any one of 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, or more phage.
  • the composition includes phage from one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phage) families, one or more orders (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phage), or one or more species (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, or more phage).
  • Compositions including one or more phage are also referred herein as “phage cocktails.” Phage cocktails are useful because they allow for targeting of a wider host range of bacteria.
  • a cocktail includes multiple phages targeting one bacterial species.
  • a cocktail includes multiple phages targeting multiple bacterial species.
  • a one-phage “cocktail” includes a single promiscuous phage (i.e. a phage with a large host range) targeting many strains within a species.
  • Suitable concentrations of the phage in the modulating agent described herein depends on factors such as efficacy, survival rate, transmissibility of the phage, number of distinct phage, and/or lysin types in the compositions, formulation, and methods of application of the composition.
  • the phage is in a liquid or a solid formulation.
  • the concentration of each phage in any of the compositions described herein is at least about any of 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 or more pfu/ml.
  • the concentration of each phage in any of the compositions described herein is no more than about any of 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 pfu/ml. In some instances, the concentration of each phage in the composition is any of about 10 2 to about 10 3 , about 10 3 to about 10 4 , about 10 4 to about 10 5 , about 10 5 to about 10 6 , about 10 7 to about 10 8 , about 10 8 to about 10 9 , about 10 2 to about 10 4 , about 10 4 to about 10 6 , about 10 6 to about 10 9 , or about 10 3 to about 10 8 pfu/ml.
  • the concentration of each type of the phages may be the same or different.
  • the concentration of one phage in the cocktail is about 10 8 pfu/ml and the concentration of a second phage in the cocktail is about 10 6 pfu/ml.
  • a modulating agent including a phage as described herein can be contacted with the target host in an amount and for a time sufficient to: (a) reach a target level (e.g., a predetermined or threshold level) of phage concentration inside a target host; (b) reach a target level (e.g., a predetermined or threshold level) of phage concentration inside a target host gut; (c) reach a target level (e.g., a predetermined or threshold level) of phage concentration inside a target host bacteriocyte; (d) modulate the level, or an activity, of one or more microorganism (e.g., endosymbiont) in the target host; or/and (e) modulate fitness of the target host.
  • a target level e.g., a predetermined or threshold level
  • a target level e.g., a predetermined or threshold level
  • a target level e.g., a predetermined or threshold level
  • a target host gut
  • phages e.g., one or more naturally occurring phage
  • polypeptides e.g., a bacteriocin, R-type bacteriocin, nodule C-rich peptide, antimicrobial peptide, lysin, or bacteriocyte regulatory peptide
  • an effective concentration of any peptide or polypeptide described herein may alter a level, activity, or metabolism of one or more microorganisms (as described herein, e.g., a Wolbachia spp.
  • a host e.g., a vector of a human pathogen, e.g., a mosquito, mite, biting louse, or tick
  • the modulation resulting in a decrease in the host's fitness e.g., as outlined herein.
  • Polypeptides included herein may include naturally occurring polypeptides or recombinantly produced variants.
  • the polypeptide may be a functionally active variant of any of the polypeptides described herein with at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, e.g., over a specified region or over the entire sequence, to a sequence of a polypeptide described herein or a naturally occurring polypeptide.
  • a modulating agent comprising a polypeptide as described herein can be contacted with the target host in an amount and for a time sufficient to: (a) reach a target level (e.g., a predetermined or threshold level) of concentration inside a target host; (b) reach a target level (e.g., a predetermined or threshold level) of concentration inside a target host gut; (c) reach a target level (e.g., a predetermined or threshold level) of concentration inside a target host bacteriocyte; (d) modulate the level, or an activity, of one or more microorganism (e.g., endosymbiont) in the target host; or/and (e) modulate fitness of the target host.
  • a target level e.g., a predetermined or threshold level
  • a target level e.g., a predetermined or threshold level
  • a target level e.g., a predetermined or threshold level
  • concentration inside a target host gut e.g.,
  • polypeptide modulating agents discussed hereinafter namely bacteriocins, lysins, antimicrobial peptides, nodule C-rich peptides, and bacteriocyte regulatory peptides, can be used to alter the level, activity, or metabolism of target microorganisms (e.g., Rickettsia or Wolbochia ) as indicated in the section for decreasing the fitness of host insects (e.g., a vector of a human pathogen, e.g., a mosquito, a mite, a biting louse, or a tick).
  • target microorganisms e.g., Rickettsia or Wolbochia
  • host insects e.g., a vector of a human pathogen, e.g., a mosquito, a mite, a biting louse, or a tick.
  • the modulating agent described herein may include a bacteriocin.
  • the bacteriocin is naturally produced by Gram-positive bacteria, such as Pseudomonas, Streptomyces, Bacillus, Staphylococcus , or lactic acid bacteria (LAB, such as Lactococcus lactis ).
  • the bacteriocin is naturally produced by Gram-negative bacteria, such as Hafnia alvei, Citrobacter freundii, Klebsiella oxytoca, Klebsiella pneumonia, Enterobacter cloacae, Serratia plymithicum, Xanthomonas campestris, Erwinia carotovora, Ralstonia solanacearum , or Escherichia coli .
  • Exemplary bacteriocins include, but are not limited to, Class I-IV LAB antibiotics (such as lantibiotics), colicins, microcins, and pyocins. Non-limiting examples of bacteriocins are listed in Table 4.
  • SLLAAAGRESIKAYLKKEIK KKGKRAVIAW (SEQ ID NO: 46) Class III b aureocin A70 Staphylococcus Broad spectrum: Gram MSWLNFLKYIAKYGKKAVSA aureus positive and Gram AWKYKGKVLEWLNVGPTLEW negative bacteria. VWQKLKKIAGL (SEQ ID NO: 47) Class IV Garvicin A Lactococcus Broad spectrum: Gram IGGALGNALNGLGTWANMMN garvieae positive and Gram GGGFVNQWQVYANKGKINQY negative bacteria.
  • RPY Unclassified Colicin V Escherichia coli Active against MRTLTLNELDSVSGGASGRD Escherichia coli (also IAMAIGTLSGQFVAGGIGAA closely related AGGVAGGAIYDYASTHKPNP bacteria); AMSPSGLGGTIKQKPEGIPS Enterbacteriaceae EAWNYAAGRLCNWSPNNLSD VCL (SEQ ID NO: 49)
  • the bacteriocin is a colicin, a pyocin, or a microcin produced by Gram-negative bacteria. In some instances, the bacteriocin is a colicin.
  • the colicin may be a group A colicin (e.g., uses the Tol system to penetrate the outer membrane of a target bacterium) or a group B colicin (e.g., uses the Ton system to penetrate the outer membrane of a target bacterium). In some instances, the bacteriocin is a microcin.
  • the microcin may be a class I microcin (e.g., ⁇ 5 kDa, has post-translational modifications) or a class II microcin (e.g., 5-10 kDa, with or without post-translational modifications).
  • the class II microcin is a class IIa microcin (e.g., requires more than one genes to synthesize and assemble functional peptides) or a class IIb microcin (e.g., linear peptides with or without post-translational modifications at C-terminus).
  • the bacteriocin is a pyocin.
  • the pyocin is an R-pyocin, F-pyocin, or S-pyocin.
  • the bacteriocin is a class I, class II, class III, or class IV bacteriocin produced by Gram-positive bacteria.
  • the modulating agent includes a Class I bacteriocin (e.g., lanthionine-containing antibiotics (lantibiotics) produced by a Gram-positive bacteria).
  • the class I bacteriocins or lantibiotic may be a low molecular weight peptide (e.g., less than about 5 kDa) and may possess post-translationally modified amino acid residues (e.g., lanthionine, ⁇ -methyllanthionine, or dehydrated amino acids).
  • the bacteriocin is a Class II bacteriocin (e.g., non-lantibiotics produced by Gram-positive bacteria). Many are positively charged, non-lanthionine-containing peptides, which unlike lantibiotics, do not undergo extensive post-translational modification.
  • the Class II bacteriocin may belong to one of the following subclasses: “pediocin-like” bacteriocins (e.g., pediocin PA-1 and carnobacteriocin X (Class IIa)); two-peptide bacteriocins (e.g., lactacin F and ABP-118 (Class IIb)); circular bacteriocins (e.g., carnocyclin A and enterocin AS-48 (Class 11c)); or unmodified, linear, non-pediocin-like bacteriocins (e.g., epidermicin NI01 and lactococcin A (Class IId)).
  • pediocin-like bacteriocins e.g., pediocin PA-1 and carnobacteriocin X (Class IIa)
  • two-peptide bacteriocins e.g., lactacin F and ABP-118
  • the bacteriocin is a Class III bacteriocin (e.g., produced by Gram-positive bacteria).
  • Class III bacteriocins may have a molecular weight greater than 10 kDa and may be heat unstable proteins.
  • the Class III bacteriocins can be further subdivided into Group IIIA and Group IIIB bacteriocins.
  • the Group IIIA bacteriocins include bacteriolytic enzymes which kill sensitive strains by lysis of the cell well, such as Enterolisin A.
  • Group IIIB bacteriocins include non-lytic proteins, such as Caseicin 80, Helveticin J, and lactacin B.
  • the bacteriocin is a Class IV bacteriocin (e.g., produced by Gram-positive bacteria).
  • Class IV bacteriocins are a group of complex proteins, associated with other lipid or carbohydrate moieties, which appear to be required for activity. They are relatively hydrophobic and heat stable. Examples of Class IV bacteriocins leuconocin S, lactocin 27, and lactocin S.
  • the bacteriocin is an R-type bacteriocin.
  • R-type bacteriocins are contractile bacteriocidal protein complexes. Some R-type bacteriocins have a contractile phage-tail-like structure. The C-terminal region of the phage tail fiber protein determines target-binding specificity. They may attach to target cells through a receptor-binding protein, e.g., a tail fiber. Attachment is followed by sheath contraction and insertion of the core through the envelope of the target bacterium. The core penetration results in a rapid depolarization of the cell membrane potential and prompt cell death. Contact with a single R-type bacteriocin particle can result in cell death.
  • R-type bacteriocin may be thermolabile, mild acid resistant, trypsin resistant, sedimentable by centrifugation, resolvable by electron microscopy, or a combination thereof.
  • Other R-type bacteriocins may be complex molecules including multiple proteins, polypeptides, or subunits, and may resemble a tail structure of bacteriophages of the myoviridae family.
  • the subunit structures may be encoded by a bacterial genome, such as that of C. difficile or P. aeruginosa and form R-type bacteriocins to serve as natural defenses against other bacteria.
  • the R-type bacteriocin is a pyocin.
  • the pyocin is an R-pyocin, F-pyocin, or S-pyocin.
  • the bacteriocin is a functionally active variant of the bacteriocins described herein.
  • the variant of the bacteriocin has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, e.g., over a specified region or over the entire sequence, to a sequence of a bacteriocin described herein or a naturally occurring bacteriocin.
  • the bacteriocin may be bioengineered, according to standard methods, to modulate their bioactivity, e.g., increase or decrease or regulate, or to specify their target microorganisms.
  • the bacteriocin is produced by the translational machinery (e.g. a ribosome, etc.) of a microbial cell.
  • the bacteriocin is chemically synthesized.
  • Some bacteriocins can be derived from a polypeptide precursor.
  • the polypeptide precursor can undergo cleavage (e.g., processing by a protease) to yield the polypeptide of the bacteriocin itself.
  • the bacteriocin is produced from a precursor polypeptide.
  • the bacteriocin includes a polypeptide that has undergone post-translational modifications, for example, cleavage, or the addition of one or more functional groups.
  • the bacteriocins described herein may be formulated in a composition for any of the uses described herein.
  • the compositions disclosed herein may include any number or type (e.g., classes) of bacteriocins, such as at least about any one of 1 bacteriocin, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, or more bacteriocins. Suitable concentrations of each bacteriocin in the compositions described herein depends on factors such as efficacy, stability of the bacteriocin, number of distinct bacteriocin types in the compositions, formulation, and methods of application of the composition. In some instances, each bacteriocin in a liquid composition is from about 0.01 ng/ml to about 100 mg/mL.
  • each bacteriocin in a solid composition is from about 0.01 ng/g to about 100 mg/g. In some instances, wherein the composition includes at least two types of bacteriocins, the concentration of each type of the bacteriocins may be the same or different. In some instances, the bacteriocin is provided in a composition including a bacterial cell that secretes the bacteriocin. In some instances, the bacteriocin is provided in a composition including a polypeptide (e.g., a polypeptide isolated from a bacterial cell).
  • Bacteriocins may neutralize (e.g., kill) at least one microorganism other than the individual bacterial cell in which the polypeptide is made, including cells clonally related to the bacterial cell and other microbial cells.
  • a bacterial cell may exert cytotoxic or growth-inhibiting effects on a plurality of microbial organisms by secreting bacteriocins.
  • the bacteriocin targets and kills one or more species of bacteria resident in the host via cytoplasmic membrane pore formation, cell wall interference (e.g., peptidoglycanase activity), or nuclease activity (e.g., DNase activity, 16S rRNase activity, or tRNase activity).
  • the bacteriocin has a neutralizing activity.
  • Neutralizing activity of bacteriocins may include, but is not limited to, arrest of microbial reproduction, or cytotoxicity.
  • Some bacteriocins have cytotoxic activity, and thus can kill microbial organisms, for example bacteria, yeast, algae, and the like.
  • Some bacteriocins can inhibit the reproduction of microbial organisms, for example bacteria, yeast, algae, and the like, for example by arresting the cell cycle.
  • the bacteriocin has killing activity.
  • the killing mechanism of bacteriocins is specific to each group of bacteriocins.
  • the bacteriocin has narrow-spectrum bioactivity.
  • Bacteriocins are known for their very high potency against their target strains. Some bacteriocin activity is limited to strains that are closely related to the bacteriocin producer strain (narrow-spectrum bioactivity). In some instances, the bacteriocin has broad-spectrum bioactivity against a wide range of genera.
  • bacteriocins interact with a receptor molecule or a docking molecule on the target bacterial cell membrane.
  • nisin is extremely potent against its target bacterial strains, showing antimicrobial activity even at a single-digit nanomolar concentration.
  • the nisin molecule has been shown to bind to lipid II, which is the main transporter of peptidoglycan subunits from the cytoplasm to the cell wall
  • the bacteriocin has anti-fungal activity.
  • a number of bacteriocins with anti-yeast or anti-fungal activity have been identified.
  • bacteriocins from Bacillus have been shown to have neutralizing activity against some yeast strains (see, for example, Adetunji and Olaoye, Malaysian Journal of Microbiology 9:130-13, 2013).
  • an Enterococcus faecalis peptide has been shown to have neutralizing activity against Candida species (see, for example, Shekh and Roy, BMC Microbiology 12:132, 2012).
  • bacteriocins from Pseudomonas have been shown to have neutralizing activity against fungi, such as Curvularia lunata, Fusarium species, Helminthosporium species, and Biopolaris species (see, for example, Shalani and Srivastava, The Internet Journal of Microbiology Volume 5 Number 2, 2008).
  • botrycidin AJ1316 and alirin B1 from B. subtilis have been shown to have antifungal activities.
  • a modulating agent including a bacteriocin as described herein can be contacted with the target host in an amount and for a time sufficient to: (a) reach a target level (e.g., a predetermined or threshold level) of bacteriocin concentration inside a target host; (b) reach a target level (e.g., a predetermined or threshold level) of bacteriocin concentration inside a target host gut; (c) reach a target level (e.g., a predetermined or threshold level) of bacteriocin concentration inside a target host bacteriocyte; (d) modulate the level, or an activity, of one or more microorganism (e.g., endosymbiont) in the target host; or/and (e) modulate fitness of the target host.
  • a target level e.g., a predetermined or threshold level
  • a target level e.g., a predetermined or threshold level
  • bacteriocin concentration inside a target host gut e.g.
  • bacteriocins e.g., colA or nisin
  • the modulating agent described herein may include a lysin (e.g., also known as a murein hydrolase or peptidoglycan autolysin). Any lysin suitable for inhibiting a bacterium resident in the host may be used. In some instances, the lysin is one that can be naturally produced by a bacterial cell. In some instances, the lysin is one that can be naturally produced by a bacteriophage. In some instances, the lysin is obtained from a phage that inhibits a bacterium resident in the host. In some instances, the lysin is engineered based on a naturally occurring lysin.
  • a lysin e.g., also known as a murein hydrolase or peptidoglycan autolysin.
  • the lysin is engineered to be secreted by a host bacterium, for example, by introducing a signal peptide to the lysin. In some instances, the lysin is used in combination with a phage holin. In some instances, a lysin is expressed by a recombinant bacterium host that is not sensitive to the lysin. In some instances, the lysin is used to inhibit a Gram-positive or Gram-negative bacterium resident in the host.
  • the lysin may be any class of lysin and may have one or more substrate specificities.
  • the lysin may be a glycosidase, an endopeptidase, a carboxypeptidase, or a combination thereof.
  • the lysin cleaves the ⁇ -1-4 glycosidic bond in the sugar moiety of the cell wall, the amide bond connecting the sugar and peptide moieties of the bacterial cell wall, and/or the peptide bonds between the peptide moieties of the cell wall.
  • the lysin may belong to one or more specific lysin groups, depending on the cleavage site within the peptidoglycan.
  • the lysin is a N-acetyl- ⁇ -D-muramidase (e.g., lysozyme), lytic transglycosylase, N-acetyl- ⁇ -D-glucosaminidase, N-acetylmuramyl-L-alanine amidase, L,D-endopeptidase, D,D-endopeptidase, D,D-carboxypeptidase, L,D-carboxypeptidase, or L,D-transpeptidase.
  • lysins and their activities are listed in Table 5.
  • Lysins Target Bacteria Producer Lysins Activity Sequence S. pneumoniae Cp1 Cpl-1 Muramidase MVKKNDLFVDVSSHNGYDITGILEQ MGTTNTIIKISESTTYLNPCLSAQV EQSNPIGFYHFARFGGDVAEAEREA QFFLDNVPMQVKYLVLDYEDDPSGD AQANTNACLRFMQMIADAGYKPIYY SYKPFTHDNVDYQQILAQFPNSLWI AGYGLNDGTANFEYFPSMDGIRWWQ YSSNPFDKNIVLLDDEEDDKPKTAG TWKQDSKGWWFRRNNGSFPYNKWEK IGGVWYYFDSKGYCLTSEWLKDNEK WYYLKDNGAMATGWVLVGSEWYYMD DSGAMVTGWVKYKNNWYYMTNERGN MVSNEFIKSGKGWYFMNTNGELADN PSFTKEPDGLITVA (SEQ ID NO:
  • aureus P68 Lys16 Endopeptidase N/A S. aureus K LysK Amidase and MAKTQAEINKRLDAYAKGTVDSPYR endopeptidase VKKATSYDPSFGVMEAGAIDADGYY HAQCQDLITDYVLWLTDNKVRTWGN AKDQIKQSYGTGFKIHENKPSTVPK KGWIAVFTSGSYEQWGHIGIVYDGG NTSTFTILEQNWNGYANKKPTKRVD NYYGLTHFIEIPVKAGTTVKKETAK KSASKTPAPKKKATLKVSKNHINYT MDKRGKKPEGMVIHNDAGRSSGQQY ENSLANAGYARYANGIAHYYGSEGY VWEAIDAKNQIAWHTGDGTGANSGN FRFAGIEVCQSMSASDAQFLKNEQA VFQFTAEKFKEWGLTPNRKTVRLHM EFVPTACPHRSMVLHTGFNPVTQGR PSQAIMNKLK
  • uberis (ATCC700407) Ply700 Amidase MTDSIQEMRKLQSIPVRYDMGDRYG prophage NDADRDGRIEMDCSSAVSKALGISM TNNTETLQQALPAIGYGKIHDAVDG TFDMQAYDVIIWAPRDGSSSLGAFG HVLIATSPTTAIHCNYGSDGITEND YNYIWEINGRPREIVFRKGVTQTQA TVTSQFERELDVNARLTVSDKPYYE ATLSEDYYVEAGPRIDSQDKELIKA GTRVRVYEKLNGWSRINHPESAQWV EDSYLVDATEM (SEQ ID NO: 61) S.
  • the lysin is a functionally active variant of the lysins described herein.
  • the variant of the lysin has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, e.g., over a specified region or over the entire sequence, to a sequence of a lysin described herein or a naturally occurring lysin.
  • the lysin may be bioengineered to modulate its bioactivity, e.g., increase or decrease or regulate, or to specify a target microorganism.
  • the lysin is produced by the translational machinery (e.g. a ribosome, etc.) of a microbial cell.
  • the lysin is chemically synthesized.
  • the lysin is derived from a polypeptide precursor.
  • the polypeptide precursor can undergo cleavage (for example, processing by a protease) to yield the polypeptide of the lysin itself.
  • the lysin is produced from a precursor polypeptide.
  • the lysin includes a polypeptide that has undergone post-translational modifications, for example, cleavage, or the addition of one or more functional groups.
  • the lysins described herein may be formulated in a composition for any of the uses described herein.
  • the compositions disclosed herein may include any number or type (e.g., classes) of lysins, such as at least about any one of 1 lysin, 2, 3, 4, 5, 10, 15, 20, or more lysins.
  • a suitable concentration of each lysin in the composition depends on factors such as efficacy, stability of the lysin, number of distinct lysin, the formulation, and methods of application of the composition.
  • each lysin in a liquid composition is from about 0.1 ng/mL to about 100 mg/mL.
  • each lysin in a solid composition is from about 0.1 ng/g to about 100 mg/g.
  • the concentration of each type of lysin may be the same or different.
  • a modulating agent including a lysin as described herein can be contacted with the target host in an amount and for a time sufficient to: (a) reach a target level (e.g., a predetermined or threshold level) of lysin concentration inside a target host; (b) reach a target level (e.g., a predetermined or threshold level) of lysin concentration inside a target host gut; (c) reach a target level (e.g., a predetermined or threshold level) of lysin concentration inside a target host bacteriocyte; (d) modulate the level, or an activity, of one or more microorganism (e.g., endosymbiont) in the target host; or/and (e) modulate fitness of the target host.
  • a target level e.g., a predetermined or threshold level
  • a target level e.g., a predetermined or threshold level
  • a target level e.g., a predetermined or threshold level
  • the modulating agent described herein may include an antimicrobial peptide (AMP). Any AMP suitable for inhibiting a microorganism resident in the host may be used. AMPs are a diverse group of molecules, which are divided into subgroups on the basis of their amino acid composition and structure.
  • the AMP may be derived or produced from any organism that naturally produces AMPs, including AMPs derived from plants (e.g., copsin), insects (e.g., drosocin, scorpion peptide (e.g., Uy192, UyCT3, D3, D10, Uy17, Uy192), mastoparan, poneratoxin, cecropin, moricin, melittin), frogs (e.g., magainin, dermaseptin, aurein), and mammals (e.g., cathelicidins, defensins and protegrins).
  • plants e.g., copsin
  • insects e.g., drosocin, scorpion peptide (e.g., Uy192, UyCT3, D3, D10, Uy17, Uy192), mastoparan, poneratoxin, cecropin, moricin, melittin), frogs (e.g., magainin, der
  • the AMP may be a scorpion peptide, such as Uy192 (5′-FLSTIWNGIKGLL-3′; SEQ ID NO: 227), UyCT3 (5′-LSAIWSGIKSLF-3; SEQ ID NO: 228), D3 (5′-LWGKLWEGVKSLI-3′; SEQ ID NO: 229), and D10 (5′-FPFLKLSLKIPKSAIKSAIKRL-3′; SEQ ID NO: 230), Uy17 (5′-ILSAIWSGIKGLL-3′; SEQ ID NO: 231), or a combination thereof.
  • Uy192 5′-FLSTIWNGIKGLL-3′; SEQ ID NO: 227)
  • UyCT3 5′-LSAIWSGIKSLF-3; SEQ ID NO: 228)
  • D3 5′-LWGKLWEGVKSLI-3′; SEQ ID NO: 229)
  • D10 5′-FPFLKLSLKIPKSAIKSAIKRL-3′; SEQ ID NO: 230
  • Uy17 5′-
  • the antimicrobial peptide may be one having at least 90% sequence identity (e.g., at least 90%, 92%, 94%, 96%, 98%, or 100% sequence identity) with one or more of the following: cecropin (SEQ ID NO: 82), melittin, copsin, drosomycin (SEQ ID NO: 93), dermcidin (SEQ ID NO: 81), andropin (SEQ ID NO: 83), moricin (SEQ ID NO: 84), ceratotoxin (SEQ ID NO: 85), abaecin (SEQ ID NO: 86), apidaecin (SEQ ID NO: 87), prophenin (SEQ ID NO: 88), indolicidin (SEQ ID NO: 89), protegrin (SEQ ID NO: 90), tachyplesin (SEQ ID NO: 91), or defensin (SEQ ID NO: 92) to a vector of a human pathogen.
  • cecropin SEQ ID NO: 82
  • the AMP may be active against any number of target microorganisms.
  • the AMP may have antibacterial and/or antifungal activities.
  • the AMP may have a narrow-spectrum bioactivity or a broad-spectrum bioactivity. For example, some AMPs target and kill only a few species of bacteria or fungi, while others are active against both gram-negative and gram-positive bacteria as well as fungi.
  • the AMP may function through a number of known mechanisms of action.
  • the cytoplasmic membrane is a frequent target of AMPs, but AMPs may also interfere with DNA and protein synthesis, protein folding, and cell wall synthesis.
  • AMPs with net cationic charge and amphipathic nature disrupt bacterial membranes leading to cell lysis.
  • AMPs may enter cells and interact with intracellular target to interfere with DNA, RNA, protein, or cell wall synthesis.
  • AMPs In addition to killing microorganisms, AMPs have demonstrated a number of immunomodulatory functions that are involved in the clearance of infection, including the ability to alter host gene expression, act as chemokines and/or induce chemokine production, inhibit lipopolysaccharide induced pro-inflammatory cytokine production, promote wound healing, and modulating the responses of dendritic cells and cells of the adaptive immune response.
  • the AMP is a functionally active variant of the AMPs described herein.
  • the variant of the AMP has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, e.g., over a specified region or over the entire sequence, to a sequence of an AMP described herein or a naturally derived AMP.
  • the AMP may be bioengineered to modulate its bioactivity, e.g., increase or decrease or regulate, or to specify a target microorganism.
  • the AMP is produced by the translational machinery (e.g. a ribosome, etc.) of a cell.
  • the AMP is chemically synthesized.
  • the AMP is derived from a polypeptide precursor.
  • the polypeptide precursor can undergo cleavage (for example, processing by a protease) to yield the polypeptide of the AMP itself.
  • the AMP is produced from a precursor polypeptide.
  • the AMP includes a polypeptide that has undergone post-translational modifications, for example, cleavage, or the addition of one or more functional groups.
  • the AMPs described herein may be formulated in a composition for any of the uses described herein.
  • the compositions disclosed herein may include any number or type (e.g., classes) of AMPs, such as at least about any one of 1 AMP, 2, 3, 4, 5, 10, 15, 20, or more AMPs.
  • the compositions may include a cocktail of AMPs (e.g., a cocktail of scorpion peptides, e.g., UyCT3, D3, D10, and Uy17).
  • a suitable concentration of each AMP in the composition depends on factors such as efficacy, stability of the AMP, number of distinct AMP in the composition, the formulation, and methods of application of the composition.
  • each AMP in a liquid composition is from about 0.1 ng/mL to about 100 mg/mL (about 0.1 ng/mL to about 1 ng/mL, about 1 ng/mL to about 10 ng/mL, about 10 ng/mL to about 100 ng/mL, about 100 ng/mL to about 1000 ng/mL, about 1 mg/mL to about 10 mg/mL, about 10 mg/mL to about 100 mg/mL).
  • each AMP in a solid composition is from about 0.1 ng/g to about 100 mg/g (about 0.1 ng/g to about 1 ng/g, about 1 ng/g to about 10 ng/g, about 10 ng/g to about 100 ng/g, about 100 ng/g to about 1000 ng/g, about 1 mg/g to about 10 mg/g, about 10 mg/g to about 100 mg/g).
  • the concentration of each type of AMP may be the same or different.
  • a modulating agent including an AMP as described herein can be contacted with the target host in an amount and for a time sufficient to: (a) reach a target level (e.g., a predetermined or threshold level) of AMP concentration inside a target host; (b) reach a target level (e.g., a predetermined or threshold level) of AMP concentration inside a target host gut; (c) reach a target level (e.g., a predetermined or threshold level) of AMP concentration inside a target host bacteriocyte; (d) modulate the level, or an activity, of one or more microorganism (e.g., endosymbiont) in the target host; or/and (e) modulate fitness of the target host.
  • a target level e.g., a predetermined or threshold level
  • AMP concentration inside a target host gut e.g., a predetermined or threshold level
  • a target level e.g., a predetermined or threshold level
  • AMPs such as scorpion peptides
  • the modulating agent described herein may include a nodule C-rich peptide (NCR peptide).
  • NCR peptides are produced in certain leguminous plants and play an important role in the mutualistic, nitrogen-fixing symbiosis of the plants with bacteria from the Rhizobiaceae family ( rhizobia ), resulting in the formation of root nodules where plant cells contain thousands of intracellular endosymbionts.
  • NCR peptides possess anti-microbial properties that direct an irreversible, terminal differentiation process of bacteria, e.g., to permeabilize the bacterial membrane, disrupt cell division, or inhibit protein synthesis.
  • NCR peptides are produced which direct irreversible differentiation of the bacteria into large polyploid nitrogen-fixing bacteroids.
  • Non-limiting examples of NCR peptides are listed in Table 7.
  • NCR Peptides NAME Peptide sequence Producer >gi
  • NCR peptide-producing plants include but are not limited to Pisum sativum (pea), Astragalus sinicus (IRLC legumes), Phaseolus vulgaris (bean), Vigna unguiculata (cowpea), Medicago truncatula (barrelclover), and Lotus japonicus .
  • pea Pisum sativum
  • IRLC Astragalus sinicus
  • Bean Phaseolus vulgaris
  • Vigna unguiculata cowpea
  • Medicago truncatula barrelclover
  • Lotus japonicus .
  • over 600 potential NCR peptides are predicted from the M. truncatula genome sequence and almost 150 different NCR peptides have been detected in cells isolated from root nodules by mass spectrometry.
  • the NCR peptides described herein may be mature or immature NCR peptides.
  • Immature NCR peptides have a C-terminal signal peptide that is required for translocation into the endoplasmic reticulum and cleaved after translocation.
  • the N-terminus of a NCR peptide includes a signal peptide, which may be cleavable, for targeting to a secretory pathway.
  • NCR peptides are generally small peptides with disulfide bridges that stabilize their structure.
  • Mature NCR peptides have a length in the range of about 20 to about 60 amino acids, about 25 to about 55 amino acids, about 30 to about 50 amino acids, about 35 to about 45 amino acids, or any range therebetween.
  • NCR peptides may include a conserved sequence of cysteine residues with the rest of the peptide sequence highly variable.
  • NCR peptides generally have about four or eight cysteines.
  • NCR peptides may be anionic, neutral, or cationic.
  • MAQFLLFVYSLIIFLSLFFGEAAFERTETRMLTIPCTSDDNCPKVISPCHTKCFDGFCGWYIEGSYEGP SEQ ID NO: 197
  • neutral and/or anionic NCR peptides such as NCR001, do not possess antimicrobial activities at a pI greater than about 8.
  • the NCR peptide is effective to kill bacteria. In some instances, the NCR peptide is effective to kill S. meliloti, Xenorhabdus spp, Photorhabdus spp, Candidatus spp, Buchnera spp, Blattabacterium spp, Baumania spp, Wigglesworthia spp, Wolbachia spp, Rickettsia spp, Orientia spp, Sodalis spp, Burkholderia spp, Cupriavidus spp, Frankia spp, Snirhizobium spp, Streptococcus spp, Wolinella spp, Xylella spp, Erwinia spp, Agrobacterium spp, Bacillus spp, Paenibacillus spp, Streptomyces spp, Micrococcus spp, Corynebacterium spp, Acetobacter
  • the NCR peptide is a functionally active variant of a NCR peptide described herein.
  • the variant of the NCR peptide has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, e.g., over a specified region or over the entire sequence, to a sequence of a NCR peptide described herein or naturally derived NCR peptide.
  • the NCR peptide may be bioengineered to modulate its bioactivity, e.g., increase or decrease or regulate, or to specify a target microorganism.
  • the NCR peptide is produced by the translational machinery (e.g. a ribosome, etc.) of a cell.
  • the NCR peptide is chemically synthesized.
  • the NCR peptide is derived from a polypeptide precursor.
  • the polypeptide precursor can undergo cleavage (for example, processing by a protease) to yield the NCR peptide itself.
  • the NCR peptide is produced from a precursor polypeptide.
  • the NCR peptide includes a polypeptide that has undergone post-translational modifications, for example, cleavage, or the addition of one or more functional groups.
  • the NCR peptide described herein may be formulated in a composition for any of the uses described herein.
  • the compositions disclosed herein may include any number or type of NCR peptides, such as at least about any one of 1 NCR peptide, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, or more NCR peptides.
  • a suitable concentration of each NCR peptide in the composition depends on factors such as efficacy, stability of the NCR peptide, number of distinct NCR peptide, the formulation, and methods of application of the composition.
  • each NCR peptide in a liquid composition is from about 0.1 ng/mL to about 100 mg/mL.
  • each NCR peptide in a solid composition is from about 0.1 ng/g to about 100 mg/g. In some instances, wherein the composition includes at least two types of NCR peptides, the concentration of each type of NCR peptide may be the same or different.
  • a modulating agent including a NCR peptide as described herein can be contacted with the target host in an amount and for a time sufficient to: (a) reach a target level (e.g., a predetermined or threshold level) of NCR peptide concentration inside a target host; (b) reach a target level (e.g., a predetermined or threshold level) of NCR peptide concentration inside a target host gut; (c) reach a target level (e.g., a predetermined or threshold level) of NCR peptide concentration inside a target host bacteriocyte; (d) modulate the level, or an activity, of one or more microorganism (e.g., endosymbiont) in the target host; or/and (e) modulate fitness of the target host.
  • a target level e.g., a predetermined or threshold level
  • a target level e.g., a predetermined or threshold level
  • a target level e.g., a predetermined or threshold level
  • the modulating agent described herein may include a bacteriocyte regulatory peptide (BRP).
  • BRPs are peptides expressed in the bacteriocytes of insects. These genes are expressed first at a developmental time point coincident with the incorporation of symbionts and their bacteriocyte-specific expression is maintained throughout the insect's life.
  • the BRP has a hydrophobic amino terminal domain, which is predicted to be a signal peptide.
  • some BRPs have a cysteine-rich domain.
  • the bacteriocyte regulatory peptide is a bacteriocyte-specific cysteine rich (BCR) protein.
  • Bacteriocyte regulatory peptides have a length between about 40 and 150 amino acids.
  • the bacteriocyte regulatory peptide has a length in the range of about 45 to about 145, about 50 to about 140, about 55 to about 135, about 60 to about 130, about 65 to about 125, about 70 to about 120, about 75 to about 115, about 80 to about 110, about 85 to about 105, or any range therebetween.
  • BRPs and their activities are listed in Table 8.
  • the BRP alters the growth and/or activity of one or more bacteria resident in the bacteriocyte of the host.
  • the BRP may be bioengineered to modulate its bioactivity (e.g., increase, decrease, or regulate) or to specify a target microorganism.
  • the BRP is produced by the translational machinery (e.g. a ribosome, etc.) of a cell.
  • the BRP is chemically synthesized.
  • the BRP is derived from a polypeptide precursor.
  • the polypeptide precursor can undergo cleavage (for example, processing by a protease) to yield the polypeptide of the BRP itself.
  • the BRP is produced from a precursor polypeptide.
  • the BRP includes a polypeptide that has undergone post-translational modifications, for example, cleavage, or the addition of one or more functional groups.
  • the variant of the BRP has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, e.g., over a specified region or over the entire sequence, to a sequence of a BRP described herein or naturally derived BRP.
  • the BRP described herein may be formulated in a composition for any of the uses described herein.
  • the compositions disclosed herein may include any number or type (e.g., classes) of BRPs, such as at least about any one of 1 BRP, 2, 3, 4, 5, 10, 15, 20, or more BRPs.
  • a suitable concentration of each BRP in the composition depends on factors such as efficacy, stability of the BRP, number of distinct BRP, the formulation, and methods of application of the composition.
  • each BRP in a liquid composition is from about 0.1 ng/mL to about 100 mg/mL.
  • each BRP in a solid composition is from about 0.1 ng/g to about 100 mg/g.
  • the concentration of each type of BRP may be the same or different.
  • a modulating agent including a BRP as described herein can be contacted with the target host in an amount and for a time sufficient to: (a) reach a target level (e.g., a predetermined or threshold level) of BRP concentration inside a target host; (b) reach a target level (e.g., a predetermined or threshold level) of BRP concentration inside a target host gut; (c) reach a target level (e.g., a predetermined or threshold level) of BRP concentration inside a target host bacteriocyte; (d) modulate the level, or an activity, of one or more microorganism (e.g., endosymbiont) in the target host; or/and (e) modulate fitness of the target host.
  • a target level e.g., a predetermined or threshold level
  • a target level e.g., a predetermined or threshold level
  • a target level e.g., a predetermined or threshold level
  • a target host gut e.g.
  • an effective concentration of any small molecule described herein may alter the level, activity, or metabolism of one or more microorganisms (as described herein) resident in a host, the alteration resulting in a decrease in the host's fitness.
  • a modulating agent comprising a small molecule as described herein can be contacted with the target host in an amount and for a time sufficient to: (a) reach a target level (e.g., a predetermined or threshold level) of a small molecule concentration inside a target host; (b) reach a target level (e.g., a predetermined or threshold level) of small molecule concentration inside a target host gut; (c) reach a target level (e.g., a predetermined or threshold level) of a small molecule concentration inside a target host bacteriocyte; (d) modulate the level, or an activity, of one or more microorganism (e.g., endosymbiont) in the target host; or/and (e) modulate fitness of the target host.
  • a target level e.g., a predetermined or threshold level
  • a target level e.g., a predetermined or threshold level
  • a target level e.g., a predetermined or threshold level
  • the small molecules discussed hereinafter can be used to alter the level, activity, or metabolism of target microorganisms as indicated in the sections for decreasing the fitness of a host insect (e.g., vector of a human pathogen), such as a mosquito, a mite, a louse, or a tick.
  • a host insect e.g., vector of a human pathogen
  • a host insect e.g., vector of a human pathogen
  • the modulating agent described herein may include an antibiotic. Any antibiotic known in the art may be used. Antibiotics are commonly classified based on their mechanism of action, chemical structure, or spectrum of activity.
  • the antibiotic described herein may target any bacterial function or growth processes and may be either bacteriostatic (e.g., slow or prevent bacterial growth) or bactericidal (e.g., kill bacteria).
  • the antibiotic is a bactericidal antibiotic.
  • the bactericidal antibiotic is one that targets the bacterial cell wall (e.g., penicillins and cephalosporins); one that targets the cell membrane (e.g., polymyxins); or one that inhibits essential bacterial enzymes (e.g., rifamycins, lipiarmycins, quinolones, and sulfonamides).
  • the bactericidal antibiotic is an aminoglycoside.
  • the antibiotic is a bacteriostatic antibiotic.
  • the bacteriostatic antibiotic targets protein synthesis (e.g., macrolides, lincosamides and tetracyclines).
  • Additional classes of antibiotics that may be used herein include cyclic lipopeptides (such as daptomycin), glycylcyclines (such as tigecycline), oxazolidinones (such as linezolid), or lipiarmycins (such as fidaxomicin).
  • examples of antibiotics include oxytetracycline, doxycycline, rifampicin, ciprofloxacin, ampicillin, and polymyxin B.
  • Other non-limiting examples of antibiotics are found in Table 9.
  • Antibiotics Antibiotics Action Penicillins, cephalosporins, Cell wall synthesis vancomycin Membrane active agent, disrupt Polymixin, gramicidin cell membrane Tetracyclines, macrolides, Inhibit protein synthesis chloramphenicol, clindamycin, spectinomycin Sulfonamides Inhibit folate-dependent pathways Ciprofloxacin Inhibit DNA-gyrase Isoniazid, rifampicin, Antimycobacterial agents pyrazinamide, ethambutol, (myambutol)l, streptomycin
  • the antibiotic described herein may have any level of target specificity (e.g., narrow- or broad-spectrum).
  • the antibiotic is a narrow-spectrum antibiotic, and thus targets specific types of bacteria, such as gram-negative or gram-positive bacteria.
  • the antibiotic may be a broad-spectrum antibiotic that targets a wide range of bacteria.
  • the antibiotics described herein may be formulated in a composition for any of the uses described herein.
  • the compositions disclosed herein may include any number or type (e.g., classes) of antibiotics, such as at least about any one of 1 antibiotic, 2, 3, 4, 5, 10, 15, 20, or more antibiotics (e.g., a combination of rifampicin and doxycycline, or a combination of ampicillin and rifampicin).
  • a suitable concentration of each antibiotic in the composition depends on factors such as efficacy, stability of the antibiotic, number of distinct antibiotics, the formulation, and methods of application of the composition. In some instances, wherein the composition includes at least two types of antibiotics, the concentration of each type of antibiotic may be the same or different.
  • a modulating agent including an antibiotic as described herein can be contacted with the target host in an amount and for a time sufficient to: (a) reach a target level (e.g., a predetermined or threshold level) of antibiotic concentration inside a target host; (b) reach a target level (e.g., a predetermined or threshold level) of antibiotic concentration inside a target host gut; (c) reach a target level (e.g., a predetermined or threshold level) of antibiotic concentration inside a target host bacteriocyte; (d) modulate the level, or an activity, of one or more microorganism (e.g., endosymbiont) in the target host; or/and (e) modulate fitness of the target host.
  • a target level e.g., a predetermined or threshold level
  • a target level e.g., a predetermined or threshold level
  • a target level e.g., a predetermined or threshold level
  • a target host gut e.g., a predetermined
  • antibiotics e.g., doxycycline, oxytetracycline, azithromycin, ciprofloxacin, or rifampicin
  • an endosymbiotic bacterium such as a Wolbachia spp.
  • an insect host e.g., an insect vector of an animal pathogen
  • a mosquito or mite or tick or biting louse e.g., an insect vector of an animal pathogen
  • antibiotics such as oxytetracycline can be used as modulating agents that target an endosymbiotic bacterium, such as a Rickettsia spp., in an insect host, such as ticks, to decrease the fitness of the host (e.g., as outlined herein).
  • the modulating agent of the compositions and methods described herein includes a secondary metabolite.
  • Secondary metabolites are derived from organic molecules produced by an organism. Secondary metabolites may act (i) as competitive agents used against bacteria, fungi, amoebae, plants, insects, and large animals; (ii) as metal transporting agents; (iii) as agents of symbiosis between microbes and plants, insects, and higher animals; (iv) as sexual hormones; and (v) as differentiation effectors.
  • Non-limiting examples of secondary metabolites are found in Table 10.
  • the secondary metabolite used herein may include a metabolite from any known group of secondary metabolites.
  • secondary metabolites can be categorized into the following groups: alkaloids, terpenoids, flavonoids, glycosides, natural phenols (e.g., gossypol acetic acid), enals (e.g., trans-cinnamaldehyde), phenazines, biphenols and dibenzofurans, polyketides, fatty acid synthase peptides, nonribosomal peptides, ribosomally synthesized and post-translationally modified peptides, polyphenols polysaccharides (e.g., chitosan), and biopolymers.
  • alkaloids e.g., gossypol acetic acid
  • enals e.g., trans-cinnamaldehyde
  • phenazines e.g., biphenols and dibenzofurans
  • Secondary metabolites useful for compositions and methods described herein include those that alter a natural function of an endosymbiont (e.g., primary or secondary endosymbiont), bacteriocyte, or extracellular symbiont.
  • one or more secondary metabolites described herein is isolated from a high throughput screening (HTS) for antimicrobial compounds.
  • HTS high throughput screening
  • a HTS screen identified 49 antibacterial extracts that have specificity against gram positive and gram negative bacteria from over 39,000 crude extracts from organisms growing in diverse ecosystems of one specific region.
  • the secondary metabolite is transported inside a bacteriocyte.
  • the small molecule is an inhibitor of vitamin synthesis.
  • the vitamin synthesis inhibitor is a vitamin precursor analog.
  • the vitamin precursor analog is pantothenol.
  • the small molecule is an amino acid analog.
  • the amino acid analog is L-canvanine, D-arginine, D-valine, D-methionine, D-phenylalanine, D-histidine, D-tryptophan, D-threonine, D-leucine, L-NG-nitroarginine, or a combination thereof.
  • the small molecule is a natural antimicrobial compound, such as propionic acid, levulinic acid, trans-cinnemaldehdye, nisin, or low molecular weight chitosan.
  • the secondary metabolite described herein may be formulated in a composition for any of the uses described herein.
  • the compositions disclosed herein may include any number or type (e.g., classes) of secondary metabolites, such as at least about any one of 1 secondary metabolite, 2, 3, 4, 5, 10, 15, 20, or more secondary metabolites.
  • a suitable concentration of each secondary metabolite in the composition depends on factors such as efficacy, stability of the secondary metabolite, number of distinct secondary metabolites, the formulation, and methods of application of the composition.
  • the concentration of each type of secondary metabolite may be the same or different.
  • a modulating agent including a secondary metabolite as described herein can be contacted with the target host in an amount and for a time sufficient to: (a) reach a target level (e.g., a predetermined or threshold level) of secondary metabolite concentration inside a target host; (b) reach a target level (e.g., a predetermined or threshold level) of secondary metabolite concentration inside a target host gut; (c) reach a target level (e.g., a predetermined or threshold level) of secondary metabolite concentration inside a target host bacteriocyte; (d) modulate the level, or an activity, of one or more microorganism (e.g., endosymbiont) in the target host; or/and (e) modulate fitness of the target host.
  • a target level e.g., a predetermined or threshold level
  • a target level e.g., a predetermined or threshold level
  • a target level e.g., a predetermined or threshold level
  • secondary metabolites e.g., gossypol
  • secondary metabolites can be used as modulating agents that target an endosymbiotic bacterium in an insect host to decrease the fitness of the host (e.g., as outlined herein).
  • small molecules such as trans-cinnemaldehyde, levulinic acid, chitosan, vitamin analogs, or amino acid transport inhibitors, can also be used as modulating agents that target an endosymbiotic bacterium in an insect host to decrease the fitness of the host (e.g., as outlined herein).
  • the modulating agent described herein includes one or more bacteria. Numerous bacteria are useful in the compositions and methods described herein. In some instances, the agent is a bacterial species endogenously found in the host. In some instances, the bacterial modulating agent is an endosymbiotic bacterial species. Non-limiting examples of bacteria that may be used as modulating agents include all bacterial species described herein in Section II of the detailed description and those listed in Table 1.
  • the modulating agent may be a bacterial species from any bacterial phyla present in insect guts, including Gammaproteobacteria, Alphaproteobacteria, Betaproteobacteria, Bacteroidetes, Firmicutes (e.g., Lactobacillus and Bacillus spp.), Clostridia, Actinomycetes, Spirochetes, Verrucomicrobia, and Actinobacteria.
  • the modulating agent is a bacterium that disrupts microbial diversity or otherwise alters the microbiota of the host in a manner detrimental to the host.
  • bacteria may be provided to disrupt the microbiota of mosquitos.
  • the bacterial modulating agent may compete with, displace, and/or reduce a population of symbiotic bacteria in a mosquito.
  • bacteria may be provided to disrupt the microbiota of mites.
  • the bacterial modulating agent may compete with, displace, and/or reduce a population of symbiotic bacteria in a mite.
  • bacteria may be provided to disrupt the microbiota of biting louse.
  • the bacterial modulating agent may compete with, displace, and/or reduce a population of symbiotic bacteria in a biting louse.
  • bacteria may be provided to disrupt the microbiota of ticks.
  • the bacterial modulating agent may compete with, displace, and/or reduce a population of symbiotic bacteria in a tick.
  • the bacterial modulating agents discussed herein can be used to alter the level, activity, or metabolism of target microorganisms as indicated in the sections for decreasing the fitness of a host insect (e.g., a vector of a human pathogen), such as a mosquito a mite, a biting louse, or a tick.
  • a host insect e.g., a vector of a human pathogen
  • a mosquito a mite e.g., a mosquito a mite, a biting louse, or a tick.
  • any of the modulating agents described herein may be fused or linked to an additional moiety.
  • the modulating agent includes a fusion of one or more additional moieties (e.g., 1 additional moiety, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more additional moieties).
  • the additional moiety is any one of the modulating agents described herein (e.g., a peptide, polypeptide, small molecule, or antibiotic).
  • the additional moiety may not act as modulating agent itself but may instead serve a secondary function.
  • the additional moiety may to help the modulating agent access, bind, or become activated at a target site in the host (e.g., at a host gut or a host bacteriocyte) or at a target microorganism resident in the host (e.g., a vector of a human pathogen, e.g., a mosquito, a mite, a biting louse, or a tick).
  • a target site in the host e.g., at a host gut or a host bacteriocyte
  • a target microorganism resident in the host e.g., a vector of a human pathogen, e.g., a mosquito, a mite, a biting louse, or a tick.
  • the additional moiety may help the modulating agent penetrate a target host cell or target microorganism resident in the host.
  • the additional moiety may include a cell penetrating peptide.
  • Cell penetrating peptides may be natural sequences derived from proteins; chimeric peptides that are formed by the fusion of two natural sequences; or synthetic CPPs, which are synthetically designed sequences based on structure-activity studies.
  • CPPs have the capacity to ubiquitously cross cellular membranes (e.g., prokaryotic and eukaryotic cellular membranes) with limited toxicity.
  • CPPs may have the capacity to cross cellular membranes via energy-dependent and/or independent mechanisms, without the necessity of a chiral recognition by specific receptors.
  • CPPs can be bound to any of the modulating agents described herein.
  • a CPP can be bound to an antimicrobial peptide (AMP), e.g., a scorpion peptide, e.g., UY192 fused to a cell penetrating peptide (e.g., YGRKKRRQRRRFLSTIWNGIKGLLFAM; SEQ ID NO: 232).
  • AMP antimicrobial peptide
  • a scorpion peptide e.g., UY192 fused to a cell penetrating peptide (e.g., YGRKKRRQRRRFLSTIWNGIKGLLFAM; SEQ ID NO: 232).
  • Non-limiting examples of CPPs are listed in Table 11.
  • CPPs Cell Penetrating Peptides
  • the additional moiety helps the modulating agent bind a target microorganism (e.g., a fungi or bacterium) resident in the host.
  • the additional moiety may include one or more targeting domains.
  • the targeting domain may target the modulating agent to one or more microorganisms (e.g., bacterium or fungus) resident in the gut of the host.
  • the targeting domain may target the modulating agent to a specific region of the host (e.g., host gut or bacteriocyte) to access microorganisms that are generally present in said region of the host.
  • the targeting domain may target the modulating agent to the foregut, midgut, or hindgut of the host.
  • the targeting domain may target the modulating agent to a bacteriocyte in the host and/or one or more specific bacteria resident in a host bacteriocyte.
  • the targeting domain may be Galanthus nivalis lectin or agglutinin (GNA) bound to a modulating agent described herein, e.g., an AMP, e.g., a scorpion peptide, e.g., Uy192.
  • the modulating agent may include a pre- or pro-amino acid sequence.
  • the modulating agent may be an inactive protein or peptide that can be activated by cleavage or post-translational modification of a pre- or pro-sequence.
  • the modulating agent is engineered with an inactivating pre- or pro-sequence.
  • the pre- or pro-sequence may obscure an activation site on the modulating agent, e.g., a receptor binding site, or may induce a conformational change in the modulating agent.
  • the modulating agent upon cleavage of the pre- or pro-sequence, the modulating agent is activated upon cleavage of the pre- or pro-sequence.
  • the modulating agent may include a pre- or pro-small molecule, e.g., an antibiotic.
  • the modulating agent may be an inactive small molecule described herein that can be activated in a target environment inside the host.
  • the small molecule may be activated upon reaching a certain pH in the host gut.
  • the modulating agent may further include a linker.
  • the linker may be a chemical bond, e.g., one or more covalent bonds or non-covalent bonds.
  • the linker may be a peptide linker (e.g., 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 20, 25, 30, 35, 40, or more amino acids longer).
  • the linker may be include any flexible, rigid, or cleavable linkers described herein.
  • a flexible peptide linker may include any of those commonly used in the art, including linkers having sequences having primarily Gly and Ser residues (“GS” linker). Flexible linkers may be useful for joining domains that require a certain degree of movement or interaction and may include small, non-polar (e.g. Gly) or polar (e.g. Ser or Thr) amino acids.
  • a peptide linker may be a rigid linker.
  • Rigid linkers are useful to keep a fixed distance between moieties and to maintain their independent functions.
  • Rigid linkers may also be useful when a spatial separation of the domains is critical to preserve the stability or bioactivity of one or more components in the fusion.
  • Rigid linkers may, for example, have an alpha helix-structure or Pro-rich sequence, (XP) n , with X designating any amino acid, preferably Ala, Lys, or Glu.
  • a peptide linker may be a cleavable linker.
  • linkers may be cleaved under specific conditions, such as the presence of reducing reagents or proteases.
  • In vivo cleavable linkers may utilize the reversible nature of a disulfide bond.
  • One example includes a thrombin-sensitive sequence (e.g., PRS) between two Cys residues.
  • PRS thrombin-sensitive sequence
  • In vitro thrombin treatment of CPRSC results in the cleavage of the thrombin-sensitive sequence, while the reversible disulfide linkage remains intact.
  • Such linkers are known and described, e.g., in Chen et al., Adv. Drug Deliv. Rev.
  • Cleavage of linkers in fusions may also be carried out by proteases that are expressed in vivo under conditions in specific cells or tissues of the host or microorganisms resident in the host. In some instances, cleavage of the linker may release a free functional, modulating agent upon reaching a target site or cell.
  • Fusions described herein may alternatively be linked by a linking molecule, including a hydrophobic linker, such as a negatively charged sulfonate group; lipids, such as a poly (—CH2-) hydrocarbon chains, such as polyethylene glycol (PEG) group, unsaturated variants thereof, hydroxylated variants thereof, amidated or otherwise N-containing variants thereof, non-carbon linkers; carbohydrate linkers; phosphodiester linkers, or other molecule capable of covalently linking two or more molecules, e.g., two modulating agents.
  • a hydrophobic linker such as a negatively charged sulfonate group
  • lipids such as a poly (—CH2-) hydrocarbon chains, such as polyethylene glycol (PEG) group, unsaturated variants thereof, hydroxylated variants thereof, amidated or otherwise N-containing variants thereof, non-carbon linkers
  • carbohydrate linkers such as polyethylene glycol (PEG) group
  • PEG polyethylene glycol
  • Non-covalent linkers may be used, such as hydrophobic lipid globules to which the modulating agent is linked, for example, through a hydrophobic region of the modulating agent or a hydrophobic extension of the modulating agent, such as a series of residues rich in leucine, isoleucine, valine, or perhaps also alanine, phenylalanine, or even tyrosine, methionine, glycine, or other hydrophobic residue.
  • the modulating agent may be linked using charge-based chemistry, such that a positively charged moiety of the modulating agent is linked to a negative charge of another modulating agent or an additional moiety.
  • compositions described herein may be formulated either in pure form (e.g., the composition contains only the modulating agent) or together with one or more additional agents (such as excipient, delivery vehicle, carrier, diluent, stabilizer, etc.) to facilitate application or delivery of the compositions.
  • additional agents such as excipient, delivery vehicle, carrier, diluent, stabilizer, etc.
  • excipients and diluents include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, saline solution, syrup, methylcellulose, methyl- and propylhydroxybenzoates, talc, magnesium stearate, and mineral oil.
  • the composition includes a delivery vehicle or carrier.
  • the delivery vehicle includes an excipient.
  • excipients include, but are not limited to, solid or liquid carrier materials, solvents, stabilizers, slow-release excipients, colorings, and surface-active substances (surfactants).
  • the delivery vehicle is a stabilizing vehicle.
  • the stabilizing vehicle includes a stabilizing excipient.
  • Exemplary stabilizing excipients include, but are not limited to, epoxidized vegetable oils, antifoaming agents, e.g. silicone oil, preservatives, viscosity regulators, binding agents and tackifiers.
  • the stabilizing vehicle is a buffer suitable for the modulating agent.
  • the composition is microencapsulated in a polymer bead delivery vehicle.
  • the stabilizing vehicle protects the modulating agent against UV and/or acidic conditions.
  • the delivery vehicle contains a pH buffer.
  • the composition is formulated to have a pH in the range of about 4.5 to about 9.0, including for example pH ranges of about any one of 5.0 to about 8.0, about 6.5 to about 7.5, or about 6.5 to about 7.0.
  • the composition may be formulated into emulsifiable concentrates, suspension concentrates, directly sprayable or dilutable solutions, coatable pastes, diluted emulsions, spray powders, soluble powders, dispersible powders, wettable powders, dusts, granules, encapsulations in polymeric substances, microcapsules, foams, aerosols, carbon dioxide gas preparations, tablets, resin preparations, paper preparations, nonwoven fabric preparations, or knitted or woven fabric preparations.
  • the composition is a liquid.
  • the composition is a solid.
  • the composition is an aerosol, such as in a pressurized aerosol can.
  • the composition is present in the waste (such as feces) of the pest.
  • the composition is present in or on a live pest.
  • the delivery vehicle is the food or water of the host. In other instances, the delivery vehicle is a food source for the host. In some instances, the delivery vehicle is a food bait for the host. In some instances, the composition is a comestible agent consumed by the host. In some instances, the composition is delivered by the host to a second host, and consumed by the second host. In some instances, the composition is consumed by the host or a second host, and the composition is released to the surrounding of the host or the second host via the waste (such as feces) of the host or the second host. In some instances, the modulating agent is included in food bait intended to be consumed by a host or carried back to its colony.
  • the modulating agent may make up about 0.1% to about 100% of the composition, such as any one of about 0.01% to about 100%, about 1% to about 99.9%, about 0.1% to about 10%, about 1% to about 25%, about 10% to about 50%, about 50% to about 99%, or about 0.1% to about 90% of active ingredients (such as phage, lysin or bacteriocin).
  • the composition includes at least any of 0.1%, 0.5%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more active ingredients (such as phage, lysin or bacteriocin).
  • the concentrated agents are preferred as commercial products, the final user normally uses diluted agents, which have a substantially lower concentration of active ingredient.
  • any of the formulations described herein may be used in the form of a bait, a coil, an electric mat, a smoking preparation, a fumigant, or a sheet.
  • compositions provided herein may be in a liquid formulation.
  • Liquid formulations are generally mixed with water, but in some instances may be used with crop oil, diesel fuel, kerosene or other light oil as a carrier.
  • the amount of active ingredient often ranges from about 0.5 to about 80 percent by weight.
  • An emulsifiable concentrate formulation may contain a liquid active ingredient, one or more petroleum-based solvents, and an agent that allows the formulation to be mixed with water to form an emulsion.
  • Such concentrates may be used in agricultural, ornamental and turf, forestry, structural, food processing, livestock, and public health pest formulations. These may be adaptable to application equipment from small portable sprayers to hydraulic sprayers, low-volume ground sprayers, mist blowers, and low-volume aircraft sprayers.
  • Some active ingredients are readily dissolve in a liquid carrier. When mixed with a carrier, they form a solution that does not settle out or separate, e.g., a homogenous solution.
  • Formulations of these types may include an active ingredient, a carrier, and one or more other ingredients. Solutions may be used in any type of sprayer, indoors and outdoors.
  • the composition may be formulated as an invert emulsion.
  • An invert emulsion is a water-soluble active ingredient dispersed in an oil carrier. Invert emulsions require an emulsifier that allows the active ingredient to be mixed with a large volume of petroleum-based carrier, usually fuel oil. Invert emulsions aid in reducing drift. With other formulations, some spray drift results when water droplets begin to evaporate before reaching target surfaces; as a result the droplets become very small and lightweight. Because oil evaporates more slowly than water, invert emulsion droplets shrink less and more active ingredient reaches the target. Oil further helps to reduce runoff and improve rain resistance. It further serves as a sticker-spreader by improving surface coverage and absorption. Because droplets are relatively large and heavy, it is difficult to get thorough coverage on the undersides of foliage. Invert emulsions are most commonly used along rights-of-way where drift to susceptible non-target areas can be a problem.
  • a flowable or liquid formulation combines many of the characteristics of emulsifiable concentrates and wettable powders. Manufacturers use these formulations when the active ingredient is a solid that does not dissolve in either water or oil. The active ingredient, impregnated on a substance such as clay, is ground to a very fine powder. The powder is then suspended in a small amount of liquid. The resulting liquid product is quite thick. Flowables and liquids share many of the features of emulsifiable concentrates, and they have similar disadvantages. They require moderate agitation to keep them in suspension and leave visible residues, similar to those of wettable powders.
  • Flowables/liquids are easy to handle and apply. Because they are liquids, they are subject to spilling and splashing. They contain solid particles, so they contribute to abrasive wear of nozzles and pumps. Flowable and liquid suspensions settle out in their containers. Because flowable and liquid formulations tend to settle, packaging in containers of five gallons or less makes remixing easier.
  • Aerosol formulations contain one or more active ingredients and a solvent. Most aerosols contain a low percentage of active ingredients. There are two types of aerosol formulations—the ready-to-use type commonly available in pressurized sealed containers and those products used in electrical or gasoline-powered aerosol generators that release the formulation as a smoke or fog.
  • Ready to use aerosol formulations are usually small, self-contained units that release the formulation when the nozzle valve is triggered.
  • the formulation is driven through a fine opening by an inert gas under pressure, creating fine droplets.
  • These products are used in greenhouses, in small areas inside buildings, or in localized outdoor areas.
  • Commercial models, which hold five to 5 pounds of active ingredient, are usually refillable.
  • Smoke or fog aerosol formulations are not under pressure. They are used in machines that break the liquid formulation into a fine mist or fog (aerosol) using a rapidly whirling disk or heated surface.
  • Dry formulations can be divided into two types: ready-to-use and concentrates that must be mixed with water to be applied as a spray. Most dust formulations are ready to use and contain a low percentage of active ingredients (less than about 10 percent by weight), plus a very fine, dry inert carrier made from talc, chalk, clay, nut hulls, or volcanic ash. The size of individual dust particles varies. A few dust formulations are concentrates and contain a high percentage of active ingredients. Mix these with dry inert carriers before applying. Dusts are always used dry and can easily drift to non-target sites.
  • the composition is formulated as granules.
  • Granular formulations are similar to dust formulations, except granular particles are larger and heavier.
  • the coarse particles may be made from materials such as clay, corncobs, or walnut shells.
  • the active ingredient either coats the outside of the granules or is absorbed into them.
  • the amount of active ingredient may be relatively low, usually ranging from about 0.5 to about 15 percent by weight.
  • Granular formulations are most often used to apply to the soil, insects living in the soil, or absorption into plants through the roots. Granular formulations are sometimes applied by airplane or helicopter to minimize drift or to penetrate dense vegetation. Once applied, granules may release the active ingredient slowly.
  • Granules require soil moisture to release the active ingredient.
  • Granular formulations also are used to control larval mosquitoes and other aquatic pests.
  • Granules are used in agricultural, structural, ornamental, turf, aquatic, right-of-way, and public health (biting insect) pest-control operations.
  • the composition is formulated as pellets. Most pellet formulations are very similar to granular formulations; the terms are used interchangeably. In a pellet formulation, however, all the particles are the same weight and shape. The uniformity of the particles allows use with precision application equipment.
  • the composition is formulated as a powder. In some instances, the composition is formulated as a wettable powder.
  • Wettable powders are dry, finely ground formulations that look like dusts. They usually must be mixed with water for application as a spray. A few products, however, may be applied either as a dust or as a wettable powder—the choice is left to the applicator. Wettable powders have about 1 to about 95 percent active ingredient by weight; in some cases more than about 50 percent. The particles do not dissolve in water. They settle out quickly unless constantly agitated to keep them suspended. They can be used for most pest problems and in most types of spray equipment where agitation is possible. Wettable powders have excellent residual activity. Because of their physical properties, most of the formulation remains on the surface of treated porous materials such as concrete, plaster, and untreated wood. In such cases, only the water penetrates the material.
  • the composition is formulated as a soluble powder.
  • Soluble powder formulations look like wettable powders. However, when mixed with water, soluble powders dissolve readily and form a true solution. After they are mixed thoroughly, no additional agitation is necessary.
  • the amount of active ingredient in soluble powders ranges from about 15 to about 95 percent by weight; in some cases more than about 50 percent. Soluble powders have all the advantages of wettable powders and none of the disadvantages, except the inhalation hazard during mixing.
  • the composition is formulated as a water-dispersible granule.
  • Water-dispersible granules also known as dry flowables, are like wettable powders, except instead of being dust-like, they are formulated as small, easily measured granules.
  • Water-dispersible granules must be mixed with water to be applied. Once in water, the granules break apart into fineparticles similar to wettable powders. The formulation requires constant agitation to keep it suspended in water. The percentage of active ingredient is high, often as much as 90 percent by weight. Water-dispersible granules share many of the same advantages and disadvantages of wettable powders, except they are more easily measured and mixed. Because of low dust, they cause less inhalation hazard to the applicator during handling
  • the composition includes a bait.
  • the bait can be in any suitable form, such as a solid, paste, pellet or powdered form.
  • the bait can also be carried away by the host back to a population of said host (e.g., a colony or hive).
  • the bait can then act as a food source for other members of the colony, thus providing an effective modulating agent for a large number of hosts and potentially an entire host colony.
  • the baits can be provided in a suitable “housing” or “trap.”
  • housings and traps are commercially available and existing traps can be adapted to include the compositions described herein.
  • the housing or trap can be box-shaped for example, and can be provided in pre-formed condition or can be formed of foldable cardboard for example. Suitable materials for a housing or trap include plastics and cardboard, particularly corrugated cardboard.
  • the inside surfaces of the traps can be lined with a sticky substance in order to restrict movement of the host once inside the trap.
  • the housing or trap can contain a suitable trough inside which can hold the bait in place.
  • a trap is distinguished from a housing because the host cannot readily leave a trap following entry, whereas a housing acts as a “feeding station” which provides the host with a preferred environment in which they can feed and feel safe from predators.
  • the composition includes an attractant (e.g., a chemoattractant).
  • the attractant may attract an adult host or immature host (e.g., larva) to the vicinity of the composition.
  • Attractants include pheromones, a chemical that is secreted by an animal, especially an insect, which influences the behavior or development of others of the same species.
  • Other attractants include sugar and protein hydrolysate syrups, yeasts, and rotting meat. Attractants also can be combined with an active ingredient and sprayed onto foliage or other items in the treatment area.
  • Attractants useful in the methods and compositions described herein include, for example, eugenol, phenethyl propionate, ethyl dimethylisobutyl-cyclopropane carboxylate, propyl benszodioxancarboxylate, cis-7,8-epoxy-2-methyloctadecane, trans-8,trans-0-dodecadienol, cis-9-tetradecenal (with cis-11-hexadecenal), trans-11-tetradecenal, cis-11-hexadecenal, (Z)-11,12-hexadecadienal, cis-7-dodecenyl acetate, cis-8-dodecenyul acetate, cis-9-dodecenyl a
  • Means other than chemoattractants may also be used to attract insects, including lights in various wavelengths or colors.
  • the composition is provided in a microencapsulated formulation.
  • Microencapsulated formulations are mixed with water and sprayed in the same manner as other sprayable formulations. After spraying, the plastic coating breaks down and slowly releases the active ingredient.
  • compositions described herein may be formulated to include the modulating agent described herein and an inert carrier.
  • carrier can be a solid carrier, a liquid carrier, a gel carrier, and/or a gaseous carrier.
  • the carrier can be a seed coating.
  • the seed coating is any non-naturally occurring formulation that adheres, in whole or part, to the surface of the seed.
  • the formulation may further include an adjuvant or surfactant.
  • the formulation can also include one or more modulating agents to enlarge the action spectrum.
  • a solid carrier used for formulation includes finely-divided powder or granules of clay (e.g. kaolin clay, diatomaceous earth, bentonite, Fubasami clay, acid clay, etc.), synthetic hydrated silicon oxide, talc, ceramics, other inorganic minerals (e.g., sericite, quartz, sulfur, activated carbon, calcium carbonate, hydrated silica, etc.), a substance which can be sublimated and is in the solid form at room temperature (e.g., 2,4,6-triisopropyl-1,3,5-trioxane, naphthalene, p-dichlorobenzene, camphor, adamantan, etc.); wool; silk; cotton; hemp; pulp; synthetic resins (e.g., polyethylene resins such as low-density polyethylene, straight low-density polyethylene and high-density polyethylene; ethylene-vinyl ester copolymers such as ethylene-vinyl acetate copoly
  • a liquid carrier may include, for example, aromatic or aliphatic hydrocarbons (e.g., xylene, toluene, alkylnaphthalene, phenylxylylethane, kerosine, gas oil, hexane, cyclohexane, etc.), halogenated hydrocarbons (e.g., chlorobenzene, dichloromethane, dichloroethane, trichloroethane, etc.), alcohols (e.g., methanol, ethanol, isopropyl alcohol, butanol, hexanol, benzyl alcohol, ethylene glycol, etc.), ethers (e.g., diethyl ether, ethylene glycol dimethyl ether, diethylene glycol monomethyl ether, diethylene glycol monoethyl ether, propylene glycol monomethyl ether, tetrahydrofuran, dioxane, etc.), esters (e.g.,
  • N-methylpyrrolidone alkylidene carbonates e.g., propylene carbonate, etc.
  • vegetable oil e.g., soybean oil, cottonseed oil, etc.
  • vegetable essential oils e.g., orange oil, hyssop oil, lemon oil, etc.
  • a gaseous carrier may include, for example, butane gas, flon gas, liquefied petroleum gas (LPG), dimethyl ether, and carbon dioxide gas.
  • LPG liquefied petroleum gas
  • the composition provided herein may include an adjuvant.
  • Adjuvants are chemicals that do not possess activity. Adjuvants are either pre-mixed in the formulation or added to the spray tank to improve mixing or application or to enhance performance. They are used extensively in products designed for foliar applications. Adjuvants can be used to customize the formulation to specific needs and compensate for local conditions. Adjuvants may be designed to perform specific functions, including wetting, spreading, sticking, reducing evaporation, reducing volatilization, buffering, emulsifying, dispersing, reducing spray drift, and reducing foaming. No single adjuvant can perform all these functions, but compatible adjuvants often can be combined to perform multiple functions simultaneously.
  • adjuvants included in the formulation are binders, dispersants and stabilizers, specifically, for example, casein, gelatin, polysaccharides (e.g., starch, gum arabic, cellulose derivatives, alginic acid, etc.), lignin derivatives, bentonite, sugars, synthetic water-soluble polymers (e.g., polyvinyl alcohol, polyvinylpyrrolidone, polyacrylic acid, etc.), PAP (acidic isopropyl phosphate), BHT (2,6-di-t-butyl-4-methylphenol), BHA (a mixture of 2-t-butyl-4-methoxyphenol and 3-t-butyl-4-methoxyphenol), vegetable oils, mineral oils, fatty acids and fatty acid esters.
  • binders specifically, for example, casein, gelatin, polysaccharides (e.g., starch, gum arabic, cellulose derivatives, alginic acid, etc.), lignin derivatives, bentonit
  • the composition provided herein includes a surfactant.
  • Surfactants also called wetting agents and spreaders, physically alter the surface tension of a spray droplet.
  • a spray droplet must be able to wet the foliage and spread out evenly over a leaf.
  • Surfactants enlarge the area of formulation coverage, thereby increasing the pest's exposure to the chemical.
  • Surfactants are particularly important when applying a formulation to waxy or hairy leaves. Without proper wetting and spreading, spray droplets often run off or fail to cover leaf surfaces adequately. Too much surfactant, however, can cause excessive runoff and reduce efficacy.
  • Surfactants are classified by the way they ionize or split apart into electrically charged atoms or molecules called ions.
  • a surfactant with a negative charge is anionic.
  • One with a positive charge is cationic, and one with no electrical charge is nonionic.
  • Formulation activity in the presence of a nonionic surfactant can be quite different from activity in the presence of a cationic or anionic surfactant. Selecting the wrong surfactant can reduce the efficacy of a pesticide product and injure the target plant.
  • Anionic surfactants are most effective when used with contact pesticides (pesticides that control the pest by direct contact rather than being absorbed systemically). Cationic surfactants should never be used as stand-alone surfactants because they usually are phytotoxic.
  • Nonionic surfactants often used with systemic pesticides, help pesticide sprays penetrate plant cuticles.
  • Nonionic surfactants are compatible with most pesticides, and most EPA-registered pesticides that require a surfactant recommend a nonionic type.
  • Adjuvants include, but are not limited to, stickers, extenders, plant penetrants, compatibility agents, buffers or pH modifiers, drift control additives, defoaming agents, and thickeners.
  • surfactants included in the compositions described herein are alkyl sulfate ester salts, alkyl sulfonates, alkyl aryl sulfonates, alkyl aryl ethers and polyoxyethylenated products thereof, polyethylene glycol ethers, polyvalent alcohol esters and sugar alcohol derivatives.
  • the modulating agent may be in a mixture with other active compounds, such as pesticidal agents (e.g., insecticides, sterilants, acaricides, nematicides, molluscicides, or fungicides; see, e.g., pesticides listed in Table 12), attractants, growth-regulating substances, or herbicides.
  • pesticidal agents e.g., insecticides, sterilants, acaricides, nematicides, molluscicides, or fungicides; see, e.g., pesticides listed in Table 12
  • attractants e.g., growth-regulating substances, or herbicides.
  • the term “pesticidal agent” refers to any substance or mixture of substances intended for preventing, destroying, repelling, or mitigating any pest.
  • a pesticide can be a chemical substance or biological agent used against pests including insects, pathogens, weeds, and microbes that compete with humans for food, destroy property, spread disease, or are a nuisance.
  • the term “pesticidal agent” may further encompass other bioactive molecules such as antibiotics, antivirals pesticides, antifungals, antihelminthics, nutrients, pollen, sucrose, and/or agents that stun or slow insect movement.
  • the modulating agent is applied to plants, a mixture with other known compounds, such as herbicides, fertilizers, growth regulators, safeners, semiochemicals, or else with agents for improving plant properties is also possible.
  • other known compounds such as herbicides, fertilizers, growth regulators, safeners, semiochemicals, or else with agents for improving plant properties is also possible.
  • a host described herein can be exposed to any of the compositions described herein in any suitable manner that permits delivering or administering the composition to the insect.
  • the modulating agent may be delivered either alone or in combination with other active or inactive substances and may be applied by, for example, spraying, microinjection, through plants, pouring, dipping, in the form of concentrated liquids, gels, solutions, suspensions, sprays, powders, pellets, briquettes, bricks and the like, formulated to deliver an effective concentration of the modulating agent.
  • Amounts and locations for application of the compositions described herein are generally determined by the habits of the host, the lifecycle stage at which the microorganisms of the host can be targeted by the modulating agent, the site where the application is to be made, and the physical and functional characteristics of the modulating agent.
  • the modulating agents described herein may be administered to the insect by oral ingestion, but may also be administered by means which permit penetration through the cuticle or penetration of the insect respiratory system.
  • the insect can be simply “soaked” or “sprayed” with a solution including the modulating agent.
  • the modulating agent can be linked to a food component (e.g., comestible) of the insect for ease of delivery and/or in order to increase uptake of the modulating agent by the insect.
  • Methods for oral introduction include, for example, directly mixing a modulating agent with the insects food, spraying the modulating agent in the insects habitat or field, as well as engineered approaches in which a species that is used as food is engineered to express a modulating agent, then fed to the insect to be affected.
  • the modulating agent composition can be incorporated into, or overlaid on the top of, the insects diet.
  • the modulating agent composition can be sprayed onto a field of crops which an insect inhabits.
  • the composition is sprayed directly onto a plant e.g., crops, by e.g., backpack spraying, aerial spraying, crop spraying/dusting etc.
  • the plant receiving the modulating agent may be at any stage of plant growth.
  • formulated modulating agents can be applied as a seed-coating or root treatment in early stages of plant growth or as a total plant treatment at later stages of the crop cycle.
  • the modulating agent may be applied as a topical agent to a plant, such that the host insect ingests or otherwise comes in contact with the plant upon interacting with the plant.
  • the modulating agent may be applied (e.g., in the soil in which a plant grows, or in the water that is used to water the plant) as a systemic agent that is absorbed and distributed through the tissues (e.g., stems or leafs) of a plant or animal host, such that an insect feeding thereon will obtain an effective dose of the modulating agent.
  • plants or food organisms may be genetically transformed to express the modulating agent such that a host feeding upon the plant or food organism will ingest the modulating agent.
  • Delayed or continuous release can also be accomplished by coating the modulating agent or a composition containing the modulating agent(s) with a dissolvable or bioerodable coating layer, such as gelatin, which coating dissolves or erodes in the environment of use, to then make the modulating agent available, or by dispersing the agent in a dissolvable or erodable matrix.
  • a dissolvable or bioerodable coating layer such as gelatin, which coating dissolves or erodes in the environment of use, to then make the modulating agent available, or by dispersing the agent in a dissolvable or erodable matrix.
  • Such continuous release and/or dispensing means devices may be advantageously employed to consistently maintain an effective concentration of one or more of the modulating agents described herein in a specific host habitat.
  • the modulating agent can also be incorporated into the medium in which the insect grows, lives, reproduces, feeds, or infests.
  • a modulating agent can be incorporated into a food container, feeding station, protective wrapping, or a hive.
  • the modulating agent may be bound to a solid support for application in powder form or in a “trap” or “feeding station.”
  • the compositions may also be bound to a solid support or encapsulated in a time-release material.
  • compositions described herein can be administered by delivering the composition to at least one habitat where a vector (e.g., a vector of a human pathogen, e.g., a mosquito, mite, biting louse, or tick) grows, lives, reproduces, feeds, or infests.
  • a vector e.g., a vector of a human pathogen, e.g., a mosquito, mite, biting louse, or tick
  • the screening assays provided herein may be effective to identify one or more modulating agents (e.g., phage) that target symbiotic microorganisms resident in the host and thereby decrease the fitness of the host.
  • modulating agents e.g., phage
  • the identified modulating agent e.g., phage
  • the identified modulating agent may be effective to decrease the viability of pesticide- or allelochemical-degrading microorganisms (e.g., bacteria e.g., a bacterium that degrades a pesticide listed in Table 12), thereby increasing the hosts sensitivity to a pesticide (e.g., sensitivity to a pesticide listed in Table 12) or allelochemical agent.
  • pesticide- or allelochemical-degrading microorganisms e.g., bacteria e.g., a bacterium that degrades a pesticide listed in Table 12
  • a phage library may be screened to identify a phage that targets a specific endosymbiotic microorganism resident in a host.
  • the phage library may be provided in the form of one or more environmental samples (e.g., soil, pond sediments, or sewage water).
  • the phage library may be generated from laboratory isolates.
  • the phage library may be co-cultured with a target bacterial strain. After incubation with the bacterial strain, phage that successfully infect and lyse the target bacteria are enriched in the culture media.
  • the phage-enriched culture may be sub-cultured with additional bacteria any number of times to further enrich for phage of interest.
  • the phage may be isolated for use as a modulating agent in any of the methods or compositions described herein, wherein the phage alters the microbiota of the host in a manner that decreases host fitness.
  • This example demonstrates the ability to kill or decrease the fitness of the Anopheles coluzzii mosquitoes and decrease the transmission rate of parasites by treatment with azithromycin, relatively broad but shallow antibacterial activity. It inhibits some Gram-positive bacteria, some Gram-negative bacteria, and many atypical bacteria.
  • the effect of azithromycin on mosquitoes is mediated through the modulation of bacterial populations endogenous to the mosquito that are sensitive to azithromycin.
  • One targeted bacterial strain is Asaia.
  • the mosquito has been described as the most dangerous animal in the world and malaria is one mosquito-borne disease that detrimentally impacts humans. There are about 3,500 mosquito species and those that transmit malaria all belong to a sub-set called the Anopheles . Approximately 40 Anopheles species are able to transmit malaria that significantly impacts human health.
  • Blood meals mixed with azithromycin solutions are formulated with final antibiotic concentrations of 0 (negative control), 0.1, 1, or 5 ⁇ g/ml in 1 mL of blood.
  • mosquitoes are grown in a lab environment and medium. Experiments are performed with female mosquitoes from an Anopheles coluzzii Ngousso colony, originally established from field mosquitoes collected in Cameroon, maintained on human blood and fed as adults with 5% fructose. Larvae are fed tetramin fish food. Temperature is maintained at 28° C. ( ⁇ 1° C.), 70-80% humidity on a 12 hr light/dark cycle.
  • Plasmodium falciparum NF54 gametocytes are cultured in RPMI medium (GIBCO) including 300 mg. L-1 L-glutamine supplemented with 50 mg/L hypoxanthine, 25 mM HEPES plus 10% heat-inactivated human serum without antibiotics. Two 25-mL cultures are started 17 and 14 days before the infection at 0.5% parasitemia in 6% v/v washed O + red blood cells (RBCs). Media is changed daily. Before mosquito infection, centrifuged RBCs are pooled and supplemented with 20% fresh washed RBCs and human serum (2:3 v/v ratio between RBCs and serum). Mosquitoes are offered a blood meal from a membrane-feeding device (2 ml Eppendorf tube) covered with Parafilm and kept at 37° C.
  • a membrane-feeding device (2 ml Eppendorf tube) covered with Parafilm and kept at 37° C.
  • Azithromycin solutions are made by dissolving azithromycin (SIGMA-ALDRICH, PZ0007) in DMSO. Different volumes of azithromycin solution are added to fresh blood to total 1 mL in preparation for blood meals. The final azithromycin concentrations in the blood are 0 (just solvent as control solution), 0.1, 1, or 5 ⁇ g/ml.
  • mosquitoes per condition For each Plasmodium infection, at least 100 age-matched, 2- to 3-day-old, mosquitoes per condition are offered a control or experimental blood meal from a membrane-feeding device (2 ml Eppendorf tube) covered with parafilm and kept at 37° C. and nonengorged mosquitoes are removed. Meals are given every four days for a total of three blood meals. Between the blood meals, mosquitoes are provided with a cotton pad moistened with distilled water for oviposition. Unfed mosquitoes are not removed after the second and later blood meals. Deaths are counted daily and carcasses are removed and stored for Asaia analysis as described herein. At least 50 mosquitoes per concentration of azithromycin are used for each replicate. At the end of the last blood meal, mosquitoes are kept for 12 hours before dissection.
  • mosquitoes are immersed in 70% ethanol for 5 minutes then rinsed 3 times in sterile phosphate-buffered saline (PBS) to kill and remove surface bacteria, thus minimizing sample contamination with cuticle bacteria during dissection.
  • PBS sterile phosphate-buffered saline
  • the midgut of each mosquitoe (control and azithromycin treatment) is removed and frozen immediately on dry ice and stored at 20° C. until processing. Midguts are only excluded from analysis if they burst and a substantial amount of the gut content is lost.
  • Samples are homogenized in phenol-chloroform in a Precellys 24 homogenizer (Bertin) using 0.5 mm-wide glass beads (Bertin) for 30 seconds at 6800 rpm and deoxy-ribonucleic acid (DNA) is extracted with phenol-chloroform.
  • the 16S ribosomal DNA (rDNA) is used for Asaia quantification and is shown as a ratio of the Anopheles housekeeping gene 40S ribosomal protein S7 (Vector-Base gene ID AGAP010592).
  • Primer sequences for Asaia are: forward 5′-GTGCCGATCTCTAAAAGCCGTCTCA-3′ (SEQ ID NO: 219) and reverse 5′-TTCGCTCACCGGCTTCGGGT-3′ (SEQ ID NO: 220), and for S7: forward 5′-GTGCGCGAGTTGGAGAAGA-3′ (SEQ ID NO: 221) and reverse 5′-ATCGGTTTGGGCAGAATGC-3′ (SEQ ID NO: 222).
  • Quantitative polymerase chain reaction qPCR is performed on a 7500 Fast Real-Time thermocycler (Applied Biosystems) using the SYBR Premix Ex Taq kit (Takara), following the manufacturer's instructions. Azithromycin treated mosquitoes show a reduction of Asaia specific genes.
  • the survival rates of mosquitoes treated with azithromycin are compared to the mosquitoes treated with the negative control.
  • the survival rate of mosquitoes treated with azithromycin solution is decreased compared to the control.
  • Example 2 Treatment of the Aedes vexans Mosquito with an Antibiotic Solution
  • This Example demonstrates the ability to kill or decrease the fitness of the Aedes vexans mosquitoes by treatment with doxycycline, a broad spectrum antibiotic that inhibits protein production.
  • the effect of doxycycline on mosquitoes is mediated through the modulation of bacterial populations endogenous to the mosquito that are sensitive to doxycycline.
  • One targeted bacterial strain is Wolbachia.
  • PRRSV porcine reproductive and respiratory syndrome virus
  • Aedes vexans is a cosmopolitan and common pest mosquito.
  • Dirofilaria immitis dog heartworm
  • Myxomatosis deadly rabbit virus disease
  • Eastern equine encephalitis deadly horse virus disease in the USA.
  • Aedes vexans is the most common mosquito in Europe, often composing more than 80% the European mosquito community. Its abundance depends upon availability of floodwater pools. In summer, mosquito traps can collect up to 8,000 mosquitoes per trap per night.
  • Blood meals mixed with doxycycline solutions are formulated with final antibiotic concentrations of 0 (negative control), 1, 10, or 50 ⁇ g/ml in 1 mL of blood
  • Doxycycline solutions are made by dissolving doxycycline (SIGMA-ALDRICH, D9891) in sterile water. Different volumes of a doxycycline solution are added to fresh blood to total 1 mL in preparation for blood meals. The final doxycycline concentrations in the blood are approximately 0 (control solution), 1, 10 or 50 ⁇ g/ml.
  • mosquitoes For each replicate, age-matched, 2- to 3-day-old mosquitoes are offered a control or experimental blood meal from a membrane-feeding device (2 ml Eppendorf tube) covered with parafilm and kept at 37° C. Nonengorged mosquitoes are discarded. Meals are given every four days for a total of three blood meals. Between the blood meals, mosquitoes are provided with a cotton pad moistened with distilled water for oviposition. Unfed mosquitoes are not removed after the second and later blood meals. Deaths are counted daily and carcasses are removed and stored for Wolbachia analysis as described herein. At least 50 mosquitoes per concentration of doxycycline are used for each replicate. At the end of the last blood meal, mosquitoes are kept for 12 hours before dissection.
  • mosquitoes are immersed in 70% ethanol for 5 minutes then rinsed 3 times in sterile phosphate-buffered saline (PBS) to kill and remove surface bacteria, thus minimizing sample contamination with cuticle bacteria during dissection.
  • PBS sterile phosphate-buffered saline
  • the midgut of each mosquito (control and doxycycline treatment) is removed and frozen immediately on dry ice and stored at 20° C. until processing. Midguts are only excluded from analysis if they burst and a substantial amount of the gut content is lost.
  • Samples are homogenized in phenol-chloroform in a Precellys 24 homogenizer (Bertin) using 0.5 mm wide glass beads (Bertin) for 30 seconds at 6800 rpm and deoxy-ribonucleic acid (DNA) is extracted with phenol-chloroform.
  • the 16S ribosomal DNA (rDNA) is used for Wolbachia quantification and is shown as a ratio of the Aedes housekeeping gene 40S ribosomal protein S7 (Vector-Base gene ID AAEL009496).
  • Primer sequences for Wolbachia are: forward primer 5′-TCAGCCACACTGGAACTGAG-3′ (SEQ ID NO: 225) and reverse primer 5′-TAACGCTAGCCCTCTCCGTA-3′ (SEQ ID NO: 226), and for S7: forward 5′-AAGGTCGACACCTTCACGTC-3′ (SEQ ID NO: 227) and reverse 5′-CCGTTTGGTGAGGGTCTTTA-3′ (SEQ ID NO: 228).
  • Quantitative polymerase chain reaction qPCR is performed on a 7500 Fast Real-Time thermocycler (Applied Biosystems) using the SYBR Premix Ex Taq kit (Takara), following the manufacturer's instructions. Doxycycline treated mosquitoes show a reduction of Wolbachia specific genes.
  • the survival rates of mosquitoes treated with doxycycline solution are compared to the mosquitoes treated with the negative control.
  • the survival rate of mosquitoes treated with doxycycline solution is decreased compared to the control.
  • Example 3 Treatment of the Dermacentor andersoni , with an Antibiotic Solution
  • This Example demonstrates the ability to kill or decrease the fitness of the tick, Dermacentor andersoni , by treatment with Liquamycin LA-200 oxytetracycline, a broad spectrum antibiotic commonly used to treat a broad range of bacterial infections in cattle.
  • Liquamycin LA-200 oxytetracycline a broad spectrum antibiotic commonly used to treat a broad range of bacterial infections in cattle.
  • the effect of Liquamycin LA-200 oxytetracycline on ticks is mediated through the modulation of bacterial populations endogenous to the tick that are sensitive to Liquamycin LA-200 oxytetracycline.
  • One targeted bacterial strain is Rickettsia.
  • Ticks are obligate hematophagous arthropods that feed on vertebrates and cause great economic losses to livestock due to their ability to transmit diseases to humans and animals.
  • ticks transmit pathogens throughout all continents and are labeled as principle vectors of zoonotic pathogens.
  • 415 new tick-borne bacterial pathogens have been discovered since Lyme disease was characterized in 1982.
  • Dermacentor andersoni the Rocky Mountain wood tick, has been labeled a ‘veritable Pandora's box of disease-producing agents’ and transmits several pathogens, including Rickettsia rickettsii and Francisella tularensis .
  • Therapeutic design A therapeutic dose (11 mg/kg of body weight) of Liquamycin LA-200 oxytetracycline injection on ⁇ 4, ⁇ 1, +3 and +5 days post application of ticks.
  • a cohort of F1 ticks are fed on either antibiotic-treated calves or untreated calves (control).
  • the antibiotic-treated calves received a therapeutic dose (11 mg/kg of body weight) of Liquamycin LA-200 oxytetracycline injections on ⁇ 4, ⁇ 1, +3 and +5 days post application of ticks, whereas untreated calves did not receive any injections (untreated control).
  • Females ticks are allowed to oviposit to continue a second generation of the untreated and treated ticks (F2 generation).
  • the F2 treated generation arose from F1 adults that are exposed to antibiotics.
  • the F2 ticks are not subjected to antibiotics.
  • F1 and F2 generation adult male ticks are fed for 7 days and then dissected within 24 h. Deaths are counted daily and ticks are removed and stored for Rickettsia analysis as described herein. Before dissection, the ticks are surface sterilized and all dissection tools are sterilized between each dissection. Tick MG and SG are dissected and pooled in groups of 30 with three biological replicates. Tissues are stored in Cell Lysis Solution (Qiagen, Valencia, Calif., USA) and Proteinase K (1.25 mg/ml). Genomic DNA is isolated using the PureGene Extraction kit (Qiagen) according to the manufacturer's specifications.
  • rickA gene copy numbers are measured using SYBR Green quantitative PCR of non-treated and antibiotic treated in F1 and F2 ticks.
  • the quantity of Rickettsia is determined using Forward (5′-TACGCCACTCCCTGTGT CA-3′; SEQ ID NO: 229) and Reverse (5′-GATGTAACGGTATTAC ACCAACAG-3′; SEQ ID NO: 230) primers.
  • the bacterial quantity is measured in F1 and F2 MG and SG of the pooled samples.
  • Quantitative polymerase chain reaction qPCR is performed on a 7500 Fast Real-Time thermocycler (Applied Biosystems) using the SYBR Premix Ex Taq kit (Takara), following the manufacturer's instructions. Liquamycin LA-200 oxytetracycline treated ticks show a reduction of Rickettsia specific genes.
  • the survival rates of ticks treated with antibiotic solution are compared to the ticks untreated.
  • the survival rate of ticks treated with Liquamycin LA-200 oxytetracycline solution is decreased compared to the untreated.
  • This Example demonstrates the ability to kill or decrease the fitness of mites by treating them with an antibiotic solution.
  • This Example demonstrates that the effect of oxytetracycline on mites is mediated through the modulation of bacterial populations endogenous, such as Bacillus , to the mites that are sensitive to oxytetracycline.
  • Sarcoptic mange is caused by mites that infest animals by burrowing deeply into the skin and laying eggs inside the burrows. The eggs hatch into the larval stage. The larval mites then leave the burrows, move up to the skin surface, and begin forming new burrows in healthy skin tissue. Development from egg to adult is completed in about 2 weeks. The lesions resulting from infestations by these mites are a consequence of the reaction of the animals' immune system to the mites' presence. Because of the intensity of the animals' immunological response, it takes only a small number of mites to produce widespread lesions and generalized dermatitis.
  • mites can be killed with chemically synthesized miticides
  • these types of chemicals must sprayed on every animal in the herd with high-pressure hydraulic spray equipment to ensure penetration by the spray into the skin.
  • these types of chemical pesticides may have detrimental ecological and/or agricultural effects.
  • Oxytetracycline solution is formulated with 0 (negative control), 1, 10, or 50 ⁇ g/ml in 10 mL of sterile water with 0.5% sucrose and essential amino acids.
  • the brood mites are collected from mite-infested pigs. Skin samples are collected by gently scraping and lifting off encrusted areas from the inner ear area of the pig with a sharpened teaspoon and subsequently examined for mites.
  • Mites are grouped per age and assayed separately. The age is determined based on the morphology and pigmentation of the larva or the pupa as follows: mites collected from spinning larvae that are small enough to move around are grouped into Group 1; mites collected from stretched larvae, which are too big to lay in the cell and start to stretch upright with their mouth in the direction of the cell opening, are grouped into Group 2; and mites collected from pupae are grouped into Group 3. Mites are stored on their host larva or pupa in glass Petri dishes until 50 units are collected. This ensures their feeding routine and physiological status remains unchanged. To prevent mites from straying from their host larva or pupa or climbing onto one another, only hosts at the same development stage are kept in the same dish.
  • the equipment a stainless steel ring (56 mm inner diameter, 2-3 mm height) and 2 glass circles (62 mm diameter)—is cleaned with acetone and hexane or pentane to form the testing arena.
  • the oxytetracycline solutions and control solution are applied on the equipment by spraying the glass disks and ring of the arena homogeneously.
  • a reservoir is loaded with 1 ml of the solutions; the distance of the sprayed surface from the bottom end of the tube is set at 11 mm and a 0.0275 inch nozzle is used.
  • the pressure is adjusted (usually in the range 350-500 hPa) until the amount of solution deposited is 1 ⁇ 0.05 mg/cm2.
  • the antibiotic solutions are poured in their respective dishes, covering the whole bottom of the dishes, and residual liquid is evaporated under a fume hood.
  • the ring is placed between the glass circles to build a cage.
  • the cages are used within 60 hr of preparation, for not more than three assays, in order to control the exposure of mites to antibiotic solutions. 10 to 15 mites are introduced in this cage and the equipment pieces are bound together with droplets of melted wax. Mites collected from spinning larvae, stretched larvae, white eyed pupae and dark eyed with white and pale body are used.
  • mites are transferred into a clean glass Petri dish (60 mm diameter) with two or three white eye pupae (4-5 days after capping) to feed on.
  • the mites are observed under a dissecting microscope at 4 hr, 24 hr, and 48 hr after being treated with the antibiotic or the control solutions, and classified according to the following categories:
  • a sterile toothpick or needle is used to stimulate the mites by touching their legs. New tooth picks or sterile needles are used for stimulating each group to avoid contamination between mite groups.
  • the assays are carried out at 32.5° C. and 60-70% relative humidity. If the mortality in the controls exceeds 30%, the replicate is excluded. Each experiment is replicated with four series of cages.
  • the status of Bacillus in mite groups is assessed by PCR.
  • Total DNA is isolated from control (non-oxytetracycline treated) and oxytetracyline treated individuals (whole body) using a DNA Kit (OMEGA, Bio-tek) according to the manufacturer's protocol.
  • the primers for Bacillus forward primer 5′-GAGGTAGACGAAGCGACCTG-3′ (SEQ ID NO: 231) and reverse primer 5′-TTCCCTCACGGTACTGGTTC-3′ (SEQ ID NO: 232), are designed based on 23S-5S rRNA sequences obtained from the Bacillus genome (Accession Number: AP007209.1) (Takeno et al., J. Bacteriol.
  • PCR amplification cycles included an initial denaturation step at 95° C. for 5 min, 35 cycles of 95° C. for 1 min, 59° C. for 1 min, and 72° C. for 2 min, and a final extension step of 5 min at 72° C.
  • Amplification products from oxytetracyline treated and control samples are analyzed on 1% agarose gels, stained with SYBR safe, and visualized using an imaging System.
  • the survival rates of mites treated with an oxytetracyline solution are compared to the Varroa mites treated with the negative control.
  • the survival rate and the mobility of mites treated with oxytetracyline solution are expected to be decreased compared to the control.
  • This Example demonstrates the acquisition of a phage collection from environmental samples.
  • Phage library collection having the following phage families: Myoviridae, Siphoviridae, Podoviridae, Lipothrixviridae, Rudiviridae, Ampullaviridae, Bicaudaviridae, Clavaviridae, Corticoviridae, Cystoviridae, Fuselloviridae, Gluboloviridae, Guttaviridae, Inoviridae, Leviviridae, Microviridae, Plasmaviridae, Tectiviridae
  • This Example demonstrates the isolation, purification, and identification of single target specific phages from a heterogeneous phage library.
  • 20-30 ml of the phage library described in Example 5 is diluted to a volume of 30-40 ml with LB-broth.
  • the target bacterial strain e.g., Buchnera
  • the target bacterial strain is added (50-200 ⁇ l overnight culture grown in LB-broth) to enrich phages that target this specific bacterial strain in the culture.
  • This culture is incubated overnight at +37° C., shaken at 230 rpm.
  • Bacteria from this enrichment culture are removed by centrifugation (3000-6000 g in Megafuge 1.0R, Heraeus, or in Eppendorf centrifuge 5702 R, 15-20 min, +4° C.) and filtered (0.2 or 0.45 ⁇ m filter).
  • 2.5 ml of the bacteria free culture is added to 2.5 ml of LB-broth and 50-100 ⁇ l of the target bacteria to enrich the phages.
  • the enrichment culture is grown overnight as above.
  • a sample from this enrichment culture is centrifuged at 13,000 g for 15 min at room temperature and 10 ⁇ l of the supernatant is plated on a LB-agar containing petri dish along with 100-300 ⁇ l of the target bacteria and 3 ml of melted 0.7% soft-agar. The plates are incubated overnight at +37° C.
  • Each of the plaques observed on the bacterial lawn are picked and transferred into 500 ⁇ l of LB-broth.
  • a sample from this plaque-stock is further plated on the target bacteria. Plaque-purification is performed three times for all discovered phages in order to isolate a single homogenous phage from the heterogeneous phage mix.
  • Lysates from plates with high-titer phages are prepared by harvesting overlay plates of a host bacterium strain exhibiting confluent lysis. After being flooded with 5 ml of buffer, the soft agar overlay is macerated, clarified by centrifugation, and filter sterilized. The resulting lysates are stored at 4° C. High-titer phage lysates are further purified by isopycnic CsCl centrifugation, as described in (Summer et al., J. Bacteriol. 192:179-190, 2010).
  • DNA is isolated from CsCl-purified phage suspensions as described in (Summer, Methods Mol. Biol. 502:27-46, 2009). An individual isolated phage is sequenced as part of two pools of phage genomes by using a 454 pyrosequencing method. Phage genomic DNA is mixed in equimolar amounts to a final concentration of about 100 ng/L. The pooled DNA is sheared, ligated with a multiplex identifier (MID) tag specific for each of the pools, and sequenced by pyrosequencing using a full-plate reaction on a Roche FLX Titanium sequencer according to the manufacturer's protocols. The pooled phage DNA is present in two sequencing reactions.
  • MID multiplex identifier
  • the trimmed FLX Titanium flow-gram output corresponding to each of the pools is assembled individually by using Newbler Assembler version 2.5.3 (454 Life Sciences), by adjusting the settings to include only reads containing a single MID per assembly.
  • the identity of individual contigs is determined by PCR using primers generated against contig sequences and individual phage genomic DNA preparations as the template.
  • Sequencher 4.8 (Gene Codes Corporation) is used for sequence assembly and editing. Phage chromosomal end structures are determined experimentally.
  • Cohesive (cos) ends for phages are determined by sequencing off the ends of the phage genome and sequencing the PCR products derived by amplification through the ligated junction of circularized genomic DNA, as described in (Summer, Methods Mol. Biol.
  • Protein-coding regions are initially predicted using GeneMark.hmm (Lukashin et al. Nucleic Acids Res. 26:1107-1115, 1998), refined through manual analysis in Artemis (Rutherford et al., Bioinformatics 16:944-945, 2000.), and analyzed through the use of BLAST (E value cutoff of 0.005) (Camacho et al., BMC Bioinformatics 10:421, 2009). Proteins of particular interest are additionally analyzed by InterProScan (Hunter et al., Nucleic Acids Res. 40:D306-D312, 2012).
  • Electron microscopy of CsCl-purified phage (>1 ⁇ 10 ⁇ circumflex over ( ) ⁇ 11 PFU/ml) that lysed the endosymbiotic bacteria, Buchnera , is performed by diluting stock with the tryptic soy broth buffer. Phages are applied onto thin 400-mesh carbon-coated Formvar grids, stained with 2% (wt/vol) uranyl acetate, and air dried. Specimens are observed on a JEOL 1200EX transmission electron microscope operating at an acceleration voltage of 100 kV. Five virions of each phage are measured to calculate mean values and standard deviations for dimensions of capsid and tail, where appropriate.
  • This Example demonstrates the ability to kill or decrease the fitness of aphids by treating them with a phage solution.
  • This Example demonstrates that the effect of phage on aphids is mediated through the modulation of bacterial populations endogenous to the aphid that are sensitive to phages.
  • One targeted bacterial strain is Buchnera with the phage identified in Example 6.
  • Aphids are representative species for testing microbiota modulating agents and effects on fitness of the aphids.
  • Phage solutions are formulated with 0 (negative control), 10 2 , 10 5 , or 10 8 plaque-forming units (pfu)/ml phage from Example 6 in 10 mL of sterile water with 0.5% sucrose and essential amino acids.
  • aphids are grown in a lab environment and medium.
  • fava bean plants are grown in a mixture of vermiculite and perlite at 24° C. with 16 h of light and 8 h of darkness.
  • 5-10 adults from different plants are distributed among 10 two-week-old plants, and allowed to multiply to high density for 5-7 days.
  • second and third instar aphids are collected from healthy plants and divided into treatments so that each treatment receives approximately the same number of individuals from each of the collection plants.
  • Phage solutions are prepared as described herein. Wells of a 96-well plate are filled with 200 ⁇ l of artificial aphid diet (Febvay et al., Canadian Journal of Zoology 66(11):2449-2453, 1988) and the plate is covered with parafilm to make a feeding sachet. Artificial diet is either mixed with sterile water and with 0.5% sucrose and essential amino acids as a negative control or phage solutions with varying concentrations of phages. Phage solutions are mixed with artificial diet to get final concentrations of phages between 10 2 to 10 8 (pfu)/ml.
  • aphids For each replicate treatment, 30-50 second and third instar aphids are placed individually in wells of a 96-well plate and a feeding sachet plate is inverted above them, allowing the insects to feed through the parafilm and keeping them restricted to individual wells. Experimental aphids are kept under the same environmental conditions as aphid colonies. After the aphids are fed for 24 hr, the feeding sachet is replaced with a new one containing sterile artificial diet and a new sterile sachet is provided every 24 h for 4 days. At the time that the sachet is replaced, aphids are also checked for mortality.
  • An aphid is counted as dead if it had turned brown or is at the bottom of the well and does not move during the observation. If an aphid is on the parafilm of the feeding sachet but not moving, it is assumed to be feeding and alive.
  • the primers for Buchnera forward primer 5′-GTCGGCTCATCACATCC-3′ (SEQ ID NO: 233) and reverse primer 5′-TTCCGTCTGTATTATCTCCT-3′ (SEQ ID NO: 234), are designed based on 23S-5S rRNA sequences obtained from the Buchnera genome (Accession Number: GCA_000009605.1) (Shigenobu et al., Nature 407:81-86, 2000) using Primer 5.0 software (Primer-E Ltd., Madison, UK).
  • the PCR amplification cycles included an initial denaturation step at 95° C. for 5 min, 35 cycles of 95° C. for 30 s, 55° C. for 30 s, and 72° C.
  • Amplification products from rifampicin treated and control samples are analyzed on 1% agarose gels, stained with SYBR safe, and visualized using an imaging System. Phage treated aphids show a reduction of Buchnera specific genes.
  • the survival rates of aphids treated with Buchnera specific phages are compared to the aphids treated with the negative control.
  • the survival rate of aphids treated with Buchnera specific phages is decreased as compared to the control treated aphids.
  • Example 8 Production of a colA Bacteriocin Solution
  • This Example demonstrates the production and purification of colA bacteriocin.
  • DNA is generated by PCR with specific primers with upstream (NdeI) and downstream (XhoI) restriction sites.
  • Forward primer GTATCTATTCCCGTCTACGAACATATGGAATTCC SEQ ID NO: 236) and reverse primer CCGCTCGAGCCATCTGACACTTCCTCCAA (SEQ ID NO: 237).
  • Purified PCR fragments (Nucleospin Extract II-Macherey Nagel) are digested with NdeI or XhoI and then the fragments are ligated.
  • the ligated DNA fragment is cloned into pcr2.1 (GenBank database accession number EY122872) vector (Anselme et al., BMC Biol. 6:43, 2008). The nucleotide sequence is systematically checked (Cogenics).
  • the plasmid with colA sequence is expressed in BL21 (DE3)/pLys.
  • Bacteria are grown in LB broth at 30° C. At an OD600 of 0.9, isopropyl ⁇ -D-1-thiogalactopyranoside (IPTG) is added to a final concentration of 1 mM and cells were grown for 6 h.
  • IPTG isopropyl ⁇ -D-1-thiogalactopyranoside
  • Bacteria are lysed by sonication in 100 mM NaCL, 1% Triton X-100, 100 mM Tris-base pH 9.5, and proteins are loaded onto a HisTrap HP column (GE Healthcare). The column is washed successively with 100 mM NaCl, 100 mM Tris-HCl pH 6.8, and PBS. Elution is performed with 0.3M imidazol in PBS.
  • Desalting PD-10 columns (GE Healthcare) are used to eliminate imidazol and PBS solubilized
  • Example 9 Treatment of Aphids with a Solution of colA Bacteriocin
  • This Example demonstrates the ability to kill or decrease the fitness of aphids by treating them with a bacteriocin solution.
  • This Example demonstrates that the effect of bacteriocins on aphids is mediated through the modulation of bacterial populations endogenous to the aphid that are sensitive to ColA bacteriocin.
  • One targeted bacterial strain is Buchnera with the bacteriocin produced in Example 8.
  • ColA solutions are formulated with 0 (negative control), 0.6, 1, 50 or 100 mg/ml of ColA from Example 8 in 10 mL of sterile water with 0.5% sucrose and essential amino acids.
  • aphids are grown in a lab environment and medium.
  • plants are grown in a mixture of vermiculite and perlite and are infested with aphids.
  • E. balteatus larvae are obtained from a mass production; the hoverflies are reared with sugar, pollen, and water; and the oviposition is induced by the introduction of infested host plants in the rearing cage during 3 h.
  • the complete life cycle takes place on the host plants that are daily re-infested with aphids.
  • Wells of a 96-well plate are filled with 200 ⁇ l of artificial aphid diet (Febvay et al., Canadian Journal of Zoology 66(11):2449-2453, 1988) and the plate is covered with parafilm to make a feeding sachet.
  • Artificial diet is either mixed with the solution of sterile water with 0.5% sucrose and essential amino acids as a negative control or ColA solutions with varying concentrations of ColA.
  • ColA solutions are mixed with artificial diet to obtain final concentrations between 0.6 to 100 mg/ml.
  • aphids For each replicate treatment, 30-50 second and third instar aphids are placed individually in wells of a 96-well plate and a feeding sachet plate is inverted above them, allowing the insects to feed through the parafilm and keeping them restricted to individual wells. Experimental aphids are kept under the same environmental conditions as aphid colonies. After the aphids are fed for 24 hr, the feeding sachet is replaced with a new one containing sterile artificial diet and a new sterile sachet is provided every 24 h for 4 days. At the time that the sachet is replaced, aphids are also checked for mortality.
  • An aphid is counted as dead if it had turned brown or is at the bottom of the well and does not move during the observation. If an aphid is on the parafilm of the feeding sachet but not moving, it is assumed to be feeding and alive.
  • the status of Buchnera in aphid samples is assessed by PCR. Aphids adults from the negative control and phage treated are first surface-sterilized with 70% ethanol for 1 min, 10% bleach for 1 min and three washes of ultrapure water for 1 min. Total DNA is extracted from each individual (whole body) using an Insect DNA Kit (OMEGA, Bio-tek) according to the manufacturer's protocol.
  • OEGA Insect DNA Kit
  • the primers for Buchnera forward primer 5′-GTCGGCTCATCACATCC-3′ (SEQ ID NO: 233) and reverse primer 5′-TTCCGTCTGTATTATCTCCT-3′ (SEQ ID NO: 234), are designed based on 23S-5S rRNA sequences obtained from the Buchnera genome (Accession Number: GCA_000009605.1) (Shigenobu, et al., Nature 200.407, 81-86) using Primer 5.0 software (Primer-E Ltd., Madison, UK).
  • the PCR amplification cycles included an initial denaturation step at 95° C. for 5 min, 35 cycles of 95° C. for 30 s, 55° C. for 30 s, and 72° C.
  • Amplification products from rifampicin treated and control samples are analyzed on 1% agarose gels, stained with SYBR safe, and visualized using an imaging System. ColA treated aphids show a reduction of Buchnera specific genes.
  • the survival rates of aphids treated with Buchnera specific ColA bacteriocin are compared to the aphids treated with the negative control.
  • the survival rate of aphids treated with Buchnera specific ColA bacteriocin is decreased as compared to the control treated aphids.
  • This Example demonstrates the ability to kill or decrease the fitness of aphids by treating them with rifampicin, a narrow spectrum antibiotic that inhibits DNA-dependent RNA synthesis by inhibiting a bacterial RNA polymerase.
  • This Example demonstrates that the effect of rifampicin on aphids is mediated through the modulation of bacterial populations endogenous to the aphid that are sensitive to rifampicin.
  • One targeted bacterial strain is Buchnera.
  • the antibiotic solutions are formulated with 0 (negative control), 1, 10, or 50 ⁇ g/ml of rifampicin in 10 mL of sterile water with 0.5% sucrose and essential amino acids.
  • aphids are grown in a lab environment and medium.
  • fava bean plants are grown in a mixture of vermiculite and perlite at 24° C. with 16 h of light and 8 h of darkness.
  • 5-10 adults from different plants are distributed among 10 two-week-old plants, and allowed to multiply to high density for 5-7 days.
  • second and third instar aphids are collected from healthy plants and divided into treatments so that each treatment receives approximately the same number of individuals from each of the collection plants.
  • Rifampicin solutions are made by dissolving rifampicin (SIGMA-ALDRICH, 557303) in sterile water with 0.5% sucrose and essential aminoacids.
  • Wells of a 96-well plate are filled with 200 ⁇ l of artificial aphid diet (Febvay et al., Canadian Journal of Zoology 66(11):2449-2453, 1988) and the plate is covered with parafilm to make a feeding sachet.
  • Artificial diet is either mixed with sterile water and with 0.5% sucrose and essential aminoacids as a negative control or a rifampicin solution with one of the concentrations of rifampicin.
  • Rifampicin solutions are mixed with artificial diet to get final concentrations of the antibiotic between 1 and 50 ⁇ g/mL.
  • aphids For each replicate treatment, 30-50 second and third instar aphids are placed individually in wells of a 96-well plate and a feeding sachet plate is inverted above them, allowing the insects to feed through the parafilm and keeping them restricted to individual wells. Experimental aphids are kept under the same environmental conditions as aphid colonies. After the aphids are fed for 24 hr, the feeding sachet is replaced with a new one containing sterile artificial diet and a new sterile sachet is provided every 24 h for four days. At the time that the sachet is replaced, aphids are also checked for mortality.
  • An aphid is counted as dead if it had turned brown or is at the bottom of the well and does not move during the observation. If an aphid is on the parafilm of the feeding sachet but not moving, it is assumed to be feeding and alive.
  • the primers for Buchnera forward primer 5′-GTCGGCTCATCACATCC-3′ (SEQ ID NO: 233) and reverse primer 5′-TTCCGTCTGTATTATCTCCT-3′ (SEQ ID NO: 234), are designed based on 23S-5S rRNA sequences obtained from the Buchnera genome (Accession Number: GCA_000009605.1) (Shigenobu et al., Nature 407:81-86, 2000) using Primer 5.0 software (Primer-E Ltd., Madison, UK).
  • the PCR amplification cycles included an initial denaturation step at 95° C. for 5 min, 35 cycles of 95° C. for 30 s, 55° C. for 30 s, and 72° C.
  • the survival rates of aphids treated with rifampicin solution are compared to the aphids treated with the negative control.
  • the survival rate of aphids treated with rifampicin solution is decreased compared to the control.
  • a HTS to identify inhibitors of targeted bacterial strains uses sucrose fermentation in pH-MMSuc medium (Ymele-Leki et al., PLoS ONE 7(2):e31307, 2012) to decrease the pH of the medium. pH indicators in the medium monitor medium acidification spectrophotometrically through a change in absorbance at 615 nm (A615).
  • a target bacterial strain, Buchnera derived from a glycerol stock, is plated on an LB-agar plate and incubated overnight at 37° C. A loopful of cells is harvested, washed three times with PBS, and then resuspended in PBS at an optical density of 0.015.
  • This step is automated and validated in the 384-well plate format using an EnVisionTM multi-well spectrophotometer to test all fractions from the library. Fractions that demonstrate delayed medium acidification by sucrose fermentation and inhibited cell growth are selected for further purification and identification.
  • Example 11 demonstrates the isolation and identification of an isolate from the fraction described in Example 11 that blocks sucrose fermentation and inhibits cell growth of Buchnera.
  • Fraction II is separated on an Agilent 1100 series HPLC with a preparative Phenyl-hexyl column (Phenomenex, Luna, 25 cm610 mm, 5 mm particle size) using an elution buffer with 20% acetonitrile/water with 0.1% formic acid at a flow rate of 2 mL/min for 50 minutes. This yields multiple compounds at different elution times.
  • Spectra for each compound is obtained on an Alpha FT-IR mass spectrometer (Bruker), an UltrospecTM 5300 pro UV/Visible Spectrophotometer (Amersham Biosciences), and an INOVA 600 MHz nuclear magnetic resonance spectrometer (Varian).
  • Example 13 Treatment of Aphids with a Solution of a Buchnera Specific Molecule
  • This Example demonstrates the ability to kill or decrease the fitness of aphids by treating them with one of the compounds identified in Example 12 through the modulation of bacterial populations endogenous to the aphid that are sensitive to this compound.
  • One targeted bacterial strain is Buchnera.
  • Each compound from Example 12 is formulated at 0 (negative control), 0.6, 1, 20 or 80 ⁇ g/ml in 10 mL of sterile water with 0.5% sucrose and essential amino acids.
  • aphids are grown in a lab environment and medium.
  • fava bean plants are grown in a mixture of vermiculite and perlite at 24° C. with 16 h of light and 8 h of darkness.
  • 5-10 adults from different plants are distributed among 10 two-week-old plants, and allowed to multiply to high density for 5-7 days.
  • second and third instar aphids are collected from healthy plants and divided into treatments so that each treatment receives approximately the same number of individuals from each of the collection plants.
  • Wells of a 96-well plate are filled with 200 ⁇ l of artificial aphid diet (Febvay et al., Canadian Journal of Zoology 66(11):2449-2453, 1988) and the plate is covered with parafilm to make a feeding sachet.
  • Artificial diet is either mixed with sterile water with 0.5% sucrose and essential amino acids as a negative control or solutions with varying concentrations of the compound.
  • aphids For each replicate treatment, 30-50 second and third instar aphids are placed individually in wells of a 96-well plate and a feeding sachet plate is inverted above them, allowing the insects to feed through the parafilm and keeping them restricted to individual wells. Experimental aphids are kept under the same environmental conditions as aphid colonies. After the aphids are fed for 24 hr, the feeding sachet is replaced with a new one containing sterile artificial diet and a new sterile sachet is provided every 24 h for 4 days. At the time that the sachet is replaced, aphids are also checked for mortality.
  • An aphid is counted as dead if it had turned brown or is at the bottom of the well and does not move during the observation. If an aphid is on the parafilm of the feeding sachet but not moving, it is assumed to be feeding and alive.
  • the primers for Buchnera forward primer 5′-GTCGGCTCATCACATCC-3′ (SEQ ID NO: 233) and reverse primer 5′-TTCCGTCTGTATTATCTCCT-3′ (SEQ ID NO: 234), are designed based on 23S-5S rRNA sequences obtained from the Buchnera genome (Accession Number: GCA_000009605.1) (Shigenobu et al., Nature 407:81-86, 2000) using Primer 5.0 software (Primer-E Ltd., Madison, UK).
  • the PCR amplification cycles included an initial denaturation step at 95° C. for 5 min, 35 cycles of 95° C. for 30 s, 55° C. for 30 s, and 72° C.
  • Amplification products from compound 1 treated and control samples are analyzed on 1% agarose gels, stained with SYBR safe, and visualized using an imaging System. Reduction of Buchnera specific genes indicates antimicrobial activity of compound 1.
  • the survival rate of aphids treated with the compound is compared to the aphids treated with the negative control. A decrease in the survival rate of aphids treated with the compound is expected to indicate antimicrobial activity of the compound.
  • This Example demonstrates the treatment of aphids with rifampicin, a narrow spectrum antibiotic that inhibits DNA-dependent RNA synthesis by inhibiting a bacterial RNA polymerase.
  • This Example demonstrates that the effect of rifampicin on a model insect species, aphids, was mediated through the modulation of bacterial populations endogenous to the insect that were sensitive to rifampicin.
  • One targeted bacterial strain is Buchnera.
  • the antibiotic solution was formulated according to the means of delivery, as follows ( FIG. 1A-1G ):
  • Leaf coating 100 ⁇ l of 0.025% nonionic organosilicone surfactant solvent Silwet L-77 in water (negative control) or 100 ⁇ l of 50 ⁇ g/ml of rifampicin formulated in solvent solution was applied directly to the leaf surface and allowed to dry.
  • injection solutions were either 0.025% nonionic organosilicone surfactant solvent Silwet L-77 in water (negative control), or 50 ⁇ g/ml of rifampicin formulated in solvent solution.
  • Topical delivery 100 ⁇ l of 0.025% nonionic organosilicone surfactant solvent Silwet L-77 (negative control), or 50 ⁇ g/ml of rifampicin formulated in solvent solution were sprayed using a 30 mL spray bottle.
  • Leaf injection method A Leaf perfusion and cutting: leaves were injected with approximately 1 ml of 50 ⁇ g/ml of rifampicin in water with food coloring or approximately 1 ml of negative control with water and food coloring. Leaves were cut into 2 ⁇ 2 cm squared pieces and aphids were placed on the leaf pieces.
  • Leaf perfusion and delivery through plant Leaves were injected with approximately 1 ml of 100 ⁇ g/ml of rifampicin in water plus food coloring or approximately 1 ml of negative control with water and food coloring. The stem of injected leaf was then placed into an Eppendorf tube with 1 ml of 100 ⁇ g/ml of rifampicin plus water and food coloring or 1 ml of negative control with only water and food coloring.
  • Combination delivery method a) Topical delivery to aphid and plant: via spraying both aphids and plants with 0.025% nonionic organosilicone surfactant solvent Silwet L-77 in water (negative control) or 100 ⁇ g/ml of rifampicin formulated in solvent solution using a 30 mL, b) Delivery through plant: water only (negative control) or 100 ⁇ g/ml of rifampicin formulated in water.
  • Aphids (LSR-1 strain, Acyrthosiphon pisum ) were grown on fava bean plants ( Vroma Vicia faba from Johnny's Selected Seeds) in a climate-controlled incubator (16 h light/8 h dark photoperiod; 60 ⁇ 5% RH; 25 ⁇ 2° C.). Prior to being used for aphid rearing, fava bean plants were grown in potting soil at 24° C. with 16 h of light and 8 h of darkness. To limit maternal effects or health differences between plants, 5-10 adults from different plants were distributed among 10 two-week-old plants, and allowed to multiply to high density for 5-7 days.
  • first instar aphids were collected from healthy plants and divided into 3 different treatment groups: 1) artificial diet alone without essential amino acids, 2) artificial diet alone without essential amino acids and 100 ⁇ g/ml rifampicin, and 3) artificial diet with essential amino acids and 100 ⁇ g/ml rifampicin).
  • Each treatment group received approximately the same number of individuals from each of the collection plants.
  • the artificial diet used was made as previously published (Akey and Beck, 1971 Continuous Rearing of the Pea Aphid, Acyrthosiphon pisum , on a Holidic Diet), with and without the essential amino acids (2 mg/ml histidine, 2 mg/ml isoleucine, 2 mg/ml leucine, 2 mg/ml lysine, 1 mg/ml methionine, 1.62 mg/ml phenylalanine, 2 mg/ml threonine, 1 mg/ml tryptophan, and 2 mg/ml valine), except neither diet included homoserine or beta-alanyltyrosine.
  • the pH of the diets was adjusted to 7.5 with KOH and diets were filter sterilized through a 0.22 ⁇ m filter and stored at 4° C. for short term ( ⁇ 7 days) or at ⁇ 80° C. for long term.
  • Rifampicin (Tokyo Chemical Industry, LTD) stock solutions were made at 25 mg/ml in methanol, sterilized by passing through a 0.22 ⁇ m syringe filter, and stored at ⁇ 20° C.
  • the appropriate amount of stock solution was added to the artificial diet with or without essential amino acids to obtain a final concentration of 100 ⁇ g/ml rifampicin.
  • the diet was then placed into a 1.5 ml Eppendorf tube with a fava bean stem with a leaf and the opening of the tube was closed using parafilm.
  • This artificial diet feeding system was then placed into a deep petri dish (Fisher Scientific, Cat# FB0875711) and aphids were applied to the leaves of the plant.
  • the developmental stage (1 st , 2 nd , 3 rd , 4 th , 5 th instar) was determined daily throughout the experiment. Once an aphid reached the 4th instar stage, they were given their own artificial feeding system in a deep petri dish so that fecundity could be monitored once they reached adulthood.
  • a DNA extraction kit Qiagen, DNeasy kit
  • the primers used for Buchnera were Buch_groES_18F (CATGATCGTGTGCTTGTTAAG; SEQ ID NO: 238) and Buch_groES_98R (CTGTTCCTCGAGTCGATTTCC; SEQ ID NO: 239) (Chong and Moran, 2016 PNAS).
  • the primers used for aphid were ApEF1a 107F (CTGATTGTGCCGTGCTTATTG; SEQ ID NO: 240) and ApEF1a 246R (TATGGTGGTTCAGTAGAGTCC; SEQ ID NO: 241) (Chong and Moran, 2016 PNAS).
  • qPCR was performed using a qPCR amplification ramp of 1.6 degrees C./s and the following conditions: 1) 95° C.
  • LSR-1 1 st instar aphids were divided into three separate treatment groups as defined in Experimental Design (above). Aphids were monitored daily and the number of aphids at each developmental stage was determined. Aphids treated with artificial diet alone without essential amino acids began reaching maturity (5th instar stage) at approximately 6 days ( FIG. 2A ). Development was delayed in aphids treated with rifampicin, and by 6 days of treatment, most aphids did not mature further than the 3 rd instar stage, even after 12 days and their size is drastically affected ( FIGS. 2A-2C ).
  • aphids treated with artificial diet with rifampicin supplemented with essential amino acids developed faster and to higher instar stages as compared to aphids treated with rifampicin alone, but not as quickly as aphids treated with artificial diet without essential amino acids ( FIGS. 2A-2C ).
  • FIGS. 2A-2C These data indicate that treatment with rifampicin impaired aphid development. Adding back essential amino acids partially rescued this defect in development.
  • aphids treated with rifampicin without essential amino acids had lower survival rates than aphids treated with artificial diet alone (p ⁇ 0.00001).
  • Rifampicin stock solution was added to 0.025% of a nonionic organosilicone surfactant solvent, Silwet L-77, to obtain a final concentration of 50 ⁇ g/ml rifampicin.
  • Aphids eNASCO strain, Acyrthosiphon pisum
  • first instar aphids were collected from healthy plants and divided into 2 different treatment groups: leaves were sprayed with 1) negative control (solvent solution only), 2) 50 ⁇ g/ml rifampicin in solvent. Solutions were absorbed onto a 2 ⁇ 2 cm piece of fava bean leaf.
  • Each treatment group received approximately the same number of individuals from each of the collection plant. For each treatment, 20 aphids were placed onto each leaf. Aphids were monitored daily for survival and dead aphids were removed when they were discovered. In addition, the developmental stage (1 st , 2 nd , 3 rd , 4 th , 5 th instar, and 5R, representing a reproducing 5th instar) was determined daily throughout the experiment. Pictures of aphids were taken throughout the experiment to evaluate size differences between treatment groups.
  • LSR-1 1 st instar aphids were divided into two separate treatment groups as defined in the Experimental Design described herein. Aphids were monitored daily and the number of aphids at each developmental stage was determined. Aphids placed on coated leaves treated with control began reaching maturity (5 th instar reproducing stage; 5R) at approximately 6 days ( FIG. 6A ). Development was delayed in aphids placed on coated leaves with rifampicin, and by 6 days of treatment, most aphids did not mature further than the 3 rd instar stage, even after 12 days, and their size is drastically affected ( FIGS. 6A and 6B ).
  • Microinjection was performed using NanoJet III Auto-Nanoliter Injector with the in-house pulled borosilicate needle (Drummond Scientific; Cat#3-000-203-G/XL).
  • Aphids eNASCO strain, Acyrthosiphon pisum ) were grown on fava bean plants as described in a previous Example. Aphids are transferred using a paint brush to a tubing system connected to vacuum ( FIG. 1C ). The injection site was at the ventral thorax of the aphid.
  • the injection solutions were either the organosilicone surfactant solvent 0.025% Silwet L-77 (Lehle Seeds, Cat No VIS-01) in water (negative control), or 50 ⁇ g/ml of rifampicin formulated in solvent solution.
  • the injection volume was 10 nl for nymph and 20 nl for adult (both at a rate of 2 nl/sec).
  • Each treatment group had approximately the same number of individuals injected from each of the collection plants. After injection, aphids were released into a petri dish with fava bean leaves, whose stems are in 2% agar.
  • Leaf Injection Method A Leaf Perfusion and Cutting
  • Aphids LSR-1 (which harbor only Buchnera ), Acyrthosiphon pisum were grown on fava bean plants ( Vroma Vicia faba from Johnny's Selected Seeds) in a climate-controlled incubator (16 h light/8 h dark photoperiod; 60 ⁇ 5% RH; 25 ⁇ 2° C.). Prior to being used for aphid rearing, fava bean plants were grown in potting soil at 24° C. with 16 h of light and 8 h of darkness. To limit maternal effects or health differences between plants, 5-10 adults from different plants were distributed among 10 two-week-old plants, and allowed to multiply to high density for 5-7 days.
  • first and second instar aphids were collected from healthy plants and divided into 2 different treatment groups: 1) negative control (leaf injected with water plus blue food coloring) and 2) leaf injected with 50 ⁇ g/ml rifampicin in water plus blue food coloring.
  • Each treatment group received approximately the same number of individuals from each of the collection plants.
  • rifampicin stock solution 25 mg/ml in 100% methanol
  • the solution was then placed into a 1.5 ml Eppendorf tube with a fava bean stem perfused with the solutions and the opening of the tube was closed using parafilm.
  • This feeding system was then placed into a deep petri dish (Fisher Scientific, Cat# FB0875711) and aphids were applied to the leaves of the plant. For each treatment, 74-81 aphids were placed onto each leaf. The feeding systems were changed every 2-3 days throughout the experiment. Aphids were monitored daily for survival and dead aphids were removed from the deep petri dish when they were discovered. In addition, the developmental stage (1 st , 2 nd , 3 rd , 4 th , 5 th and 5R (5 th that has reproduced) instar) was determined daily throughout the experiment.
  • LSR-1 1st and 2nd instar aphids were divided into two separate treatment groups as defined in Leaf injection method A—Leaf perfusion and cutting Experimental Design (described herein). Aphids were monitored daily and the number of aphids at each developmental stage was determined. Aphids treated with water plus food coloring began reaching maturity (5th instar stage) at approximately 6 days ( FIG. 11 ). Development was delayed in aphids feeding on rifampicin injected leaves, and by 6 days of treatment, most aphids did not mature further than the 4th instar stage. Even after 8 days, the development of aphids feeding on rifampicin injected leaves was drastically delayed ( FIG. 11 ). These data indicate that rifampicin treatment via leaf perfusion impaired aphid development.
  • Aphids LSR-1 (which harbor only Buchnera ), Acyrthosiphon pisum were grown on fava bean plants ( Vroma Vicia faba from Johnny's Selected Seeds) in a climate-controlled incubator (16 h light/8 h dark photoperiod; 60 ⁇ 5% RH; 25 ⁇ 2° C.). Prior to being used for aphid rearing, fava bean plants were grown in potting soil at 24° C. with 16 h of light and 8 h of darkness.
  • first and second instar aphids were collected from healthy plants and divided into 2 different treatment groups: 1) aphids placed on leaves injected with the negative control solution (water and food coloring) and placed into an Eppendorf tube with the negative control solution, or 2) aphids placed on leaves that were injected with 100 ug/ml rifampicin in water plus food coloring and put into an Eppendorf tube with 100 ug/ml rifampicin in water.
  • Each treatment group received approximately the same number of individuals from each of the collection plants.
  • rifampicin stock solution 25 mg/ml in 100% methanol
  • rifampicin stock solution 25 mg/ml in 100% methanol
  • the solution was then placed into a 1.5 ml Eppendorf tube with a fava bean stem with a leaf also perfused with the solutions and the opening of the tube was closed using parafilm.
  • This feeding system was then placed into a deep petri dish (Fisher Scientific, Cat# FB0875711) and aphids were applied to the leaves of the plant.
  • LSR-1 1 st and 2 nd instar aphids were divided into two separate treatment groups as defined in Leaf perfusion and delivery through plant Experimental Design (described herein). Aphids were monitored daily and the number of aphids at each developmental stage was determined. Aphids treated with the control solution (water plus food coloring only) began reaching maturity (5 th instar stage) at approximately 6 days ( FIG. 14 ).
  • Aphids LSR-1 (which harbor only Buchnera ), Acyrthosiphon pisum were grown on fava bean plants ( Vroma Vicia faba from Johnny's Selected Seeds) in a climate-controlled incubator (16 h light/8 h dark photoperiod; 60 ⁇ 5% RH; 20 ⁇ 2° C.). Prior to being used for aphid rearing, fava bean plants were grown in potting soil at 24° C. with 16 h of light and 8 h of darkness. To limit maternal effects or health differences between plants, 5-10 adults from different plants were distributed among 10 two-week-old plants, and allowed to multiply to high density for 5-7 days.
  • first and second instar aphids were collected from healthy plants and divided into 2 different treatment groups: 1) treatment with Silwet-L77 or water control solutions or 2) treatment with rifampicin diluted in silwet L-77 or water.
  • Each treatment group received approximately the same number of individuals from each of the collection plants.
  • the combination of delivery methods was as follows: a) Topical delivery to aphid and plant by spraying 0.025% nonionic organosilicone surfactant solvent Silwet L-77 (negative control) or 100 ⁇ g/ml of rifampicin formulated in solvent solution using a 30 mL spray bottle and b) Delivery through plant with either water (negative control) or 100 ⁇ g/ml of rifampicin formulated in water.
  • rifampicin stock solution 25 mg/ml in 100% methanol
  • Silwet L-77 for topical treatment to aphid and coating the leaf
  • water for delivery through plant.
  • the solution was then placed into a 1.5 ml Eppendorf tube with a fava bean stem with a leaf also perfused with the solutions and the opening of the tube was closed using parafilm.
  • This feeding system was then placed into a deep petri dish (Fisher Scientific, Cat# FB0875711) and aphids were applied to the leaves of the plant.
  • aphids were placed onto each leaf.
  • the feeding systems were changed every 2-3 days throughout the experiment. Aphids were monitored daily for survival and dead aphids were removed from the deep petri dish when they were discovered.
  • LSR-1 1 st and 2 nd instar aphids were divided into two separate treatment groups as defined in Combination delivery method Experimental Design (described herein). Aphids were monitored daily and the number of aphids at each developmental stage was determined. Control treated aphids began reaching maturity (5 th instar stage) at approximately 6 days ( FIG. 17 ). Development was delayed in aphids treated with rifampicin, and by 6 days of treatment, most aphids did not mature further than the 3 rd instar stage, even after 7 days their development was drastically delayed ( FIG. 17 ). These data indicate that a combination of rifampicin treatments impaired aphid development.
  • Example 15 Insects Treated with a Natural Antimicrobial Polysacharide
  • This Example demonstrates the treatment of aphids with Chitosan, a natural cationic linear polysaccharide of deacetylated beta-1,4-D-glucosamine derived from chitin.
  • Chitin is the structural element in the exoskeleton of insects, crustaceans (mainly shrimp and crabs) and cell walls of fungi, and the second most abundant natural polysaccharide after cellulose.
  • This Example demonstrates that the effect of chitosan on insects was mediated through the modulation of bacterial populations endogenous to the insect that were sensitive to chitosan.
  • One targeted bacterial strain is Buchnera aphidicola.
  • the chitosan solution was formulated using a combination of leaf perfusion and delivery through plants ( FIG. 20 ).
  • the control solution was leaf injected with water+blue food coloring and water in tube.
  • the treatment solution with 300 ug/ml chitosan in water (low molecular weight) via leaf injection (with blue food coloring) and through plant (in Eppendorf tube).
  • Aphids LSR-1 (which harbor only Buchnera ), Acyrthosiphon pisum were grown on fava bean plants ( Vroma Vicia faba from Johnny's Selected Seeds) in a climate-controlled incubator (16 h light/8 h dark photoperiod; 60 ⁇ 5% RH; 25 ⁇ 2° C.). Prior to being used for aphid rearing, fava bean plants were grown in potting soil at 24° C. with 16 h of light and 8 h of darkness. To limit maternal effects or health differences between plants, 5-10 adults from different plants were distributed among 10 two-week-old plants, and allowed to multiply to high density for 5-7 days.
  • first and second instar aphids were collected from healthy plants and divided into 2 different treatment groups: 1) negative control (water treated), 2) The treatment solution included 300 ug/ml chitosan in water (low molecular weight). Each treatment group received approximately the same number of individuals from each of the collection plants.
  • Chitosan (Sigma, catalog number 448869-50G) stock solution was made at 1% in acetic acid, sterilized autoclaving, and stored at 4° C.
  • the appropriate amount of stock solution was diluted with water to obtain the final treatment concentration of chitosan.
  • the solution was then placed into a 1.5 ml Eppendorf tube with a fava bean stem with a leaf also perfused with the solutions and the opening of the tube was closed using parafilm. This feeding system was then placed into a deep petri dish (Fisher Scientific, Cat# FB0875711) and aphids were applied to the leaves of the plant.
  • aphids were placed onto each leaf.
  • the feeding systems were changed every 2-3 days throughout the experiment. Aphids were monitored daily for survival and dead aphids were removed from the deep petri dish when they were discovered.
  • a DNA extraction kit Qiagen, DNeasy kit
  • the primers used for Buchnera were Buch_groES_18F (CATGATCGTGTGCTTGTTAAG; SEQ ID NO: 238) and Buch_groES_98R (CTGTTCCTCGAGTCGATTTCC; SEQ ID NO: 239) (Chong and Moran, 2016 PNAS).
  • the primers used for aphid were ApEF1a 107F (CTGATTGTGCCGTGCTTATTG; SEQ ID NO: 240) and ApEF1a 246R (TATGGTGGTTCAGTAGAGTCC; SEQ ID NO: 241) (Chong and Moran, 2016 PNAS).
  • qPCR was performed using a qPCR amplification ramp of 1.6 degrees C./s and the following conditions: 1) 95° C.
  • LSR-1 A pisum 1 st and 2 nd instar aphids were divided into two separate treatment groups as defined in Experimental Design (above). Aphids were monitored daily and the number of aphids at each developmental stage was determined. Aphids treated with the negative control alone began reaching maturity (5th instar stage) at approximately 6 days ( FIG. 21 ). Development was delayed in aphids treated with chitosan solution, and by 6 days of treatment with chitosan, most aphids did not mature further than the 4 rd instar stage. These data indicate that treatment with chitosan delayed and stopped progression of aphid development.
  • Example 16 Insects Treated with Nisin, a Natural Antimicrobial Peptide
  • This Example demonstrates the treatment of aphids with the natural, “broad spectrum,” polycyclic antibacterial peptide produced by the bacterium Lactococcus lactis that is commonly used as a food preservative.
  • the antibacterial activity of nisin is mediated through its ability to generate pores in the bacterial cell membrane and interrupt bacterial cell-wall biosynthesis through a specific lipid II interaction.
  • This Example demonstrates that the negative effect of nisin on insect fitness is mediated through the modulation of bacterial populations endogenous to the insect that were sensitive to nisin.
  • One targeted bacterial strain is Buchnera aphidicola.
  • Nisin was formulated using a combination of leaf perfusion and delivery through plants.
  • the control solution (water) or treatment solution (nisin) was injected into the leaf and placed in the Eppendorf tube.
  • the treatment solutions consisted of 1.6 or 7 mg/ml nisin in water.
  • LSR-1 aphids Acyrthosiphon pisum were grown on fava bean plants (Vroma Vicia faba from Johnny's Selected Seeds) in a climate-controlled incubator (16 h light/8 h dark photoperiod; 60 ⁇ 5% RH; 25 ⁇ 2° C.). Prior to being used for aphid rearing, fava bean plants were grown in potting soil at 24° C. with 16 h of light and 8 h of darkness. To limit maternal effects or health differences between plants, 5-10 adults from different plants were distributed among 10 two-week-old plants, and allowed to multiply to high density for 5-7 days.
  • first and second instar aphids were collected from healthy plants and divided into 2 different treatment groups: 1) negative control (water treated), 2) nisin treated with either 1.6 or 7 mg/ml nisin in water. Each treatment group received approximately the same number of individuals from each of the collection plants.
  • nisin (Sigma, product number: N5764) solution was made at 1.6 or 7 mg/ml (w/v) in water and filter sterilized using a 0.22 um syringe filter. The solution was then injected into the leaf of the plant and the stem of the plant was placed into a 1.5 ml Eppendorf tube. The opening of the tube was closed using parafilm. This feeding system was then placed into a deep petri dish (Fisher Scientific, Cat# FB0875711) and aphids were applied to the leaves of the plant.
  • aphids were placed onto each leaf.
  • the feeding systems were changed every 2-3 days throughout the experiment. Aphids were monitored daily for survival and dead aphids were removed from the deep petri dish when they were discovered.
  • a DNA extraction kit Qiagen, DNeasy kit
  • the primers used for Buchnera were Buch_groES_18F (CATGATCGTGTGCTTGTTAAG; SEQ ID NO: 238) and Buch_groES_98R (CTGTTCCTCGAGTCGATTTCC; SEQ ID NO: 239) (Chong and Moran, 2016 PNAS).
  • the primers used for aphid were ApEF1a 107F (CTGATTGTGCCGTGCTTATTG; SEQ ID NO: 240) and ApEF1a 246R (TATGGTGGTTCAGTAGAGTCC; SEQ ID NO: 241) (Chong and Moran, 2016 PNAS).
  • qPCR was performed using a qPCR amplification ramp of 1.6 degrees C./s and the following conditions: 1) 95° C.
  • LSR-1 A pisum 1st and 2nd instar aphids were divided into three separate treatment groups as defined in Experimental Design (above). Aphids were monitored daily and the number of aphids at each developmental stage was determined. Aphids treated with the negative control solution (water) began reaching maturity (5th instar stage) at approximately 6 days, and reproducing (5R stage) by 7 days ( FIG. 24 ). Development was severely delayed in aphids treated with 7 mg/ml nisin. Aphids treated with 7 mg/ml nisin only developed to the 2nd instar stage by day 3, and by day 6, all aphids in the group were dead ( FIG. 24 ).
  • Example 17 Insects Treated with Levulinic Acid Decreases Insect Fitness
  • This Example demonstrates the treatment of aphids with levulinic acid, a keto acid produced by heating a carbohydrate with hexose (e.g., wood, starch, wheat, straw, or cane sugar) with the addition of a dilute mineral acid reduces insect fitness.
  • Levulinic acid has previously been tested as an antimicrobial agent against Escherichia coli and Salmonella in meat production (Carpenter et al., 2010, Meat Science; Savannah G. Hawkins, 2014, University of Tennessee Honors Thesis).
  • This Example demonstrates that the effect of levulinic acid on insects was mediated through the modulation of bacterial populations endogenous to the insect that were sensitive to levulinic acid.
  • One targeted bacterial strain is Buchnera aphidicola.
  • the levulinic acid was formulated using a combination of leaf perfusion and delivery through plants.
  • the control solution was leaf injected with water and water was placed in the Eppendorf tube.
  • the treatment solutions included 0.03 or 0.3% levulinic acid in water via leaf injection and through plant (in Eppendorf tube).
  • eNASCO aphids Acyrthosiphon pisum were grown on fava bean plants ( Vroma Vicia faba from Johnny's Selected Seeds) in a climate-controlled incubator (16 h light/8 h dark photoperiod; 60 ⁇ 5% RH; 25 ⁇ 2° C.). Prior to being used for aphid rearing, fava bean plants were grown in potting soil at 24° C. with 16 h of light and 8 h of darkness. To limit maternal effects or health differences between plants, 5-10 adults from different plants were distributed among 10 two-week-old plants, and allowed to multiply to high density for 5-7 days.
  • first and second instar aphids were collected from healthy plants and divided into 2 different treatment groups: 1) negative control (water treated), 2) The treatment solution included 0.03 or 0.3% levulinic acid in water. Each treatment group received approximately the same number of individuals from each of the collection plants.
  • levulinic acid (Sigma, product number: W262706) was diluted to either 0.03 or 0.3% in water. The solution was then placed into a 1.5 ml Eppendorf tube with a fava bean stem with a leaf also perfused with the solutions and the opening of the tube was closed using parafilm. This feeding system was then placed into a deep petri dish (Fisher Scientific, Cat# FB0875711) and aphids were applied to the leaves of the plant.
  • 57-59 aphids were placed onto each leaf.
  • the feeding systems were changed every 2-3 days throughout the experiment. Aphids were monitored daily for survival and dead aphids were removed from the deep petri dish when they were discovered.
  • a DNA extraction kit Qiagen, DNeasy kit
  • the primers used for Buchnera were Buch_groES_18F (CATGATCGTGTGCTTGTTAAG; SEQ ID NO: 238) and Buch_groES_98R (CTGTTCCTCGAGTCGATTTCC; SEQ ID NO: 239) (Chong and Moran, 2016 PNAS).
  • the primers used for aphid were ApEF1a 107F (CTGATTGTGCCGTGCTTATTG; SEQ ID NO: 240) and ApEF1a 246R (TATGGTGGTTCAGTAGAGTCC; SEQ ID NO: 241) (Chong and Moran, 2016 PNAS).
  • qPCR was performed using a qPCR amplification ramp of 1.6 degrees C./s and the following conditions: 1) 95° C.
  • eNASCO A pisum 1 st and 2 nd instar aphids were divided into three separate treatment groups as defined in Experimental Design (above). Aphids were monitored daily and the number of aphids at each developmental stage was determined. Aphids treated with the negative control alone began reaching maturity (5 th instar stage) at approximately 7 days ( FIG. 27 ). Development was delayed in aphids treated with levulinic acid and by 11 days post-treatment, nearly all control treated aphids reached maturity while ⁇ 23 and 63% aphids treated with 0.03 and 0.3% levulinic acid, respectively, did not mature further than the 4 rd instar stage. These data indicate that treatment with levulinic acid delayed and stopped progression of aphid development and this delay in development is dependent on the dose of levulinic acid administered.
  • Example 18 Insects Treated with a Plant Derived Secondary Metabolite Solution
  • This Example demonstrates the treatment of aphids with gossypol acetic acid, a natural phenol derived from the cotton plant (genus Gossypium ) that permeates cells and acts as an inhibitor for several dehydrogenase enzymes.
  • gossypol acetic acid a natural phenol derived from the cotton plant (genus Gossypium ) that permeates cells and acts as an inhibitor for several dehydrogenase enzymes.
  • This Example demonstrates that the effect of gossypol on insects was mediated through the modulation of bacterial populations endogenous to the insect that were sensitive to gossypol.
  • One targeted bacterial strain is Buchnera aphidicola.
  • the gossypol solution was formulated depending on the delivery method:
  • injection solutions were either 0.5% of gossypol or artificial diet only (negative control).
  • Aphids either eNASCO (which harbor both Buchnera and Serratia primary and secondary symbionts, respectively) or LSR-1 (which harbor only Buchnera ) strains, Acyrthosiphon pisum ) were grown on fava bean plants ( Vroma Vicia faba from Johnny's Selected Seeds) in a climate-controlled incubator (16 h light/8 h dark photoperiod; 60 ⁇ 5% RH; 25 ⁇ 2° C.). Prior to being used for aphid rearing, fava bean plants were grown in potting soil at 24° C. with 16 h of light and 8 h of darkness.
  • first and second instar aphids were collected from healthy plants and divided into 4 different treatment groups: 1) artificial diet alone without essential amino acids, 2) artificial diet alone without essential amino acids and 0.05% of gossypol, 3) artificial diet alone without essential amino acids and 0.25% of gossypol, and 4) artificial diet alone without essential amino acids and 0.5% of gossypol.
  • Each treatment group received approximately the same number of individuals from each of the collection plants.
  • the artificial diet used was made as previously published (Akey and Beck, 1971 Continuous Rearing of the Pea Aphid, Acyrthosiphon pisum , on a Holidic Diet), with and without the essential amino acids (2 mg/ml histidine, 2 mg/ml isoleucine, 2 mg/ml leucine, 2 mg/ml lysine, 1 mg/ml methionine, 1.62 mg/ml phenylalanine, 2 mg/ml threonine, 1 mg/ml tryptophan, and 2 mg/ml valine), except neither diet included homoserine or beta-alanyltyrosine.
  • the pH of the diets was adjusted to 7.5 with KOH and diets were filter sterilized through a 0.22 ⁇ m filter and stored at 4° C. for short term ( ⁇ 7 days) or at ⁇ 80° C. for long term.
  • Gossypol acetic acid (Sigma, Cat#G4382-250MG) stock solution was made at 5% in methanol and sterilized by passing through a 0.22 ⁇ m syringe filter, and stored at 4° C.
  • the appropriate amount of stock solution was added to the artificial diet to obtain the different final concentrations of gossypol.
  • the diet was then placed into a 1.5 ml Eppendorf tube with a fava bean stem with a leaf and the opening of the tube was closed using parafilm. This feeding system was then placed into a deep petri dish (Fisher Scientific, Cat# FB0875711) and aphids were applied to the leaves of the plant.
  • the developmental stage (1 st , 2 nd , 3 rd , 4 th , 5 th , and 5R (5 th that has reproduced) instar) was determined daily throughout the experiment. Once an aphid reached the 4th instar stage, they were given their own artificial feeding system in a deep petri dish so that fecundity could be monitored once they reached adulthood.
  • a DNA extraction kit Qiagen, DNeasy kit
  • the primers used for Buchnera were Buch_groES_18F (CATGATCGTGTGCTTGTTAAG; SEQ ID NO: 238) and Buch_groES_98R (CTGTTCCTCGAGTCGATTTCC; SEQ ID NO: 239) (Chong and Moran, 2016 PNAS).
  • the primers used for aphid were ApEF1a 107F (CTGATTGTGCCGTGCTTATTG; SEQ ID NO: 240) and ApEF1a 246R (TATGGTGGTTCAGTAGAGTCC; SEQ ID NO: 241) (Chong and Moran, 2016 PNAS).
  • qPCR was performed using a qPCR amplification ramp of 1.6 degrees C./s and the following conditions: 1) 95° C.
  • eNASCO and LSR-1 A pisum 1 st and 2 nd instar aphids were divided into four separate treatment groups as defined in Experimental Design (described herein). Aphids were monitored daily and the number of aphids at each developmental stage was determined. Aphids treated with artificial diet alone began reaching maturity (5 th instar stage) at approximately 3 days ( FIG. 30A ). Development was delayed in aphids treated with gossypol, and by 5 days of treatment with 0.5% of gossypol, most aphids did not mature further than the 3 rd instar stage, and their size is also affected ( FIGS. 30A and 30B ). These data indicate that treatment with gossypol delayed and stopped progression of aphid development, and that this response was dose dependent.
  • 0.5% gossypol-treated aphids began dying after 2 days of treatment and all aphids succumbed to treatment by 4 days. Aphids treated with 0.25% survived a bit longer than those treated with 0.5% but were also drastically affected.
  • Microinjection was performed using NanoJet III Auto-Nanoliter Injector with the in-house pulled borosilicate needle (Drummond Scientific; Cat#3-000-203-G/XL).
  • Aphids LSR-1 strain, A. pisum
  • Each treatment group had approximately the same number of individuals injected from each of the collection plants.
  • Nymph aphids ( ⁇ 3 rd instar stage) were transferred using a paint brush to a tubing system connected to vacuum and microinjected into the ventral thorax with 20 nl of either artificial diet without essential amino acids (negative control) or 0.05% of gossypol solution in artificial diet without essential amino acids.
  • aphids were placed in a deep petri dish with a fava bean leaf with stem in 2% agar.
  • trans-cinnemaldehyde a natural aromatic aldehyde that is the major component of bark extract of cinnamon ( Cinnamomum zeylandicum ) results in decreased fitness.
  • Trans-cinnemaldehyde has been shown to have antimicrobial activity against both gram-negative and gram-positive organisms, although the exact mechanism of action of its antimicrobial activity remains unclear.
  • Trans-cinnemaldehyde may damage bacterial cell membranes and inhibit of specific cellular processes or enzymes (Gill and Holley, 2004 Applied Environmental Microbiology).
  • This Example demonstrates that the effect of trans-cinnemaldehyde on insects was mediated through the modulation of bacterial populations endogenous to the insect that were sensitive to trans-cinnemaldehyde.
  • One targeted bacterial strain is Buchnera aphidicola.
  • Trans-cinnemaldehyde was diluted to 0.05%, 0.5%, or 5% in water and was delivered through leaf perfusion ( ⁇ 1 ml was injected into leaves) and through plants.
  • Aphids (LSR-1 (which harbor only Buchnera ) strains, Acyrthosiphon pisum ) were grown on fava bean plants ( Vroma Vicia faba from Johnny's Selected Seeds) in a climate-controlled incubator (16 h light/8 h dark photoperiod; 60 ⁇ 5% RH; 25 ⁇ 2° C.). Prior to being used for aphid rearing, fava bean plants were grown in potting soil at 24° C. with 16 h of light and 8 h of darkness. To limit maternal effects or health differences between plants, 5-10 adults from different plants were distributed among 10 two-week-old plants, and allowed to multiply to high density for 5-7 days.
  • first and second instar aphids were collected from healthy plants and divided into four different treatment groups: 1) water treated controls, 2) 0.05% trans-cinnemaldehyde in water, 3) 0.5% trans-cinnemaldehyde in water, and 4) 5% trans-cinnemaldehyde in water.
  • Each treatment group received approximately the same number of individuals from each of the collection plants.
  • Trans-cinnemaldehyde (Sigma, Cat#C80687) was diluted to the appropriate concentration in water (see Therapeutic design), sterilized by passing through a 0.22 ⁇ m syringe filter, and stored at 4° C. Fava bean leaves were injected with approximately 1 ml of the treatment and then the leaf was placed in a 1.5 ml Eppendorf tube containing the same treatment solution. The opening of the tube where the fava bean stem was placed was closed using parafilm. This treatment feeding system was then placed into a deep petri dish (Fisher Scientific, Cat# FB0875711) and aphids were applied to the leaves of the plant.
  • a DNA extraction kit Qiagen, DNeasy kit
  • the primers used for Buchnera were Buch_groES_18F (CATGATCGTGTGCTTGTTAAG; SEQ ID NO: 238) and Buch_groES_98R (CTGTTCCTCGAGTCGATTTCC; SEQ ID NO: 239) (Chong and Moran, 2016 PNAS).
  • the primers used for aphid were ApEF1a 107F (CTGATTGTGCCGTGCTTATTG; SEQ ID NO: 240) and ApEF1a 246R (TATGGTGGTTCAGTAGAGTCC; SEQ ID NO: 241) (Chong and Moran, 2016 PNAS).
  • qPCR was performed using a qPCR amplification ramp of 1.6 degrees C./s and the following conditions: 1) 95° C.
  • LSR-1 A pisum 1 st and 2 nd instar aphids were divided into four separate treatment groups as defined in Experimental Design (described herein). Aphids were monitored daily and the number of aphids at each developmental stage was determined. Aphids treated with water alone began reaching the 3 rd instar stage at 3 days post-treatment ( FIG. 35 ). Development was delayed in aphids treated with trans-cinnemaldehyde, and by 3 days of treatment with each the three of the trans-cinnemaldehyde concentrations, none of the aphids matured past the second instar stage ( FIG. 35 ).
  • trans-cinnemaldehyde treatment decreased Buchnera levels, and that this decrease was concentration dependent.
  • This Example demonstrates the treatment of aphids with multiple scorpion antimicrobial peptides (AMP), of which several are identified AMPs in the venom gland transcriptome of the scorpion Urodacus yaschenkoi (Luna-Ramirez et al., 2017, Toxins).
  • AMPs typically have a net positive charge and hence, a high affinity for bacterial membranes.
  • This Example demonstrates that the effect of the AMP on insects was mediated through the modulation of bacterial populations endogenous to the insect that were sensitive to AMP peptides.
  • One targeted bacterial strain is Buchnera aphidicola , an obligate symbiont of aphids.
  • the Uy192 solution was formulated using a combination of leaf perfusion and delivery through plants.
  • the control solution was leaf injected with water+blue food coloring and water in tube.
  • the treatment solution consisted of 100 ug/ml Uy192 in water via leaf injection (with blue food coloring) and through plant (in Eppendorf tube).
  • Aphids LSR-1 (which harbor only Buchnera ), Acyrthosiphon pisum were grown on fava bean plants ( Vroma Vicia faba from Johnny's Selected Seeds) in a climate-controlled incubator (16 h light/8 h dark photoperiod; 60 ⁇ 5% RH; 20 ⁇ 2° C.). Prior to being used for aphid rearing, fava bean plants were grown in potting soil at 24° C. with 16 h of light and 8 h of darkness. To limit maternal effects or health differences between plants, 5-10 adults from different plants were distributed among 10 two-week-old plants, and allowed to multiply to high density for 5-7 days.
  • first and second instar aphids were collected from healthy plants and divided into 2 different treatment groups: 1) negative control (water treated), 2) The treatment solution of 100 ug/ml AMP in water. Each treatment group received approximately the same number of individuals from each of the collection plants.
  • Uy192 was synthesized by Bio-Synthesis at >75% purity. 1 mg of lyophilized peptide was reconstituted in 500 ul of 80% acetonitrile, 20% water, and 0.1% TFA, 100 ul (100 ug) was aliquoted into 10 individual Eppendorf tubes, and allowed to dry. For treatment (see Therapeutic design), 1 ml of water was added to a 100 ug aliquot of peptide to obtain the final concentration of Uy192 (100 ug/ml). The solution was then placed into a 1.5 ml Eppendorf tube with a fava bean stem with a leaf also perfused with the solutions and the opening of the tube was closed using parafilm. This feeding system was then placed into a deep petri dish (Fisher Scientific, Cat# FB0875711) and aphids were applied to the leaves of the plant.
  • aphids were placed onto each leaf.
  • the feeding systems were changed every 2-3 days throughout the experiment. Aphids were monitored daily for survival and dead aphids were removed from the deep petri dish when they were discovered.
  • a DNA extraction kit Qiagen, DNeasy kit
  • the primers used for Buchnera were Buch_groES_18F (CATGATCGTGTGCTTGTTAAG; SEQ ID NO: 238) and Buch_groES_98R (CTGTTCCTCGAGTCGATTTCC; SEQ ID NO: 239) (Chong and Moran, 2016 PNAS).
  • the primers used for aphid were ApEF1a 107F (CTGATTGTGCCGTGCTTATTG; SEQ ID NO: 240) and ApEF1a 246R (TATGGTGGTTCAGTAGAGTCC; SEQ ID NO: 241) (Chong and Moran, 2016 PNAS).
  • qPCR was performed using a qPCR amplification ramp of 1.6 degrees C./s and the following conditions: 1) 95° C.
  • LSR-1 A pisum 1 st and 2 nd instar aphids were divided into two separate treatment groups as defined in Experimental Design (above). Aphids were monitored daily and the number of aphids at each developmental stage was determined. Aphids treated with the negative control alone began reaching maturity (5th instar stage) at approximately 6 days ( FIG. 38 ). Development was delayed in aphids treated with Uy192, and after 8 days of treatment, aphids did not mature further than the 4 rd instar stage. These data indicate that treatment with Uy192 delayed and stopped progression of aphid development.
  • This Example demonstrates the treatment of aphids with several scorpion antimicrobial peptides (AMPs) D10, D3, Uyct3, and Uy17, which have been recently identified AMPs in the venom gland transcriptome of the scorpion Urodacus yaschenkoi (Luna-Ramirez et al., 2017, Toxins).
  • AMPs typically have a net positive charge and hence, a high affinity for bacterial membranes.
  • This Example demonstrates that the effect of the AMPs on insects was mediated through the modulation of bacterial populations endogenous to the insect that were sensitive to AMP peptides.
  • One targeted bacterial strain is Buchnera aphidicola , an obligate symbiont of aphids.
  • Aphids are agricultural insect pests causing direct feeding damage to the plant and serving as vectors of plant viruses.
  • aphid honeydew promotes the growth of sooty mold and attracts nuisance ants.
  • the indicated peptide or peptide cocktail was directly microinjected into aphids or delivered using a combination of leaf perfusion and delivery through plants.
  • aphids were microinjected with water (for microinjection experiments) or placed on leaves injected with water and water in tube (for leaf perfusion and plant delivery experiments).
  • the treatment solutions consisted of 20 nl of 5 ⁇ g/ ⁇ l of D3 or D10 dissolved in water (for aphid microinjections) or 40 ⁇ g/ml of a cocktail of D10, Uy17, D3, and UyCt3 in water via leaf injection and through plant (in Eppendorf tube).
  • Microinjection was performed using NanoJet III Auto-Nanoliter Injector with the in-house pulled borosilicate needle (Drummond Scientific; Cat#3-000-203-G/XL).
  • Aphids LSR-1 strain, Acyrthosiphon pisum
  • fava bean plants Vroma Vicia faba from Johnny's Selected Seeds
  • a climate-controlled incubator (16 h light/8 h dark photoperiod; 60 ⁇ 5% RH; 25 ⁇ 2° C.).
  • fava bean plants Prior to being used for aphid rearing, fava bean plants were grown in potting soil at 24° C. with 16 h of light and 8 h of darkness.
  • aphids were microinjected into the ventral thorax with 20 nl of either water or 100 ng (20 ul of a 5 ug/ml solution of peptide D3 or D10. The microinjection rate as 5 nl/sec. After injection, aphids were placed in a deep petri dish containing a fava bean leaf with stem in 2% agar.
  • Peptides were synthesized by Bio-Synthesis at >75% purity. 1 mg of lyophilized peptide was reconstituted in 500 ⁇ l of 80% acetonitrile, 20% water, and 0.1% TFA, 100 ⁇ l (100 ⁇ g) was aliquoted into 10 individual Eppendorf tubes, and allowed to dry.
  • a DNA extraction kit Qiagen, DNeasy kit
  • the primers used for Buchnera were Buch_groES_18F (CATGATCGTGTGCTTGTTAAG; SEQ ID NO: 238) and Buch_groES_98R (CTGTTCCTCGAGTCGATTTCC; SEQ ID NO: 239) (Chong and Moran, 2016 PNAS).
  • the primers used for aphid were ApEF1a 107F (CTGATTGTGCCGTGCTTATTG; SEQ ID NO: 240) and ApEF1a 246R (TATGGTGGTTCAGTAGAGTCC; SEQ ID NO: 241) (Chong and Moran, 2016 PNAS).
  • qPCR was performed using a qPCR amplification ramp of 1.6 degrees C./s and the following conditions: 1) 95° C.
  • LSR-1 A pisum 1 st and 2 nd instar aphids were divided into three separate treatment groups as defined in Experimental Design (described herein). Aphids were monitored daily and survival rate was determined. After 5 days of treatment, the aphids injected with the scorpion peptides had lower survival rates compared to water injected controls (9, 35, and 45% survival for injection with D3, D10, and water, respectively) ( FIG. 41 ). These data indicate that there was a decrease in survival upon treatment with the scorpion AMPs D3 and D10.
  • eNASCO Aphids which harbor Buchnera and Serratia
  • Acyrthosiphon pisum were grown on fava bean plants ( Vroma Vicia faba from Johnny's Selected Seeds) as described above.
  • first and second instar aphids were collected from healthy plants and divided into 2 different treatment groups: 1) negative control (water treated), 2) The treatment solution consisted of 40 ⁇ g/ml of each D10, Uy17, D3, and UyCt3 in water. Each treatment group received approximately the same number of individuals from each of the collection plants.
  • Peptides were synthesized, dissolved, and aliquoted, as described above.
  • water was added to a 100 ⁇ g aliquot of peptide to obtain the desired final concentration (40 ⁇ g/ml).
  • the four peptides were combined to make the peptide cocktail solution. This solution was used to perfuse into leaves via injection. Following injection, the stems of the injected leaves were placed into a 1.5 ml Eppendorf tube which was then sealed with parafilm. This feeding system was then placed into a deep petri dish (Fisher Scientific, Cat# FB0875711) and aphids were applied to the leaves of the plant.
  • Example 22 Insects Treated with an Antimicrobial Peptide Fused to a Cell Penetrating Peptide
  • This Example demonstrates the treatment of aphids with a fused scorpion antimicrobial peptide (AMP) (Uy192) to a cell penetrating peptide derived from a virus.
  • AMP scorpion antimicrobial peptide
  • Uy192 is one of several recently identified AMPs in the venom gland transcriptome of the scorpion Urodacus yaschenkoi (Luna-Ramirez et al., 2017, Toxins).
  • AMPs typically have a net positive charge and hence, a high affinity for bacterial membranes.
  • the peptide was synthesized fused to a portion of the transactivator of transcription (TAT) protein of human immunodeficiency virus I (HIV-1).
  • TAT transactivator of transcription
  • HAV-1 human immunodeficiency virus I
  • the scorpion peptide conjugated to the cell penetrating peptide and fluorescently tagged with 6FAM was formulated using a combination of leaf perfusion and delivery through plants.
  • the control solution (water) or treatment solution (Uy192+CPP+FAM) was injected into the leaf and placed in the Eppendorf tube.
  • the treatment solution included 100 ⁇ g/ml Uy192+CPP+FAM in water.
  • LSR-1 aphids Acyrthosiphon pisum were grown on fava bean plants ( Vroma Vicia faba from Johnny's Selected Seeds) in a climate-controlled incubator (16 h light/8 h dark photoperiod; 60 ⁇ 5% RH; 25 ⁇ 2° C.). Prior to being used for aphid rearing, fava bean plants were grown in potting soil at 24° C. with 16 h of light and 8 h of darkness. To limit maternal effects or health differences between plants, 5-10 adults from different plants were distributed among 10 two-week-old plants, and allowed to multiply to high density for 5-7 days.
  • Uy192+CPP+FAM amino acid sequence: YGRKKRRQRRRFLSTIWNGIKGLL-FAM
  • Bio-Synthesis at >75% purity. 5 mg of lyophilized peptide was reconstituted in 1 ml of 80% acetonitrile, 20% water, and 0.1% TFA, 50 ⁇ l (100 ⁇ g) was aliquoted into individual Eppendorf tubes, and allowed to dry.
  • 1 ml of sterile water was added to a 100 ⁇ g aliquot of peptide to obtain the final concentration of Uy192+CPP+FAM (100 ⁇ g/ml).
  • the solution was then injected into the leaf of the plant and the stem of the plant was placed into a 1.5 ml Eppendorf tube. The opening of the tube was closed using parafilm. This feeding system was then placed into a deep petri dish (Fisher Scientific, Cat# FB0875711) and aphids were applied to the leaves of the plant. Epi fluorescence imaging of the leaf confirmed that the leaves contained the green fluorescently tagged peptide in their vascular system.
  • a DNA extraction kit Qiagen, DNeasy kit
  • the primers used for Buchnera were Buch_groES_18F (CATGATCGTGTGCTTGTTAAG; SEQ ID NO: 238) and Buch_groES_98R (CTGTTCCTCGAGTCGATTTCC; SEQ ID NO: 239) (Chong and Moran, 2016 PNAS).
  • the primers used for aphid were ApEF1a 107F (CTGATTGTGCCGTGCTTATTG; SEQ ID NO: 240) and ApEF1a 246R (TATGGTGGTTCAGTAGAGTCC; SEQ ID NO: 241) (Chong and Moran, 2016 PNAS).
  • qPCR was performed using a qPCR amplification ramp of 1.6 degrees C./s and the following conditions: 1) 95° C.
  • LSR-1 A pisum 1st instar aphids were divided into three separate treatment groups as defined in Experimental Design (above). Aphids were monitored daily and the number of aphids at each developmental stage was determined. Development for both aphids treated with water and those treated with the scorpion peptide fused to the cell penetrating peptide was similar for days 0 and 1 ( FIG. 44 ). By day 2, however, control treated aphids developed to either in the second or third instar stage, while some aphids treated with the scorpion peptide fused to the cell penetrating peptide remained in the first instar stage ( FIG. 44 ).
  • isolated bacteriocytes were directly exposed to the fusion protein and imaged.
  • the bacteriocytes were dissected from the aphids in Schneider's medium supplemented with 1% w/v BSA (Schneider-BSA medium), and placed in an imaging well containing 20 ul of schneider's medium.
  • a 100 ug lyophilized aliquot of the scorpion peptide was resuspended in 100 ul of the schneider's medium to produce 1 mg/ml solution, and 5 ul of this solution was mixed in to the well containing bacteriocytes.
  • this data demonstrates the ability to kill and decrease the development, longevity, and endogenous bacterial populations, e.g., fitness, of aphids by treating them with the scorpion antimicrobial peptide Uy192 fused to a cell penetrating peptide.
  • pantothenol which is the alcohol analog of pantothenic acid (Vitamin B5).
  • Aphids have obligate endosymbiont bacteria, Buchnera , that help them make essential amino acids and vitamins, including Vitamin B5.
  • pantothenol inhibits the growth of Plasmodium falciparium by inhibition of the specific parasite kinases (Saliba et al., 2005). It was hypothesized that treating insects with pantothenol would be detrimental for the bacterial-insect symbiosis therefore affecting insect fitness. This Example demonstrates that the treatment with pantothenol decreased insect fitness.
  • Pantothenol solutions were formulated depending on the delivery method:
  • pantothenol formulated in an artificial diet (based on Akey and Beck, 1971; see Experimental Design) without essential amino acids (2 mg/ml histidine, 2 mg/ml isoleucine, 2 mg/ml leucine, 2 mg/ml lysine, 1 mg/ml methionine, 1.62 mg/ml phenylalanine, 2 mg/ml threonine, 1 mg/ml tryptophan, and 2 mg/ml valine).
  • Leaf coating 100 ⁇ l of 0.025% nonionic organosilicone surfactant solvent Silwet L-77 in water (negative control) or 100 ⁇ l of 50 ⁇ g/ml of rifampicin formulated in solvent solution was applied directly to the leaf surface and allowed to dry.
  • Aphids were grown on fava bean plants ( Vroma Vicia faba from Johnny's Selected Seeds) in a climate-controlled incubator (16 h light/8 h dark photoperiod; 60 ⁇ 5% RH; 25 ⁇ 2° C.). Prior to being used for aphid rearing, fava bean plants were grown in potting soil at 24° C. with 16 h of light and 8 h of darkness. To limit maternal effects or health differences between plants, 5-10 adults from different plants were distributed among 10 two-week-old plants, and allowed to multiply to high density for 5-7 days.
  • first and second instar aphids were collected from healthy plants and divided into 3 different treatment groups: 1) artificial diet alone without essential amino acids, 2) artificial diet alone without essential amino acids and 10 uM pantothenol, and 3) artificial diet alone without essential amino acids and 100 uM pantothenol.
  • Each treatment group received approximately the same number of individuals from each of the collection plants.
  • the artificial diet used was made as previously published (Akey and Beck, 1971 Continuous Rearing of the Pea Aphid, Acyrthosiphon pisum , on a Holidic Diet), with and without the essential amino acids (2 mg/ml histidine, 2 mg/ml isoleucine, 2 mg/ml leucine, 2 mg/ml lysine, 1 mg/ml methionine, 1.62 mg/ml phenylalanine, 2 mg/ml threonine, 1 mg/ml tryptophan, and 2 mg/ml valine), except neither diet included homoserine or beta-alanyltyrosine.
  • the pH of the diets was adjusted to 7.5 with KOH and diets were filter sterilized through a 0.22 ⁇ m filter and stored at 4° C. for short term ( ⁇ 7 days) or at ⁇ 80° C. for long term.
  • Pantothenol (Sigma Cat#295787) solutions were made at 10 uM and 100 uM in artificial diet without essential amino acids, sterilized by passing through a 0.22 ⁇ m syringe filter, and stored at ⁇ 20° C.
  • the appropriate amount of stock solution was added to the artificial diet without essential amino acids to obtain a final concentration of 10 or 100 uM pantothenol.
  • the diet was then placed into a 1.5 ml Eppendorf tube with a fava bean stem with a leaf and the opening of the tube was closed using parafilm. This artificial diet feeding system was then placed into a deep petri dish (Fisher Scientific, Cat# FB0875711) and aphids were applied to the leaves of the plant.
  • the developmental stage (1st, 2nd, 3rd, 4th, 5th instar) was determined daily throughout the experiment. Once an aphid reached the 4th instar stage, they were given their own artificial feeding system in a deep petri dish so that fecundity could be monitored once they reached adulthood.
  • a DNA extraction kit Qiagen, DNeasy kit
  • the primers used for Buchnera were Buch_groES_18F (CATGATCGTGTGCTTGTTAAG; SEQ ID NO: 238) and Buch_groES_98R (CTGTTCCTCGAGTCGATTTCC; SEQ ID NO: 239) (Chong and Moran, 2016 PNAS).
  • the primers used for aphid were ApEF1a 107F (CTGATTGTGCCGTGCTTATTG; SEQ ID NO: 240) and ApEF1a 246R (TATGGTGGTTCAGTAGAGTCC; SEQ ID NO: 241) (Chong and Moran, 2016 PNAS).
  • qPCR was performed using a qPCR amplification ramp of 1.6 degrees C./s and the following conditions: 1) 95° C.
  • eNASCO 1st and 2nd instar aphids were divided into three separate treatment groups as defined in Plant Delivery Experimental Design (described herein). Aphids were monitored daily and the number of aphids at each developmental stage was determined. Aphids treated with artificial diet alone without essential amino acids began reaching maturity (5th instar stage) at approximately 5 days ( FIG. 48A ). Development was delayed in aphids treated with pantothenol, especially at days two and three post-treatment ( FIG. 48A ), indicating that treatment with pantothenol impairs aphid development. Eventually, most aphids from each treatment group reached maturity and began reproducing.
  • aphids were also imaged and aphid area was determined. All aphids were the same size after 1 day of treatment, however, by 3 days post-treatment, aphids treated with pantothenol were smaller in area than untreated controls. Moreover, aphids treated with pantothenol had much less of an increase in body size (as determined by area) over the course of the experiment, compared to aphids treated with artificial diet alone without essential amino acids ( FIG. 48B ).
  • Fecundity was also monitored in aphids during the treatments. The fraction of aphids surviving to maturity and reproducing was determined. Approximately one quarter of aphids treated with artificial diet without essential amino acids survived to reach maturity by 8 days post-treatment ( FIG. 50A ). In contrast, only a little over 1/10th of aphids treated with 10 or 100 uM pantothenol survived to reach maturity and reproduce by 8 days post-treatment. The mean day aphids in each treatment group began reproducing was also measured and for all treatment groups, the mean day aphids began reproducing was 7 days ( FIG. 50B ). Additionally, the mean number of offspring per day produced by mature, reproducing aphids was also monitored.
  • Pantothenol Treatment does not Affect Buchnera in Aphids
  • Pantothenol powder was added to 0.025% of a nonionic organosilicone surfactant solvent, Silwet
  • Each treatment group received approximately the same number of individuals from each of the collection plant. For each treatment, 20 aphids were placed onto each leaf. Aphids were monitored daily for survival and dead aphids were removed when they were discovered. In addition, the developmental stage (1st, 2nd, 3rd, 4th, 5th instar, and 5R, representing a reproducing 5th instar) was determined daily throughout the experiment.
  • Pantothenol treatment delivered through leaf coating does not affect aphid development
  • eNASCO 1st instar aphids were divided into two separate treatment groups as defined in the Experimental Design described herein. Aphids were monitored daily and the number of aphids at each developmental stage was determined. Aphids placed on coated leaves treated with either the control or pantothenol solution matured at similar rates up to two days post-treatment ( FIG. 52 ). These data indicate that leaf coating with pantothenol did not affect aphid development.
  • Example 24 Insects Treated with a Cocktail of Amino Acid Transporters Inhibitors
  • This Example demonstrates the treatment of aphids with a cocktail of amino acid analogs.
  • the objective of this treatment was to inhibit uptakes of glutamine into the bacteriocytes through the ApGLNT1 glutamine transporter. It has previously been shown that arginine inhibits glutamine uptake by the glutamine transporter (Price et al., 2014 PNAS), and we hypothesized that treatment with analogs of arginine, or other amino acid analogs, may also inhibit uptake of essential amino acids into the aphid bacteriocytes.
  • This Example demonstrates that the decrease in fitness upon treatment was mediated through the modulation of bacterial populations endogenous to the insect that were sensitive to amino acid analogs.
  • One targeted bacterial strain is Buchnera.
  • the amino acid cocktail was formulated for delivery through leaf perfusion and through the plant. This delivery method consisted of injecting leaves with approximately 1 ml of the amino acid cocktail in water (see below for list of components in the cocktail) or 1 ml of the negative control solution containing water only.
  • Aphids LSR-1 (which harbor only Buchnera ), Acyrthosiphon pisum were grown on fava bean plants ( Vroma Vicia faba from Johnny's Selected Seeds) in a climate-controlled incubator (16 h light/8 h dark photoperiod; 60 ⁇ 5% RH; 25 ⁇ 2° C.). Prior to being used for aphid rearing, fava bean plants were grown in potting soil at 24° C. with 16 h of light and 8 h of darkness. To limit maternal effects or health differences between plants, 5-10 adults from different plants were distributed among 10 two-week-old plants, and allowed to multiply to high density for 5-7 days.
  • amino acid cocktail contained each of the following agents at the indicated final concentrations: 330 ⁇ M L-NNA (N-nitro-L-Arginine; Sigma), 0.1 mg/ml L-canavanine (Sigma), 0.5 mg/ml D-arginine (Sigma), 0.5 mg/ml D-phenylalanine (Sigma), 0.5 mg/ml D-histidine (Sigma), 0.5 mg/ml D-tryptophan (Sigma), 0.5 mg/ml D-threonine (Sigma), 0.5 mg/ml D-valine (Sigma), 0.5 mg/ml D-methionine (Sigma), 0.5 mg/ml D-leucine, and 6 ⁇ M L-NMMA (citrate) (Cayman Chemical).
  • L-NNA N-nitro-L-Arginine
  • Sigma 0.1 mg/ml L-canavanine
  • 0.5 mg/ml D-arginine Sigma
  • ⁇ 1 ml of the treatment solution was perfused into the fava bean leaf via injection and the stem of the plant was put into a 1.5 ml Eppendorf tube containing the treatment solution. The opening of the tube was closed using parafilm.
  • This feeding system was then placed into a deep petri dish (Fisher Scientific, Cat# FB0875711) and aphids were applied to the leaves of the plant. For each treatment, a total of 56-58 aphids were placed onto each leaf (each treatment consisted of two replicates of 28-31 aphids). Each treatment group received approximately the same number of individuals from each of the collection plants. The feeding systems were changed every 2-3 days throughout the experiment.
  • Aphids were monitored daily for survival and dead aphids were removed from the deep petri dish when they were discovered.
  • the aphid developmental stage (1st, 2nd, 3rd, 4th, and 5th instar) was determined daily throughout the experiment and microscopic images were taken of the aphids on day 5 to determine aphid area measurements.
  • Stock solutions of L-NNA were made at 5 mM in water, sterilized by passing through a 0.22 ⁇ m syringe filter, and stored at ⁇ 20° C.
  • Stock solutions of L-canavanine were made at 100 mg/ml in water, sterilized by passing through a 0.22 ⁇ m syringe filter, and stored at 4° C.
  • Stock solutions of D-arginine and D-threonine were made at 50 mg/ml in water, sterilized by passing through a 0.22 ⁇ m syringe filter, and stored at 4° C.
  • Stock solutions of D-valine and D-methionine were made at 25 mg/ml in water, sterilized by passing through a 0.22 ⁇ m syringe filter, and stored at 4° C.
  • Stock solutions of D-leucine were made at 12 mg/ml in water, sterilized by passing through a 0.22 ⁇ m syringe filter, and stored at 4° C.
  • Stock solutions of D-phenylalanine and D-histidine were made at 50 mg/ml in 1M HCl, sterilized by passing through a 0.22 ⁇ m syringe filter, and stored at 4° C.
  • Stock solutions of D-tryptophan were made at 50 mg/ml in 0.5M HCl, sterilized by passing through a 0.22 ⁇ m syringe filter, and stored at 4° C.
  • Stock solutions of L-NMMA were made at 6 mg/ml in sterile PBS, sterilized by passing through a 0.22 ⁇ m syringe filter, and stored at ⁇ 20° C.
  • the appropriate amount of stock solution was added to water to obtain the final concentration of the agent in the cocktail as indicated above.
  • a DNA extraction kit Qiagen, DNeasy kit
  • the primers used for Buchnera were Buch_groES_18F (CATGATCGTGTGCTTGTTAAG; SEQ ID NO: 238) and Buch_groES_98R (CTGTTCCTCGAGTCGATTTCC; SEQ ID NO: 239) (Chong and Moran, 2016 PNAS).
  • the primers used for aphid were ApEF1a 107F (CTGATTGTGCCGTGCTTATTG; SEQ ID NO: 240) and ApEF1a 246R (TATGGTGGTTCAGTAGAGTCC; SEQ ID NO: 241) (Chong and Moran, 2016 PNAS).
  • qPCR was performed using a qPCR amplification ramp of 1.6 degrees C./s and the following conditions: 1) 95° C.
  • LSR-1 1st instar aphids were divided into two separate treatment groups as defined in Leaf perfusion and delivery through plants experimental design (described herein). Aphids were monitored daily and the number of aphids at each developmental stage was determined. Aphids treated with water began reaching maturity (5th instar stage) at day 5 post-treatment ( FIG. 54A ). By 6 days post-treatment, ⁇ 20 percent of aphids treated with water reached the 5th instar stage. In contrast, less than 3 percent of the aphids treated with the amino acid cocktail reached the 5th instar stage, even after 6 days ( FIG. 54A ). This delay in development upon treatment with the amino acid cocktail was further exemplified by aphid size measurements taken at 5 days post-treatment.
  • this data demonstrates the ability to decrease the development and endogenous bacterial populations, e.g., fitness, of aphids by treating them with a cocktail of amino acid analogs.
  • Example 25 Insects Treated with a Combination of Agents (Antibiotic, Peptide, and Natural Antimicrobial)
  • This Example demonstrates the treatment of insects with a combination of three antimicrobial agents—an antibiotic (rifampicin), a peptide (the scorpion peptide Uy192), and a natural antimicrobial (low molecular weight chitosan).
  • an antibiotic rifampicin
  • a peptide the scorpion peptide Uy192
  • a natural antimicrobial low molecular weight chitosan
  • each of these agents administered individually resulted in decreased aphid fitness and reduced endosymbiont levels.
  • This Example demonstrates that through the delivery of a combination of treatments, insect fitness and endosymbiont levels were reduced as well as, or better than, treatment with each individual agent alone.
  • the combination treatment was formulated for delivery through leaf perfusion and through the plant.
  • This delivery method consisted of injecting leaves with approximately 1 ml of the combination treatment in water (with final concentrations of 100 ⁇ g/ml rifampicin, 100 ⁇ g/ml Uy192, and 300 ⁇ g/ml chitosan) or 1 ml of the negative control solution containing water only.
  • Aphids LSR-1 (which harbor only Buchnera ), Acyrthosiphon pisum were grown on fava bean plants ( Vroma Vicia faba from Johnny's Selected Seeds) in a climate-controlled incubator (16 h light/8 h dark photoperiod; 60 ⁇ 5% RH; 25 ⁇ 2° C.). Prior to being used for aphid rearing, fava bean plants were grown in potting soil at 24° C. with 16 h of light and 8 h of darkness. To limit maternal effects or health differences between plants, 5-10 adults from different plants were distributed among 10 two-week-old plants, and allowed to multiply to high density for 5-7 days.
  • first instar aphids were collected from healthy plants and divided into 2 different treatment groups: 1) negative control (water treatment) and 2) a combination of 100 ⁇ g/ml rifampicin, 100 ⁇ g/ml Uy192, and 300 ⁇ g/ml chitosan treatment.
  • ⁇ 1 ml of the treatment solution was perfused into the fava bean leaf via injection and the stem of the plant was put into a 1.5 ml Eppendorf tube containing the treatment solution. The opening of the tube was closed using parafilm.
  • This treatment system was then placed into a deep petri dish (Fisher Scientific, Cat# FB0875711) and aphids were applied to the leaves of the plant.
  • each treatment consisted of two replicates of 28 aphids.
  • Each treatment group received approximately the same number of individuals from each of the collection plants.
  • the feeding systems were changed every 2-3 days throughout the experiment. Aphids were monitored daily for survival and dead aphids were removed from the deep petri dish when they were discovered.
  • the aphid developmental stage (1 st 2 nd , 3 rd , 4 th , and 5 th instar) was determined daily throughout the experiment and microscopic images were taken of the aphids on day 5 to determine aphid area measurements.
  • Rifampicin (Tokyo Chemical Industry, LTD) stock solution was made at 25 mg/ml in methanol, sterilized by passing through a 0.22 ⁇ m syringe filter, and stored at ⁇ 20° C. For treatment, the appropriate amount of stock solution was added to water to obtain a final concentration of 100 ⁇ g/ml rifampicin.
  • Uy192 was synthesized by Bio-Synthesis at >75% purity. 1 mg of lyophilized peptide was reconstituted in 500 ⁇ l of 80% acetonitrile, 20% water, and 0.1% TFA. 100 ⁇ l (100 ⁇ g) was aliquoted into 10 individual Eppendorf tubes and allowed to dry.
  • a DNA extraction kit Qiagen, DNeasy kit
  • the primers used for Buchnera were Buch_groES_18F (CATGATCGTGTGCTTGTTAAG; SEQ ID NO: 238) and Buch_groES_98R (CTGTTCCTCGAGTCGATTTCC; SEQ ID NO: 239) (Chong and Moran, 2016 PNAS).
  • the primers used for aphid were ApEF1a 107F (CTGATTGTGCCGTGCTTATTG; SEQ ID NO: 240) and ApEF1a 246R (TATGGTGGTTCAGTAGAGTCC; SEQ ID NO: 241) (Chong and Moran, 2016 PNAS).
  • qPCR was performed using a qPCR amplification ramp of 1.6 degrees C./s and the following conditions: 1) 95° C.
  • LSR-1 1 st instar aphids were divided into two separate treatment groups as defined in Leaf perfusion and delivery through plants experimental design (described herein). Aphids were monitored daily and the number of aphids at each developmental stage was determined. Aphids treated with water began reaching maturity (5th instar stage) at day 5 post-treatment ( FIG. 56A ). By 6 days post-treatment, ⁇ 20 percent of aphids treated with water reached the 5th instar stage. In contrast, no aphids treated with the combination of three agents reached the 5th instar stage, even after 6 days ( FIG. 56A ). This delay in development upon combination treatment was further exemplified by aphid size measurements taken at 5 days post-treatment.
  • this data demonstrates the ability to decrease the development and endogenous bacterial populations, e.g., fitness, of aphids by treating them with a combination of a peptide, antibiotic, and natural antimicrobial.
  • This Example demonstrates the effects of treatment of weevils with ciprofloxacin, a bactericidal antibiotic that inhibits the activity of DNA gyrase and topoisomerase, two enzymes essential for DNA replication.
  • This Example demonstrates that the phenotypic effect of ciprofloxacin on another model insect, weevils, was mediated through the modulation of bacterial populations endogenous to the insects that were sensitive to ciprofloxacin.
  • One targeted bacterial strain is Sitophilus primary endosymbiont (SPE, Candidatus Sodalis pierantonius ).
  • Sitophilus maize weevils Sitophilus zeamais were reared on organic corn at 27.5° C. and 70% relative humidity. Prior to being used for weevil rearing, corn was frozen for 7 days and then tempered to 10% humidity with sterile water. For experiments, adult male/female mating pairs were divided into 3 different treatment groups that were done in triplicate: 1) water control, 2) 250 ⁇ g/ml ciprofloxacin, and 3) 2.5 mg/ml ciprofloxacin. Ciprofloxacin (Sigma) stock solutions were made at 25 mg/ml in 0.1 N HCl, sterilized by passing through a 0.22 ⁇ m syringe filter, and stored at ⁇ 20° C. For treatments, the appropriate amount of stock solution was diluted in sterile water.
  • the weevils were subjected to three successive treatments:
  • weevil survival was monitored daily for 18 days, after which DNA was extracted from the remaining weevils in each group. Briefly, the weevil body was surface sterilized by dipping the weevil into a 6% bleach solution for approximately 5 seconds. Weevils were then rinsed in sterile water and DNA was extracted from each individual aphid using a DNA extraction kit (Qiagen, DNeasy kit) according to manufacturer's instructions. DNA concentration was measured using a nanodrop nucleic acid quantification, and SPE and weevil DNA copy numbers were measured by qPCR.
  • a DNA extraction kit Qiagen, DNeasy kit
  • the primers used for SPE were qPCR_Sod_F (ATAGCTGTCCAGACGCTTCG; SEQ ID NO: 242) and qPCR_Sod_R (ATGTCGTCGAGGCGATTACC; SEQ ID NO: 243).
  • the primers used for weevil ( ⁇ -actin) were SACT144_FOR (GGTGTTGGCGTACAAGTCCT; SEQ ID NO: 244) and SACT314_REV (GAATTGCCTGATGGACAGGT; SEQ ID NO: 245) (Login et al., 2011).
  • qPCR was performed using a qPCR amplification ramp of 1.6 degrees C./s and the following conditions: 1) 95° C. for 10 minutes, 2) 95° C.
  • corn kernels were soaked in water without antibiotics or water with 2.5 or 0.25 mg/ml ciprofloxacin (as described above). A concentrated culture of E. coli was then spread onto LB plates and one of the coated kernels was then placed onto the center of the plate. The plates were incubated overnight, and bacterial growth was assessed the next day.
  • FIG. 56A A lawn of bacteria grew on the entire plate with the corn kernel that had been coated in water without any antibiotics. In contrast, no bacteria grew on plates with entire corn kernels that had been soaked in either of the two concentrations of ciprofloxacin ( FIG. 56B , left panels). These data show that the coating method employed in these experiments allowed for ciprofloxacin to successfully coat the surface of corn kernels and inhibit bacterial growth.
  • corn kernels soaked in 2.5 or 0.25 mg/ml ciprofloxacin were cut in half and placed cut side down on an LB plate with a concentrated culture of E. coli . The plates were incubated overnight and the next day bacterial growth was assessed. No bacterial growth was present on the plates with the kernels soaked in either concentration of antibiotic, indicating that ciprofloxacin penetrated the corn kernel ( FIG. 56B , right panels). Together, these data indicate that the method of corn kernel soaking used for these experiments successfully coated and penetrated the kernels with the antibiotic.
  • Antibiotic Treatment Decreases SPE Levels in the F0 Generation.
  • this data demonstrate the ability to kill and decrease the development, reproductive ability, longevity, and endogenous bacterial populations, e.g., fitness, of weevils by treating them with an antibiotic through multiple delivery methods.
  • This Example demonstrates the ability to kill, decrease the fitness of two-spotted spider mites by treating them with rifampicin, a narrow spectrum antibiotic that inhibits DNA-dependent RNA synthesis by inhibiting a bacterial RNA polymerase, and doxycycline, a broad-spectrum antibiotic that prevents bacterial reproduction by inhibiting protein synthesis.
  • rifampicin and doxycycline a narrow spectrum antibiotic that inhibits DNA-dependent RNA synthesis by inhibiting a bacterial RNA polymerase
  • doxycycline a broad-spectrum antibiotic that prevents bacterial reproduction by inhibiting protein synthesis.
  • the effect of rifampicin and doxycycline on mites was mediated through the modulation of bacterial populations endogenous to the mites that were sensitive to the antibiotics.
  • Insects such as mosquitoes
  • arachnids such as ticks
  • Vector-borne diseases cause millions of human deaths every year.
  • vector-borne diseases that infect animals, such as livestock represent a major global public health burden.
  • Two-spotted spider mites are arachnids in the same subclass as ticks. Therefore, this Example demonstrates methods and compositions used to decrease the fitness of two-spotted spider mites and provide insight into decreasing tick fitness.

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