BR112019014730B1 - METHOD TO INCREASE THE FITNESS OF A HONEY BEE - Google Patents

Abstract

The present invention relates to agents, compositions and methods for agricultural use, for example, for altering the level, activity or metabolism of one or more resident microorganisms in a nematode or arthropod host (for example, honeybee or honeybee). silk), the alteration resulting in an increase in host fitness. The invention features a composition that includes an agent (e.g., phage, peptide, small molecule, antibiotic, or combinations thereof) that can alter the host microbiota in a way that is beneficial to the host. By promoting favorable microbial levels, microbial activity, microbial metabolism and/or microbial diversity, the agents described here can be used to increase the fitness of a variety of beneficial nematodes or arthropods, such as bees and silkworms, used in agriculture and business.

Description

CROSS-REFERENCE TO RELATED ORDERS

[001] This order claims priority for US Provisional Order No. 62/450,017 filed on January 24, 2017, and US Provisional Order No. 62/583,736 filed on November 9, 2017, the contents of which are incorporated here for reference in its entirety.

BACKGROUND

[002] Certain invertebrates, such as nematodes and arthropods (e.g. insects, e.g. European honey bees (Apis melliferia) or silkworms (Bombyx mori)), are used in agriculture for pollination and pest control efforts as well as in trade for the production of commercial products such as honey or silk. To cultivate beneficial nematodes and arthropods for use in agricultural or commercial industries, ways of promoting the growth and fitness of beneficial invertebrates are needed in the art.

SUMMARY OF THE INVENTION

[003] Compositions and methods for modulating the fitness of invertebrates for agriculture or commerce are disclosed herein. The composition 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 of the fitness of the host.

[004] In one aspect, there is provided herein a method of increasing the fitness of a honey bee, the method including administering to the honey bee a composition comprising an effective amount of an organophosphate insecticide metabolizing bacteria formulated with an insect edible carrier.

[005] In some embodiments, administration involves distributing the composition into a honey bee hive or at least a habitat where the honey bee grows, lives, breeds or feeds.

[006] In some embodiments, the composition may be a liquid, a solid, an aerosol, a paste, a gel or a gas.

[007] In some embodiments, the organophosphate insecticide may be fenitrothion.

[008] In some embodiments, the carrier may be a seed coating.

[009] In some embodiments, the honey bee may be in a honey bee colony.

[0010] In another aspect, there is provided herein a composition including an effective amount of an organophosphate insecticide metabolizing bacteria formulated with an insect edible carrier in the form of a liquid, a solid, an aerosol, a paste, a gel or a gas .

[0011] In some embodiments of the second aspect, organophosphate insecticide metabolizing bacteria metabolize fenitrothion.

[0012] In some embodiments of the second aspect, the carrier is a seed coating.

[0013] In some embodiments of the second aspect, the organophosphate insecticide metabolizing bacteria are present at a concentration of at least 100,000 cells/mL (e.g., at least about 100,000 cells/mL, at least about 150,000 cells/mL /ml, at least about 200,000 cells/ml, at least about 250,000 cells/ml, at least about 300,000 cells/ml, at least about 350,000 cells/ml, at least about 400,000 cells /ml, at least about 450,000 cells/ml, or at least about 500,000 cells/ml).

[0014] In yet another aspect, the composition includes an agent that alters a level, activity, or metabolism of one or more resident microorganisms in an insect host, the alteration resulting in an increase in the fitness of the insect host.

[0015] In another instance, the composition includes an agent that alters a level, activity, or metabolism of one or more microorganisms residing in a host nematode, the alteration resulting in an increase in the fitness of the host nematode.

[0016] In some embodiments of any of the above compositions, the one or more microorganisms may be a bacteria or fungus resident in the host. In some embodiments, the host-resident 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, Acetobacter spp, Cyanobacteria spp, Salmonella spp, Rhodococcus spp, Pseudomonas spp, Lactobacillus spp, Enterococcus spp, Alcaligenes spp, Klebsiella spp, Paenibacillus spp, Arthrobacter spp, Corynebacterium spp, Brevibacterium spp, Thermus spp, Pseudomonas spp, Clostridium spp, and Escherichia spp. In some embodiments, the host-resident fungus is at least one selected from the group consisting of Candida, Metschnikowia, Debaromyces, Starmerella, Pichia, Cryptococcus, Pseudozyma, Symbiotaphrina bucneri, Symbiotaphrina kochii, Scheffersomyces shehatae, Scheffersomyces stipites, Cryptococcus, Trichosporon, Amylostereum areolatum , Epichloe spp, Pichia pinus, Hansenula capsulate, Daldinia decipien, Ceratocytis spp, Ophiostoma spp, and Attamyces bromatificus.

[0017] In any of the above compositions, the agent, which hereinafter may also be referred to as a modulating agent, can alter the growth, division, viability, metabolism and/or longevity of the microorganism residing in the host. In any of the above embodiments, the modulating agent can decrease the viability of the one or more microorganisms resident in the host. In some embodiments, the modulating agent enhances the growth or viability of the one or more microorganisms resident in the host.

[0018] In any of the above embodiments, the modulating agent is a phage, a polypeptide, a small molecule, an antibiotic, a bacterium, or any combination thereof.

[0019] In some embodiments, the phage binds to a cell surface protein on a host-resident bacterium. In some embodiments, the phage is virulent to a resident bacteria 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 .

[0020] In some embodiments, the polypeptide is at least one of a bacteriocin, R-type bacteriocin, nodule-specific C-rich peptide, antimicrobial peptide, lysine, or bacteriocyte regulatory peptide.

[0021] In some embodiments, the small molecule is a metabolite.

[0022] In some embodiments, the antibiotic is a broad-spectrum antibiotic.

[0023] In some embodiments, the modulating agent is a naturally occurring bacteria. In some embodiments, the bacterium 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. In some embodiments, 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, Acetobacter spp, Cyanobacteria spp, Salmonella spp, Rhodococcus spp, Ps eudomonas spp, Lactobacillus spp, Enterococcus spp, Alcaligenes spp, Klebsiella spp, Paenibacillus spp, Arthrobacter spp, Corynebacterium spp, Brevibacterium spp, Thermus spp, Pseudomonas spp, Clostridium spp, and Escherichia spp. In certain cases, the bacteria is a naturally occurring bacteria that is capable of degrading pesticides such as organophosphate insecticides (eg phosphorothioate, eg fenitrothion).

[0024] In any of the above compositions, the fitness of the host can be measured by the survival, reproduction or metabolism of the host. In some embodiments, the modulating agent modulates host fitness by decreasing the host's pesticide susceptibility (e.g., susceptibility to a pesticide listed in Table 12). In some embodiments, the pesticide susceptibility is bactericide or fungicide susceptibility. In some embodiments, the pesticide susceptibility is insecticide or nematicide susceptibility.

[0025] In any of the above compositions, the composition may include a plurality of different modulating agents. In some embodiments, the composition includes a modulating agent and a pesticidal agent (e.g., a pesticide listed in Table 12). In some embodiments, the pesticidal agent is a bactericidal or fungicidal agent. In some embodiments, the pesticidal agent is an insecticidal or nematicidal agent.

[0026] In any of the above compositions, the composition may include a modulating agent and a crop growth enhancing agent.

[0027] In any of the above compositions, the modulating agent may be attached to a second moiety. In some embodiments, the second moiety is a modulating agent.

[0028] In any of the above compositions, the modulating agent may be linked to a targeting domain. In some embodiments, the targeting domain directs the modulating agent to a target site in the host. In some embodiments, the targeting domain directs the modulating agent to the one or more microorganisms resident in the host.

[0029] In any of the above compositions, the modulating agent may include a pre- or pro-inactivating sequence, thereby forming a precursor modulating agent. In some embodiments, the precursor modulating agent is converted to an active form in the host.

[0030] In any of the above compositions, the modulating agent may include a binder. In some embodiments, the linker is a cleavable linker.

[0031] In any of the above compositions, the composition may additionally include a carrier. In some cases, the carrier may be an agriculturally acceptable carrier.

[0032] In any of the above compositions, the composition may additionally include a host bait, a sticking agent or a combination thereof. In some embodiments, the bait for the host is an edible agent and/or a chemoattractant.

[0033] In any of the above compositions, the composition may be at a dose effective to modulate host fitness.

[0034] In any of the above compositions, the composition may be formulated for administration to a microorganism inhabiting the intestine of the host.

[0035] In any of the above compositions, the composition may be formulated for administration to a microorganism inhabiting a bacteriocyte of the host and/or the intestine of the host. In some embodiments, the composition can be formulated for administration to a plant. In some embodiments, the composition can be formulated for use in a host feeding station.

[0036] In any of the above compositions, the composition can be formulated as a liquid, a powder, granules or nanoparticles. In some embodiments, the composition is formulated as one selected from the group consisting of a liposome, polymer, bacterial secretion peptide, and synthetic nanocapsule. In some embodiments, the synthetic nanocapsule delivers the composition to a target site in the host. In some embodiments, the target site is the intestine of the host. In some embodiments, the target site is a bacteriocyte in the host.

[0037] In a further aspect, hosts that include any of the above compositions are also provided herein. In some embodiments, the host is an insect. In some embodiments, the insect assists in pest control, pollination, generation of a commercial product, or a combination thereof. In some embodiments, the insect is a species belonging to Coccinellidae, Carabidae, Mantidae, Syrphidae, Lampyridae, Myrmeliontidase, Chrysopidae, Hemerobiidae, Brachonidae, Ichneumonidae or Odonata. In some embodiments, the insect is a species belonging to the Andrenidae, Apidae, Colletidae, Halicitdae, or Megahlidae. In some embodiments, the insect is a species belonging to Bombycidae or Saturniidae. In certain embodiments, the insect is a honeybee or silkworm.

[0038] In some embodiments, the host is a nematode. In some embodiments, the nematode is a species belonging to Heterorhabditis or Steinernema.

[0039] In yet a further aspect, also provided herein is a system for modulating the fitness of a host comprising a modulating agent that targets a microorganism that is required for the fitness of a host, wherein the system is effective to modulate the fitness of the host, and wherein the host is an insect or nematode. The modulating agent can include any of the compositions described herein. In some embodiments, the modulating agent is formulated as a powder. In some embodiments, the modulating agent is formulated as a solvent. In some embodiments, the modulating agent is formulated as a concentrate. In some embodiments, the modulating agent is formulated as a diluent. In some embodiments, the modulating agent is prepared for administration by combining any of the foregoing compositions with a carrier.

[0040] In another aspect, also provided herein are methods for modulating the fitness of an insect or nematode using any of the compositions described herein. In one case, the method of modulating the fitness of an insect or nematode host includes administering the composition of any one of the preceding claims to the host, wherein the modulating agent targets the one or more microorganisms resident in the host, and thereby modulates the fitness of the host. In another instance, the method of modulating microbial diversity in an insect or nematode host includes administering the composition of any one of the preceding claims to the host, wherein the modulating agent targets the one or more microorganisms resident in the host, and thereby modulates the microbial diversity in the host.

[0041] In some embodiments of any of the above methods, the modulating agent can 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 can alter the function of the one or more microorganisms resident in the host. In some embodiments, the one or more microorganisms can 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.

[0042] In some embodiments of any of the above methods, the administration step may include providing the modulating agent at a dose and time sufficient to affect the one or more microorganisms, thereby modulating microbial diversity in the host. In some embodiments, the administering step includes topically applying any of the foregoing compositions to a plant. In some embodiments, the administering step includes providing the modulating agent via a genetically engineered plant. In some embodiments, the administering step includes providing the modulating agent to the host in the form of an edible product. In some embodiments, the administering step includes providing a host containing the modulating agent. In some embodiments, the host containing the modulating agent can transmit the modulating agent to one or more additional hosts.

[0043] In some embodiments of any of the above methods, the composition is effective to enhance the health and/or survival of the host. In some embodiments, the composition is effective to increase host fitness, increase host lifespan, increase effective pollination, increase generation of a host product, increase host reproduction, or a combination thereof. In some embodiments, the composition is effective to decrease host sensitivity to a pesticidal agent (e.g., a pesticide listed in Table 12). In certain embodiments, the pesticidal agent is a neonicotinoid (e.g., imidacloprid). In certain embodiments, the pesticidal agent is an organophosphate insecticide (e.g., a phosphorothioate, e.g., fenitrothion). In some embodiments, the composition is effective to increase host resistance to an allelochemical agent produced by a plant. In some embodiments, the allelochemical agent is toxic to the host prior to administration of the composition. In some embodiments, the allelochemical agent is caffeine, cystatin from soybean N, monoterpenes, diterpene acids, or phenolic compounds.

[0044] In some embodiments of any of the above methods, the host is an insect. In some embodiments, the insect assists in pest control, pollination, generation of a commercial product, waste degradation, or a combination thereof. In some embodiments, the insect is a species belonging to Coccinellidae, Carabidae, Mantidae, Syrphidae, Lampyridae, Myrmeliontidase, Chrysopidae, Hemerobiidae, Brachonidae, Ichneumonidae or Odonata. In some embodiments, the insect is a species belonging to the Andrenidae, Apidae, Colletidae, Halicitdae, or Megahlidae. In some embodiments, the insect is a species belonging to Bombycidae or Saturniidae. In certain embodiments, the insect is a honeybee or silkworm.

[0045] In some embodiments, the host is a nematode. In some embodiments, the nematode is a species belonging to Heterorhabditis or Steinernema.

[0046] In some embodiments of any of the above methods, the administering step includes administering any of the foregoing compositions to a plant. In some embodiments, the plant is an agricultural crop. In some embodiments, the culture is a non-harvested culture at the time of administration. In some embodiments, the culture is a culture taken at the time of administration. In some embodiments, the crop comprises harvested fruits or vegetables. In some embodiments, the composition is administered in an amount and for a period of time effective to enhance culture growth. In some embodiments, the crop includes corn, soybean or wheat plants.

[0047] In another aspect, screening assays for identifying a modulating agent that modulates the fitness of a host are also provided herein. In one case, the screening assay for identifying a modulating agent that modulates the fitness of a host includes the steps of (a) exposing a microorganism that may reside in the host to one or more candidate modulating agents and (b) identifying a modulating agent which increases host fitness.

[0048] In some embodiments of the screening assay, the modulating agent is a host-resident microorganism. In some embodiments, the microorganism is a bacterium. In some embodiments, the bacterium, when resident in the host, increases the fitness of the host. In some embodiments, the bacteria degrades a pesticide (eg, a pesticide listed in Table 12). In certain embodiments, the pesticide is a neonicotinoid (e.g., imidacloprid) or an organophosphate insecticide (e.g., a phosphorothioate, e.g., fenitrothion). In some embodiments, the bacterium secretes an amino acid. In certain embodiments, wherein the amino acid is methionine.

[0049] In some embodiments of the screening assay, the modulating agent affects an allelochemical degradation microorganism. In some embodiments, the modulating agent is a phage, an antibiotic or a test compound. In certain embodiments, the antibiotic is timentin or azithromycin.

[0050] In some embodiments of the screening assay, the host may be an invertebrate. In some embodiments, the invertebrate is an insect or a nematode. In certain embodiments, the insect is a honey bee. In other particular embodiments, the insect is a silkworm.

[0051] In any of the above embodiments of the screening assay, the fitness of the host can be modulated by altering the microbiota of the host.

Definitions

[0052] As used herein, the term "bacteriocin" refers to a peptide or polypeptide that has antimicrobial properties. Naturally occurring bacteriocins are produced by certain prokaryotes and act against organisms related to the producing strain, but not against the producing strain itself. Bacteriocins contemplated herein include, but are not limited to, naturally occurring bacteriocins, such as bacteriocins produced by bacteria, or derivatives thereof, such as engineered bacteriocins, recombinantly expressed bacteriocins, or chemically synthesized bacteriocins. In some cases, the bacteriocin is a functionally active variant of the bacteriocins described here. In some cases, the bacteriocin variant 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% of identity, for example, over a specified region or over the entire sequence, with a sequence of a bacteriocin described herein or a naturally occurring bacteriocin.

[0053] As used herein, the term "bacteriocyte" refers to a specialized cell present in certain insects where intracellular bacteria reside with symbiotic bacterial properties.

[0054] As used herein, the term "effective amount" refers to an amount of a modulating agent (e.g., a phage, lysine, bacteriocin, small molecule or antibiotic) or composition including said agent sufficient to effect the indicated result , for example, to increase or promote the fitness of a host organism (eg, insect); to achieve a target level (e.g., a predetermined or threshold level) of concentration of a modulating agent within a target host; to achieve a target level (e.g., a predetermined or threshold level) of concentration of a modulating agent within the gut of a target host; to achieve a target level (e.g., a predetermined or threshold level) of concentration of a modulating agent within a target host bacteriocyte; to modulate the level or activity of one or more microorganisms (eg, endosymbiont) in the target host.

[0055] As used herein, the term "fitness" refers to the ability of a host organism to survive, grow, and/or to produce surviving progeny. The fitness of an organism can be measured by one or more parameters, including, but not limited to, lifespan, reproductive rate, mobility, body weight and/or metabolic rate. Fitness can be additionally measured based on measures of activity (eg pollination) or product production (eg honey or silk).

[0056] As used herein, the term "gut" refers to any portion of the intestine of a host, including the foregut, midgut, or hindgut of the host.

[0057] As used herein, the term "host" refers to an organism (eg, insect) containing resident microorganisms (eg, endogenous microorganisms, endosymbiotic microorganisms (eg, primary or secondary endosymbionts), commensal organisms, and/or pathogenic microorganisms).

[0058] As used herein, "increasing host fitness" or "promoting host fitness" refers to any favorable alteration of host physiology or any activity performed 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) increase a population of a host by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80 %, 90%, 95%, 99%, 100% or more; (2) increase the reproductive rate of a host (e.g. insect, e.g. bee or silkworm) by about 10%, 20%, 30%, 40%, 50%, 60%, 70% , 80%, 90%, 95%, 99%, 100% or more; (3) increase the mobility of a host (e.g. insect, e.g. bee or silkworm) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (4) increase the body weight of a host (e.g., insect, e.g., bee or silkworm) by about 10%, 20%, 30%, 40%, 50%, 60%, 70 %, 80%, 90%, 95%, 99%, 100% or more; (5) increase the metabolic rate or activity of a host (e.g. insect, e.g. bee or silkworm) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (6) increase pollination (e.g. number of plants pollinated in a given time period) by a host (e.g. insect, e.g. bee or silkworm) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (7) increase the production of by-products (e.g. honey from a honey bee or silk from a silkworm) from a host (e.g. insect, e.g. bee or silkworm) by about of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (8) increase host (e.g., insect) nutrient content (e.g., protein, fatty acids, or amino acids) by about 10%, 20%, 30%, 40%, 50%, 60%, 70% , 80%, 90%, 95%, 99%, 100% or more; or (9) increase host resistance to a pesticide (eg, a neonicotinoid (eg, imidacloprid) or an organophosphate insecticide (eg, a phosphorothioate, eg, fenitrothion)) by about 10%, 20%, 30 %, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more. An increase in host fitness can be determined compared to a host organism to which the modulating agent has not been administered.

[0059] The term "insect" includes any organism belonging to the phylum Arthropoda and the class Insecta or the class Arachnida, at any stage of development, that is, immature or adult insects.

[0060] As used herein, "lysine", 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 bacteria. Lysines contemplated herein include, but are not limited to, naturally occurring lysines, such as lysines produced by phages, lysines produced by bacteria, or derivatives thereof, such as engineered lysines, recombinantly expressed lysines, or chemically synthesized lysines. A functionally active bacteriocin variant can 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% of identity, for example, over a specified region or over the entire sequence, with a sequence of a synthetic, recombinant, or naturally derived bacteriocin, including any described herein.

[0061] As used herein, the term "microorganism" refers to bacteria or fungi. Microorganisms may refer to microorganisms residing in a host organism (eg, endogenous microorganisms, endosymbiotic microorganisms (eg, primary or secondary endosymbionts)) or microorganisms exogenous to the host, including those that can act as modulating agents. As used herein, the term "target microorganism" refers to a microorganism that resides in the host and is impacted by a modulating agent, either directly or indirectly.

[0062] As used herein, the term "agent" or "modulating agent" refers to an agent that is capable of altering the levels and/or functioning of microorganisms residing in a host organism (e.g., insect), and thereby modulate (eg, increase) the fitness of the host organism (eg, insect).

[0063] As used herein, the term "pesticide" or "pesticide agent" refers to a substance that can be used in the control of agricultural, environmental, or household/household pests, such as insects, fungi, bacteria, or viruses. The term "pesticide" is understood to encompass naturally occurring or synthetic insecticides (larvicides or adulticides), insect growth regulators, acaricides (miticides), nematicides, ectoparasiticides, bactericides, fungicides or herbicides (substances that may be used in agriculture to control or modify plant growth). Additional examples of pesticides or pesticidal agents are listed in Table 12. In some cases, the pesticide is an allelochemical. As used herein, "allelochemical" or "allelochemical agent" is a substance produced by one organism that can effect a physiological function (e.g., germination, growth, survival, or reproduction) of another organism (e.g., an insect or nematode host ).

[0064] As used herein, the term "peptide," "protein," or "polypeptide" encompasses any chain of naturally occurring or unnaturally occurring amino acids (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, for example, of one or more non-aminoacyl groups (for example, sugar, lipid, etc.) covalently attached to the peptide, and includes, for example, natural proteins, polypeptides and synthetic peptides, or recombinant, hybrid molecules, peptoids or peptidomimetics.

[0065] As used herein, "percent identity" between two sequences is determined by the BLAST 2.0 algorithm, which is described in Altschul et al., J. Mol. Biol. 215:403-410 1990). Software to perform BLAST analyzes is publicly available from the National Center for Biotechnology Information.

[0066] As used herein, the term "phage" or "bacteriophage" refers to a virus that infects and replicates in bacteria. Bacteriophages replicate within bacteria after injecting their genome into the cytoplasm, and they 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 can be a naturally occurring phage isolate, or an engineered phage, including vectors, or nucleic acids encoding a partial phage genome (e.g., including at least all of the essential genes needed to drive the phage life cycle within a host bacterium) or the complete genome of the phage.

[0067] As used herein, the term "plant" refers to whole plants, plant organs, plant tissues, seeds, plant cells, seeds, and progeny thereof. Plant cells include, without limitation, seed cells, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, or microspores. Parts of plants include differentiated or 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., isolated cells, protoplasts, embryos or callus tissue). The plant tissue can be in a plant or in a plant organ, tissue or cell culture. Additionally, a plant can 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.

[0068] The terms "obtainable by", "produced by" or similar are used to indicate that a claim or embodiment refers to a compound, composition, product, etc. per se, i.e. that the compound, composition, product, etc. can be obtained or produced by a method that is described for the manufacture of the compound, composition, product, etc., but that the compound, composition, product, etc. it can also be obtained or produced by other methods than described. The terms "obtained by", "produced by", or the like indicate that the compound, composition, product, is obtained or produced by a specific disclosed method. It should be understood that the terms "obtainable by", "produced by" and the like also disclose the terms "obtainable by", "produced by" and the like as a preferred embodiment of "obtainable by", "produced by" and the like.

[0069] Other features and advantages of the invention will be clear from the following Detailed Description and Claims.

BRIEF DESCRIPTION OF THE FIGURES

[0070] The Figures are intended to be illustrative of one or more features, aspects or embodiments of the invention and are not intended to be limiting.

[0071] Figure 1 is a graph showing the time to reach the adult stage of embryos in Drosophila melanogaster. Drosophila melanogaster embryos were reared on a diet seeded with Corynebacterium glutamicum (a strain that produces glutamate - C. glutamicum Glu) or on an axenic diet without any bacteria. The percentage of adults emerging from their pupae was measured every 12 hours from the time of emergence of the first adult. Organisms reared on a bacteria-supplemented diet reached adulthood faster than their non-bacteria counterparts.

[0072] Figure 2A is a graph showing the effects of male gender on developmental rate differences in Drosophila melanogaster. The adults emerging from Figure 1 were classified according to sex and their emergence rate was graphically represented.

[0073] Figure 2B is a graph showing the effects of female gender on developmental rate differences in Drosophila melanogaster. The adults emerging from Figure 1 were classified according to sex and their emergence rate was graphically represented. The enhancement of development rate in females due to the presence of bacteria in the diet is significantly greater than in their male counterparts. The benefits of the presence of bacteria in the fly's diet are higher in females compared to males.

[0074] Figure 3 is a graph showing which strains of C. glutamicum promoted larval biomass. Larvae reared on a diet supplemented with C. glutamicum strains producing glutamate or methionine are larger than those reared on a sterilized diet or a diet supplemented with Escherichia coli. Larvae areas are measured as the number of pixels in the larval images. Medians and 95% confidence intervals are shown as lines on the graph.

[0075] Figure 4 is a graph panel showing the results of a Seahorse flow assay for bacterial respiration. Bacteria were grown to log phase and loaded onto Seahorse XFe96 plates for temporal measurements of rate of oxygen consumption (OCR) and rate of extracellular acidification (ECAR) as described in methods. Treatments were injected into the wells after approximately 20 minutes and the bacteria were monitored for changes in growth. Rifampicin = 100 µg/mL; Chloramphenicol = 25 µg/mL; Phage (T7 for E. coli and ΦSmVL-C1 for Serratia marcescens) lysates were diluted 1:2 or 1:100 in SM Buffer. Markers on each line are only provided as indicators of the condition each line corresponds to, and are not data point indicators.

[0076] Figure 5 is a graph showing that a phage against S. marcescens reduced fly mortality. The flies that were bitten with S. marcescens all died within a one-day period, whereas a sizable portion of the flies that were bitten with both S. marcescens and the phage survived for five days after treatment. Almost all of the control flies that were not treated at all survived to the end of the experiment. Log-rank test was used to compare curves for statistical significance, asterisk designates p<0.0001.

DETAILED DESCRIPTION

[0077] Methods and compositions for agricultural use are provided herein, for example, for altering a level, activity or metabolism of one or more resident microorganisms in a nematode or arthropod host (for example, honeybee or silkworm), the change resulting in an increase in host fitness. 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 microbiota in a way that is beneficial to the host. By promoting favorable microbial levels, microbial activity, microbial metabolism and/or microbial diversity, the modulating agent described here can be used to increase the fitness of a variety of beneficial nematodes or arthropods, such as bees and silkworms, used in agriculture and trade.

[0078] The methods and compositions described here are based in part on examples that illustrate how different agents, for example, microorganisms that degrade imidacloprid, microorganisms that degrade fenitrothion, and different phages can be used on host insects, such as honey bees or Drosophila, to indirectly improve the health of these hosts by altering the level, activity or metabolism of microorganisms within these hosts. Microorganisms that degrade imidacloprid are a representative example of microorganisms that degrade neonicotinoids and more generally are representative of microorganisms that degrade insecticides or pesticides. Similarly, microorganisms that degrade fenitrothion are a representative example of microorganisms that degrade organophosphate insecticides and more generally are representative of microorganisms that degrade insecticides or pesticides. On this basis, the present disclosure describes a variety of different approaches to using agents that alter a level, activity or metabolism of one or more microorganisms residing in a host, the alteration resulting in a modulation of host fitness.

I. Hosts i. Host

[0079] The host of any of the compositions or methods described herein may be any organism belonging to the phyla Nematoda (e.g., nematodes, e.g., beneficial nematodes) or Arthropoda (e.g., insects, e.g., beneficial insects), including any arthropods described here. In some cases, the host is a beneficial insect or nematode (eg, a pollinator, a natural competitor of a pest, or a producer of substances useful to humans). The term "beneficial insect" or "beneficial nematode", as used herein, refers to an insect or nematode that confers a benefit (eg, economic and/or ecological) to humans, animals, an ecosystem and/or the environment. For example, the host can be an insect that is involved in the production of a commercial product, including, but not limited to, insects cultivated to produce food (e.g., honey from honey bees, e.g., Apis mellifera), materials ( such as silk from Bombyx mori) and/or substances (eg lacquer from Laccifer lacca or pigments from Dactylopius coccus and Cynipidae). Additionally, the host may include insects or nematodes that are used in agricultural applications, including insects that aid in crop pollination, seed dissemination, or pest control. Also, in some cases, the host may be an insect that is useful for waste disposal and/or organic recycling (eg earthworms, termites or Diptera larvae).

[0080] In some cases, the host produces a usable product (eg honey, silk, beeswax or shellac). In some cases, the host is a bee. Exemplary bee genera include, but are not limited to, Apis, Bombus, Trigona, and Osmia. In some cases, the bee is a honeybee (for example, an insect belonging to the genus Apis). In some cases, the honey bee is the species Apis mellifera (the European or Western honey bee), Apis cerana (the Asian, Eastern or Himalayan honey bee), Apis dorsata (the "giant" honey bee), Apis florea (the "red dwarf" honey bee), Apis andreniformis (the "black dwarf" honey bee), or Apis nigrocincta. In some cases, the host is a silkworm. The silkworm may be a species in the Bombycidae or Saturniidae family. In some cases, the silkworm is Bombyx mori. In some cases, the host is a lacquer mealybug. The lacquer cochineal may be a species in the Kerriidae family. In some cases, the lacquer cochineal is Kerria lacca.

[0081] In some cases, the host assists the pollination of a plant (eg, bees, beetles, wasps, flies, butterflies, or moths). In some examples, the host aiding pollination of a plant is a beetle. In some cases, the beetle is a species of the family Buprestidae, Cantharidae, Cerambycidae, Chrysomelidae, Cleridae, Coccinellidae, Elateridae, Melandryidae, Meloidae, Melyridae, Mordellidae, Nitidulidae, Oedemeridae, Scarabaeidae or Staphyllinidae. In some cases, the host aiding the pollination of a plant is a butterfly or moth (eg, Lepidoptera). In some cases, the butterfly or moth is a species in the family Geometriidae, Hesperiidae, Lycaenidae, Noctuidae, Nymphalidae, Papilionidae, Pieridae or Sphingidae. In some cases, the host assisting the pollination of a plant is a fly (eg Diptera). In some cases, the fly is from the family Anthomyiidae, Bibionidae, Bombyliidae, Calliphoridae, Cecidomiidae, Certopogonidae, Chrionomidae, Conopidae, Culicidae, Dolichopodidae, Empididae, Ephydridae, Lonchopteridae, Muscidae, Mycetophilidae, Phoridae, Simuliidae, Stratiomyidae or Syrphidae. In some cases, the host aiding pollination is an ant (eg Formicidae), hornet (eg Tenthredinidae) or wasp (eg Sphecidae or Vespidae). In some cases, the host aiding pollination of a plant is a bee. In some cases, the bee is from the family Andrenidae, Apidae, Colletidae, Halictidae or Megachilidae.

[0082] In some cases, the host assists pest control. In some cases, the host aiding pest control is a predatory nematode. In particular examples, the nematode is a species of Heterorhabditis or Steinernema. In some cases, the host aiding pest control is an insect. For example, the host assisting pest control could be a species belonging to the family Braconidae (for example, parasitoid wasps), Carabidae (for example, ground beetles), Chrysopidae (for example, green scavengers), Coccinellidae (for e.g. ladybugs), Hemerobiidae (e.g. brown fireflies), Ichneumonidae (e.g. Ichneumon wasps), Lampyridae (e.g. fireflies), Mantidae (e.g. praying mantis), Myrmeleontidae (e.g. lion ants), Odonata (e.g. dragonflies and zygopterans) or Syrphidae (e.g. syrphid). In other cases, the host aiding pest control is an insect that competes with an insect that is considered a pest (for example, an agricultural pest). For example, the Mediterranean fruit fly, Ceratitis capitata, is a common pest of fruits and vegetables worldwide. One way to control C. capitata is to release the sterilized male insect into the environment to compete with wild males to mate with females. In these cases, the host may be a sterilized male belonging to a species that is typically considered a pest.

[0083] In some cases, the host assists the degradation of waste or organic material. In some examples, the host aiding the degradation of waste or organic material belongs to Coleoptera or Diptera. In some cases, the host belonging to Diptera is from the family Calliphoridae, Curtonotidae, Drosophilidae, Fanniidae, Heleomyzidae, Milichiidae, Muscidae, Phoridae, Psychodidae, Scatopsidae, Sepsidae, Sphaeroceridae, Stratiomyidae, Syrphidae, Tephritidae or Ulidiidae. In some cases, the host belonging to Coleoptera is from the family Carabidae, Hydrophilidae, Phalacaridae, Ptiliidae or Staphylinidae.

[0084] In particular cases, the modulating agents disclosed herein can be used to increase the fitness of host honey bees or silkworms.

ii. Host Aptitude

[0085] The methods and compositions provided herein can be used to enhance the fitness of any of the hosts described herein. Increased fitness can arise from any changes in microorganisms resident in the host, where the changes are a consequence of administration of a modulating agent and have beneficial or advantageous effects on the host.

[0086] In some cases, increased host fitness may manifest as an improvement in host physiology (eg, improved health or survival) as a result of administration of a modulating agent. In some cases, the fitness of an organism may be measured by one or more parameters, including, but not limited to, reproductive rate, life expectancy, motility, fecundity, body weight, metabolic rate or activity, or survival in comparison with a host organism to which the modulating agent has not been administered. For example, the methods or compositions provided herein can be effective to improve the overall health of the host or to improve the overall survival of the host compared to a host organism to which the modulating agent has not been administered. In some cases, the improved host survival is about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more than than 100% greater than a reference level (eg, a level present in a host not receiving a modulating agent). In some cases, the methods and compositions are effective in improving the reproduction (e.g., reproductive rate) of the host compared to a host organism to which the modulating agent has not been administered. In some cases, the methods and compositions are effective in increasing other physiological parameters, such as mobility, body weight, lifespan, 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 (eg, a level present in a host not receiving a modulating agent).

[0087] In some cases, the increased fitness of the host may manifest itself as an increased production of a product generated by said host compared to a host organism to which the modulating agent has not been administered. In some cases, the methods or compositions provided herein may be effective in increasing the production of a host-generated product as described herein (e.g. honey, beeswax, bee bread, propolis, silk or lacquer) by about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more than 100% relative to a reference level (for example, a level present in a host that does not receive a modulating agent).

[0088] In some cases, increased host fitness may manifest as an increase in the frequency or effectiveness of a desired activity performed by the host (e.g., pollination, pest predation, seed dissemination, or degradation of waste or material organic) compared to a host organism to which the modulating agent was not administered. In some cases, the methods or compositions provided herein may be effective in increasing the frequency or effectiveness of a desired activity performed by the host (e.g., pollination, pest predation, seed spreading, or degradation of waste or organic material) by about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more than 100% relative to a reference level (e.g. , a level present in a host that does not receive a modulating agent).

[0089] In some cases, increased host fitness may manifest as an increased production of one or more nutrients in the host (e.g., vitamins, carbohydrates, amino acids, or polypeptides) compared to a host organism to which the agent modulator was not administered. In some cases, the methods or compositions provided herein may be effective in increasing host nutrient production (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 present in a host not receiving a modulating agent) . In some cases, the methods or compositions provided herein can increase nutrients in the host by increasing nutrient production by one or more microorganisms (e.g., endosymbiont) in the host.

[0090] In some cases, increased host fitness may manifest as a decrease in host sensitivity to a pesticidal agent (e.g., a pesticide listed in Table 12) and/or an increase in host resistance to an agent pesticide (e.g., a pesticide listed in Table 12) compared to a host organism to which the modulating agent was not administered. In some cases, the methods or compositions provided herein can be effective in decreasing host 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 present in a host not receiving a modulating agent ). In some cases, host sensitivity to the pesticidal agent is altered by administration of a modulating agent that degrades a pesticidal agent (e.g., a bacterium that degrades pesticides, e.g., a bacterium that degrades neonicotinoids, or a bacterium that degrades organophosphate insecticides). . The pesticidal agent can be any pesticidal agent known in the art, including insecticidal agents. In some cases, the pesticidal agent is a neonicotinoid (eg imidacloprid) or an organophosphate insecticide (eg a phosphorothioate, eg fenitrothion). In some cases, the methods or compositions provided herein may decrease the host's sensitivity to a pesticidal agent (eg, a pesticide listed in Table 12) by increasing the host's ability to metabolize or degrade the pesticidal agent into usable substrates.

[0091] In some cases, host sensitivity to the pesticidal agent is altered by administration of a modulating agent that detoxifies a xenobiotic.

[0092] In some cases, increased host fitness may manifest as a decrease in host sensitivity to an allelochemical agent and/or an increase in host resistance to an allelochemical agent compared to a host organism to which the agent modulator was not administered. In some cases, the methods or compositions provided herein may be effective in increasing host 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 (eg, a level present in a host that does not receive a modulating agent). In some cases, the allelochemical agent is caffeine, cystatin from N soybean seeds, monoterpenes, diterpene acids, or phenolic compounds. In some cases, the methods or compositions provided herein can decrease the host's sensitivity to an allelochemical agent by increasing the host's ability to metabolize or degrade the allelochemical agent into usable substrates.

[0093] In some cases, the methods or compositions provided herein may be effective in increasing host resistance to parasites or pathogens (e.g., fungal, bacterial, or viral pathogens; or parasitic mites (e.g., Varroa destructor mite on honey bees) )) compared to a host organism to which the modulating agent was not administered. In some cases, the methods or compositions provided herein may be effective in increasing host resistance to a pathogen or parasite (e.g., fungal, bacterial, or viral pathogens; or parasitic mites (e.g., Varroa destructor mite on honeybees)) by about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more than 100% relative to a reference level (eg, a level present in a host that does not receive a modulating agent).

[0094] In some cases, increased host fitness may manifest itself as other fitness advantages, such as improved tolerance to certain environmental factors (e.g., a high or low temperature tolerance), improved ability to survive in certain habitats, or an improved ability to maintain a certain diet (eg, an improved ability to metabolize soy versus corn) compared to a host organism to which the modulating agent was not administered. In some cases, the methods or compositions provided herein may be effective in increasing host fitness in any of the plurality of ways described herein. Furthermore, the modulating agent can increase host fitness in any number of host classes, orders, families, genera, or species (e.g., host species 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 host species). In some cases, the modulating agent acts on a single host class, order, family, genus, or species.

[0095] Host fitness can be assessed using any standard methods of the art. In some cases, host fitness can be assessed by evaluating an individual host. Alternatively, host fitness can be assessed by evaluating a host population. For example, an increase in host fitness may manifest itself as an increase in successful competition against other insects, thereby leading to an increase in host population size.

iii. Invertebrate hosts in agriculture

[0096] By enhancing the fitness of nematodes or beneficial insects, the modulating agents provided herein may be effective in promoting the growth of plants that benefit from said hosts. The modulating agent can be administered using any formulations and methods of administration described herein, in an amount and for a period of time effective to increase the fitness of the hosts of interest and thereby benefit the plant (e.g., increase crop growth, increase crop yields, decrease pest infestation and/or decrease plant damage). This may or may not involve direct application of the modulating agent to the plant. For example, in cases where the primary host habitat is different from the region of plant growth, the modulating agent can be applied to the primary host habitat, the plants of interest, or a combination of both.

[0097] In some cases, the plant may be an agricultural crop, such as a cereal, grain, legume, fruit, or vegetable crop. The compositions described herein can be administered to the crop at any time before or after harvesting the cereal, grain, legume, fruit or vegetable. Crop yield is an often used measurement, for example, for a cereal, grain or legume and is usually measured in metric tons per hectare (or kilograms per hectare). Crop yield can also refer to the actual seed generation of the plant. In some cases, the modulating agent may be effective in increasing crop yields (e.g., increasing metric tons of cereal, grain, legume, fruit or vegetable per hectare and/or increasing seed generation) by about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more compared to a reference level (for example, a crop to which the modulating agent was not administered).

[0098] In some cases, the plant (eg crop) may be at risk of developing a pest infestation (eg, by an insect) or may have already developed a pest infestation. The methods and compositions described herein can be used to reduce or prevent pest infestation on such crops by promoting the fitness of beneficial insects that attack agricultural pests. In some cases, the modulating agent may be effective in reducing crop infestation (e.g., reducing the number of infested plants, reducing pest population size, or reducing plant injury) by about 2%, 5%, 10 %, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more compared to a reference level (e.g., a culture to which the modulating agent has not been administered). In other cases, the modulating agent may be effective in preventing or reducing the likelihood of crop infestation by about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more compared to a reference level (eg, a culture to which the modulating agent has not been administered).

[0099] Any suitable plant tissue can benefit from the compositions and methods described herein, including, but not limited to, somatic embryos, pollen, leaves, stems, calluses, stolons, microtubers, and shoots. The methods described here may include the treatment of angiosperms and gymnosperms such as acacia, alfalfa, apple, apricot, artichoke, ash, asparagus, avocado, banana, barley, beans, beet, birch, beech, blackberry, blueberry, broccoli, cabbage Brussels sprouts, cabbage, canola, cantaloupe, carrot, cassava, cauliflower, cedar, a cereal, celery, chestnut, cherry, Chinese cabbage, citrus, clementine, clover, coffee, corn, cotton, cowpea, cucumber , cypress, eggplant, elm, endive, eucalyptus, broad bean, fennel, figs, fir, geranium, grape, grapefruit, mendubis, physalis, hemlock, walnut, collard greens, kiwi, kohlrabi, larch, lettuce, leek, lemon, lime, carob, pine, maidenhair, maize, mango, maple, melon, millet, mushroom, mustard, dried fruit, oak, oats, okra, onion, orange, an ornamental plant or flower or tree, papaya, palm, parsley parsnip, pea, peach, peanut, pear, peat, pepper, persimmon, guinea pea, pine, pineapple, plantain, plum, pomegranate, potato, pumpkin, chicory, radish, rapeseed, raspberry, rice, rye , sorghum, willow, soybean, spinach, spruce, zucchini, strawberry, sugar beet, sugar cane, sunflower, sweet potato, sweet corn, tangerine, tea, tobacco, tomato, trees, triticale, lawns, turnips, a vine , walnut, watercress, watermelon, wheat, yams, yew and courgette.

II. Target microorganisms

[00100] The 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. Host-resident microorganisms may include, for example, symbiotic (eg, endosymbiotic microorganisms that provide beneficial nutrients or enzymes to the host), commensal, pathogenic, or parasitic microorganisms. A symbiotic microorganism (e.g., bacteria or fungi) may be an obligate host symbiont or a facultative host symbiont. Host-resident microorganisms can be acquired by any mode of transmission, including vertical, horizontal, or multiple sources of transmission.

I. Bacteria

[00101] Exemplary bacteria that can be targeted according to 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, Cyanobacteria spp, Salmonella spp, Rhodococcus spp, Pseudomonas spp, Lactobacillus spp, Enterococcus spp, Alcaligenes spp, Klebsiella spp, Paenibacillus spp, Arthrobacter spp, Corynebacterium spp, Brevibacterium spp, Thermus spp, Pseudomonas spp, Clostridium spp, and Escherichia spp. Non-limiting examples of bacteria that can be targeted by the methods and compositions provided herein are shown in Table 1. Table 1: Examples of Target Bacteria and Insect Hosts

[00102] Any number of bacterial species can be targeted by the compositions or methods described herein. For example, in some cases, the modulating agent may target a single bacterial species. In some cases, the modulating agent can 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. In some instances, the modulating agent can 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 50. 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. In some cases, the modulating agent can 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.

[00103] In some cases, the modulating agent can increase a population of one or more bacteria (e.g., symbiotic bacteria) by at least about any of 10%, 20%, 30%, 40%, 50%, 60% , 70%, 80%, 90% or more in the host compared to a host organism to which the modulating agent was not administered. In some cases, the modulating agent can reduce the population of one or more bacteria (e.g., pathogenic or parasitic bacteria) by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in the host compared to a host organism to which the modulating agent was not administered. In some cases, the modulating agent can eradicate the population of a bacteria (eg, a pathogenic or parasitic bacteria) in the host. In some cases, the modulating agent can increase 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 and/or decreases 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 compared to a host organism to which the modulating agent has not been administered.

[00104] In some cases, the modulating agent may change the bacterial diversity and/or bacterial composition of the host. In some cases, the modulating agent can increase 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 compared to a host organism to which the modulating agent was not administered. In some cases, the modulating agent can decrease 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 compared to a host organism to which the modulating agent was not administered.

[00105] In some cases, the modulating agent can alter the function, activity, growth and/or division of one or more bacterial cells. For example, the modulating agent can alter the expression of one or more genes in bacteria. In some cases, the modulating agent can alter the function of one or more proteins in the bacteria. In some cases, the modulating agent can alter the function of one or more cellular structures (eg, cell wall, outer or inner membrane) in bacteria. In some cases, the modulating agent can kill (eg, lyse) the bacteria.

[00106] The target bacteria can reside in one or more parts of the insect. Furthermore, the target bacteria can be intracellular or extracellular. In some cases, the bacteria reside in or on one or more parts of the host's gut, including, for example, the foregut, midgut, and/or hindgut. For example, in honeybees (eg, Apis mellifera), bacterial symbionts confined to the hindguts of adults are acquired within the first few days after emergence of adults from the pupal stage, through social interactions with other adult worker bees in the colony. The inhabitants of the intestines of honey bees belong to a small number of distinctive lineages present only in honey bees and also in other Apis species and in Bombus species (bumblebees). In some cases, the target bacteria reside on a honeybee. In some cases, one or more bacteria targeted on the honey bee is a Snodgrassella spp. (eg Snodgrasella alvi), a Gilliamella spp. (eg Gilliamella apicola), a Bartonella spp. (e.g. Bartonella apis), a Parasaccharibacter spp. (for example Parasaccharibacter apium) or a Lactobacillus spp. (e.g. Lactobacillus kunkeei, Lactobacillus Firm-4).

[00107] In some cases, the bacteria reside as intracellular bacteria within an insect host cell. In some cases, the bacteria reside in a bacteriocyte of the host insect.

[00108] Changes in bacterial populations in the host can 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 (eg, FISH), spectrophotometry, matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS), and DNA sequencing. In some cases, a sample of the host treated with a modulating agent is sequenced (eg, by metagenomic sequencing of rRNA or 16S rDNA) to determine the microbiome of the host following delivery or administration of the modulating agent. In some cases, a sample from a host that has not received the modulating agent is also sequenced to provide a reference.

II. fungi

[00109] Exemplary fungi that can be targeted according to 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 present in insects include Candida, Metschnikowia, Debaromyces, Scheffersomyces shehatae and Scheffersomyces stipites, Starmerella, Pichia, Trichosporon, Cryptococcus, Pseudozyma, and yeast-like symbionts of the subphylum Pezizomycotina (e.g., Symbiotaphrina bucneri and Symbiotaphrina kochii). Non-limiting examples of yeast that can be targeted by the methods and compositions herein are listed in Table 2. Table 2: Examples of Yeast on Insects

[00110] Any number of fungal species can be targeted by the compositions or methods described herein. For example, in some cases, the modulating agent may target a single fungal species. In some cases, the modulating agent can 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. In some instances, the modulating agent can 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 50. 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. In some cases, the modulating agent can 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.

[00111] In some cases, the modulating agent can increase a 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 compared to a host organism to which the modulating agent was not administered. In some cases, the modulating agent can reduce the 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 compared to a host organism to which the modulating agent was not administered. In some cases, the modulating agent can eradicate the population of a fungus (eg, pathogenic or parasitic fungi) in the host. In some cases, the modulating agent can 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 pathogenic fungi by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in the host compared to a host organism to which the modulating agent was not administered.

[00112] In some cases, the modulating agent can change the fungal diversity and/or fungal composition of the host. In some cases, the modulating agent can increase 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 compared to a host organism to which the modulating agent was not administered. In some cases, the modulating agent can decrease 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 compared to a host organism to which the modulating agent was not administered.

[00113] In some cases, the modulating agent can alter the function, activity, growth and/or division of one or more fungi. For example, the modulating agent can alter the expression of one or more genes in the fungus. In some cases, the modulating agent can alter the function of one or more proteins in the fungus. In some cases, the modulating agent can alter the function of one or more cellular components in the fungus. In some cases, the modulating agent can kill the fungus.

[00114] In addition, the target fungus may reside in one or more parts of the insect. In some cases, the fungus resides in or on one or more parts of the insect's gut, including, for example, the foregut, midgut, and/or hindgut. In some cases, the fungus lives extracellularly in the hemolymph, adipose bodies or in specialized structures in the host.

[00115] Changes in the fungal population in the host can 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 (eg, FISH), spectrophotometry, matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS), and DNA sequencing. In some cases, a host sample treated with a modulating agent is sequenced (eg, by metagenomic sequencing) to determine the host's microbiome after delivery or administration of the modulating agent. In some cases, a sample from a host that has not received the modulating agent is also sequenced to provide a reference.

III. Modulating Agents

[00116] 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, or any combination thereof.

Phage

[00117] The modulating agent described herein may include a phage (e.g., a lytic phage or a non-lytic phage). In some cases, an effective concentration of any phage described herein may alter a level, activity, or metabolism of one or more microorganisms (as described herein) residing in a host described herein, the alteration resulting in an increase in host fitness. In some cases, the modulating agent includes at least one phage selected from the order Tectiviridae, Myoviridae, Siphoviridae, Podoviridae, Caudovirales, Lipothrixviridae, Rudiviridae or Ligamenvirales. In some cases, 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 . Other non-limiting examples of phages useful in the methods and compositions are listed in Table 3. Table 3: Examples of Phages and Target Bacteria

[00118] In some cases, a modulating agent includes a lytic phage. Thus, upon administration of the lytic phage to a resident bacterial cell in the host, the phage causes lysis of the target bacterial cell. In some cases, the lytic phage targets and kills a resident bacterium on an insect host to increase the host's fitness. Alternatively or additionally, the modulating agent phage can be a non-lytic phage (also referred to as lysogenic or temperate phage). Thus, after administration of the non-lytic phage to a bacterial cell residing in the host, the bacterial cell may remain viable and be able to stably maintain the expression of genes encoded in the phage genome. In some cases, a non-lytic phage is used to alter gene expression in a resident bacterium in an insect host to increase host fitness. In some cases, the modulating agent includes a mixture of lytic and non-lytic phage.

[00119] The modulating agent described herein may include a phage with a narrow or broad range of bacterial targets. In some cases, the phage has a narrow range of bacterial targets. In some cases, the phage is a promiscuous phage with a wide range of bacterial targets. For example, the promiscuous phage can target at least about any 5, 10, 20, 30, 40, 50 or more bacteria resident in the host. A phage with a narrow bacterial targeting range can target a specific bacterial strain in the host without affecting other, eg non-target, bacteria in the host. For example, the phage can target no more than about any 50, 40, 30, 20, 10, 8, 6, 4, 2 or 1 bacteria resident in the host. For example, the compositions described here may target the bacterial pathogen Paenibacillus larvae in honey bees but may not target other bacteria (e.g., symbiotic bacteria) including Lactobacillus Firm4, Lactobacillus Firm5, Bifidobacterium sp, Snodgrassella alvi, Gilliamella apicola, Bartonella apis, Parasaccharibacter apium or Lactobacillus kunkeei. In some cases, the phage infects one or more specific bacteria by binding to a cell surface protein (eg OmpF, LamB, BtuB, TolC, etc.) or a different recognition molecule (eg glycolipids, LPS, lipoteichoic acid, etc.).

[00120] The compositions described herein can include any number of phages, such as at least about any one of 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100 or more phages. In some cases, the composition includes phages from one or more families (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more phage families), one or more orders (for example, 2 , 3, 4, 5, 6, 7, 8, 9, 10 or more phage orders) or one or more species (e.g. 1, 2, 3, 4, 5, 10, 15, 20, 30, 40 , 50, 100 or more phage species). Compositions including one or more phages are also referred to herein as "phage cocktails". Cocktails of phages are useful because they allow you to target a wider range of bacterial hosts. Furthermore, they allow each bacterial strain (i.e., subspecies) to be targeted by multiple orthogonal phages, thereby preventing or significantly delaying the emergence of resistance. In some cases, a cocktail includes multiple phages targeting one bacterial species. In some cases, a cocktail includes multiple phages targeting multiple bacterial species. In some cases, a phage "cocktail" includes a single promiscuous phage (ie, a phage with a wide host range) targeting many strains within a species.

[00121] Appropriate phage concentrations in the modulating agent described herein depend on factors such as efficacy, survival rate, phage transmissibility, number of distinct phages and/or types of lysine in the compositions, formulation, and methods of applying the composition. In some cases, the phage is in a liquid or solid formulation. In some cases, the concentration of each phage in any of the compositions described herein is at least about any of 102, 103, 104, 105, 106, 107, 108, 109, 1010 or more pfu/ml. In some cases, the concentration of each phage in any of the compositions described herein is no more than about any of 102, 103, 104, 105, 106, 107, 108, 109 pfu/ml. In some instances, the concentration of each phage in the composition is any from about 102 to about 103, about 103 to about 104, about 104 to about 105, about 105 to about 106, about 107 to about 108, about 108 to about 109, about 102 to about 104, about 104 to about 106, about 106 to about 109, or about 103 to about 108 pfu/mL. In some cases, where the composition includes at least two types of phage, the concentration of each type of phage can be the same or different. For example, in some cases, the concentration of one phage in the cocktail is about 108 pfu/ml and the concentration of a second phage in the cocktail is about 106 pfu/ml.

[00122] A modulating agent including a phage as described herein may be contacted with the target host in an amount and for a time sufficient to: (a) achieve a target level (e.g., a predetermined or threshold level) of concentration of the phage inside a target host; (b) achieving a target level (e.g., a predetermined or threshold level) of phage concentration within the gut of a target host; (c) achieving a target level (e.g., a predetermined or threshold level) of phage concentration within a bacteriocyte of a target host; (d) modulate the level, or an activity, of one or more microorganisms (eg, endosymbiont) in the target host; or/and (e) modulate the fitness of the target host.

[00123] As illustrated by Example 7 and 11, phages can be used as modulating agents by eliminating bacterial pathogens such as Serratia marcescens, Erwinia catotovora, and Pseudomonas enzomophila from host insects such as honey bees or silkworms.

ii. Polypeptides

[00124] Numerous polypeptides (eg, a bacteriocin, R-type bacteriocin, nodule-specific C-rich peptide, antimicrobial peptide, lysine, or bacteriocyte regulatory peptide) can be used in the compositions and methods described herein. In some cases, an effective concentration of any peptide or polypeptide described herein can alter a level, activity, or metabolism of one or more microorganisms (as described herein) residing in a host, the alteration resulting in an increase in host fitness. Polypeptides included herein can include naturally occurring polypeptides or recombinantly produced variants. For example, the polypeptide can 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, for example, over a specified region or over the entire sequence, with a sequence of a polypeptide described herein or a naturally occurring polypeptide.

[00125] A modulating agent comprising a polypeptide as described herein may be contacted with the target host in an amount and for a time sufficient to: (a) achieve a target level (e.g., a predetermined or threshold level) of concentration inside a target host; (b) achieving a target level (e.g., a predetermined or threshold level) of concentration within the gut of a target host; (c) achieving a target level (eg, a predetermined or threshold level) of concentration within a bacteriocyte of a target host; (d) modulate the level, or an activity, of one or more microorganisms (eg, endosymbiont) in the target host; or/and (e) modulate the fitness of the target host.

[00126] The polypeptide modulating agents discussed hereinafter, namely bacteriocins, lysines, antimicrobial peptides, nodule-specific C-rich peptides, and bacteriocyte regulatory peptides, can be used to alter the level, activity, or metabolism of microorganisms- target as indicated in the section for increasing the fitness of insects, such as honeybees or silkworms. (a) Bacteriocins

[00127] The modulating agent described herein may include a bacteriocin. In some cases, bacteriocin is naturally produced by Gram-positive bacteria such as Pseudomonas, Streptomyces, Bacillus, Staphylococcus, or lactic acid bacteria (LAB, such as Lactococcus lactis). In some cases, 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, LAB Class I-IV antibiotics (such as lantibiotics), colicins, microcins, and pyocins. Non-limiting examples of bacteriocins are listed in Table 4. Table 4: Examples of Bacteriocins

[00128] In some cases, the bacteriocin is a colicin, a pyocin, or a microcin produced by Gram-negative bacteria. In some cases, the bacteriocin is a colicin. The colicin can be a group A colicin (eg, uses the Tol system to penetrate the outer membrane of a target bacterium) or a group B colicin (eg, uses the Ton system to penetrate the outer membrane of a bacterium - target). In some cases, the bacteriocin is a microcin. The microkine can be a class I microkine (eg, <5 kDa, has post-translational modifications) or a class II microkine (eg, 5-10 kDa, with or without post-translational modifications). In some cases, the class II microkine is a class IIa microkine (eg, requires more than one gene to synthesize and assemble functional peptides) or a class IIb microkine (eg, linear peptides with or without post-translational modifications). translation at the C terminal). In some cases, the bacteriocin is a pyocin. In some cases, the piocin is an R-piocin, F-piocin or S-piocin.

[00129] In some cases, the bacteriocin is a class I, class II, class III, or class IV bacteriocin produced by Gram-positive bacteria. In some cases, the modulating agent includes a Class I bacteriocin (eg, lanthionine-containing antibiotics (lantibiotics) produced by a Gram-positive bacterium). The class I bacteriocin or lantibiotic may be a low molecular weight peptide (e.g., less than about 5 kDa) and may have post-translationally modified amino acid residues (e.g., lanthionine, β-methyl-lanthionine or dehydrated amino acids).

[00130] In some cases, the bacteriocin is a Class II bacteriocin (eg, not lantibiotics produced by Gram-positive bacteria). Many are positively charged peptides, containing no lanthionine, which, unlike lantibiotics, do not undergo extensive post-translational modification. Class II bacteriocins may belong to one of the following subclasses: "pediocin-like" bacteriocins (eg, pediocin PA-1 and carnobacteriocin X (Class IIa)); two-peptide bacteriocins (eg, lactacin F and ABP-118 (Class IIb)); circular bacteriocins (eg, carnocycline A and enterocin AS-48 (Class IIc)); or unmodified, linear, non-pediocin-type bacteriocins (eg, epidermycin NI01 and lactococcin A (Class IId)).

[00131] In some cases, the bacteriocin is a Class III bacteriocin (eg, produced by Gram-positive bacteria). Class III bacteriocins can have a molecular weight greater than 10 kDa and can be heat-labile proteins. Class III bacteriocins can be further subdivided into Group IIIA and Group IIIB bacteriocins. Group IIIA bacteriocins include bacteriolytic enzymes that kill susceptible strains by lysing the cell wall, such as Enterolysin A. Group IIIB bacteriocins include non-lytic proteins, such as Caseicin 80, Helveticin J, and lactacin B.

[00132] In some cases, the bacteriocin is a Class IV bacteriocin (eg, produced by Gram-positive bacteria). Class IV bacteriocins are a group of complex proteins, associated with other lipid or carbohydrate moieties, that appear to be required for activity. They are relatively hydrophobic and heat stable. Examples of Class IV bacteriocins are leuconocin S, lactocin 27, and lactocin S.

[00133] In some cases, 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 can bind to target cells through a receptor-binding protein, for example, a tail fiber. Ligation is followed by contraction of the sheath and insertion of the core through the envelope of the target bacteria. Penetration of the nucleus results in a rapid depolarization of the cell membrane potential and immediate cell death. Contact with a single R-type bacteriocin particle can result in cell death. An R-type bacteriocin, for example, may be thermolabile, resistant to weak acids, resistant to trypsin, sedimentable by centrifugation, resolvable by electron microscopy, or a combination of these. Other R-type bacteriocins can be complex molecules including multiple proteins, polypeptides or subunits, and can resemble a tail structure of bacteriophages of the Myoviridae family. In naturally occurring R-type bacteriocins, the subunit structures can be encoded by a bacterial genome, such as that of C. difficile or P. aeruginosa, and form R-type bacteriocins to act as natural defenses against other bacteria. In some cases, the R-type bacteriocin is a pyocin. In some cases, the piocin is an R-piocin, F-piocin or S-piocin.

[00134] In some cases, the bacteriocin is a functionally active variant of the bacteriocins described here. In some cases, the bacteriocin variant 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% of identity, for example, over a specified region or over the entire sequence, with a sequence of a bacteriocin described herein or a naturally occurring bacteriocin.

[00135] In some cases, bacteriocin can be biomanipulated, according to standard methods, to modulate its bioactivity, for example, increase or decrease or regulate, or to specify its target microorganisms. In other cases, the bacteriocin is produced by the translation machinery (eg, a ribosome, etc.) of a microbial cell. In some cases, bacteriocin is synthesized chemically. Some bacteriocins can be derived from a polypeptide precursor. The polypeptide precursor may undergo cleavage (eg, processing by a protease) to yield the bacteriocin polypeptide itself. As such, in some cases, the bacteriocin is produced from a precursor polypeptide. In a few different instances, the bacteriocin includes a polypeptide that has undergone post-translational modifications, for example, cleavage or the addition of one or more functional groups.

[00136] The bacteriocins described herein may be formulated into a composition for any of the uses described herein. The compositions disclosed herein can 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 depend on factors such as efficacy, stability of the bacteriocin, number of distinct types of bacteriocins in the compositions, formulation, and methods of application of the composition. In some cases, each bacteriocin in a liquid composition ranges from about 0.01 ng/ml to about 100 mg/ml. In some cases, each bacteriocin in a solid composition ranges from about 0.01 ng/g to about 100 mg/g. In some cases, where the composition includes at least two types of bacteriocins, the concentration of each type of 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).

[00137] Bacteriocins can neutralize (e.g., kill) at least one microorganism other than the individual bacterial cell in which the polypeptide is produced, including cells clonally related to the bacterial cell and other microbial cells. As such, a bacterial cell can exert cytotoxic or growth inhibitory effects on a variety of microbial organisms by secreting bacteriocins. In some cases, the bacteriocin targets and kills one or more species of bacteria residing in the host via pore formation in the cytoplasmic membrane, cell wall interference (e.g., peptidoglycanase activity), or nuclease activity (e.g., DNase activity, 16S rRNase activity or tRNase activity).

[00138] In some cases, bacteriocin has a neutralizing activity. Neutralizing activity of bacteriocins can 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 cell cycle arrest.

[00139] In some cases, bacteriocin has exterminating activity. The mechanism of killing of bacteriocins is specific to each group of bacteriocins. In some cases, bacteriocin has small-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 producing strain (small-spectrum bioactivity). In some cases, the bacteriocin has broad-spectrum bioactivity against a wide range of genera.

[00140] In some cases, bacteriocins interact with a receptor molecule or an anchoring molecule on the target bacterial cell membrane. For example, nisin is extremely potent against its target bacterial strains, exhibiting antimicrobial activity even at single-digit nanomolar concentrations. 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.

[00141] In some cases, bacteriocin has antifungal activity. Some bacteriocins with anti-yeast or anti-fungal activity have been identified. For example, Bacillus bacteriocins have been shown to have neutralizing activity against some strains of yeast (see, for example, Adetunji and Olaoye, Malaysian Journal of Microbiology 9:130-13, 2013). In another example, a peptide from Enterococcus faecalis was shown to have neutralizing activity against Candida species (see, for example, Shekh and Roy, BMC Microbiology 12:132, 2012). In another example, 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 Issue 2, 2008). In another example, botrycidin AJ1316 and allyrin B1 from B. subtilis were shown to have antifungal activities.

[00142] A modulating agent including a bacteriocin as described herein may be contacted with the target host in an amount and for a period of time sufficient to: (a) reach a target level (e.g., a predetermined or threshold level) concentration of bacteriocin within a target host; (b) achieving a target level (e.g., a predetermined or threshold level) of bacteriocin concentration within the gut of a target host; (c) achieving a target level (e.g., a predetermined or threshold level) of bacteriocin concentration within a bacteriocyte of a target host; (d) modulate the level, or an activity, of one or more microorganisms (eg, endosymbiont) in the target host; or/and (e) modulate the fitness of the target host.

(b) Lysines

[00143] The modulating agent described herein may include a lysine (eg also known as a murein hydrolase or autolysin to peptidoglycan). Any lysine suitable for inhibiting a resident bacteria in the host can be used. In some cases, the lysine is such that it can be naturally produced by a bacterial cell. In some cases, the lysine is such that it can be naturally produced by a bacteriophage. In some cases, the lysine is obtained from a phage that inhibits a resident bacteria in the host. In some cases, the lysine is engineered based on a naturally occurring lysine. In some cases, lysine is engineered to be secreted by a host bacterium, for example, by introducing a signal peptide into lysine. In some cases, lysine is used in combination with a phage holin. In some cases, a lysine is expressed by a recombinant host bacterium that is not sensitive to lysine. In some cases, lysine is used to inhibit a Gram-positive or Gram-negative bacteria residing in the host.

[00144] Lysine may consist of any class of lysine and may have one or more substrate specificities. For example, lysine can be a glycosidase, an endopeptidase, a carboxypeptidase or a combination thereof. In some cases, lysine cleaves the β-1-4 glycosidic bond in the sugar portion of the cell wall, the amide bond connecting the sugar and peptide portions of the bacterial cell wall, and/or the peptide bonds between the peptide portions. of the cell wall. Lysine may belong to one or more lysine-specific groups, depending on the site of cleavage within the peptidoglycan. In some cases, lysine is an N-acetyl-β-D-muramidase (eg, lysozyme), lytic transglycosylase, N-acetyl-β-D-glucosaminidase, N-acetylmuramyl-L-alanine amidase, L, D-endopeptidase, D,D-endopeptidase, D,D-carboxypeptidase, L,D-carboxypeptidase or L,D-transpeptidase. Non-limiting examples of lysines and their activities are listed in Table 5. Table 5: Examples of Lysines

[00145] In some cases, lysine is a functionally active variant of the lysines described here. In some cases, the lysine variant 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% of identity, for example, over a specified region or over the entire sequence, with a sequence of a lysine described herein or a naturally occurring lysine.

[00146] In some cases, lysine can be biomanipulated to modulate its bioactivity, for example, increase or decrease or regulate or to specify a target microorganism. In some cases, lysine is produced by the translation machinery (eg, a ribosome, etc.) of a microbial cell. In some cases, lysine is chemically synthesized. In some cases, lysine is derived from a polypeptide precursor. The polypeptide precursor can undergo cleavage (eg, processing by a protease) to yield the polypeptide from lysine itself. As such, in some cases, lysine is produced from a precursor polypeptide. In some cases, lysine includes a polypeptide that has undergone post-translational modifications, for example, cleavage or the addition of one or more functional groups.

[00147] The lysines described herein can be formulated into a composition for any of the uses described herein. The compositions disclosed herein can include any number or type (e.g., grades) of lysines, such as at least about any one of 1 lysine, 2, 3, 4, 5, 10, 15, 20 or more lysines. An appropriate concentration of each lysine in the composition depends on factors such as efficacy, lysine stability, number of distinct lysines, the formulation, and methods of application of the composition. In some cases, each lysine in a liquid composition ranges from about 0.1 ng/ml to about 100 mg/ml. In some cases, each lysine in a solid composition ranges from about 0.1 ng/g to about 100 mg/g. In some cases, where the composition includes at least two types of lysines, the concentration of each type of lysine can be the same or different.

[00148] A modulating agent including a lysine as described herein may be contacted with the target host in an amount and for a period of time sufficient to: (a) reach a target level (e.g., a predetermined or threshold level) of lysine concentration within a target host; (b) achieving a target level (e.g., a predetermined or threshold level) of lysine concentration within the gut of a target host; (c) achieving a target level (e.g., a predetermined or threshold level) of lysine concentration within a bacteriocyte of a target host; (d) modulate the level, or an activity, of one or more microorganisms (eg, endosymbiont) in the target host; or/and (e) modulate the fitness of the target host.

(c) Antimicrobial Peptides

[00149] The modulating agent described herein may include an antimicrobial peptide (AMP). Any AMP suitable for inhibiting a host-resident microorganism can be used. AMPs are a diverse group of molecules, which are divided into subgroups based on their amino acid composition and structure. AMP can be derived from or produced from any organism that naturally produces AMP, including AMP derived from plants (e.g. copsin), insects (e.g. drosocin, scorpion peptide (e.g. Uy192, UyCT3, D3, D10 , Uy17, Uy192), mastoparan, poneratoxin, cecropine, moricin, melittin), frogs (e.g., magainin, dermaseptin, aurein), and mammals (e.g., cathelicidins, defensins, and protegrins). For example, the AMP can be a scorpion peptide such as Uy192 (5'-FLSTIWNGIKGLL-3'; SEQ ID NO: 193), UyCT3 (5-LSAIWSGIKSLF-3; SEQ ID NO: 194'), D3 ( 5'- LWGKLWEGVKSLI-3'; SEQ ID NO: 195), and D10 (5'- FPFLKLSLKIPKSAIKSAIKRL-3'; SEQ ID NO: 196), Uy17 (5'- ILSAIWSGIKGLL-3'; SEQ ID NO: 197). Other non-limiting examples of AMP are listed in Table 6. Table 6: Examples of Antimicrobial Peptides

[00150] AMP can be active against any number of target microorganisms. In some cases, AMP may have antibacterial and/or antifungal activities. In some cases, AMP can have a small-spectrum bioactivity or a broad-spectrum bioactivity. For example, some AMPs target and kill only certain species of bacteria or fungi, while others are active against gram-negative and gram-positive bacteria as well as fungi.

[00151] In addition, AMP can act through some known mechanisms of action. For example, the cytoplasmic membrane is a frequent target of AMP, but AMP can also interfere with DNA and protein synthesis, protein folding, and cell wall synthesis. In some cases, AMP with a net cationic charge and amphipathic nature disrupt bacterial membranes, leading to cell lysis. In some cases, AMP can enter cells and interact with an intracellular target to interfere with DNA, RNA, protein, or cell wall synthesis. In addition to killing microorganisms, AMPs have been shown to have some immunomodulatory functions that are involved in clearing an infection, including the ability to alter host gene expression, act as chemokines and/or induce chemokine production, inhibit the production of pro- inflammatory reactions induced by lipopolysaccharides, promote wound healing, and modulate dendritic cell and adaptive immune cell responses.

[00152] In some cases, AMP is a functionally active variant of the AMPs described here. In some cases, the AMP variant 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% of identity, for example, over a specified region or over the entire sequence, with a sequence of an AMP described herein or a naturally derived AMP.

[00153] In some cases, AMP can be bioengineered to modulate its bioactivity, for example, increase or decrease or regulate or to specify a target microorganism. In some cases, AMP is produced by the translation machinery (eg, a ribosome, etc.) of a cell. In some cases, AMP is synthesized chemically. In some cases, AMP is derived from a polypeptide precursor. The precursor polypeptide can undergo cleavage (eg, processing by a protease) to yield the AMP polypeptide itself. As such, in some cases, AMP is produced from a precursor polypeptide. In some cases, AMP includes a polypeptide that has undergone post-translational modifications, for example, cleavage or the addition of one or more functional groups.

[00154] The AMPs described herein can be formulated into a composition for any of the uses described herein. The compositions disclosed herein can include any number or type (e.g., grades) of AMP, such as at least about any one of 1 AMP, 2, 3, 4, 5, 10, 15, 20 or more AMP. An appropriate concentration of each AMP in the composition depends on factors such as effectiveness, stability of the AMP, number of distinct AMPs in the composition, the formulation, and methods of application of the composition. In some instances, each AMP in a liquid composition ranges from about 0.1 ng/ml to about 100 mg/ml. In some cases, each AMP in a solid composition ranges from about 0.1 ng/g to about 100 mg/g. In some cases, where the composition includes at least two types of AMP, the concentration of each type of AMP may be the same or different.

[00155] A modulating agent including an AMP as described herein may be contacted with the target host in an amount and for a period of time sufficient to: (a) reach a target level (e.g., a predetermined or threshold level) AMP concentration within a target host; (b) achieving a target level (e.g., a predetermined or threshold level) of AMP concentration within the gut of a target host; (c) achieving a target level (e.g., a predetermined or threshold level) of AMP concentration within a bacteriocyte of a target host; (d) modulate the level, or an activity, of one or more microorganisms (eg, endosymbiont) in the target host; or/and (e) modulate the fitness of the target host.

(d) Nodule-specific C-rich peptides

[00156] The modulating agent described here may include a nodule-specific 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 plants with bacteria of the Rhizobiaceae family (rhizobia), resulting in the formation of root nodules where plant cells contain thousands of intracellular endosymbionts. NCR peptides possess antimicrobial properties that direct an irreversible, terminal differentiation process of bacteria, for example to permeabilize the bacterial membrane, disrupt cell division or inhibit protein synthesis. For example, in cells of Medicago truncatula nodules infected with Sinorhizobium meliloti, hundreds of NCR peptides are produced that direct the irreversible differentiation of bacteria into large polyploid nitrogen-fixing bacteroides. Non-limiting examples of NCR peptides are listed in Table 7. Table 7: Examples of NCR Peptides

[00157] Any NCR peptide known in the art is suitable for use in the methods or compositions described herein. Plants producing NCR peptides include, but are not limited to, Pisum sativum (pea), Astragalus sinicus (IRLC legume), Phaseolus vulgaris (bean), Vigna unguiculata (cowpea), Medicago truncatula (cut lucerne), and Lotus japonicus. For example, more than 600 potential NCR peptides are predicted from the genome sequence of M. truncatula and nearly 150 different NCR peptides have been detected in cells isolated from root nodules by mass spectrometry.

[00158] 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 is cleaved after translocation. The N-terminus of an NCR peptide includes a signal peptide, which may be cleavable, for targeting a secretory pathway. NCR peptides are generally small peptides with disulfide bridges that stabilize their structure. Mature NCR peptides range in length from 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 in between. those. NCR peptides can include a conserved sequence of cysteine residues with the remainder of the peptide sequence being highly variable. NCR peptides usually have about four or eight cysteines.

[00159] NCR peptides can be anionic, neutral or cationic. In some cases, synthetic cationic NCR peptides having a pI greater than about eight have antimicrobial activities. For example, NCR247 (pI = 10.15, RNGCIVDPRCPYQQCRRPLYCRRR; SEQ ID NO: 164) and NCR335 (pI = 11.22) are both effective against gram-negative and gram-positive bacteria as well as fungi. In some cases, neutral and/or anionic NCR peptides, such as NCR001 (MAQFLLFVYSLIIFLSLFFGEAAFERTETRMLTIPCTSDDDNCPKVISPCH TKCFDGFCGWYIEGSYEGP; SEQ ID NO: 165), do not have antimicrobial activities at a pI greater than about 8.

[00160] In some cases, the NCR peptide is effective in killing bacteria. In some cases, 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 spp, Cyanobacteria spp, Salmonella spp, Rhodococcus spp, Pseudomonas spp, Lactobacillus spp, Enterococcus spp, Alcaligenes spp, Klebsiella spp, Paenibacillus spp, Arthrobacter spp, Corynebacterium spp, Brevibacterium spp, Thermus spp, Pseudomonas spp, Clostridium spp, or Escherichia spp.

[00161] In some cases, the NCR peptide is a functionally active variant of an NCR peptide described here. In some cases, the NCR peptide variant 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, with a sequence of an NCR peptide described herein or naturally derived NCR peptide.

[00162] In some cases, the NCR peptide can be biomanipulated to modulate its bioactivity, for example, increase or decrease or regulate or to specify a target microorganism. In some cases, the NCR peptide is produced by the translation machinery (eg, a ribosome, etc.) of a cell. In some cases, the NCR peptide is chemically synthesized. In some cases, the NCR peptide is derived from a polypeptide precursor. The polypeptide precursor can undergo cleavage (eg, processing by a protease) to yield the NCR peptide itself. As such, in some cases, the NCR peptide is produced from a precursor polypeptide. In some cases, the NCR peptide includes a polypeptide that has undergone post-translational modifications, for example, cleavage or the addition of one or more functional groups.

[00163] The NCR peptide described herein can be formulated into a composition for any of the uses described herein. Compositions disclosed herein can 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 peptides NCR. An appropriate concentration of each NCR peptide in the composition depends on factors such as efficacy, stability of the NCR peptide, number of distinct NCR peptides, the formulation, and methods of applying the composition. In some instances, each NCR peptide in a liquid composition ranges from about 0.1 ng/ml to about 100 mg/ml. In some cases, each NCR peptide in a solid composition ranges from about 0.1 ng/g to about 100 mg/g. In some cases, where the composition includes at least two types of NCR peptides, the concentration of each type of NCR peptide may be the same or different.

[00164] A modulating agent including an NCR peptide as described herein may be contacted with the target host in an amount and for a period of time sufficient to: (a) reach a target level (e.g., a predetermined or threshold level ) NCR peptide concentration within a target host; (b) achieving a target level (e.g., a predetermined or threshold level) of NCR peptide concentration within the gut of a target host; (c) achieving a target level (e.g., a predetermined or threshold level) of NCR peptide concentration within a bacteriocyte of a target host; (d) modulate the level, or an activity, of one or more microorganisms (eg, endosymbiont) in the target host; or/and (e) modulate the fitness of the target host.

(e) Bacteriocyte Regulatory Peptides

[00165] The modulating agent described herein may include a bacteriocyte regulatory peptide (BRP). BRP are peptides expressed in insect bacteriocytes. These genes are first expressed at a time of development coinciding with the incorporation of symbionts and their specific expression in bacteriocytes is maintained throughout the life of the insect. In some cases, BRP has a hydrophobic amino terminal domain, which is predicted to be a signal peptide. Additionally, some BRPs have a cysteine-rich domain. In some cases, the bacteriocyte regulatory peptide is a bacteriocyte-specific cysteine-rich protein (BCR). Bacteriocyte regulatory peptides are between about 40 and 150 amino acids in length. In some instances, 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. Non-limiting examples of BRP and their activities are listed in Table 8. Table 8: Examples of Bacteriocyte Regulatory Peptides

[00166] In some cases, BRP alters the growth and/or activity of one or more resident bacteria in the host bacteriocyte. In some cases, BRP can be bioengineered to modulate its bioactivity (eg, increase, decrease, or regulate) or to specify a target microorganism. In some cases, BRP is produced by the translation machinery (eg, a ribosome, etc.) of a cell. In some cases, BRP is chemically synthesized. In some cases, BRP is derived from a polypeptide precursor. The polypeptide precursor may undergo cleavage (eg, processing by a protease) to yield the BRP polypeptide itself. As such, in some cases, BRP is produced from a precursor polypeptide. In some cases, the BRP includes a polypeptide that has undergone post-translational modifications, for example, cleavage or the addition of one or more functional groups.

[00167] Functionally active variants of the BRPs as described herein are also useful in the compositions and methods described herein. In some cases, the BRP variant 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% of identity, for example, over a specified region or over the entire sequence, with a sequence from a BRP described herein or naturally derived BRP.

[00168] The BRP described herein can be formulated into a composition for any of the uses described herein. The compositions disclosed herein can include any number or type (e.g. grades) of BRP, such as at least about any one of 1 BRP, 2, 3, 4, 5, 10, 15, 20 or more BRP. An appropriate concentration of each BRP in the composition depends on factors such as effectiveness, stability of the BRP, number of different BRPs, the formulation, and methods of application of the composition. In some cases, each BRP in a liquid composition ranges from about 0.1 ng/mL to about 100 mg/mL. In some cases, each BRP in a solid composition ranges from about 0.1 ng/g to about 100 mg/g. In some cases, where the composition includes at least two types of BRP, the concentration of each type of BRP may be the same or different.

[00169] A modulating agent including a BRP as described herein may be contacted with the target host in an amount and for a period of time sufficient to: (a) reach a target level (e.g., a predetermined level or threshold) BRP concentration within a target host; (b) achieving a target level (e.g., a predetermined or threshold level) of BRP concentration within the gut of a target host; (c) achieving a target level (e.g., a predetermined or threshold level) of BRP concentration within a bacteriocyte of a target host; (d) modulate the level, or an activity, of one or more microorganisms (eg, endosymbiont) in the target host; or/and (e) modulate the fitness of the target host.

iii. Small Molecules

[00170] Numerous small molecules (eg, an antibiotic or a metabolite) can be used in the compositions and methods described herein. In some cases, an effective concentration of any small molecule described herein can alter the level, activity, or metabolism of one or more microorganisms (as described herein) residing in a host, the alteration resulting in an increase in host fitness.

[00171] A modulating agent comprising a small molecule as described herein may be contacted with the target host in an amount and for a period of time sufficient to: (a) reach a target level (e.g., a predetermined or threshold level ) the concentration of a small molecule inside a target host; (b) achieving a target level (e.g., a predetermined or threshold level) of concentration of a small molecule within the gut of a target host; (c) achieving a target level (e.g., a predetermined or threshold level) of concentration of a small molecule within a bacteriocyte of a target host; (d) modulate the level, or an activity, of one or more microorganisms (eg, endosymbiont) in the target host; or/and (e) modulate the fitness of the target host.

[00172] The small molecules discussed hereafter, namely antibiotics and secondary metabolites, can be used to alter the level, activity, or metabolism of target microorganisms as indicated in the sections for increasing the fitness of insects such as honey bees and honey bees -silk.

antibiotics

[00173] The modulating agent described herein may include an antibiotic. Any antibiotic known in the art can be used. Antibiotics are usually classified based on their mechanism of action, chemical structure or spectrum of activity.

[00174] The antibiotic described here can target any bacterial function or growth processes and can be either bacteriostatic (eg, slows down or prevents bacterial growth) or bactericidal (eg, kills bacteria). In some cases, the antibiotic is a bactericidal antibiotic. In some cases, the bactericidal antibiotic is one that targets the bacterial cell wall (eg, penicillins and cephalosporins); one that targets the cell membrane (eg, polymyxins); or one that inhibits essential bacterial enzymes (for example, rifamycins, lipiarmycins, quinolones, and sulfonamides). In some cases, the bactericidal antibiotic is an aminoglycoside. In some cases, the antibiotic is a bacteriostatic antibiotic. In some cases, the bacteriostatic antibiotic targets protein synthesis (eg, macrolides, lincosamides, and tetracyclines). Additional classes of antibiotics that may be used here include cyclic lipopeptides (such as daptomycin), glycylcyclines (such as tigecycline), oxazolidinones (such as linezolid), or lipiarmycins (such as fidaxomicin). Examples of antibiotics include rifampicin, ciprofloxacin, doxycycline, ampicillin, and polymyxin B. Non-limiting examples of antibiotics are shown in Table 9. Table 9: Examples of Antibiotics

[00175] The antibiotic described here may have any level of target specificity (eg, narrow or broad spectrum). In some cases, the antibiotic is a small-spectrum antibiotic, and thus targets specific types of bacteria, such as gram-negative or gram-positive bacteria. Alternatively, the antibiotic can be a broad-spectrum antibiotic that targets a broad range of bacteria.

[00176] The antibiotics described herein may be formulated into a composition for any of the uses described herein. The compositions disclosed herein can 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). An appropriate concentration of each antibiotic in the composition depends on factors such as efficacy, antibiotic stability, number of different antibiotics, the formulation, and methods of application of the composition. In some cases, where the composition includes at least two types of antibiotics, the concentration of each type of antibiotic may be the same or different.

[00177] A modulating agent including an antibiotic as described herein may be contacted with the target host in an amount and for a period of time sufficient to: (a) reach a target level (e.g., a predetermined or threshold level) of antibiotic concentration within a target host; (b) achieving a target level (e.g., a predetermined or threshold level) of antibiotic concentration within the gut of a target host; (c) achieving a target level (e.g., a predetermined or threshold level) of antibiotic concentration within a bacteriocyte of a target host; (d) modulate the level, or an activity, of one or more microorganisms (eg, endosymbiont) in the target host; or/and (e) modulate the fitness of the target host. (B)

Secondary metabolites

[00178] In some cases, the modulating agent of the compositions and methods described herein includes a minor metabolite. Secondary metabolites are derived from organic molecules produced by an organism. Secondary metabolites can act (i) as competitive agents used against bacteria, fungi, amoebae, plants, insects, and large animals; (ii) as metal carriers; (iii) as agents of symbiosis between microbes and plants, nematodes, insects, and higher animals; (iv) as sex hormones; and (v) as differentiation effectors. Non-limiting examples of secondary metabolites are found in Table 10. Table 10: Examples of Secondary Metabolites

[00179] The secondary metabolite used herein may include a metabolite from any known group of secondary metabolites. For example, secondary metabolites can be classified 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, non-ribosomal peptides, ribosomally synthesized and post-translationally modified peptides, polyphenols, polysaccharides (eg, chitosan), and biopolymers. For an in-depth review of secondary metabolites see, eg, Vining, Annu. Rev. Microbiol. 44:395-427, 1990.

[00180] 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. In some cases, one or more minor metabolites described herein are isolated from a high-throughput screen (HTS) for antimicrobial compounds. For example, an HTS screen identified 49 antibacterial extracts that have specificity against gram-positive and gram-negative bacteria from over 39,000 raw extracts of organisms growing in diverse ecosystems in a specific region. In some cases, the secondary metabolite is transported within a bacteriocyte.

[00181] In some cases, the small molecule is an inhibitor of vitamin synthesis. In some cases, the vitamin synthesis inhibitor is an analogue of a vitamin precursor. In certain cases, the analogue of a vitamin precursor is pantothenol.

[00182] In some cases, the small molecule is an amino acid analogue. In certain cases, the amino acid analogue 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.

[00183] In some cases, the small molecule is a natural antimicrobial compound, such as propionic acid, levulinic acid, trans-cinemaldehyde, nisin or low molecular weight chitosan. The secondary metabolite described herein can be formulated into a composition for any of the uses described herein. The compositions disclosed herein can include any number or type (e.g., classes) of minor metabolites, such as at least about any one of 1 minor metabolite, 2, 3, 4, 5, 10, 15, 20 or more minor metabolites. A suitable concentration of each minor metabolite in the composition depends on factors such as efficacy, stability of the minor metabolite, number of distinct minor metabolites, the formulation, and methods of application of the composition. In some cases, where the composition includes at least two types of secondary metabolites, the concentration of each type of secondary metabolite may be the same or different.

[00184] A modulating agent including a secondary metabolite as described herein may be brought into contact with the target host in an amount and for a period of time sufficient to: (a) reach a target level (e.g., a predetermined or threshold level ) secondary metabolite concentration within a target host; (b) achieving a target level (e.g., a predetermined or threshold level) of secondary metabolite concentration within the gut of a target host; (c) achieving a target level (e.g., a predetermined or threshold level) of secondary metabolite concentration within a bacteriocyte of a target host; (d) modulate the level, or an activity, of one or more microorganisms (eg, endosymbiont) in the target host; or/and (e) modulate the fitness of the target host.

iv. Bacteria as modulating agents

[00185] In some cases, the modulating agent described here includes one or more bacteria. Numerous bacteria are useful in the compositions and methods described herein. In some cases, the agent is a bacterial species endogenously present in the host. In some cases, the bacterial modulating agent is an endosymbiotic bacterial species. Non-limiting examples of bacteria that can be used as modulating agents include all bacterial species described here in Section II of the detailed description and those listed in Table 1 beginning on page 15. For example, the modulating agent can be a bacterial species from any phyla bacteria present in the intestines of insects, including Gammaproteobacteria, Alphaproteobacteria, Betaproteobacteria, Bacteroidetes, Firmicutes (eg, Lactobacillus and Bacillus spp.), Clostridia, Actinomycetes, Spirochetes, Verrucomicrobia, and Actinobacteria.

[00186] In some cases, the modulating agent is a bacterium that promotes microbial diversity or otherwise alters the host microbiota in a favorable manner. In one case, bacteria can be provided to promote microbiome development in honey bees. For example, the modulating agent may include, for example, Bartonella apis, Parasaccharibacter apium, Frischella perrara, Snodgrassella alvi, Gilliamela apicola, Bifidobacterium spp or Lactobacillus spp.

[00187] The bacterial modulating agents discussed here can be used to alter the level, activity or metabolism of target microorganisms as indicated in the sections for increasing the fitness of insects such as honeybees and silkworms.

[00188] In some cases, such bacterial modulating agents are bacteria that are capable of degrading pesticides as shown in Table 12 including insecticides. Such insecticides include neonicotinoids such as imidacloprid or organophosphate insecticides such as fenitrothion. In some instances, pesticide metabolizing bacteria are present at a concentration of at least 100,000 cells/mL (e.g., at least about 100,000 cells/mL, at least about 150,000 cells/mL, at least about 200,000 cells/mL, at least about 250,000 cells/mL, at least about 300,000 cells/mL, at least about 350,000 cells/mL, at least about 400,000 cells/mL, at least about 450,000 cells/ml or at least about 500,000 cells/ml).

[00189] Examples 1 through 3, 5, and 6 describe how microorganisms can be identified that degrade imidacloprid and fenitrothion which can then be used as modulating agents in host insects such as honey bees, providing the treated insect hosts with a competitive advantage. It is understood that the administration of such microorganisms that degrade pesticides, for example, microorganisms that degrade imidacloprid or fenitrothion, to host insects such as honey bees is encompassed by altering a level, activity or metabolism of one or more resident microorganisms in the host.

[00190] In some cases, such bacterial modulating agents are bacteria that are capable of producing nutrients, including amino acids (eg, methionine or glutamate). The nutrient producing bacteria can be naturally occurring bacteria, for example naturally occurring bacteria exogenous to the host insect. Such bacteria can be isolated from a population of bacteria, such as those present in an environmental sample. Bacteria that produce one or more amino acids can be isolated in a manner that increases the production of amino acids in the host relative to a host to which the amino acid producing bacteria has not been administered. Amino acids that can be produced by bacteria in the host include methionine, alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glutamate, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine or valine. In certain cases, the amino acid producing bacteria are methionine producing bacteria.

[00191] In some cases, nutrient-producing bacteria (e.g., amino acid-producing bacteria, e.g., methionine-producing bacteria) are present at a concentration of at least 100 000 cells/mL (e.g., at least about 100,000 cells/mL, at least about 150,000 cells/mL, at least about 200,000 cells/mL, at least about 250,000 cells/mL, at least about 300,000 cells/mL, at least about 350,000 cells/ml, at least about 400,000 cells/ml, at least about 450,000 cells/ml, or at least about 500,000 cells/ml).

[00192] Examples 8, 9, and 10 describe how methionine-producing microorganisms can be identified which can then be used as modulating agents in host insects such as honey bees or the model organism Drosophila to increase host fitness (e.g. for example, increase the amino acid content (eg methionine content or glutamate content).

v. Modifications to modulating agents Mergers

[00193] Any of the modulating agents described herein can be fused or linked to an additional moiety. In some cases, 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). In some cases, the additional moiety is any of the modulating agents described herein (eg, a peptide, polypeptide, small molecule, or antibiotic). Alternatively, the additional moiety itself may not act as a modulating agent but may instead have a secondary function. For example, the additional moiety can help the modulating agent access, bind, or become activated at a target site in the host (e.g., in a host gut or a host bacteriocyte) or at a target microorganism residing in the host. (e.g. honeybee or silkworm).

[00194] In some cases, the additional portion may help the modulating agent to penetrate a target host cell or target microorganism residing in the host. For example, the additional portion can include a cell penetrating peptide. Cell-penetrating peptides (CPP) can be natural sequences derived from proteins; chimeric peptides that are formed by the fusion of two natural sequences; or synthetic CPP, which are synthetically engineered sequences based on structure-activity studies. In some cases, CPP have the ability to ubiquitously cross cell membranes (eg, prokaryotic and eukaryotic cell membranes) with limited toxicity. Furthermore, CPP may have the ability to cross cell membranes via energy-dependent and/or energy-independent mechanisms, without the need for chiral recognition by specific receptors. CPP can be linked to any of the modulating agents described herein. For example, a CPP can be linked to an antimicrobial peptide (AMP), for example, a scorpion peptide, for example, UY192 fused to a cell penetrating peptide (for example, YGRKKRRQRRRFLSTIWNGIKGLLFAM; SEQ ID NO: 198). Non-limiting examples of CPP are listed in Table 11. Table 11: Examples of Cell Penetrating Peptides (CPP)

[00195] In other cases, the additional portion helps the modulating agent bind to a target microorganism (eg, a fungus or bacteria) residing in the host. The additional portion may include one or more targeting domains. In some cases, the targeting domain can direct the modulating agent to one or more microorganisms (eg bacteria or fungus) residing in the gut of the host. In some cases, the targeting domain may direct the modulating agent to a specific host region (e.g., intestine or host bacteriocyte) to access microorganisms that are generally present in said host region. For example, the targeting domain can direct the modulating agent to the host's foregut, midgut, or hindgut. In other cases, the targeting domain may direct the modulating agent to a bacteriocyte in the host and/or one or more specific bacteria resident in a bacteriocyte of the host. For example, the targeting domain can be Galanthus nivalis lectin or agglutinin (GNA) linked to a modulating agent described herein, for example an AMP, for example a scorpion peptide, for example Uy192.

(b) Pre- or Pro-domains

[00196] In some cases, the modulating agent may include a pre- or pro-amino acid sequence. For example, the modulating agent can be an inactive protein or peptide that can be activated by cleavage or post-translational modification of a pre- or pro-sequence. In some cases, the modulating agent is manipulated with a pre- or pro-inactivating sequence. For example, the pre- or pro-sequence can obscure an activation site on the modulating agent, for example a receptor binding site, or it can induce a conformational change in the modulating agent. Thus, by cleaving the pre- or pro-sequence, the modulating agent is activated.

[00197] Alternatively, the modulating agent may include a small pre- or pro-molecule, for example an antibiotic. The modulating agent can be an inactive small molecule described here that can be activated in a targeted environment within the host. For example, the small molecule can be activated when a certain pH is reached in the host gut.

(c) Binders

[00198] In cases where the modulating agent is connected to an additional moiety, the modulating agent may additionally include a linker. For example, the linker can be a chemical bond, for example, one or more covalent bonds or non-covalent bonds. In some cases 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 in length) . The linker can include any flexible, rigid or cleavable linkers described herein.

[00199] A flexible peptide linker can include any of those commonly used in the art, including linkers having sequences that have mostly Gly and Ser residues ("GS linker"). Flexible linkers can be useful for joining domains that require some degree of movement or interaction and can include small, non-polar (eg Gly) or polar (eg Ser or Thr) amino acids.

[00200] Alternatively, a peptide linker may be a rigid linker. Rigid linkers are useful for maintaining a fixed distance between portions and for maintaining their independent functions. Rigid linkers can also be useful when a spatial separation of domains is critical to retaining the stability or bioactivity of one or more components of 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.

[00201] In still other cases, a peptide linker may be a cleavable linker. In some cases, linkers can be cleaved under specific conditions, such as the presence of reducing reagents or proteases. In vivo cleavable linkers can utilize the reversible nature of a disulfide bond. An example includes a thrombin-sensitive sequence (e.g., PRS) between two Cys residues. In vitro thrombin treatment of CPRSC results in cleavage of the thrombin-sensitive sequence, while the reversible disulfide bond remains intact. Such linkers are known and described, for example, in Chen et al., Adv. Drug Deliv. Rev. 65(10):1357-1369, 2013. Cleavage of linkers in fusions can also be effected by proteases that are expressed in vivo under conditions in specific host cells or tissues or resident microorganisms in the host. In some cases, ligand cleavage can release a functional free modulating agent upon reaching a target site or cell.

[00202] Fusions described herein may alternatively be linked by a linker molecule, including a hydrophobic linker, such as a negatively charged sulfonate group; lipids, such as poly(--CH2--) hydrocarbon chains, such as the polyethylene glycol (PEG) group, its unsaturated variants, its hydroxylated variants, its amidated or otherwise N-containing variants, non-carbon linkers; carbohydrate binders; phosphodiester linkers or another molecule capable of covalently linking two or more molecules, for example, two modulating agents. Non-covalent linkers can be used, such as hydrophobic lipid globules to which the modulating agent is attached, 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 another hydrophobic residue. The modulating agent may be linked using charge-based chemistry, such that a positively charged portion of the modulating agent is linked to a negatively charged portion of another modulating agent or an additional portion.

IV. Formulations and Compositions

[00203] The compositions described here can be formulated in pure form (for example, 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 the application or administration of the compositions. Examples of suitable 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, syrup, methylcellulose, methyl and propyl hydroxybenzoates, talc, magnesium stearate, and mineral oil.

[00204] In some cases, the composition includes an administration vehicle or carrier. In some cases, the delivery vehicle includes an excipient. Exemplary excipients include, but are not limited to, solid or liquid carrier materials, solvents, stabilizers, slow release excipients, colorants, and surface active substances (surfactants). In some cases, the delivery vehicle is a stabilizing vehicle. In some cases, the stabilizing vehicle includes a stabilizing excipient. Exemplary stabilizing excipients include, but are not limited to, epoxidized vegetable oils, antifoaming agents, for example, silicone oil, preservatives, viscosity regulators, binding agents, and tackifiers. In some cases, the stabilizing vehicle is a suitable buffer for the modulating agent. In some instances, the composition is microencapsulated in a polymeric bead delivery vehicle. In some cases, the stabilizing vehicle protects the modulating agent against UV and/or acidic conditions. In some cases, the delivery vehicle contains a pH buffer. In some instances, 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 anywhere from about 5.0 to about 8.0, about 6.5 to about 7.5 or about 6.5 to about 7.0.

[00205] Depending on the intended objectives and prevailing circumstances, the composition can be formulated into emulsifiable concentrates, suspension concentrates, directly sprayable or dilutable solutions, coatable pastes, dilute emulsions, sprayable 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 preparations or knitted or woven preparations. In some cases, the composition is a liquid. In some cases, the composition is a solid. In some cases, the composition is an aerosol, as in a pressurized aerosol can. In some cases, the composition is present in the residues (such as faeces) of the pest. In some cases, the composition is present in or on a living pest.

[00206] In some cases, the vehicle of administration is the host's food or water. In other cases, the delivery vehicle is a food source for the host. In some cases, the delivery vehicle is a food bait for the host. In some cases, the composition is an edible agent consumed by the host. In some cases, the composition is administered by the host to a second host, and is consumed by the second host. In some instances, the composition is consumed by the host or a second host, and the composition is released into the vicinity of the host or second host via the waste products (such as feces) of the host or second host. In some cases, the modulating agent is included in a food bait intended to be consumed by a host or transported back to its colony.

[00207] In some cases, the modulating agent may make up about 0.1% to about 100% of the composition, such as any from 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% active ingredients (such as phage, lysine or bacteriocin). In some cases, 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, lysine or bacteriocin). In some cases concentrated agents are preferred as commercial products, the end user typically uses diluted agents, which have a substantially lower concentration of active ingredient.

[00208] Any of the formulations described here can be used in the form of a bait, a coil, an electric mat, a fumigant preparation, a fumigant or a sheet.

Liquid Formulations

[00209] The compositions provided herein may be in a liquid formulation. Liquid formulations are generally mixed with water, but in some cases can 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 weight percent.

[00210] 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 can be used in formulations for agricultural, ornamental and grass pests, forestry, structural, food processing, livestock, and public health. These can be adapted to application equipment from small portable sprayers to hydraulic sprayers, low volume ground sprayers, mist blowers, and low volume aerial sprayers. Some active ingredients readily dissolve in a liquid carrier. When mixed with a carrier, they form a solution that does not settle or separate, for example, a homogeneous solution. Formulations of these types can include an active ingredient, a carrier, and one or more other ingredients. Solutions can be used on any type of sprayer, indoors and outdoors.

[00211] In some cases, the composition can be formulated as an inverse emulsion. An inverse emulsion is a water-soluble active ingredient dispersed in an oil carrier. Inverse emulsions require an emulsifier that allows the active ingredient to be mixed with a large volume of petroleum-based carrier, usually fuel oil. Inverse emulsions help reduce drift. With other formulations some spray drift occurs when water droplets begin to evaporate before reaching target surfaces; as a result, the droplets become very small and light. Since oil evaporates more slowly than water, inverse emulsion droplets shrink less and more active ingredient reaches the target. The oil also helps reduce runoff and improve rain resistance. It also serves as a fixer-spreader by improving surface coverage and absorption. Since droplets are relatively large and heavy, it is difficult to obtain extensive coverage on the undersides of the foliage. Inverse emulsions are most commonly used along routes where drift to different susceptible target areas could be a problem.

[00212] A fluid 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 water or oil. The active ingredient, impregnated in a clay-like substance, is ground into a very fine powder. The powder is then suspended in a small amount of liquid. The resulting liquid product is quite thick. Fluids and liquids share many of the characteristics of emulsifiable concentrates, and have similar disadvantages. They require moderate agitation to keep them in suspension and leave visible residues, similar to wettable powders.

[00213] Fluids/liquids are easy to handle and apply. Since they are liquid, they are subject to spills and splashes. They contain solid particles, so they contribute to abrasive wear on nozzles and pumps. Fluid and liquid suspensions settle in their containers. Since fluid and liquid formulations tend to settle out, packaging in five gallon containers or less facilitates remixing.

[00214] 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 gasoline-powered or electric aerosol generators that release the formulation as a smoke or mist.

[00215] Ready-to-use aerosol formulations are usually small, self-contained units that release the formulation when the nozzle valve is activated. 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, can usually be refilled.

[00216] Smoke or fog aerosol formulations are not under pressure. They are used in machines that separate the liquid formulation into a mist or fine mist (aerosol) using a rapidly swirling heated disc or surface.

ii. Dry or Solid Formulations

[00217] 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 powder 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 shells or volcanic ash. The size of individual dust particles varies. Some powder formulations are concentrates and contain a high percentage of active ingredients. Mix these with dry inert carriers before application. Dusts are always used dry and can easily drift to sites other than the target.

iii. Formulations in Granules or Pellets

[00218] In some cases, the composition is formulated as granules. Granular formulations are similar to dust formulations, with the exception that the granular particles are larger and heavier. Coarse particles can be made from materials such as clay, corn cobs, or walnut shells. The active ingredient coats the outside of the granules or is absorbed into them. The amount of active ingredient can be relatively low, usually ranging from about 0.5 to about 15 weight percent. Granular formulations are most often used for application to soil, soil-dwelling insects or nematodes, or absorption into plants through the roots. Granular formulations are sometimes applied by plane or helicopter to minimize drift or to penetrate dense vegetation. Once applied, the granules can release the active ingredient slowly. Some granules require soil moisture to release the active ingredient. Granular formulations are also used to control mosquito larvae and other aquatic pests. Granules are used in agricultural, structural, ornamental, turf, aquatic, road, and public health (biting insects) pest control operations.

[00219] In some cases, the composition is formulated as pellets. Most pellet formulations are very similar to granular formulations; the terms are used interchangeably. However, in a pellet formulation, all particles are the same weight and shape. Particle uniformity allows use with precision application equipment.

iv. Post

[00220] In some cases, the composition is formulated as a powder. In some cases, the composition is formulated as a wettable powder. Wettable powders are dry, finely ground formulations that look like dust. They usually must be mixed with water for application as a spray. However, some products can be applied 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. Particles do not dissolve in water. They settle quickly unless constantly agitated to keep them suspended. They can be used for most pest problems and on most types of spray equipment where agitation is possible. Wettable powders have excellent residual activity. Due to its 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 water penetrates the material.

[00221] In some cases, the composition is formulated as a soluble powder. Soluble powder formulations appear to be wettable powders. However, when mixed with water, soluble powders readily dissolve and form a true solution. After being thoroughly mixed, no further agitation is required. The amount of active ingredient in soluble powders ranges from about 15 to about 95 weight percent; in some cases more than about 50 percent. Soluble powders have all the advantages of wettable powders and none of the disadvantages, except the danger of inhalation during the mixing process.

[00222] In some cases, the composition is formulated as a water-dispersible granule. Water-dispersible granules, also known as dry fluids, are like wettable powders, except that instead of being dusty types, they are formulated as small, easily measured granules. Water dispersible granules must be mixed with water to apply. Once in water, the granules separate into fine particles similar to wettable powders. The formulation requires constant stirring 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 as wettable powders, with the exception that they are easier to measure and mix. Due to the low dustiness, they have less danger of inhalation for the applicator during handling.

v. Bait

[00223] In some cases, the composition includes a bait. The bait may be in any suitable form such as a solid, paste, pellet or powder form. The bait can also be carried by the host back into a population of said host (e.g. a colony or hive). The bait can then act as a food source for other colony members, thereby providing an effective modulating agent for a large number of hosts and potentially an entire colony of hosts.

[00224] The baits can be provided in a suitable "compartment" or "trap". Such compartments and traps are commercially available, and existing traps can be adapted to include the compositions described here. The compartment or trap can be shaped like a box, for example, and can be provided in a pre-formed state or can be formed from folding cardboard, for example. Suitable materials for a compartment or trap include plastics and paperboard, particularly corrugated paperboard. The inside surfaces of traps can be coated with a sticky substance to restrict the host's movement once inside the trap. The compartment or trap may contain a suitable depression the interior of which may hold the bait in place. A trap is distinct from a compartment in that the host cannot easily leave a trap once it has entered it, whereas a compartment acts as a "nurture station" that provides the host with a preferred environment in which it can feed and feel. predator insurance.

saw. Attractors

[00225] In some cases, the composition includes an attractor (eg, a chemoattractant). The attractor can attract an adult host or immature host (e.g. larva) into the vicinity of the composition. Attractors include pheromones, a chemical that is secreted by an animal, especially an insect, that influences the behavior or development of others of the same species. Other attractants include sugar and protein hydrolysate syrups, yeast, and rotten meat. Attractants can also be combined with an active ingredient and sprayed onto foliage or other items in the treatment area.

[00226] Several attractors are known to influence host behavior, such as a host's search for food, oviposition, or mating sites or mating partners. Attractants useful in the methods and compositions described herein include, for example, eugenol, phenethyl propionate, ethyl dimethylisobutylcyclopropanecarboxylate, propyl benzodioxancarboxylate, cis-7,8-epoxy-2-methyloctadecane, trans-8,trans-O-dodecadienol , cis-9-tetradecenal (with cis-11-hexadecenal), trans-11-tetradecenal, cis-11-hexadecenal, (Z)-11,12-hexadecadienal, cis-7-dodecenyl acetate, cis-8 acetate -dodecenyl, cis-9-dodecenyl acetate, cis-9-tetradecenyl acetate, cis-11-tetradecenyl acetate, trans-11-tetradecenyl acetate (with cis-11), cis-9,trans-11 acetate -tetradecadienyl (with cis-9,trans-12), cis-9,trans-12-tetradecadienyl acetate, cis-7,cis-11-hexadecadienyl acetate (with cis-7,trans-11), cis acetate -3,cis-13-octadecadienyl acetate, trans-3,cis-13-octadecadienyl acetate, anethole and isoamyl salicylate.

[00227] Means other than chemoattractants can also be used to attract insects, including lights of various wavelengths or colors.

vii. Nanocapsules/Microencapsulation/Liposomes

[00228] In some cases, 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 degrades and slowly releases the active ingredient.

viii. Conveyors

[00229] Any of the compositions described herein may be formulated to include the modulating agent described herein and an inert carrier. Such a carrier can be a solid carrier, a liquid carrier, a gel carrier and/or a gaseous carrier. In certain cases, the carrier may be a seed coat. Seed coating is any non-naturally occurring formulation that adheres, in whole or in part, to the surface of the seed. The formulation can additionally include an adjuvant or surfactant. The formulation can also include one or more modulating agents to broaden the spectrum of action.

[00230] A solid carrier used for formulation includes finely divided powder or clay granules (eg kaolin clay, diatomaceous earth, bentonite, Fubasami clay, acid clay, etc.), synthetic hydrated silicon oxide, talc, ceramics, other minerals inorganic (e.g. sericite, quartz, sulfur, activated carbon, calcium carbonate, hydrated silica, etc.), a substance that can be sublimed and is in solid form at room temperature (e.g., 2,4,6-tri -isopropyl-1,3,5-trioxane, naphthalene, p-dichlorobenzene, camphor, adamantane, etc.); there; silk; cotton; hemp; pulp; synthetic resins (for example, polyethylene resins such as low-density polyethylene, linear low-density polyethylene, and high-density polyethylene; ethylene-vinyl ester copolymers such as ethylene-vinyl acetate copolymers; ethylene-methacrylic acid ester copolymers as ethylene methyl methacrylate copolymers and ethylene ethyl methacrylate copolymers; ethylene acrylic acid ester copolymers as ethylene methyl acrylate copolymers and ethylene ethyl acrylate copolymers; ethylene vinylcarboxylic acid copolymers as copolymers ethylene-acrylic acid copolymers; ethylene-tetracyclododecene copolymers; polypropylene resins such as propylene homopolymers and propylene-ethylene copolymers; poly-4-methylpentene-1, polybutene-1, polybutadiene, polystyrene; acrylonitrile-styrene resins; styrene such as acrylonitrile-butadiene-styrene resins, styrene-conjugated diene block copolymers, and styrene-conjugated diene block copolymer hydrides; fluororesins; acrylic resins such as poly(methyl methacrylate); polyamide resins such as nylon 6 and nylon 66; polyester resins such as polyethylene terephthalate, polyethylene naphthalate, polybutylene terephthalate, and polycyclohexylenedimethylene terephthalate; polycarbonates, polyacetals, polyacrylsulfones, polyarylates, hydroxybenzoic acid polyesters, polyetherimides, polyester carbonates, polyphenylene ether resins, polyvinyl chloride, polyvinylidene chloride, polyurethane, and porous resins (such as polyurethane foam, polypropylene foam or ethylene, etc.), glass, metals, ceramics, fibers, cloths, knitted fabrics, foils, papers, yarn, foam, porous substances, and multifilaments.

[00231] A liquid carrier may include, for example, aromatic or aliphatic hydrocarbons (for example, xylene, toluene, alkylnaphthalene, phenylxylylethane, kerosene, gasoil, hexane, cyclohexane, etc.), halogenated hydrocarbons (for example, chlorobenzene, dichloromethane, dichloroethane, trichloroethane, etc.), alcohols (e.g. methanol, ethanol, isopropyl alcohol, butanol, hexanol, benzyl alcohol, ethylene glycol, etc.), ethers (e.g. diethyl ether, dimethyl ether ethylene glycol, diethylene glycol monomethyl ether, diethylene glycol monoethyl ether, propylene glycol monomethyl ether, tetrahydrofuran, dioxane, etc.), esters (e.g. ethyl acetate, butyl acetate, etc.), ketones (e.g. e.g. acetone, methyl ethyl ketone, methyl isobutyl ketone, cyclohexanone, etc.), nitriles (e.g., acetonitrile, isobutyronitrile, etc.), sulfoxides (e.g., dimethyl sulfoxide, etc.), amides (e.g., N,N-dimethylformamide , N,N-dimethylacetamide), cyclic imides (e.g. N-methylpyrrolidone), alkylidene carbonates (e.g. propylene carbonate, etc.), vegetable oil (e.g. soybean oil, cottonseed oil, etc.), vegetable essences (eg orange essence, hyssop essence, lemon essence, etc.) or water.

[00232] A carrier gas may include, for example, butane gas, Flon gas, liquefied petroleum gas (LPG), dimethyl ether, and carbon dioxide gas.

ix. Adjuvants

[00233] In some cases, the composition provided herein may include an adjuvant. Adjuvants are chemicals that have no activity. Adjuvants are pre-mixed into the formulation or added to the spray tank to improve the mixing or application process 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 can 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 of these functions, but compatible adjuvants can often be combined to perform multiple functions simultaneously.

[00234] Among non-limiting examples of adjuvants included in the formulation are binders, dispersants and stabilizers, specifically, for example, casein, gelatin, polysaccharides (for example, starch, gum arabic, cellulose derivatives, alginic acid, etc.), lignin derivatives, bentonite, sugars, water-soluble synthetic 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.

x. Surfactants

[00235] In some cases, the composition provided herein includes a surfactant. Surfactants, also called wetting agents and spreading agents, physically change the surface tension of a spray droplet. For a formulation to do its job properly, a spray droplet must be able to wet the foliage and spread evenly over a leaf. Surfactants increase the coverage area of the formulation, thereby increasing the exposure of the pest 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 adequately cover leaf surfaces. However, too much surfactant can cause excessive flow and reduce effectiveness.

[00236] Surfactants are classified by the way they are ionized or separated 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. The activity of a formulation in the presence of a nonionic surfactant can be quite different from the activity in the presence of a cationic or anionic surfactant. Selecting the wrong surfactant can reduce the effectiveness of a pesticide product and damage the target plant. Anionic surfactants are very 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 are usually phytotoxic.

[00237] 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, adhesives, extenders, plant penetrants, compatibility agents, pH buffers or modifiers, drift control additives, antifoam agents, and thickeners.

[00238] Non-limiting examples of surfactants included in the compositions described herein include salts of alkylsulfate esters, alkylsulfonates, alkylarylsulfonates, alkylaryl ethers and their polyoxyethylenated products, polyethylene glycol ethers, esters of polyvalent alcohols and sugar alcohol derivatives.

xi. combinations

[00239] In formulations and forms of use prepared from these formulations, the modulating agent may be in a mixture with other active compounds, such as pesticidal agents (for example, insecticides, sterilants, acaricides, nematicides, molluscicides or fungicides; see, eg pesticides listed in Table 12), attractants, growth regulating substances or herbicides. As used herein, the term "pesticidal agent" refers to any substance or mixture of substances intended to prevent, destroy, repel or mitigate any pest. A pesticide can be a chemical or biological agent used against pests including insects, molluscs, pathogens, weeds, nematodes, and microbes that compete with humans for food, destroy property, spread disease, or are a nuisance. The term "pesticidial agent" may also encompass other bioactive molecules such as antibiotics, antiviral pesticides, antifungals, anthelmintics, nutrients, pollen, sucrose and/or agents that stun or slow down the movement of insects.

[00240] In cases where the modulating agent is applied to plants, it is also possible to mix it with other known compounds, such as herbicides, fertilizers, growth regulators, phytoprotectors, semiochemicals or else with agents to improve plant properties.

V. Administration

[00241] A host described herein may be exposed to any of the compositions described herein in any suitable manner that allows delivery or administration of the composition to the insect. The modulating agent can be administered alone or in combination with other active or inactive substances and can be applied, for example, by spraying, microinjection, through plants, pouring, immersion, 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.

[00242] Amounts and locations for applying the compositions described here are generally determined by the habits of the host, the stage of the life cycle in which the host's microorganisms can be targeted by the modulating agent, the site where the application should be carried out, and the physical and functional characteristics of the modulating agent. The modulating agents described herein can be administered to the insect by oral ingestion, but they can also be administered by means that allow penetration through the cuticle or penetration into the insect's respiratory system.

[00243] In some cases, the insect can simply be "soaked" or "sprayed" with a solution including the modulating agent. Alternatively, the modulating agent may be attached to a food (e.g., edible) component of the insect for ease of administration and/or to enhance the insect's uptake of the modulating agent. Methods for oral introduction include, for example, direct mixing of a modulating agent with the insect's food, spraying the modulating agent into the insect's habitat or field, as well as manipulated approaches where a species that is used as food is manipulated to express a modulating agent, then the insect to be affected is fed that one. In some cases, for example, the modulating agent composition can be incorporated into, or superimposed on, the insect's diet. For example, the modulating agent composition can be sprayed onto a field of crops that an insect inhabits.

[00244] In some cases, the composition is sprayed directly onto a plant, e.g. crops, e.g. by dorsal spraying, aerial spraying, crop spraying/dusting, etc. In cases where the modulating agent is administered to a plant, the plant receiving the modulating agent can be at any stage of plant growth. For example, formulated modulating agents can be applied as a seed dressing or root treatment in early stages of plant growth or as a total plant treatment in later stages of the crop cycle. In some cases, the modulating agent may be applied as a topical agent to a plant, such that the host insect ingests or otherwise contacts the plant by interacting with the plant.

[00245] In addition, the modulating agent can be applied (for example, in the soil where a plant grows or in the water that is used to water the plant) as a systemic agent that is absorbed and distributed through tissues (for example, stems or leaves) of a plant or animal host such that an insect feeding on them will obtain an effective dose of the modulating agent. In some cases, plants or food organisms can be genetically engineered to express the modulating agent such that a host that feeds on the plant or food organism will ingest the modulating agent.

[00246] Delayed or continuous release can also be achieved by coating the modulating agent or a composition with the modulating agent(s) with a dissolvable or bioerodible coating layer, such as gelatin, which coating dissolves or undergoes erosion in the environment of use, to then make the modulating agent available or by dispersing the agent in a dissolvable or erodible matrix. Such continuous delivery and/or delivery devices can be advantageously employed to consistently maintain an effective concentration of one or more of the modulating agents described herein in a specific host habitat.

[00247] The modulating agent can also be incorporated into the medium where the insect grows, lives, reproduces, feeds or infests. For example, a modulating agent can be incorporated into a food container, feeding station, protective enclosure, or a hive. For some applications, the modulating agent can be bound to a solid support for application in powder form or in a "trap" or "nutrition station". As an example, for applications where the composition is intended to be used in a trap or as a bait for a particular host insect, the compositions can also be bound to a solid support or encapsulated in a time release material. For example, in cases where the host is a honeybee, the compositions described herein can be administered by administering the composition to a honeybee hive or at least one habitat where a honeybee grows, lives, breeds or feeds.

SAW. Tracking

[00248] Included herein are screening assays to identify a modulating agent, where the modulating agent is effective to alter the microbiota of a host and thereby increase host fitness (e.g., insect or nematode fitness). For example, the screening assay can be used to identify one or more modulating agents that target specific microorganisms and/or specific hosts. In addition, screening assays can be used to identify one or more microorganisms with enhanced functionality. For example, the screening assay can be effective in isolating one or more microorganisms with enhanced ability to metabolize (e.g., degrade) a pesticide (e.g., a pesticide listed in Table 12 or an insecticide known in the art, e.g., a neonicotinoid (e.g., imidacloprid) or an organophosphate insecticide (e.g., a phosphorothioate, e.g., fenitrothion)) or plant allelochemical (e.g., caffeine, cystatin from N soybean seeds, monoterpenes, diterpene acids, or phenolic compounds) . Administration and colonization of an isolated microorganism in the host can increase the host's resistance to the pesticide (eg, a pesticide listed in Table 12) or plant allelochemical, thereby increasing the fitness of the host. The methods may also be useful for isolating microorganisms with enhanced ability to colonize any of the hosts described herein.

[00249] For example, to screen for microorganisms that increase a host's resistance to a pesticide (e.g., a pesticide listed in Table 12), a starter culture that includes microorganisms (e.g., bacteria) and elevated concentrations of a pesticide (for example, a pesticide listed in Table 12 or an insecticide known in the art, for example, a neonicotinoid (for example, imidacloprid) or an organophosphate insecticide (for example, a phosphorothioate, for example, fenitrothion)) . In some cases, the pesticide may be provided in the form of a pesticide-enriched environmental sample (e.g., a soil sample). Alternatively, the pesticide (for example, a pesticide listed in Table 12) can be provided in pure form or in combination with other carriers. Furthermore, the one or more microorganism isolates can be inoculated directly into the medium (eg, from a laboratory strain) or can be an environmental sample including one or more microorganism species. The growth medium can be liquid or solid. In some cases, the pesticide of interest is the only source of carbon or nitrogen for the microorganisms present in the medium. The culture can be subcultured (eg, inoculated into fresh medium with high levels of the pesticide) any number of times to enrich for and/or isolate microbial strains (eg, bacterial strains) capable of metabolizing the pesticide. The original culture or subcultures can be evaluated using any methods known in the art to test for changes (eg, decrease) in pesticide levels in the sample (eg, using HPLC). Isolates that reduce the concentration of the pesticide (for example, a pesticide listed in Table 12 or a pesticide known in the art, for example, a neonicotinoid (for example, imidacloprid) or an organophosphate insecticide (for example, a phosphorothioate, for example, fenitrothion) )) can be isolated for use as a modulating agent in any of the methods or compositions described herein.

[00250] The methods can be used to further select for microorganisms described here, including those isolated from a screening assay, with enhanced ability to colonize and survive in a host (eg insect, eg bee). For example, a host can be inoculated with a bacterial isolate (eg, one with the ability to degrade a pesticide). The host can then be tested at regular intervals for the presence of the bacterial isolate (eg via culture or 16s RNA from intestines isolated from the host (eg honey bee)). Bacterial isolates that survive in the host (e.g., in the midgut of a honey bee) can be isolated for use as a modulating agent in any of the methods or compositions described herein. Table 12. Pesticides

EXAMPLES

[00251] The following is an example of the methods of the invention. It is understood that several different embodiments can be practiced, given the general description provided above.

Example 1: Isolation of microorganisms that degrade imidacloprid, a nicotinoid

[00252] This example demonstrates the acquisition of a library of microorganisms capable of degrading imidacloprid, a neonicotinoid. Experimental planning:

[00253] Four soil samples with high concentrations of imidacloprid are collected as described in Bonmatin et al. (Environ. Sci. Pollut. Res. Int. 22:35-67, 2015) and diluted in Kaufman and Kearney Minimal Salts Medium (MSM) for enrichment for microorganisms that degrade imidacloprid. Three crop variations for each soil sample include: 50 mL carbon-limited MSM with 83 mg/L imidacloprid as the sole carbon source; 50 mL nitrogen-limited MSM with imidacloprid as sole nitrogen source and sodium citrate and sucrose added as supplemental carbon sources; and 50 mL of MSM broth, with all components plus sodium citrate, sucrose and imidacloprid. Each culture is inoculated with 2 g of the collected soil.

[00254] The cultures are maintained on a shaker at 100 rpm and 27°C. Subcultures (1 mL of cultured solution diluted in 25 mL of fresh medium) are prepared monthly to obtain soilless enrichments. Aliquots of cultures are periodically removed from shaker flasks for analysis by HPLC as described in Tago et al. (Microbes Environ. 21:58-64, 2006). For the HPLC assay, ~7.0 x 10 7 microorganisms are incubated in 1.0 mL of 20 mM sodium-potassium phosphate buffer (pH 7.0) with 1.0 M imidacloprid. After 20 min of incubation at 30 °C, 1.0 mL of methanol is added. Imidacloprid and its partial degradation products are quantitatively analyzed on an HPLC system (600E pump and 2487 dual absorbance detector; Waters). The retention times and peak areas of the HPLC profiles are compared with those of known standards ( Bonmatin et al. Environ. Sci. Pollut. Res. Int. 22:35-67, 2015 ).

[00255] Mixed soil enrichment cultures that exhibit a decrease of imidacloprid via HPLC analysis during incubation are spread-plated using 10-1 dilutions to 10-6 nitrogen-limited agar plates with 36 mg/kg of imidacloprid and streptomycin.

[00256] After a one-week incubation at 27°C, the plates are screened for colonies that appear to be morphologically different from each other (size differences). Individual colonies are grown on new agar plates with imidacloprid as the sole nitrogen source. After three weeks, isolated colonies are placed in 25 mL of tryptic soy broth (TSB) with 25 mg/L of imidacloprid.

[00257] After growth for three days, each of the isolates is centrifuged in conical Falcon tubes for 10 min at 6700 x g. The supernatant is removed, and the isolates are resuspended in sterile phosphate buffer to a total volume of 10 mL. A 2 mL sample of each isolate is inoculated into a nitrogen-limited MSM culture and a carbon-limited MSM culture; both cultures with 30 mg/L of imidacloprid for a total of 25 mL. Uninoculated controls are also prepared by inoculating 2 mL of phosphate buffer into each of the media types. All samples are wrapped in aluminum foil and placed on a shaker operated at 27°C and 100 rpm. Samples are then analyzed for imidacloprid concentration by HPLC.

[00258] After seven days of incubation, 25 mL of methanol are added to the flasks. Samples are then sonicated for six minutes each at 50% duty cycle and centrifuged at 6700 x g. The supernatant is then analyzed and imidacloprid is quantitatively measured by HPLC as described here.

[00259] Isolates that reduce the concentration of imidacloprid are stored at -80°C in a 50% glycerol solution.

Example 2: Selection of isolates that degrade imidacloprid that survive in the midgut of bees

[00260] This example demonstrates the ability to select a microorganism that degrades imidacloprid from isolates described in Example 1 that are able to survive in the midgut of bees.

[00261] Therapeutic planning: Ten isolates described in Example 1 are formulated with 0 (negative control), 102, 105 or 108 cfu/mL in 10 mL of 50% sucrose syrup.

Experimental planning:

[00262] About fifty bees per bacterial isolate are collected and kept in a wooden cage. Bacterial strains isolated in the previous example are cultured and prepared for feeding as follows. Cells from each isolate are washed and diluted to 0.1 OD600 in MSM supplemented with 50% sucrose syrup (w/v) and incubated at 30°C for cell growth. The four concentrations of fresh bacterial cultures are fed to bees as feed (between 0-108 cfu/ml) as follows. The different concentrations of isolates are placed in a 15 mL tube with six small holes drilled where the bees project their proboscis and consume the solutions containing isolates. Fresh cultures containing isolates are prepared daily for use as feed and bee mortality is scored each day throughout the (7 days) experiment by counting and removing dead bees. After 7 days, the solution containing the isolate is replaced by 15% sucrose (w/v) for 48 h.

[00263] Ten bees are removed from each replicate every 24 hours for bacterial analysis; the first sample is removed before the start of the experiment, followed by seven additional sampling points. After each sampling, the bees are anesthetized on ice and the midgut is removed. Repeats for each time point are pooled and homogenized in 150 µL 1*PBS, then serially diluted and plated on nitrogen limited agar plates with 36 mg/kg imidacloprid to grow bacteria under selective growth conditions at 30°C for 2 days.

[00264] The bacterial isolates that survive in the midgut of bees are selected for administration with the purpose of increasing the resistance of bees to neonicotinoids.

Example 3: Increased resistance of bees to neonicotinoids through the administration of bacteria that degrade imidacloprid

[00265] This example demonstrates the ability to increase the resistance of bees to insecticides, and more specifically to neonicotinoids. Bees play an important role in the pollination of flowering plants, and they are the main type of pollinator in many ecosystems that contain flowering plants. It is estimated that one third of the human food supply depends on pollination by insects, birds and bats, most of which is achieved by bees, especially the domesticated European honey bee. The number of colonies maintained by beekeepers has declined, through urbanization, systematic use of pesticides, and tracheal and Varroa mites. Through direct consumption of contaminated nectar and pollen from insecticide-treated plants, neonicotinoids in insecticides affect foraging, learning, and memory in worker bees. By introducing bacteria that degrade imidacloprid into their microbiome, bees are expected to increase their resistance to neonicotinoids through improved behavior such as foraging, grooming behavior, and increased survival.

[00266] Therapeutic planning: The bacterial isolate selected in Example 2 and identified as surviving in the midgut of bees is formulated to 108 organisms in 10 mL of 50% sucrose syrup.

Experimental planning:

[00267] The isolate selected in Example 2 is prepared at a concentration of 108 cells/mL (see Example 2) and placed in a 15 mL tube with six small holes drilled where the bees project their proboscis and consume the solution containing the isolate . Fresh cultures containing isolate are prepared daily for use as feed. The pretreatment lasts seven days in total.

[00268] Approximately 1500, 3000 or 7000 worker hives, estimated by the weight of worker bees, Apis mellifera (0.5, 1, and 2 pounds, respectively or 0.23, 0.45, and 0.1 kg) are pretreated with the isolate selected in Example 2 prepared at a concentration of 108 cells/mL and placed in a 15 mL tube with six small holes drilled where the bees protrude their proboscis and consume the solution containing the isolate. Fresh cultures containing isolate are prepared daily for use as feed. The pretreatment lasts seven days in total.

[00269] After pre-treatment for 7 days, each worker hive is fed 2 to 4 grams of pollen supplement and sugar syrup (1:1) for 1-2 days before imidacloprid treatment. Smaller colonies (1500 and 3000 bees) are placed in observation hives with glass walls. Larger colonies (7000 bees) are placed in Ulster observation hives (Brushy Mountain Bee Supply, NC) containing a box at the base holding 4 standard frames and a split plate feeder where treatment syrup is provided. All observation hives are housed in huts maintained at constant temperature and relative humidity (23-25°C and 70%). Additionally, all hives contain an entrance leading to the outside allowing bees to freely forage for food in surrounding agricultural fields and urban residential neighborhoods.

[00270] When egg laying is confirmed visually, each colony is randomly assigned a treatment with imidacloprid (0, 10, 20, 50, and 100 ppm) provided in 50% sucrose syrup. The colonies receive, through a dispensing system similar to that described in Example 2, proportional amounts of sucrose solution with imidacloprid: 80, 160 and 320 mL for colonies with 1500, 3000, and 7000 bees, respectively. Syrup is replenished every other day for 3 weeks. The amounts of syrup are designed to supplement, but not sustain, the colonies, so it is necessary for the bees to forage freely on other resources. Imidacloprid stock solutions (100 ppm) are prepared using technical grade 99.5 ±0.5% imidacloprid purchased from Chem Service, Inc (PS-2086) dissolved with stirring in 50% sucrose overnight. Stock solutions are prepared every two weeks and treatment solutions are prepared every week. Samples of treatment solutions (3-6 per dose) are randomly selected and tested for residue concentrations to confirm dosing accuracy.

Measurements of behavioral phenotypes:

[00271] Foraging activity is measured by recording the number of workers entering and leaving the bee colony entrance during one-minute observations twice a day. Hygiene behavior is used as a measure to assess worker bee activity within the hive, and is defined as the ability of worker bees to detect and remove diseased and mite-infested progeny, thereby limiting the intra-colony transmission of pathogens and parasites. Grooming behavior is measured using a freeze-killed progeny assay, in which a 3-inch (7.6 cm) polyvinyl chloride tube is gently pushed into a section of a honeycomb containing a large area of pupae cells. sealed (collected from non-experimental field colonies). The number of empty cells is counted and recorded before pouring 400 mL of liquid nitrogen to freeze kill approximately 160 pupae. The frame is then temporarily placed within the bee colony and the proportion of pupae completely or partially removed from the cells is quantified after 24 hours to assess hygienic behavior. Removal of dead progeny by freezing is correlated with removal of diseased and parasitic progeny.

[00272] Bees pre-treated with the bacterial isolate selected in Example 2 demonstrate higher activity than bees treated with the control.

Example 4: Treatment of Varroa mites that infect bees with oxytetracycline solutions

[00273] This example demonstrates the ability to kill, decrease the fitness of Varroa mites by treating them with an antibiotic solution. This example demonstrates that the effect of oxytetracycline on Varroa mites is mediated through switching endogenous bacterial populations, such as Bacillus, to Varroa mites that are sensitive to oxytetracycline.

[00274] Varroa mites are believed to be a major cause for the widespread spread of Colony Collapse Disorder (CCD) that decimates colonies of domesticated honey bees of Apis mellifera throughout the world. They attach themselves to the abdomen of the bees and suck their blood, depriving them of nutrients and eventually killing them. Although Varroa mites can be killed with chemically synthesized acaricides, these types of chemicals must be kept away from edible honey.

[00275] Therapeutic planning: Oxytetracycline solution is formulated with 0 (negative control), 1, 10 or 50 μg/mL in 10 mL of sterilized water with 0.5% sucrose and essential amino acids.

Experimental planning:

[00276] To determine whether adult Varroa mites in the reproductive stage have different susceptibility compared to phoretic mites or their offspring once their cuticle is not hardened, Varroa mites living on adult bees, Apis melifera, and mites associated with larvae and pupae are collected. This assay tests antibiotic solutions on different types of mites and determines how their fitness is altered when targeting endogenous microbes such as Bacillus.

[00277] Progeny mites are collected from combs (or pieces of combs) from bee colonies infested with Varroa mites. Mites are collected from cells where bee larvae develop.

[00278] Varroa mites are grouped by the age of their progeny host and evaluated separately. The age of the offspring is determined based on the morphology and pigmentation of the larva or pupa as follows: Varroa mites collected from spinner larvae that are small enough to move in their cell are grouped in Group 1; Varroa mites collected from elongated larvae, which are too large to settle in the cell and begin to stretch forward with their mouth towards the cell opening, are grouped in Group 2; and Varroa mites collected from pupae are grouped in Group 3. Mites are stored in their host larva or pupa in glass petri dishes until 50 units are collected. This ensures that your feeding routine and physiological state remain unchanged. To prevent mites from being strayed from their host larvae or pupae or from climbing over each other, only hosts at the same developmental stage are kept on the same dish.

[00279] The equipment - a stainless steel ring (56 mm in internal diameter, 2-3 mm in height) and 2 glass circles (62 mm in diameter) - is cleaned with acetone and hexane or pentane to form the arena of test. The oxytetracycline and control solution solutions are applied to the equipment by spraying the glass disks and arena ring homogeneously. For this purpose, a reservoir is filled with 1 ml of the solutions; the distance between the sprayed surface and 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 deposited solution is 1 ± 0.05 mg/cm2. The antibiotic solutions are poured into their respective plates, covering the entire base of the plates, and the residual liquid is evaporated under a hood. The ring is placed between the glass circles to build a cage. The cages are used within 60 hr of preparation, for no more than three trials, for the purpose of controlling exposure of mites to antibiotic solutions. 10 to 15 Varroa mites are introduced into this cage and the pieces of equipment are joined together with droplets of molten wax. Mites collected from spinner larvae, elongated larvae, white-eyed and dark-eyed pupae with pale white bodies are used.

[00280] After 4 hours, the mites are transferred to a clean glass Petri dish (60 mm in diameter) to feed on two or three white-eyed pupae (4-5 days after being covered). Mites are observed under a dissecting microscope at 4 hr, 24 hr, and 48 hr after being treated with the antibiotic or control solutions, and are classified according to the following categories:

[00281] Movable: move when standing up, after being pushed with a needle or not.

[00282] Paralyzed: move one or more appendages, not stimulated or after stimulation, but unable to move.

[00283] Dead: motionless and unresponsive to 3 subsequent stimulations.

[00284] A sterilized toothpick or needle is used to stimulate the mites by touching their paws. New sterilized sticks or needles are used to stimulate each group to avoid contamination between groups of mites.

[00285] The tests are carried out at 32.5°C and 60-70% relative humidity. If mortality in controls exceeds 30%, repetition is excluded. Each experiment is repeated with four series of cages.

[00286] Bacillus status in groups of Varroa mites is assessed by PCR. Total DNA is isolated from control (non-oxytetracycline-treated) and oxytetracycline-treated (whole body) individuals 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: 186) and reverse primer 5'-TTCCCTCACGGTACTGGTTC -3' (SEQ ID NO: 187), are designed based on 23S rRNA sequences -5S obtained from the genome of Bacillus (Accession Number: AP007209.1) ( Takeno et al., J. Bacteriol. 194(17):4767-4768, 2012 ) using Primer 5.0 software (Primer-E Ltd., Plymouth , UK). 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 an extension step final 5 min at 72°C. Amplification products from oxytetracycline-treated and control samples are analyzed on 1% agarose gels, stained with SYBR Safe, and visualized using an Imaging System.

[00287] The survival rates of Varroa mites treated with an oxytetracycline solution are compared with those of Varroa mites treated with the negative control.

[00288] The survival rate and mobility of Varroa mites treated with oxytetracycline solution are decreased compared to the control.

Example 5: Isolation of microorganisms that degrade fenitrothion, an organophosphate insecticide

[00289] This Example demonstrates the acquisition of a library of microorganisms capable of degrading fenitrothion, an organophosphate insecticide.

Experimental planning:

[00290] Soil samples are obtained from various regions where fenitrothion was previously used for insect control. Bacteria that degrade fenitrothion will be isolated from soil samples as described in (Microbes Environ. Vol. 21, No. 1,58-64, 2006). Briefly, 1 g of the soil sample is mixed extensively with 9 mL of sterilized distilled water. The soil particles are then centrifuged at 1000 rcf for 5 min, and 100 µL of the supernatant is then plated on fenitrothion with mineral salt medium (0.4 g/L yeast extract, 0.4 g/L fenitrothion , 15 g/L of bacteriological agar). Plates are cloudy when prepared as fenitrothion is an emulsion.

[00291] Colonies that develop clear zones around them and are likely to be degrading or metabolizing fenitrothion, and these colonies are isolated and recultured on LB agar, nutrient agar, yeast glucose agar, TSA agar, BHI agar, or MRS agar. Once single bacterial colonies are identified, their genomes are extracted using a genomic DNA extraction kit (Qiagen, DNeasy Blood and Tissue Kit) following the manufacturer's protocol.

[00292] The 16S rRNA gene is amplified using universal bacterial primers 27F (5'-AGAGTTTGATCMTGGCTCAG-3'; SEQ ID NO: 188) and 1492R (5'-TACCTTGTTACGACTT-3'; SEQ ID NO: 189), and using PCR conditions of 94°C for 2 min, 30 cycles of 94°C for 1 min, 56°C for 1 min, and 72°C for 2 min, and a final extension of 72°C for 5 min. Gel electrophoresis is used to confirm that the amplicons are the correct size (~1500 bp), and the products are excised from the gel and purified using a gel extraction kit (Qiagen, QIAquick) following the manufacturer's protocol. Correctly sized amplicons are sequenced by Sanger and BLAST is used to match the sequence against multiple 16s rRNA gene sequence repositories to identify bacteria.

[00293] To determine whether the isolated bacteria degrade fenitrothion, ~107 bacterial cells are incubated in 1 mL of 20 mM sodium-potassium phosphate buffer (pH 7) with 1 mM fenitrothion, as described in PNAS, Vol. 109, N 22, 8618-8622, 2012. After 30 min of incubation at 30 °C, the reaction is stopped by adding an equal volume of methanol. Then fenitrothion and its metabolite, 3-methyl-4-nitrophenol, are analyzed by HPLC. The retention times and peak areas of the HPLC profiles are compared with known standards.

[00294] Single bacterial isolates that have fenitrothion degradation capabilities are then stored frozen in glycerol at -80°C.

Example 6: Increased resistance of Drosophila melanogaster to fenitrothion by administering fenitrothion-degrading bacteria

[00295] This Example demonstrates the ability to protect a model insect, Drosophila melanogaster, from one or more negative effects of insecticides in its diet, more specifically fenitrothion. The next approach is a substitute for other insects such as bees or other insects disclosed here, for example, insects that are important pollinators for many flowering plant and vegetable crops. Many insecticides including fenitrothion have been shown to be toxic to bees.

Experimental planning:

[00296] Therapeutic planning: The bacterial isolates selected in Example 5 are formulated to 109 organisms in 100 μL of feed medium for flies with and without fenitrothion.

[00297] The medium used to raise flies is corn bran, molasses and yeast medium (11 g/L yeast, 54 g/L yellow corn bran, 5 g/L agar, 66 mL/L molasses , and 4.8 mL/L of propionic acid). For experimental procedures, fenitrothion at 0, 10, and 100 p.p.m. or phosphate buffered saline as negative controls are infused into sterilized fly chow medium.

Development rate test

[00298] On day one, 109 fenitrothion-degrading bacteria described in Example 5 are suspended in 100 µL of phosphate buffered saline or equal volumes of saline (negative controls) are added to sterilized fly chow medium with or without fenitrothion . All are allowed to dry for one day at 25°C as described in Appl. Environ. Microbiol. Vol. 82, No. 20, 6204-6213, 2016.

[00299] On day two, fertilized embryos collected from flies are treated with 2% hypochlorite solution for 5 min and then washed with sterile water to remove any extracellular microbes from the embryos. 10 µL of the embryo suspension in water (1:3 embryo suspension:water) is added to the fly chow seeded with bacteria or negative control with or without fenitrothion. The fly feed cohorts with the embryos are maintained at 25°C on a 12 h light/dark cycle for the remainder of the experiment.

[00300] The time to puparium formation and the number of pupae formed are measured in each cohort. The time to emergence of adults and the rate of emergence are measured in each sample. From the time the first adult emerges from the pupa, the number of emerging adult flies is counted every 12 hours and the emergence rate is calculated.

[00301] Embryos in fenitrothion-infused fly feed seeded with fenitrothion-degrading bacteria are expected to develop more rapidly than embryos in fenitrothion-infused fly feed without the fenitrothion-degrading bacteria.

survival test

[00302] About 12 days prior to day one, sterilized embryos are generated as previously described and grown in sterilized fly chow. Adults begin to emerge from their pupae 11 days after embryo collection when reared on sterilized fly chow without fenitrothion at 25°C with a 12 h light/12 h dark cycle.

[00303] On day one, 109 of the bacteria that degrade fenitrothion in phosphate buffered saline are added to sterilized fly feed medium as described in a previous Example.

[00304] On day two, 10 newly emerged sterilized adult males and females are introduced to fly chow seeded with bacteria or negative control with or without fenitrothion. The fly chow with the flies is maintained at 25°C with a 12 h light and 12 h dark cycle for the remainder of the experiment. The number of surviving male and female flies is counted each day until all flies are dead. Survival analysis is performed to assess the fitness benefit of bacteria that degrade fenitrothion on fly survival.

[00305] Flies reared on fenitrothion-infused fly chow seeded with bacteria that degrade fenitrothion are expected to survive longer than flies reared on fenitrothion-infused chow without the bacteria that degrade fenitrothion.

Example 7: Elimination of entomopathogenic bacteria from Drosophila melanogaster using naturally occurring phages

[00306] This Example demonstrates the ability to eliminate bacterial insect pathogens (such as Serratia marcescens, Erwinia carotovora, and Pseudomonas entomophila) from Drosophila melanogaster using naturally occurring phages. This procedure may be useful as a substitute assay to eliminate bacteria in other insect species, such as bees.

Experimental planning:

[00307] Therapeutic planning: Collections of phage libraries having the following phage families are used: Myoviridae, Siphoviridae, Podoviridae, Lipothrixviridae, Rudiviridae, Ampullaviridae, Bicaudaviridae, Clavaviridae, Corticoviridae, Cystoviridae, Fuselloviridae, Gluboloviridae, Guttaviridae, Inoviridae, Leviviridae, Microviridae, Plasmaviridae, Tectiviridae.

[00308] Multiple environmental samples (soil, pond sediments, and sewage) are collected in sterilized 1 L flasks over a period of two weeks and are immediately processed after collection and then stored at 4°C. Solid samples are homogenized in sterilized dual potency Difco Luria (LB) broth or tryptic soy broth (TSB) supplemented with 2 mM CaCl 2 to a final volume of 100 mL. pH and phosphate levels are measured using phosphate test strips. For purification, all samples are centrifuged at 3000-6000 g for 10-15 min at 4°C, and filtered through 0.2 µm low protein binding filters to remove all remaining bacterial cells. The supernatant containing the phage library is then stored at 4°C in the presence of chloroform in a glass bottle.

[00309] 20-30 ml of the phage library is diluted to a volume of 30-40 ml with LB broth. The target bacterial strain (eg, Serratia marcescens, Erwinia carotovora, and Pseudomonas entomophila) is added (50-200 µl 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, agitated at 230 rpm. Bacteria from this enrichment culture are removed by centrifugation (3000-6000 g, 15-20 min, 4°C) and filtered (0.2 or 0.45 µm filter). 2.5 ml of the culture without bacteria is added to 2.5 ml of LB broth and 50-100 µl of the target bacteria is added back to the culture to further enrich the phages. The enrichment culture is grown overnight as above. A sample of this enrichment culture is centrifuged at 13,000 g for 15 min at room temperature and 10 μL of the supernatant is plated on a Petri dish with LB-agar along with 100300 μL of the target bacteria and 3 mL of 0 soft agar, 7% cast. Plates are incubated overnight at 37°C.

[00310] Each of the plaques observed on the bacterial lawn is selected and transferred to 500 μL of LB broth. A sample of this plaque stock is further plated on the target bacteria. Plaque purification is performed three times for all phage discovered to isolate a single homogeneous phage from the heterogeneous phage mixture.

[00311] Plaque lysates with high titer phage (>1x 10A10 PFU/ml) are prepared by harvesting coated plaques from a host bacterial strain exhibiting confluent lysis. After being flooded with 5 mL of buffer, the soft agar overlay is macerated, clarified by centrifugation, and sterile filtered. 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.

[00312] DNA is isolated from CsCl-purified phage suspensions as described in Summer, Methods Mol. Biol. 502:27-46, 2009. An isolated individual phage is sequenced as part of two pools of phage genomes using a pyrosequencing method 454 . Briefly, genomic phage 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 to each of the pools, and sequenced by pyrosequencing using a full plate reaction on a sequencer (Roche) according to the manufacturer's protocols. The pooled phage DNA is present in two sequencing reactions. The result corresponding to each of the pools is individually assembled using software (454 Life Sciences), by adjusting the parameters to include only readings with a single MID per pool. The identity of individual contigs is determined by PCR using primers generated against contig sequences and genomic DNA preparations from individual phages as a template. Sequence software (Gene Codes Corporation) is used for sequence assembly and editing.

[00313] Phage chromosomal terminal structures are determined experimentally. Cohesive ends (cos) for phages are determined by sequencing the ends of the phage genome and sequencing the PCR products derived by ligated-junction amplification of circularized genomic DNA, as described in Summer, Methods Mol. Biol. 502:27-46, 2009. Protein coding regions are initially predicted using gene prediction software (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 using BLAST (E cutoff value of 0.005) ( Camacho et al., BMC Bioinformatics 10:421, 2009 ). Proteins of particular interest are further analyzed by sequence search software (Hunter et al., Nucleic Acids Res. 40:D306-D312, 2012).

[00314] Electron microscopy of phage purified with CsCl (>1 x10A11 PFU/mL) that lysed pathogenic bacterial species of Drosophila is performed by diluting phage stock with tryptic soy broth buffer. Phages are spotted onto 400 mesh carbon-coated thin grids, stained with 2% (wt/vol) uranyl acetate, and air-dried. Specimens are observed in a transmission electron microscope operating at an accelerating voltage of 100 kV. Five virions from each phage are measured to calculate mean values and standard deviations for capsid and tail dimensions, where appropriate.

Incorporating phages into a feed

[00315] The medium used to raise flies is corn bran, molasses and yeast medium (11 g/L yeast, 54 g/L yellow corn bran, 5 g/L agar, 66 mL/L molasses , and 4.8 mL/L of propionic acid). Phage solutions are infused into the fly chow to obtain final phage concentrations between 0 and 108 pfu/ml.

[00316] The bacteria S. marcescens, Erwinia carotovora, and Pseudomonas entomphila are grown at 30 °C in nutrient broth or LB broth.

[00317] Sterilized fly embryos are generated by treating fertilized embryos collected from flies with 2% hypochlorite solution for 5 min and then washed with sterilized water to remove any extracellular microbes. Fly larvae with the target bacteria are generated by seeding 10 9 CFU of bacteria in sterilized fly chow and adding sterilized fly embryos to this chow. After 2 days, ten first instar fly larvae infected with S. marcescens are added to the fly feed with a range (0-108 pfu/ml) of phage concentrations. The larvae are allowed to grow in the feed with the phages for 3 days until they reach the third instar. The larvae are then collected and individually homogenized in nutrient broth or LB broth, and plated on nutrient agar or LB agar plates, and incubated at 30°C. The number of S. marcescens CFU obtained from larvae in fly chow with varying concentrations of phages is recorded. This shows the number of live bacteria that were present on the flies.

[00318] It is expected that the number of live bacteria will be reduced in the larvae that were grown on fly feed with the phages against the bacteria.

Example 8: Generation of a natural microbe library

[00319] This Example demonstrates the isolation of soil bacteria that naturally produce the amino acid methionine.

[00320] The medium used for the isolation of microorganisms is Starch-Casein-Nitrate agar (Starch, 10.0 g; Casein, 0.003 g; KNO3, 0.02 g; NaCl, 0.02 g; MgSO4, 0.5 mg; CaCO3, 0.2 mg; FeSO4, 0.1 mg; Agar, 12.0 g; H2O, 1 L; pH 7.0) (Kuster and Williams, 1964). Each environmental soil sample (1.0 g) is suspended in 9 mL of sterilized water, and 1 mL of the suspension is serially diluted ten times in sterilized distilled water. One milliliter of the 10-5 dilution is inoculated onto the agar medium and incubated for 7 days at 30°C. At the end of this period, the plates are observed for growth. Discreet, leathery white colonies are screened and grown on fresh Starch-Casein-Nitrate agar plates to create a library of isolates. After 7 days of growth at 30°C, the plates are kept at 4°C.

Example 9: Screening for methionine producing isolates

[00321] This Example demonstrates the screening assay for isolates from Example 8 that naturally produce the amino acid methionine. 'pScreen for methionine production:

[00322] A modified basal medium (K2HPO4, 0.3 g; KH2PO4, 0.7 g; Na2CO3, 1.0 g; CaCl2, 5.0 mg; MgSO4, 0.3 g; FeSO4, 1.0 mg; H2O, 1 L) with sucrose (20.0 g) and NH4Cl (10.0 g) is used for the fermentation (Chay, B.P., Galvez, F.C.F., and Padolina, W.G.P.U.L.B.P. (1992). "Methionine production by batch from fermentation various carbohydrates". ASEAN Food Journal (Malaysia)). The pH of the medium is 7.2.

[00323] Cultivation conditions: Two calibrated loops of the 7-day culture of isolates from Example 8 are inoculated in a 250 mL Erlenmeyer flask with 30 mL of the fermentation medium. Methionine production is evaluated after incubating the flask for 5 days on a rotary shaker (160 rpm) at 30°C. Duplicate flasks are prepared and uninoculated flasks served as controls in all experiments.

[00324] The presence of methionine in the culture broths of the isolates is examined by paper chromatography following a modified method of Khanna and Nag (Khanna et al., "Production of amino acids in vitro tissue culture", Indian Journal of Experimental Biology ( 1973)). The culture broth is centrifuged at 5000 x g for 20 min and 2 µl of the supernatant is applied 1.5 cm above one end of Whatman No. 1 paper filter, with dimensions of 18 cm x 22 cm. 1 μL volume of a standard methionine solution (0.1 mg/mL) is applied along with the supernatant, and the chromatogram is developed in a solvent mixture of n-butanol, acetic acid and water (4: 1: 1 ) for 18 h. The chromatogram is air dried at room temperature, sprayed with 0.15% ninhydrin solution in butanol and dried again before heating at 60°C for 5 min in an oven. The ninhydrin positive spot value (bluish violet) of the supernatant that corresponds to the methionine standard solution value indicates the presence of methionine in the broth culture. The concentration of methionine produced in the broth culture of the isolate is estimated as follows. The ninhydrin-positive spot of the isolate supernatant on the chromatogram elutes in 10% ethanol and the spectrophotometric reading of the eluate at 520 nm is recorded. The methionine concentration in the supernatant is determined from a standard curve. A graph of optical density values against varying concentrations (0.1 to 0.9 mg/mL) of a methionine solution serves as the methionine standard curve.

[00325] Methionine-producing isolates are maintained on fresh agar plates and a stock solution is created by suspending two calibrated loops of microorganism in an aliquot of 50% glycerol solution.

Example 10: Administration of amino acid-producing bacteria strain to Drosophila melanogaster via diet to increase its rate of development

[00326] This example demonstrates the ability to treat Drosophila melanogaster with amino acid-producing bacteria to increase their rate of development when raised on nutrient-deprived diets. This experimental design can be extended to aid the development of other insects, such as honey bees, which may lack nutrients in their diets.

Therapeutic planning:

[00327] Isolated Corynebacterium glutamicum bacteria that are glutamate or methionine producers are formulated with a solution of 109 colony forming units (CFU) mixed with the feed substrate for Drosophila flies.

Experimental planning:

[00328] Strains of Corynebacterium glutamicum that produce glutamate or methionine were grown in nutrient broth at 30°C.

[00329] The medium used to raise flies is corn bran, molasses and yeast medium (11 g/L yeast, 54 g/L yellow corn bran, 5 g/L agar, 66 mL/L molasses , and 4.8 mL/L of propionic acid). All diet components except propionic acid are heated together to 80°C in deionized water with constant mixing for 30 minutes and allowed to cool to 60°C. The propionic acid is then mixed into the system and 50 mL of the diet is aliquoted into individual bottles and allowed to cool and solidify. Flies are reared at 26°C, 16:8 hour light:dark cycle, at about 60% humidity.

[00330] For the experimental setup to measure the larval growth rate a defined diet was used (Piper et al., 2014, Nature Methods). The defined diet eliminates the effects of batch-to-batch variation in the ingredients used for the corn bran diet. Additionally, the defined diet allows for the exclusion of individual components to test their effects on fly development.

Development rate test

[00331] On day one, 100 uL of a suspension of Corynebacterium glutamicum in nutrient broth consisting of 109 colony forming units (CFU) was added to five replicates of fly chow. As controls, nutrient broth without the bacteria was added to five more bottles of fly food. Fertilized embryos collected from fruit flies were treated with 2% hypochlorite solution for five minutes and then washed with sterile water to remove any extracellular microbes from the embryos. 10 ul of the embryos in water suspension (suspension one:three embryos:water) was added to all seeded bacteria and control fly chow bottles. The fly chow with the embryos was maintained at 26°C, 16:8 hour light:dark cycle, at about 60% humidity for the remainder of the experiment. The time to emergence of adults and the rate of emergence were measured at each replicate. From the time the first adult emerges from the pupa, the number of emerging adult flies was counted every 12 hours and the emergence rate was calculated.

Larval mass test

[00332] To test whether the presence of amino acid-producing bacteria can increase the body mass of developing larvae when reared on a defined diet, we produce larvae that are axenic, and monoassociated with a single strain of bacteria. For these assays, three different bacteria were used, Corynebacterium glutamicum - a strain that produces glutamate, Corynebacterium glutamicum - a strain that produces methionine, and E. coli.

[00333] First, axenic embryos were generated. Fertilized embryos were harvested from fruit flies over a 6 hour period on yeast grape juice agar plates. To eliminate any bacterial contamination, the embryos were treated with a 2% hypochlorite solution for five minutes and then washed three times with sterilized water. One volume of embryos was then suspended in 3 volumes of water.

[00334] The defined diet was transferred in aliquots to flasks and nine replicates were used for each condition being tested. Conditions were: No bacteria added to feed

[00335] Feed containing C. glutamicum, a strain that produces glutamate (C.glu-Glu)

[00336] Feed containing C. glutamicum, a strain that produces methionine (C.glu-Met) Feed containing E. coli

[00337] To each vial of the rations that were under conditions 2, 3, and 4, 100 uL of overnight stationary phase cultures were added.

[00338] To each of the nine repetitions in each condition, 10 uL of the suspension of sterilized embryos+water were added. The flasks were then incubated at 26°C, 60% humidity, 16:8 light:dark cycle.

[00339] After 13 days, 10-15 randomly chosen larvae from each replicate were sampled, and their areas were measured, as an approximation to their biomass and weight. Larvae were picked from the feed with a sterilized spatula, rinsed in water to clean the feed from their bodies, and an image of each sampled larvae was acquired individually for each replicate in each condition. An Image J script was used to identify, delineate, and measure the area of larvae in each image. Treatment with amino acid-producing bacteria increases the rate of insect development.

[00340] Embryos that developed on a diet that was seeded with the amino acid-producing strain of bacteria reached adulthood significantly faster than those that were raised on the sterilized diet (Figure 1). Furthermore, this effect was slightly stronger in female flies than in male flies (Figs. 2A and 2B). Treatment with amino acid-producing bacteria increases larval body mass.

[00341] Larvae on the diet supplemented with C.glu-Met exhibited the largest body size on average, followed by those on the diet with C.glu-Glu, E.coli, and no bacteria (Figure 3). This shows that supplementing the insect diet with bacteria that produce amino acids produced insect biomass faster than the unsupplemented diet.

[00342] Taken together, these data demonstrate that supplementing the insect diet with bacteria that were able to produce amino acids produced insect biomass more rapidly than the unsupplemented diet. Extending this to other insects such as bees by supplementing their diet with bacteria that are capable of producing methionine can increase their fitness.

Example 11: Insects treated with a purified phage solution

[00343] This Example demonstrates the isolation and purification of phages from environmental samples that targeted specific insect bacteria. This Example also demonstrates the efficacy of isolated phages against target bacteria in vitro by plaque assays, by measuring their rate of oxygen consumption, and the rate of extracellular acidification. Finally, this Example demonstrates the effectiveness of phages in vivo by measuring the ability of the phage to target bacteria from flies by treating them with a phage isolate against the bacteria. This Example demonstrates that a pathogenic bacteria that has decreased the fitness of an insect can be eliminated using a phage to target the bacteria. Specifically, Serratia marcescens, which is a pathogenic bacterium in flies, can be killed using a phage that has been isolated from garden manure.

[00344] There are several beneficial and commercially useful insects that are affected by naturally occurring bacterial pathogens. One such example is the bacterium Paenibacillus larvae which is the cause of foulbrood disease in honey bees. Using a phage to treat foulbrood disease would be of great value in mitigating huge losses due to foulbrood disease.

Experimental planning

[00345] Isolation of specific bacteriophages from natural samples:

[00346] Bacteriophages against target bacteria were isolated from material from an environmental source. Briefly, a saturated culture of Serratia marcescens was diluted in fresh double potency tryptic soy broth (TSB) and grown for ~120 minutes for the initial log phase at 24-26°C or in Luria-Bertani broth (LB) of double potency and grown for ~90 min at 37°C. Garden manure was prepared by homogenization in PBS and sterilized by filtration at 0.2 µm. Crude waste was sterilized by filtration at 0.2 µm. A volume of filtered source material was added to log phase bacterial cultures and incubation was continued for 24 h. Enriched source material was prepared by pelleting cultures and filtering the supernatant fluid through 0.45 µm membranes.

[00347] Phages were isolated by plating samples onto bacterial lawns in double agar. Stationary bacterial cultures were combined with molten LB or 0.6% TSB agar and poured onto LB or 1.5% TSB agar plates. After solidification, 2.5 µL of phage sample dilutions were spotted onto double agar plates and allowed to absorb. Plates were then wrapped and incubated overnight at 25°C (TSA) or 37°C (LB), then evaluated for visible plaque formation. Newly isolated plaques were purified by serially passing individual plaques in the target strain by sorting plates into SM Buffer (50 mM Tris-HCl [pH 7.4], 10 mM MgSO4, 100 mM NaCl) and incubating for 15 min. at 55°C, then repeating the double agar spotting method above using the plate suspension.

[00348] Bacteriophages were successfully isolated from litter and compost, as detailed above. Plaque formation was clearly evident after spotting samples onto lawns of the S. marcescens bacteria used for the enrichments.

Passage, quantification, and propagation of bacteriophages:

[00349] Propagation and generation of phage lysates for use in subsequent experiments were performed using bacteriophage isolated and purified as above. Briefly, saturated bacterial cultures were diluted 100-fold in fresh medium and grown for 60-120 minutes to reach an initial logarithmic growth state for effective phage infection. Phage suspensions or lysates were added to early log phase cultures and incubation was continued until elimination of broth turbidity, indicative of phage propagation and bacterial lysis, was observed or until 24 h post-infection. Lysates were collected by pelleting cells at 7197 x g for 20 min, then filtering the supernatant fluid through 0.45 or 0.2 µm membranes. Filtered lysates were stored at 4°C.

[00350] Enumeration of infectious phage particles was performed using the double agar blotting method. Briefly, a series of 1:10 dilutions of samples were performed in PBS and the dilutions were spotted onto solidified double agar plates prepared with the host bacteria as above. Plaque forming units (PFU) were counted after overnight incubation to determine the approximate titer of the samples.

In vitro analysis of isolated phages measuring bacterial respiration:

[00351] A Seahorse XFe96 Analyzer (Agilent) was used to measure the effects of phages on bacteria by monitoring the rate of oxygen consumption (OCR) and rate of extracellular acidification (ECAR) during infection. XFe96 plates were coated the day before experiments by 15 µL of 1 mg/mL poly-L-lysine stock per well and dried overnight at 28°C, and XFe96 probes were equilibrated by placing in wells containing 200 μL of XF Calibrator and incubation in the dark at room temperature. The following day, poly-L-lysine coated plates were washed twice with ddH2O. Saturated overnight cultures of E. coli BL21 (LB, 37°C) or S. marcescens (TSB, 25°C) were subcultured at 1:100 in the same medium and grown with aeration for ~2.5 h at 30°C . Cultures were then diluted to O.D. 600 nm ~ 0.02 using the same medium. Treatments were prepared by diluting stocks in SM Buffer to 10x final concentration and loading 20 µL of the 10x solutions into the appropriate injection ports of the probe plate. While the probes were equilibrating on the XFe96 Flow Analyzer, plaques were prepared by adding 90 µL of bacterial suspensions or media controls and rotating at 3000 rpm for 10 min. After centrifugation, an additional 90 μL of the appropriate medium was gently added to the wells so as not to disturb bacterial adherence, bringing the total volume to 180 μL per well.

[00352] The XFe96 Flow Analyzer was operated at ~30°C, after an application of Mix, Wait, Read cycles of 1:00, 0:30, 3:00. Four cycles were completed to allow bacteria to equilibrate/normalize, then 20 µL treatments were injected and cycling continued as above for a total time period of approximately 6 h. Data were analyzed using the Seahorse XFe96 Wave software package.

[00353] The effects of isolated bacteriophages were evaluated by measuring the oxygen consumption rate (OCR) and the extracellular acidification rate (ECAR) of bacteria with a Seahorse XFe96 Analyzer. When E. coli was infected with T7 phage and S. marcescens infected with the de novo isolate ΦSmVL-C1, dramatic decreases in OCR were observed after brief bursts of this rate (Fig. 4). For both phages with the two host organisms, the Seahorse assay allowed the detection of successful phage infection without the need for plaque assays. Thus, this method is applicable for detecting phage infection of a host organism not conducive to traditional phage detection methods.

Transduction Assay with SYBR Gold for Identification of Infection:

[00354] Bacteriophage preparations have been produced for staining by pretreatment with nucleases in order to remove extraviral nucleic acids that may interfere with the interpretation of fluorescent signals. Briefly, MgCl2 was added to 10 ml of phage lysate to a final concentration of 10 mM, and RNase A (Qiagen) and DNase I (Sigma) were both added to final concentrations of 10 µg/ml. Samples were incubated for 1 h at room temperature. After treatment with nucleases, 5 mL of lysates were combined with 1 µL of SYBR Gold (Thermo, 10,000x) and incubated at room temperature for ~1.5 h. Excess dye was subsequently removed from samples using Amicon ultrafiltration columns. Briefly, Amicon columns (15 ml, molecular weight cutoff 10 k) were washed by adding 10 ml Buffer SM and rotating at 5000 x g, 4°C for 5 min. Tagged phage samples were then rotated through the columns at 5,000 x g, 4°C until the volume had decreased approximately 10-fold (15-30 min). To wash samples, 5 mL of Buffer SM was added to each well and the rotation was repeated, followed by two more washes. After the third wash, the retained samples were pipetted from the Amicon wells and made up to approximately 1 mL using Buffer SM. To remove larger contaminants, washed and labeled phage samples were spun at 10,000 x g for 2 min, and the supernatants were subsequently filtered through 0.2 µm membranes into black microtubes and stored at 4°C.

[00355] Saturated bacterial cultures (E. coli MG1655 grown in LB at 37°C, S. marcescens and S. symbiotica grown in TSB at 26°C) were prepared by rotating 1 mL aliquots and washing once with 1 mL of PBS before a final resuspension using 1 mL of PBS. Positive control labeled bacteria were stained by combining 500 µL of washed bacteria with 1 µL of SYBR Gold and incubating for one hour in the dark at room temperature. Bacteria were pelleted by spinning at 8000 x g for 5 min and washed twice with an equal volume of PBS followed by resuspension in a final volume of 500 µL of PBS. A 25 µL volume of stained bacteria was combined with 25 µL of Buffer SM in a black microtube, to which 50 µL of 10% formalin (5% final volume, ~2% formaldehyde) was added and mixed with a flick. Samples were fixed at room temperature for ~3 h and then washed using Amicon ultrafiltration columns. Briefly, 500 µL of picopure water was added to Amicon columns (0.5 mL, molecular weight cutoff 100 k) and spun at 14,000 x g for 5 min to wash membranes. Fixed samples were diluted by adding 400 µL of PBS and then transferred to prewashed spin columns and spun at 14,000 x g for 10 min. Columns were transferred to fresh collection tubes, and 500 µL of PBS was added to dilute fixative remaining in the retentate. Subsequently, two additional dilutions with PBS were performed, for a total of three washes. Final retentates were diluted to approximately 100 µL, then columns were inverted into fresh collection tubes and spun at 1000 x g for 2 min to collect samples. Washed samples were transferred to black microtubes and stored at 4°C.

[00356] For transduction and control experiments, 25 μL of bacteria (or PBS) and 25 μL of phage labeled with SYBR Gold (or SM Buffer) were combined in black microtubes and incubated statically for 15-20 min at room temperature environment to allow phage adsorption and injection into recipient bacteria. Immediately after incubation, 50 µL of 10% formalin was added to samples and fixation was performed at room temperature for ~4 h. Samples were washed with PBS using Amicon columns as above.

[00357] Injection of bacteriophage nucleic acid was required for a phage to successfully infect a bacterial host cell. Coliphage P1kc labeled with SYBR Gold and co-incubated with S. marcescens revealed the presence of fluorescent bacteria by microscopy, validating the use of this assay in a chained architecture of phage isolation. As with the Seahorse assay, this approach provided an alternative to traditional phage methods to allow expansion for organisms not conducive to a plaque assay. Additionally, the SYBR Gold transduction assay did not require bacterial growth, so it is applicable to phage analysis targeting difficult to culture or even non-cultivable organisms, including endosymbionts such as Buchnera.

In vivo test of phage efficacy against S. marcescens in Drosophila melanogaster flies

[00358] Cultures of S. marcescens were grown in Tryptic Soy Broth (TSB) at 30°C with constant agitation at 200 rpm.

[00359] The medium used to create fly stocks was corn bran, molasses and yeast medium (11 g/L yeast, 54 g/L yellow corn bran, 5 g/L agar, 66 mL/L of molasses, and 4.8 mL/L of propionic acid). All diet components except propionic acid were heated together to 80°C in deionized water with constant mixing for 30 minutes and allowed to cool to 60°C. The propionic acid was then mixed into the system and 50 mL of the diet was transferred in aliquots to individual bottles and allowed to cool and solidify. Flies were reared at 26°C, 16:8 hour light:dark cycle, at about 60% humidity.

[00360] To infect the flies with S. marcescens, a fine needle (tip about 10 µm wide) was immersed in a dense stationary phase culture overnight and the thorax of the flies was punctured. For this experiment, four replicates of 10 males and 10 females each were infected with S. marcescens using the needle stick method as a positive control for fly mortality. For the treatment group, four replicates of 10 males and 10 females each were pricked with S. marcescens and a phage solution containing about 108 phage particles/ml. Finally, two replicates of 10 males and 10 females each that were neither bitten nor treated in any way were used as negative controls for mortality.

[00361] Flies in all conditions were placed in food bottles and incubated at 26°C, 16:8 light:dark cycle, at 60% humidity. The numbers of live and dead flies were counted each day for four days after the bites. All flies bitten with S. marcescens alone were dead within 24 hours of treatment. In comparison, more than 60% of the flies in the phage treatment group, and all the flies in the untreated control group, were alive at this time (Fig. 5). Furthermore, most of the flies in the phage treatment group and the negative control group survived for an additional four days when the experiment was terminated.

[00362] To ascertain the reason for the death of the flies, dead flies from both flies bitten with S. marcescens and S. marcescens + phage were homogenized and plated. Four dead flies from each of the four replicates of both S. marcescens and S. marcescens + phage treatments were homogenized in 100 uL of TSB. A 1:100 dilution was also produced by diluting the homogenate in TSB. 10 ul of the concentrated homogenate as well as the 1:100 dilution were plated on TSA plates, and incubated overnight at 30°C. Upon inspection of the plates for bacterial growth, all plates from dead flies bitten with S. marcescens displayed a lawn of bacteria growing on them, whereas plates from dead flies bitten with S. marcescens + phage showed no bacteria on top of them. the same. This shows that, in the absence of the phage, S. marcescens probably induced septic shock in the flies, leading to their fatality. However, in the presence of the phage, mortality may have been due to needle stick injury.

OTHER MODALITIES

[00363] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the descriptions and examples should not be considered limiting the scope of the invention. The disclosures of all patents and scientific literature cited herein are expressly incorporated in their entirety by reference.

Claims (5)

1. Method for increasing the fitness of a honey bee, the method characterized in that it comprises: administering to the honey bee a liquid, gel or paste composition comprising at least 100,000 cells/mL of organophosphate insecticide metabolizing bacteria formulated with a edible transporter by insects, and the organophosphate insecticide metabolizing bacteria are selected from the group consisting of Bartonella apis, Parasaccharibacter apium, Frischella perrara, Snodgrassella alvi, Bifidobacterium spp and Lactobacillus spp and are able to colonize the intestine of the honey bee, increasing the aptitude of the honey bee. 2. Method according to claim 1, characterized in that the administration comprises administering the composition to a hive of honey bees or at least a habitat where the honey bee grows, lives, reproduces or feeds. 3. Method according to claim 1, characterized in that the organophosphate insecticide is fenitrothion. 4. Method according to claim 1, characterized in that the carrier is a seed coat. 5. Method according to claim 1, characterized in that the honey bee is in a colony of honey bees.