WO2016154602A1

WO2016154602A1 - Compositions and methods for pest control

Info

Publication number: WO2016154602A1
Application number: PCT/US2016/024371
Authority: WO
Inventors: Nicholas Ryan CONLEY; Zachary Alan HOBBS
Original assignee: Epibiome, Inc.
Priority date: 2015-03-26
Filing date: 2016-03-25
Publication date: 2016-09-29

Abstract

The present invention provides methods of preparing a phage composition or a bacteriocin composition for killing or degrading fitness of a pest. Also provided are compositions prepared by the methods, kits, and methods of killing or degrading fitness of a pest using the compositions. The phage compositions or bacteriocin compositions of the present invention target one or more symbiotic bacteria of the pest.

Description

COMPOSITIONS AND METHODS FOR PEST CONTROL

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority benefit of U. S. Provisional Application No.

62/138,539, filed March 26, 2015, the content of which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[0002] The present invention relates to compositions and methods for killing or reducing the fitness of pests. The compositions and methods are useful for a variety of applications, including pest control.

BACKGROUND OF THE INVENTION

[0003] Pests are any organisms that are detrimental to humans or human concerns, such as agriculture or livestock production. In the broadest sense, pests can belong to any kingdom, but most fall within the Animalia, Plantae, or Fungi kingdoms. Examples of animal pests include bed bugs, cockroaches, termites, ants, rodents, flies, mosquitos, fleas, spiders, snakes, ticks, beetles, caterpillars, apbids, gnats, earwigs, bees, wasps, stink bugs, millipedes, weevils, fruit flies, and mites. Traps, poisons, predators, physical barriers, and deterrents have historically been used to control these pests. Examples of plant pests include Russian olive, Cajeput tree, purple loosestrife, tree-of-heaven, Japanese honeysuckle, kudzu, leafy spurge, rnile-a-minute, tamarisk, and cogon grass. Strategies to combat plant pests include herbicides, coverings, manual removal, goat grazing, tilling, and thermal methods. Examples of fungal pests include Cladosporium, Penicillium, Aspergillus, Alternaria, and Stachybotrys. Fungal pests are typically controlled by dehumidification, biocides, and physical removal. While often effective, many of these control methods are non-specific and pose inconvenience, annoyance, economic hardship, or worse yet, harm to humans.

[0004] Many common pests rely on symbiotic relationships with other organisms for increased fitness or survival. In its broadest sense, symbiosis constitutes all species interactions, but in many cases, it involves a close and long-term interaction between two or more different biological species. Symbiotic relationships can fall into one of several categories: mutualistic, where both individuals benefit; commensal, where one individual benefits, but the other receives neither benefit nor harm; and parasitic, where one individual benefits and the other is harmed. For the benefitting individual, the relationship can be further classified as obligate (i.e., necessary for survival) or facultative (i.e., discretionary).

[0005] Land fungi, ocean animals, and land plants appeared about 1300, 760, and 700 million years ago, respectively. These organisms coevolved interdependencies with bacteria, which had already existed on the planet for more than 2 billion years, and symbioses flourished. Bacteria gained mobility and/or access to a nutrient source, and in exchange, they provided their symbionts with new functions that conferred survival advantages, such as nitrogen fixation, antibiotic production, digestive capability, and vitamin synthesis. In the absence of a selection pressure compelling these early plants, animals, and fungi to genetically encode the machinery allowing them to carry out such functions themselves, they readily ceded them to their bacterial symbionts. This created an "Achilles heel" that likely extends to every eukaryote alive today.

BRIEF SUMMARY OF THE INVENTION

[0006] The present invention provides methods for preparing a phage composition or bacteriocin composition that kill or reduce fitness of a pest

[0007] One aspect of the present application provides a method of preparing a phage composition for killing or degrading fitness of a pest, comprising: (a) identifying a symbiotic bacterium in a specimen of the pest; (b) obtaining a phagic agent (such as phage or lysin) that inhibits the symbiotic bacterium; and (c) formulating a composition comprising the phagic agent (such as phage or lysin) to provide the phage composition. In some embodiments, the symbiotic bacterium is identified by sequencing the specimen of the pest In some embodiments, the symbiotic bacterium is identified by comparative sequencing of a first specimen of the pest comprising one or more pest individuals affected by an antibacterial treatment (such as applying a pool of phages, or antibiotics), and a second specimen of the pest comprising one or more pest individuals unaffected by the antibacterial treatment. In some embodiments, the sequencing or comparative sequencing is metagenomics sequencing, such as 16S rRNA sequencing.

[0008] In some embodiments according to any one of the methods of preparing a phage composition described above, the specimen (including the specimen of the pest, the first specimen of the pest, and/or the second specimen of the pest) is derived from the pest. In some embodiments, the specimen (including the specimen of the pest, the first specimen of the pest, and/or the second specimen of the pest) is derived from the surroundings of the pest. [0009] In some embodiments according to any one of the methods or preparing a phage composition described above, wherein the symbiotic bacterium is identified by comparative sequencing, the antibacterial treatment comprises applying a pool of phages to the one or more pest individuals affected by the antibacterial treatment and the one or more pest individuals unaffected by the antibacterial treatment In some embodiments, the pool of phages is present in an environmental sample. In some embodiments, the phage is identified from the results of the comparative sequencing. In some embodiments, the phagic agent (such as phage or lysin) is obtained from the pool of phages.

[0010] In some embodiments according to any one of the methods of preparing a phage composition described above, the method further comprises isolating the symbiotic bacterium from the specimen.

[0011] In some embodiments according to any one of the methods of preparing a phage composition described above, the phagic agent (such as phage or lysin) is obtained from a specimen of the pest (such as the specimen of the pest). In some embodiments, the phagic agent (such as phage or lysin) is obtained from an environmental sample.

[0012] In some embodiments according to any one of the methods of preparing a phage composition described above, the method further comprises assessing the inhibitory activity

(such as killing or reduction of fitness) of the phagic agent (such as phage or lysin) against the symbiotic bacterium (such as in vitro, or in vivo, i.e., in the pest).

[0013] In some embodiments according to any one of the methods of preparing a phage composition described above, wherein the phagic agent is a phage, the method further comprises engineering the phage to expand the host range of the phage.

[0014] In some embodiments according to any one of the methods of preparing a phage composition described above, wherein the phagic agent is a phage, the method further comprises propagating the phage.

[0015] In some embodiments according to any one of the methods of preparing a phage composition described above, the method further comprises assessing the inhibitory activity (such as killing or reduction of fitness) of the phage composition against the pest and adjusting the phage composition based on the inhibitory activity.

[0016] In some embodiments according to any one of the methods of preparing a phage composition described above, step (c) comprises incorporating the phage composition into a delivery vehicle. In some embodiments, the delivery vehicle is a bacterial vector. In some embodiments, the delivery vehicle is a food bait for the pest. In some embodiments, the delivery vehicle comprises an excipient.

[0017] In some embodiments according to any one of the methods of preparing a phage composition described above, the symbiotic bacterium is an obligate symbiont of the pest In some embodiments, the symbiotic bacterium is a facultative symbiont of the pest.

[0018] In some embodiments according to any one of the methods of preparing a phage composition described above, wherein the phagic agent is a phage, the phage is a

promiscuous phage with a large host range.

[0019] In some embodiments according to any one of the methods of preparing a phage composition described above, wherein the phagic agent is a phage, the phage is lytic. In some embodiments, the phage is temperate.

[0020] In some embodiments according to any one of the methods of preparing a phage composition described above, the pest is an animal. In some embodiments, the pest is a termite. In some embodiments, the symbiotic bacterium is selected from the group consisting of Treponema, Bacteroides, Desulfovibrio, Citrobacter, Enterobacter, Enterococcus, Lactococcus saAMethanobrevibacter. In some embodiments, the pest is a plant. In some embodiments, the pest is a lichenized fungus.

[0021] In some embodiments according to any one of the methods of preparing a phage composition described above, the method comprises obtaining a plurality of phagic agents (such as phages and/or lysins) that each inhibits the symbiotic bacterium.

[0022] In some embodiments according to any one of the methods of preparing a phage composition described above, the method comprises identifying a plurality of symbiotic bacteria of the pest, and isolating one or more phagic agents (such as phages and/or lysins) that inhibit each symbiotic bacterium.

[0023] One aspect of the present application provides a phage composition prepared by any one of the methods of preparing a phage composition described above. In some embodiments, the phage composition is a liquid. In some embodiments, wherein the phagic agent is a phage, the concentration of the phage in the phage composition is at least about 10³ pfu/ml. hi some embodiments, the phage composition is a solid. In some embodiments, wherein the phagic agent is a phage, the concentration of the phage in the phage composition is at least about 10³ pfu/g. In some embodiments, the phage composition is an aerosol.

[0024] In some embodiments according to any of the phage compositions described above, the phage composition is present in a pest extract. In some embodiments, the phage composition is present in a live pest. In some embodiments, the phage composition is present in the feces secreted by the pest.

[0025] In some embodiments according to any of the phage compositions described above, the phage composition further comprises a bacteiiocin mat inhibits a symbiotic bacterium of the pest.

[0026] One aspect of the present application provides a kit for killing or degrading fitness of a pest, comprising any one of the phage compositions described above, and an instruction.

[0027] One aspect of the present application provides a method of killing or degrading fitness of a pest, comprising applying an effective amount of any one of the phage compositions described above. In some embodiments, the phage composition is applied directly to the pest In some embodiments, the phage composition is applied to the surroundings of the pest In some embodiments, the phage composition is applied to a second pest, wherein the second pest closely associates with the pest, and wherein the second pest delivers the phage composition to the pest. In some embodiments, the phage composition is applied via a sprayer.

[0028] One aspect of the present application provides a method of preparing a bacteriocin composition for killing or degrading fitness of a pest, comprising: (a) identifying a symbiotic bacterium in a specimen of the pest; (b) obtaining a bacteriocin that inhibits the symbiotic bacterium; and (c) formulating a composition comprising the bacteriocin to provide the bacteriocin composition. In some embodiments, the symbiotic bacterium is identified by sequencing the specimen of the pest. In some embodiments, the symbiotic bacterium is identified by comparative sequencing of a first specimen of the pest comprising one or more pest individuals affected by an antibacterial treatment (such as applying antibiotics), and a second specimen of the pest comprising one or more pest individuals unaffected by the antibacterial treatment In some embodiments, the sequencing or comparative sequencing is metagenomics sequencing, such as 16S rRNA sequencing.

[0029] In some embodiments according to any one of the methods of preparing a bacteriocin composition described above, the specimen (including the specimen of the pest, the first specimen of the pest, and/or the second specimen of the pest) is derived from the pest In some embodiments, the specimen (including the specimen of the pest, the first specimen of the pest, and/or the second specimen of the pest) is derived from the surroundings of the pest. [0030] In some embodiments according to any one of the methods of preparing a bacteriocin composition described above, the method further comprises isolating the symbiotic bacterium from the specimen.

[0031] In some embodiments according to any one of the methods of preparing a bacteriocin composition described above, the bacteriocin is obtained from a natural source. In some embodiments, the bacteriocin is engineered.

[0032] In some embodiments according to any one of the methods of preparing a bacteriocin composition described above, the method further comprises assessing the inhibitory activity (such as killing or reduction of fitness) of the bacteriocin composition against the pest and adjusting the bacteriocin composition based on the inhibitory activity.

[0033] In some embodiments according to any one of the methods of preparing a bacteriocin composition described above, step (c) comprises incorporating the bacteriocin composition into a delivery vehicle. In some embodiments, the delivery vehicle is a bacterial vector. In some embodiments, the delivery vehicle is a food bait for the pest hi some embodiments, the delivery vehicle comprises an excipient.

[0034] In some embodiments according to any one of the methods of preparing a bacteriocin composition described above, the symbiotic bacterium is an obligate symbiont of the pest In some embodiments, the symbiotic bacterium is a facultative symbiont of the pest.

[0035] In some embodiments according to any one of the methods of preparing a bacteriocin composition described above, the pest is an animal. In some embodiments, the pest is a termite. In some embodiments, the symbiotic bacterium is selected from the group consisting of Treponema, Bacteroides, Desulfovibrio, Citrobacter, Enterobacter,

Enterococcus, Lactococcus and Methanobrevibacter. In some embodiments, the pest is a plant. In some embodiments, the pest is a lichenized fungus.

[0036] In some embodiments according to any one of the methods of preparing a bacteriocin composition described above, the method comprises obtaining a plurality of bacteriocins that each inhibits the symbiotic bacterium.

[0037] In some embodiments according to any one of the methods of preparing a bacteriocin composition described above, the method comprises identifying a plurality of symbiotic bacteria of the pest, and isolating one or more bacteriocins that inhibit each symbiotic bacterium

[0038] One aspect of the present application provides a bacteriocin composition prepared by any one of the methods of preparing a bacteriocin composition described above. In some embodiments, the bacteriocin is a pyocin. In some embodiments, the bacteriocin is engineered (such as based on pyocin).

[0039] In some embodiments according to any of the bacteriocin compositions described above, the bacteriocin composition is a liquid. In some embodiments, the bacteriocin composition is a solid. In some embodiments, the bacteriocin composition is an aerosol.

[0040] In some embodiments according to any of the bacteriocin compositions described above, the bacteriocin composition is present in a pest extract. In some embodiments, the bacteriocin composition is present in a live pest In some embodiments, the bacteriocin composition is present in the feces secreted by the pest

[0041] In some embodiments according to any of the bacteriocin compositions described above, the bacteriocin composition further comprises a phagic agent (such as phage or lysin) that inhibits a symbiotic bacterium of the pest In some embodiments, the bacteriocin composition comprises a plurality of phagic agents (such as phages and/or lysins), each inhibiting a symbiotic bacterium of the pest.

[0042] One aspect of the present application provides a kit for killing or degrading fitness of a pest, comprising any one of the bacteriocin compositions described above, and an instruction.

[0043] One aspect of the present application provides a method of killing or degrading fitness of a pest, comprising applying an effective amount of any one of the bacteriocin compositions described above. In some embodiments, the bacteriocin composition is applied directly to the pest. In some embodiments, the bacteriocin composition is applied to the surroundings of the pest. In some embodiments, the bacteriocin composition is applied to a second pest, wherein the second pest closely associates with the pest, and wherein the second pest delivers the bacteriocin composition to the pest. In some embodiments, the bacteriocin composition is applied via a sprayer.

[0044] Further provided are kits, and articles of manufacture comprising any one of the compositions described above.

[0045] All publications, patents, patent applications and published patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that embodiments of the present disclosure are not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0046] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

BRIEF DESCRIPTION OF THE DRAWINGS

[0047] FIG. 1 depicts an exemplary workflow for developing a pest-control composition.

[0048] FIG. 2 depicts an alternative exemplary workflow for developing a pest-control composition.

DETAILED DESCRIPTION OF THE INVENTION

[0049] The present invention discloses a method of preparing a phage or bacteriocin composition for killing or degrading fitness of a pest by targeting one or more symbiotic bacteria of the pest When a pest acts as an obligate symbiont in a mutualistic, commensal, or parasitic relationship, its fate is tied to the fate of its symbiont. Thus, an opportunity exists to kill the pest by killing its non-pest symbiont Even in cases where the pest is a benefiting facultative symbiont, its fitness may be substantially degraded by killing its non- pest symbiont. The symbiotic bacteria of the pest can be identified by sequencing a specimen of the pest The phagic agents (such as phages and/or lysins) in the phage composition and bacteriocins in the bacteriocin composition can be obtained from a natural source, such as a specimen of the pest or an environmental sample. Compositions prepared by the methods and methods of killing or degrading fitness of a pest using the compositions are further provided.

[0050] Exploitation of the dependence of eukaryotes on bacteria is an unconventional strategy to kill or reduce the fitness of pests. In principle, small-molecule antibiotics could be used to target a pest's bacterial symbionts, but their medical utility would be severely diminished by the proliferation of antibiotic-resistant bacteria associated with widespread antibiotic use, and these resistant bacteria would support the pest's continued survival, rendering the strategy ineffective. Additionally, the antibiotic would need to be delivered to every pest, which would be difficult in the case of large numbers of colonizing pests, especially those which do not readily accept bait and live in areas that are difficult to treat or cannot be readily located.

[0051] Deployment of bacterial viruses, also known as "bacteriophages" or "phages," represents an attractive strategy to exploit the dependence of pests on bacteria. Phages are abundant in nature (~10³¹), encompass enormous genetic diversity (~10⁸ unique phages), and kill about half of the bacteria on the planet every two days. They act by binding to specific receptors on the surface of bacteria. Once bound, "lytic" phages infect the bacterial host by injecting their genetic material into the cell, which hijacks the bacteria's own cellular machinery to produce tens or hundreds of copies of daughter phages. This process, which culminates in the bursting of the bacterial cell to release the infectious daughter phages into the surroundings, is complete within about twenty minutes. Phage propagation lends itself well to targeting colonizing pests because once phages infect bacterial endosymbionts living in the gut of an animal pest, they can spread through feces to the guts of other pests living in the colony. These phages may persist in the environment and exert a beneficial latent effect Although bacteria can develop resistance to individual phages by modifying their surface receptors or through their own adaptive immune systems, the emergence of resistance can be effectively suppressed using phage cocktails, in which a bacterial strain is targeted by at least two orthogonal phages. The use of phages to kill a pest's important bacterial symbionts, leading to the death or reduction in fitness of the pest, represents a convenient and safe alternative to conventional methods of pest control.

[0052] Accordingly, in some embodiments, there is provided a method of preparing a phage composition for killing or degrading fitness of a pest, comprising: (a) identifying a symbiotic bacterium in a specimen of the pest; (b) obtaining a phage mat inhibits the symbiotic bacterium; and (c) formulating a composition comprising the phage to provide the phage composition.

[0053] In some embodiments, mere is provided a method of preparing a phage composition for killing or degrading fitness of a pest, comprising: (a) identifying a symbiotic bacterium in a specimen of the pest; (b) obtaining a lysin that inhibits the symbiotic bacterium; and (c) formulating a composition comprising the lysin to provide the phage composition.

[0054] In some embodiments, there is provided a method of preparing a bacteriocin composition for killing or degrading fitness of a pest, comprising: (a) identifying a symbiotic bacterium in a specimen of the pest; (b) obtaining a bacteriocin that inhibits the symbiotic bacterium; and (c) formulating a composition comprising the bacteriocin to provide the bacteriocin composition.

I. Definition

[0055] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0056] As used herein, "fitness" refers to the ability of an organism to survive, and/or to produce surviving offspring. Fitness of an organism may be measured by one or more parameters, including, but not limited to, reproductive rate, mobility, body weight, and metabolic rate.

[0057] As used herein, "effective amount" refers to an amount of an agent (such as lysin, phage, or bacteriocin) or composition (such as phage composition or bacteriocin composition) sufficient to kill or reduce the fitness of a pest, such as producing any one or more of the following desired effects: (1) killing at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9% or more of a population of the pest; (2) reducing the reproductive rate of the pest by about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9% or more; (3) reducing the mobility of the pest by about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more; (4) reducing the body weight of the pest by about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more; and/or (5) reducing the metabolic rate of the pest by about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more.

[0058] As used herein, "bacterium" refers to a bacterium isolate in which members of the isolate has substantially the same genetic makeup, such as sharing at least about any of 90%, 95%, 99%, 99.9% or more sequence identity in their genome. "Bacterium" may refer to the same bacterium genus, species, strain, or clone. "Bacterium" refers to the parent bacterium as well as the progeny or derivatives (such as genetically engineered versions) thereof.

[0059] As used herein, "phage" refers to a bacteriophage isolate in which members of the isolate has substantially the same genetic makeup, such as sharing at least about any of 90%, 95%, 99%, 99.9% or more sequence identity in the genome. "Phage" refers to the parent phage as well as the progeny or derivatives (such as genetically engineered versions) thereof. The phage can be a naturally occurring phage isolate, or an engineered phage, including vectors, or nucleic acids that encode at least all essential genes, or the full genome of a phage to carry out the life cycle of the phage inside a host bacterium.

[0060] As used herein, "phagic agent" refers to a phage or a phage component, such as a phage enzyme, (hat can inhibit the host bacterium of the phage, or a derivative thereof.

[0061] As used herein, "lysin," also known as endolysin or murein hydrolase, refers to a hydrolytic enzyme that can lyse a bacterium by cleaving peptidoglycan in the cell wall of the bacterium. Lysins contemplated herein include naturally occurring lysins, such as lysins produced by phages, and derivatives thereof, such as engineered lysins, recombinantly expressed lysins, chemically synthesized lysins, etc.

[0062] As used herein, "phage composition" refers to a composition comprising one or more phagic agents.

[0063] As used herein, "phage cocktail" refers to a composition comprising one or more phages.

[0064] As used herein, "bacteriocin cocktail" refers to a composition comprising one or more bacteriocins.

[0065] As used herein, "metagenomics sequencing" refers to the method of sequencing genes or genomes of multiple organisms present in a given complex sample. Any suitable sequencing methods may be used to carry out metagenomics sequencing, including, but not limited to, capillary sequencing, PCR-based sequencing, and next-generation sequencing (NGS) methods. "16S rRNA sequencing" refers to an amplicon sequencing method that sequences only (he 16S rRNA gene or its transcripts that are selected amplified from the sample prior to the sequencing.

[0066] As used herein, "comparative sequencing" refers to sequencing of two or more samples, and compare the sequencing results of the two or more samples to identify sequences mat are differentially present in the two samples, including sequences that are present in one sample but absent in another sample, and sequences (hat are present at a relatively high level in one sample but are present at a relatively low level in another sample.

[0067] The terms "specimen" and "sample" are used interchangeably herein to refer to a sample of one or more substances, materials, organisms, compositions, and mixtures thereof for examination or study.

[0068] The terms "degrade" and "reduce" are used interchangeably herein to refer to decrease in a level or quantity, such as fitness of a pest [0069] As used herein, "target" or "inhibit" a symbiotic bacterium refers to killing, slowing down, or stopping the growth of the symbiotic bacterium.

[0070] The term "affected pest individuals" or "pest individuals affected by..." refer to pest individuals that are killed or exhibit reduced fitness after an antibacterial treatment. The term "unaffected pest individuals" or "pest individuals unaffected by..." refer to pest individuals that are not killed nor do not exhibit reduced fitness after an antibacterial treatment.

"Antibacterial treatment" refers to the application of any agent that causes a microbial imbalance (e.g., change in the types or population of one or more bacteria) on or inside the pest. Suitable agents may include, but not limited to, antibiotics, phages, phage components (such as phage en2ymes, for example, lysins), and bacteriocins.

[0071] It is noted that, as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

[0072] It is understood that aspect and embodiments of the invention described herein include "consisting" and/or "consisting essentially of aspects and embodiments.

[0073] Where a range of values is provided, it is understood mat each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within embodiments of the present disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within embodiments of the present disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in embodiments of the present disclosure.

[0074] Reference to "about" a value or parameter herein includes (and describes) variations that are directed to mat value or parameter per se. For example, description referring to "about X" includes description of "X".

[0075] The term "about X-Y" used herein has the same meaning as "about X to about Y."

[0076] As used herein, reference to "not" a value or parameter generally means and describes "other than" a value or parameter. [0077] It is to be understood that aspects of the present disclosure are not limited to the particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since (he scope of embodiments of the present disclosure will be defined only by the appended claims.

Π. Methods of preparation

Methods of preparing a phage composition

[0078] The present invention provides a method of preparing a phage composition for killing or degrading fitness of a pest, comprising: (a) identifying a symbiotic bacterium in a specimen of the pest; (b) obtaining a phagic agent (such as phage or lysin) that inhibits the symbiotic bacterium; and (c) formulating a composition comprising the phagic agent (such as phage or lysin) to provide the phage composition. In some embodiments, the method comprises obtaining a plurality of phagic agents (such as phages and/or lysins) that each inhibits the symbiotic bacterium In some embodiments, the method comprises identifying a plurality of symbiotic bacteria of the pest, and obtaining one or more phagic agents (such as phages and/or lysins) that inhibit each symbiotic bacterium In some embodiments, (he phagic agent (such as phage or lysin) is obtained from a specimen of the pest, or from an environmental sample.

[0079] In some embodiments, there is provided a method of preparing a phage composition for killing or degrading fitness of a pest, comprising: (a) identifying a symbiotic bacterium in a specimen of the pest; and (b) formulating a composition comprising a phagic agent (such as phage or lysin) that inhibits the symbiotic bacterium to provide the phage composition. In some embodiments, the method comprises formulating a composition comprising a plurality of phagic agents (such as phages and/or lysins) that each inhibits the symbiotic bacterium In some embodiments, (he method comprises identifying a plurality of symbiotic bacteria of the pest, and formulating a composition comprising a plurality of phagic agents (such as phages and/or lysins), wherein each symbiotic bacterium is inhibited by one or more phagic agents (such as phages and/or lysins) among the plurality of phagic agents (such as phages and/or lysins). In some embodiments, the phagic agent (such as phage or lysin) is obtained from a specimen of the pest, or from an environmental sample. [0080] In some embodiments, there is provided a method of preparing a phage composition for killing or degrading fitness of a pest, comprising: (a) identifying a symbiotic bacterium in a specimen of the pest; and (b) obtaining a composition comprising a phagic agent (such as phage or lysin) that inhibits the symbiotic bacterium to provide (he phage composition. In some embodiments, the method comprises obtaining a composition comprising a plurality of phagic agents (such as phages and/or lysins) that each inhibits the symbiotic bacterium. In some embodiments, the method comprises identifying a plurality of symbiotic bacteria of the pest, and obtaining a composition comprising a plurality of phagic agents (such as phages and/or lysins), wherein each symbiotic bacterium is inhibited by one or more phagic agents (such as phages and/or lysins) among the plurality of phagic agents (such as phages and/or lysins). In some embodiments, the phagic agent (such as phage or lysin) is obtained from a specimen of the pest, or from an environmental sample.

[0081] In some embodiments, there is provided a method of preparing a phage composition for killing or degrading fitness of a pest, comprising: (a) identifying a symbiotic bacterium in a specimen of the pest; (b) obtaining a phagic agent (such as phage or lysin) that inhibits the symbiotic bacterium; and (c) formulating a composition comprising the phagic agent (such as phage or lysin) to provide the phage composition, wherein the symbiotic bacterium is identified by sequencing (such as metagenomics sequencing) the specimen of the pest In some embodiments, the method comprises obtaining a plurality of phagic agents (such as phages and/or lysins) that each inhibits the symbiotic bacterium. In some embodiments, the method comprises identifying a plurality of symbiotic bacteria of the pest, and obtaining one or more phagic agents (such as phages and/or lysins) that inhibit each symbiotic bacterium In some embodiments, the phagic agent (such as phage or lysin) is obtained from a specimen of the pest, or from an environmental sample.

[0082] In some embodiments, there is provided a method of preparing a phage composition for killing or degrading fitness of a pest, comprising: (a) identifying a symbiotic bacterium in a specimen of the pest; (b) obtaining a phagic agent (such as phage or lysin) that inhibits the symbiotic bacterium; and (c) formulating a composition comprising the phagic agent (such as phage or lysin) to provide the phage composition, wherein the symbiotic bacterium is identified by comparative sequencing (such as metagenomics sequencing) of a first specimen of the pest comprising one or more pest individuals affected by an antibacterial treatment (such as applying a pool of phages to the pest), and a second specimen of the pest comprising one or more pest individuals unaffected by the antibacterial treatment. In some embodiments, the method comprises obtaining a plurality of phagic agents (such as phages and/or lysins) that each inhibits the symbiotic bacterium In some embodiments, the method comprises identifying a plurality of symbiotic bacteria of the pest, and obtaining one or more phagic agents (such as phages and/or lysins) that inhibit each symbiotic bacterium. In some embodiments, the phage is identified from results of the comparative sequencing. In some embodiments, the phagic agent (such as phage or lysin) is obtained from the pool of phages.

[0083] In some embodiments, any of the above methods of preparing a phage composition may further comprise any one or more (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13) of the following steps: (i) obtaining a specimen of the pest; (ii) sequencing the specimen (including comparative sequencing of two or more specimen, such as metagenomics sequencing); (iii) obtaining the symbiotic bacterium (such as from a cell bank, or by isolating the symbiotic bacterium from the specimen); (iv) isolating or culturing the symbiotic bacterium; (v) determining the symbiotic relationship between the symbiotic bacterium and the pest; (vi) identifying the phage from the sequencing results; (vii) obtaining a sample containing phages (such as a specimen of the pest, or an environmental sample); (viii) isolating the phagic agent (such as phage or lysin) from the sample; (ix) assessing the inhibitory activity (such as killing or reduction of fitness) of the phagic agent (such as phage or lysin) against the symbiotic bacterium (such as in vitro, or in vivo, i.e., in the pest); (x) expanding the host range of the phage; (xi) propagating the phage; and (xii) assessing the inhibitory activity (such as killing or reduction of fitness) of the phage composition against the pest and adjusting the phage composition based on the inhibitory activity.

[0084] Thus, in some embodiments, there is provided a method of preparing a phage composition for killing or degrading fitness of a pest, comprising: (a) sequencing a specimen of the pest to identify a symbiotic bacterium of the pest; (b) obtaining a phagic agent (such as phage or lysin) that inhibits the symbiotic bacterium; and (c) formulating a composition comprising the phagic agent (such as phage or lysin) to provide the phage composition. In some embodiments, the method comprises obtaining a plurality of phagic agents (such as phages and/or lysins) that each inhibits the symbiotic bacterium. In some embodiments, the method comprises identifying a plurality of symbiotic bacteria of the pest, and obtaining one or more phagic agents (such as phages and/or lysins) that inhibit each symbiotic bacterium. In some embodiments, the phagic agent (such as phage or lysin) is obtained from a specimen of the pest, or from an environmental sample. [0085] In some embodiments, there is provided a method of preparing a phage composition for killing or degrading fitness of a pest, comprising: (a) applying a pool of phages to a plurality of the pest to obtain a first specimen of the pest comprising one or more affected pest individuals and a second specimen comprising one or more unaffected pest individuals; (b) comparatively sequencing (such as metagenomics sequencing) the first specimen and the second specimen to identify a symbiotic bacterium from the sequencing results; (c) obtaining a phagic agent (such as phage or lysin) that inhibits the symbiotic bacterium; and (d) formulating a composition comprising the phagic agent (such as phage or lysin) to provide the phage composition. In some embodiments, the method comprises obtaining a plurality of phagic agents (such as phages and/or lysins) that each inhibits the symbiotic bacterium. In some embodiments, the method comprises identifying a plurality of symbiotic bacteria of the pest, and obtaining one or more phagic agents (such as phages and/or lysins) that inhibit each symbiotic bacterium. In some embodiments, the phage is identified from results of the comparative sequencing. In some embodiments, the phagic agent (such as phage or lysin) is obtained from the pool of phages.

[0086] In some embodiments, there is provided a method of preparing a phage composition for killing or degrading fitness of a pest, comprising: (a) sequencing a specimen of the pest to identify a symbiotic bacterium of the pest; (b) isolating a phage that inhibits the symbiotic bacterium from a sample (such as a specimen of the pest, or an environmental sample); and (c) formulating a composition comprising the phage to provide the phage composition. In some embodiments, the method comprises obtaining a plurality of phages that each inhibits the symbiotic bacterium. In some embodiments, the method comprises identifying a plurality of symbiotic bacteria of the pest, and obtaining one or more phages that inhibit each symbiotic bacterium

[0087] In some embodiments, there is provided a method of preparing a phage composition for killing or degrading fitness of a pest, comprising: (a) applying a pool of phages to a plurality of the pest to obtain a first specimen of the pest comprising one or more affected pest individuals and a second specimen comprising one or more unaffected pest individuals; (b) comparatively sequencing (such as metagenomics sequencing) the first specimen and the second specimen to identify a symbiotic bacterium from the sequencing results; (c) isolating a phage that inhibits the symbiotic bacterium from the pool of phages; and (d) formulating a composition comprising the phage to provide the phage composition. In some embodiments, the method comprises obtaining a plurality of phages that each inhibits the symbiotic bacterium. In some embodiments, the method comprises identifying a plurality of symbiotic bacteria of the pest, and obtaining one or more phages that inhibit each symbiotic bacterium.

[0088] In some embodiments, there is provided a method of preparing a phage composition for killing or degrading fitness of a pest, comprising: (a) sequencing a specimen of the pest to identify a symbiotic bacterium of the pest; (b) isolating a phage mat inhibits the symbiotic bacterium from a sample (such as a specimen of the pest, or an environmental sample); (c) engineering the phage to expand the host range of the phage; and (d) formulating a composition comprising the phage to provide the phage composition. In some embodiments, the method comprises obtaining a plurality of phages that each inhibits the symbiotic bacterium. In some embodiments, the method comprises identifying a plurality of symbiotic bacteria of (he pest, and obtaining one or more phages that inhibit each symbiotic bacterium.

[0089] In some embodiments, there is provided a method of preparing a phage composition for killing or degrading fitness of a pest, comprising: (a) applying a pool of phages to a plurality of the pest to obtain a first specimen of the pest comprising one or more affected pest individuals and a second specimen comprising one or more unaffected pest individuals; (b) comparatively sequencing (such as metagenomics sequencing) the first specimen and the second specimen to identify a symbiotic bacterium from the sequencing results; (c) isolating a phage that inhibits the symbiotic bacterium from the pool of phages; (d) engineering the phage to expand the host range of the phage; and (e) formulating a composition comprising the phage to provide the phage composition. In some embodiments, the method comprises obtaining a plurality of phages that each inhibits the symbiotic bacterium. In some embodiments, the method comprises identifying a plurality of symbiotic bacteria of the pest, and obtaining one or more phages that inhibit each symbiotic bacterium.

[0090] In some embodiments, mere is provided a method of preparing a phage composition for killing or degrading fitness of a pest, comprising: (a) sequencing a specimen of the pest to identify a symbiotic bacterium of the pest; (b) isolating a phage mat inhibits the symbiotic bacterium from a sample (such as a specimen of the pest, or an environmental sample); optionally (c) engineering the phage to expand the host range of the phage; (d) propagating the phage; and (e) formulating a composition comprising the phage to provide the phage composition. In some embodiments, the method comprises obtaining a plurality of phages that each inhibits the symbiotic bacterium In some embodiments, the method comprises identifying a plurality of symbiotic bacteria of the pest, and obtaining one or more phages that inhibit each symbiotic bacterium. [0091] In some embodiments, there is provided a method of preparing a phage composition for killing or degrading fitness of a pest, comprising: (a) applying a pool of phages to a plurality of the pest to obtain a first specimen of the pest comprising one or more affected pest individuals and a second specimen comprising one or more unaffected pest individuals; (b) comparatively sequencing (such as metagenomics sequencing) the first specimen and the second specimen to identify a symbiotic bacterium from the sequencing results; (c) isolating a phage that inhibits the symbiotic bacterium from the pool of phages; optionally (d) engineering the phage to expand the host range of the phage; (e) propagating the phage; and (f) formulating a composition comprising the phage to provide the phage composition. In some embodiments, the method comprises obtaining a plurality of phages that each inhibits the symbiotic bacterium In some embodiments, the method comprises identifying a plurality of symbiotic bacteria of the pest, and obtaining one or more phages that inhibit each symbiotic bacterium.

[0092] In some embodiments, there is provided a method of preparing a phage composition for killing or degrading fitness of a pest, comprising: (a) sequencing a specimen of the pest to identify a symbiotic bacterium of the pest; (b) isolating a phage that inhibits the symbiotic bacterium from a sample (such as a specimen of the pest, or an environmental sample); optionally (c) engineering the phage to expand the host range of the phage; optionally (d) propagating the phage; (e) formulating a composition comprising the phage to provide the phage composition; and (f) assessing the inhibitory activity (such as killing or reduction of fitness) against the pest and adjusting the phage composition based on the inhibitory activity. In some embodiments, the method comprises obtaining a plurality of phages that each inhibits the symbiotic bacterium In some embodiments, the method comprises identifying a plurality of symbiotic bacteria of the pest, and obtaining one or more phages that inhibit each symbiotic bacterium

[0093] In some embodiments, there is provided a method of preparing a phage composition for killing or degrading fitness of a pest, comprising: (a) applying a pool of phages to a plurality of the pest to obtain a first specimen of the pest comprising one or more affected pest individuals and a second specimen comprising one or more unaffected pest individuals; (b) comparatively sequencing (such as metagenomics sequencing) the first specimen and the second specimen to identify a symbiotic bacterium from the sequencing results; (c) isolating a phage that inhibits the symbiotic bacterium from the pool of phages; optionally (d) engineering the phage to expand the host range of the phage; optionally (e) propagating the phage; (f) formulating a composition comprising the phage to provide the phage composition; and (g) assessing the inhibitory activity (such as killing or reduction of fitness) against the pest and adjusting the phage composition based on the inhibitory activity. In some embodiments, the method comprises obtaining a plurality of phages that each inhibits the symbiotic bacterium. In some embodiments, the method comprises identifying a plurality of symbiotic bacteria of the pest, and obtaining one or more phages that inhibit each symbiotic bacterium.

[0094] In some embodiments, there is provided a method of preparing a phage composition (or phage cocktail) for killing or degrading fitness of a pest, comprising: (a) obtaining a specimen of the pest; (b) performing metagenomics sequencing on the specimen of the pest to identify bacteria present on or in the pest; (c) culture the bacteria from the pest to obtain bacterial isolates; (d) determining the identity of the bacterial isolates; (e) identifying a symbiotic bacterium from the bacterial isolates; (f) identifying a phage that kills the symbiotic bacterial isolate; and (g) including the phage in the phage composition. In some embodiments, the bacterial isolate is a symbiotic bacterium of the pest if the bacterial isolate has been reported to enhance the pest's fitness or enable the pest's survival. In some embodiments, the method further comprises performing functional assays or measuring gene expression levels in the bacterial isolates to identify a symbiotic bacterial isolate. In some embodiments, the method further comprises administering the phage to the pest to assess killing activity against the symbiotic bacterium In some embodiments, the phage is included in the phage composition if the phage kills the symbiotic bacterium when administered to the pest. In some embodiments, wherein the phage composition kills or degrades the fitness of the pest, the phage composition is incorporated into a suitable delivery vehicle for pest control.

[0095] In some embodiments, there is provided a method of preparing a phage composition (or phage cocktail) for killing or degrading fitness of a pest, comprising: (a) obtaining a specimen of the pest; (b) performing metagenomics sequencing on the specimen of the pest to identify bacteria present on or in the pest; (c) obtaining bacterial isolates from a cell bank; (d) sequencing the bacterial isolates to confirm identity of the bacterial isolates; (e) identifying a symbiotic bacterium from the bacterial isolates; (i) identifying a phage that kills the symbiotic bacterial isolate; and (g) including the phage in the phage composition. In some embodiments, the bacterial isolate is a symbiotic bacterium of the pest if the bacterial isolate has been reported to enhance the pest's fitness or enable the pest's survival. In some embodiments, the method further comprises performing functional assays or measuring gene expression levels in the bacterial isolates to identify a symbiotic bacterial isolate. In some embodiments, the method further comprises administering the phage to the pest to assess killing activity against the symbiotic bacterium. In some embodiments, the phage is included in the phage composition if the phage kills the symbiotic bacterium when administered to the pest. In some embodiments, wherein the phage composition kills or degrades the fitness of the pest, the phage composition is incorporated into a suitable delivery vehicle for pest control.

[0096] Exemplary methods of identifying the symbiotic bacterium, obtaining a phagic agent (such as phage or lysin), expanding the host range of the phage, propagating the phage, obtaining lysins, assessing and adjusting the phage composition, and formulating the phage compositions are described in detail below.

Phagic agent

[0097] The phage composition of the present application comprises one or more phagic agents, such as phages, and/or phage components (such as phage enzyme, for example, lysin). In some embodiments, the method comprises obtaining a phage that inhibits the symbiotic bacterium. In some embodiments, the method comprises obtaining a lysin that inhibits the symbiotic bacterium. In some embodiments, the method comprises obtaining a phage that inhibits the symbiotic bacterium, and isolating a lysin from the phage, and formulating a composition comprising the lysin to provide the phage composition. In some embodiments, the phage composition comprises one or more phages only. In some embodiments, the phage composition comprises one or more lysins only. In some embodiments, the phage composition comprises both phage(s) and lysin(s).

[0098] In some embodiments, the phagic agent (such as phage or lysin) is obtained from a phage source, such as an environmental sample, or a specimen of the pest In general, phages can be found anywhere their bacterial hosts exist. Accordingly, pest extracts and samples from the environment in which the pest lives are suitable sources from which to isolate phages. Untreated sewage and wastewater streams also represent attractive sources since they have large numbers of diverse bacteria, and by implication, phages.

[0099] In some embodiments, the phagic agent (such as phage or lysin) is obtained from an environmental sample. In some embodiments, the phagic agent (such as phage or lysin) is obtained from the surroundings (such as the habitat) of the pest. In some embodiments, the environmental sample is from the food or water source of the pest In some embodiments, the environmental sample is an untreated sewer sample. In some embodiments, the

environmental sample is a soil sample. In some embodiments, an untreated sewer sample from the Silicon Valley Clean Water (SVCW) Wastewater Treatment Plant is used as the phage source. In some embodiments, a water sample from Alameda Creek is used as the phage source.

[0100] In some embodiments, the phagic agent (such as phage or lysin) is obtained from a specimen of the pest. The specimen can be the same specimen that is used for the sequencing, or it can be a different specimen. In some embodiments, the specimen is the pest In some embodiments, the specimen is a pest exhibiting reduced fitness. In some embodiments, the specimen is a pest extract In some embodiments, the specimen is from the feces or other secretion of the pest. In some embodiments, the specimen is the stomach content of the pest In some embodiments, homogenized aphids are used as the phage source.

[0101] It is important to note that pests that exhibit reduced fitness or are killed after exposure to phage pools, with or without enrichment, may themselves serve as enriched sources of phages, as phages that kill critical symbiotic bacteria can propagate within the pest. Many bacteria cannot be cultured in the laboratory using known practices, and it may therefore be desirable to use the pest as a vehicle for propagation of the pest-killing phages. In some embodiments, a 100-ml water sample from the Ganges River in India is mixed with 100 homogenized Argentine ants (Linepithema humile) and incubated at 26°C for 10 h. The suspension is then subjected to centrifugation at 10,000 x g for 15 min, followed by filtration of the supernatant through a 0.5 um syringe filter and a 0.2 μηι syringe filter to isolate an enriched pool of phages. A collection of about 10,000 Argentine ants are treated with the enriched phage supernatant by pouring it onto their colony. After 48 hours, the ants are harvested, homogenized in the presence of 100 ml phage buffer, and the phages are isolated by size-exclusion filtration.

[0102] In some embodiments, the phage is extracted, enriched, and/or isolated from the environmental sample or the specimen of the pest In some embodiments, the environmental sample or the specimen of the pest is first processed to obtain a phage extract. For example, the environmental sample or the specimen of the pest can be agitated, mixed, centrifuged, filtered, fractionated, and/or suspended in an appropriate buffer. In some embodiments, the phage extract is enriched for phages. In some embodiments, the phage extract is screened against the symbiotic bacterium to isolate phage that inhibits the growth of the symbiotic bacterium. One or more phages that infect, propagate within, and lyse the symbiotic bacterium are selected and included in the phage composition to target the symbiotic bacterium. In some embodiments, the selected phage reaches a concentration of at least any of 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10ⁿ, or more pfu/ml in the screening mixture of the phage and the symbiotic bacterium in an overnight assay. In some embodiments, the phage is isolated from the environmental sample or the specimen of the pest by any of the methods known in the art, such as chloroform extraction to lyse phage-infected bacteria. In some embodiments, the isolated phage is further purified.

[0103] For example, in some embodiments, a phage-containing soil sample is agitated for 1 h with phage buffer (10 mM Tris, pH 7.5, 10 mM MgCl₂, 68 mM NaCl, and 1 mM CaCl₂) to extract phages. A 10-ml aliquot of this suspension is added to Sx brain heart infusion (BHI) broth for a final lx concentration, inoculated with a bacterial isolate of interest, and incubated with shaking overnight at 37°C. In this manner, the bacteria of interest grow in the presence of a diverse pool of phages. This method selects for those phages that infect, propagate within, and lyse the bacteria, as the concentration of "successful" phages increase rapidly (to about 10⁶-10ⁿ plaque-forming units/ml). The suspension is then treated with chloroform to lyse phage-infected bacteria and centrifuged, removing sediment and the majority of the bacteria while leaving phages in the supernatant. The phage suspension is then spotted onto agar plates that have an overlay of a low percentage nutrient agar with bacterial culture (the bacteria of interest) added. If, after overnight incubation, the spotted regions show clearing of the bacterial lawn, the phage suspension is likely to contain active phages against the bacteria. Phage suspensions mat are positive for the spot test are diluted and re-plated so that individual phage plaques can be observed, and individual plaques are purified and amplified three times to ensure a clonal phage isolate is obtained. Phage purification may be

accomplished by precipitation with polyethylene glycol, size-exclusion chromatography, centrifugal filtration, dialysis, or any other method known to a person of ordinary skill in the art.

[0104] Lysine have been described, for example, see, Young R (1992). "Bacteriophage lysis: mechanism and regulation". Microbiological Reviews 56 (3): 430-81. Any lysin suitable for inhibiting the symbiotic bacterium can be used in the methods and phage compositions described herein. In some embodiments, the lysin is obtained from a phage that inhibits the symbiotic bacterium. In some embodiments, the lysin is identified by sequencing the phage. In some embodiments, the lysin is cloned from the phage. In some embodiments, the lysin is engineered based on a naturally occurring lysin. In some embodiments, the lysin is engineered to be secreted by a host bacterium, for example, by introducing a signal peptide to the lysin. In some embodiments, the lysin is used in combination with a holin in the phage composition. In some embodiments, a lysin is expressed by a recombinant bacterium host that is not sensitive to the lysin. In some embodiments, the lysin is used to inhibit a Gram- positive symbiotic bacterium

[0105] In some cases, it may be desirable to use lysins, instead of phages. Although lysine are proteins rather than viruses, and therefore lack the replication function of phages, they can also be used to kill a host's bacterial symbionts. Lysins may be used in lieu of phages in any of the embodiments of methods of preparing a phage composition or methods of killing or degrading the fitness of a pest described herein for which phage replication is not required to kill a host's bacterial symbionts. For example, lysins can be applied directly to a pest or the environment in which the pest lives.

[0106] In some embodiments, the inhibitory activity of the phagic agent (such as phage or lysin) against the symbiotic bacterium is assessed. In some embodiments, the phagic agent (such as phage or lysin) is applied to the symbiotic bacterium in vitro, such as in a bacterial culture, to assess the inhibitory activity. In some embodiments, the phagic agent (such as phage or lysin) is applied to the symbiotic bacterium in vivo to assess the inhibitory activity. For example, the phagic agent (such as phage or lysin) is applied to the pest, and changes to the population of the symbiotic bacterium in the pest in response to the treatment are determined. Changes to the population of the symbiotic bacterium in the pest may be determined by any methods known in the art, such as microarray, polymerase chain reaction (PCR), real-time PCR, matrix-assisted laser desorption ionization-mass spectrometry

(MALDI-MS), and DNA sequencing. In some embodiments, a sample of the pest treated with the phagic agent (such as phage or lysin) is sequenced (such as by metagenomics sequencing) to determine the microbiome of the pest after the treatment. In some embodiments, a sample of untreated pest is also sequenced to provide a reference. The phagic agent (such as phage or lysin) is selected for inclusion in the phage composition if the phagic agent (such as phage or lysin) reduces the population of the symbiotic bacterium by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in the pest In some embodiments, the phagic agent (such as phage or lysin) is selected for inclusion in the phage composition if the phagic agent (such as phage or lysin) eradicates the population of the symbiotic bacterium in the pest. [0107] For example, in some embodiments, western drywood termites (Incisitermes minor) from a single colony are split into a control and an experimental group. Termites in the control group are misted with phage buffer, while termites in the experimental group are misted with phage buffer containing 10⁹ pfu/ml of phage targeting a symbiotic bacterium in the termite hindgut After 24 h, each group of termites is washed with distilled water, homogenized, and subjected to shotgun metagenomics sequencing to determine if the symbiotic bacteria in the hindgut have been eliminated in the experimental group.

[0108] In some embodiments, Bermuda grass (Cynodon daetylon) is partitioned into a control and an experimental group, each of equal area (10 cm x 10 cm), using a waterproof physical barrier that extends 0.5 m above and 1 m below the soil. The topsoil of the control group is treated with 500 ml phage buffer, while topsoil in the experimental group is treated with 500 ml of phage buffer containing 10⁸ pfu/ml of phage targeting a symbiotic nitrogen- fixing bacterium in the soil. After 12 h, 5 g of rhizomes from each group of Bermuda grass are collected, bacteria are collected from the surface by washing, and bacterial DNA is extracted and characterized by 16S rDNA or 16S rRNA amplicon sequencing to detennine if the symbiotic nitrogen-fixing bacteria on the rhizome have been eliminated in the experimental group.

[0109] The isolated phages can be stored as stocks for future use. In some embodiments, the phage is stored at 4 °C in phage buffer. In some embodiments, the phage is lyophilized. In some embodiments, the phage is stored in phage buffer with 50% glycerol, frozen rapidly in liquid nitrogen, and then stored at -80 °C. In other embodiments, the phage is stored within its infected bacterial host, which is frozen at -80 °C in liquid culture media containing 25% glycerol.

In vivo workflow

[0110] The present invention also includes in vivo workflows for discovering symbiotic bacteria necessary for the survival of the pest. A diverse pool of phages is introduced into or onto one or more living pests or its immediate surroundings in an in vivo experiment. Those pests that die or exhibit reduced fitness are collected and subjected to sequencing (such as metagenomics sequencing) analysis, either individually or in aggregate, and compared to the sequencing results of their unaffected peers, either individually or in aggregate. Comparative sequencing analysis (such as metagenomics analysis) of the pest's bacterial consortia before and after treatment allows for the discovery of symbiotic bacteria necessary for the survival of the pest, since the death or reduced fitness of the pest can be correlated with the elimination of previously colonizing bacteria. Additionally, comparative metagenomics of the viral consortia allows for the identification of phages that propagated within the less-fit or killed pest.

[0111] In some embodiments, the symbiotic bacterium is identified by: (a) applying a pool of phages to a plurality of the pest to obtain a first specimen of the pest comprising one or more affected pest individuals and a second specimen comprising one or more unaffected pest individuals; (b) comparatively sequencing (such as metagenomics sequencing) the first specimen and the second specimen to identify a symbiotic bacterium from the sequencing results. In some embodiments, the symbiotic bacterium is identified by: (a) applying a pool of phages to a plurality of the pest to obtain a first specimen of the pest comprising one or more affected pest individuals and a second specimen comprising one or more unaffected pest individuals; (b) comparatively sequencing (such as metagenomics sequencing) the first specimen and the second specimen to identify a symbiotic bacterium from the sequencing results, wherein the phage is obtained (such as isolated) from the pool of phages. In some embodiments, the affected individuals are killed by the pool of phages. In some embodiments, the fitness (such as reproduction rate, mobility, body weight or metabolic rate) of the affected individuals is degraded by the pool of phages. In some embodiments, the method further comprises obtaining a lysin from the phage, and formulating a composition comprising the lysin to provide the phage composition.

[0112] Suitable specimens, methods of sequencing the specimens, and methods of identifying symbiotic bacteria from sequencing results are described in the section

"Symbiotic bacteria" below. Whole pest or portions of a pest (such as fruiting body, stomach, or hindgut) can be used for the sequencing experiments (such as metagenomics sequencing studies). Matching specimens should be used for the comparative sequencing studies. For example, the same portions of affected and unaffected pest individuals, or affected and unaffected pest individuals of the same developmental stage and gender should be used for the comparative sequencing studies in order identify candidate bacteria that is reduced or eliminated in response to the antibacterial treatment with the pool of phages. Bacteria genus, species or strains that are present in the unaffected individuals, but absent or reduced in affected individuals may be selected as candidate symbiotic bacteria, and may be further obtained (such as from a cell bank, or isolated from the specimen of the unaffected individual), and assessed to confirm the symbiotic relationship between each candidate symbiotic bacterium and the pest, as described in the section "Symbiotic bacteria." In some embodiments, the specimens are subjected to size-exclusion filtration to obtain a sample enhanced with bacterial genetic material and/or phage genetic material. In some embodiments, the sequencing is metagenomics sequencing, such as shotgun sequencing of all genes, or the 16S rRNA amplicon sequencing. In some embodiments, the metagenomics sequencing is carried out by next-generation sequencing.

[0113] The pool of phages can be obtained from any suitable phage source. Any of the phage sources including environmental samples or specimens of the pest as described in the "Phagic agent" section can be used. In some embodiments, the pool of phages is present in an environmental sample, such as an untreated sewer sample, or a soil sample. The pool of phages may be enriched (such as by filtering through a size exclusion filter), extracted, fractioned, purified and/or propagated from the phage source or sample before application to the pest individuals.

[0114] For example, in some embodiments, an untreated sewer sample from the Silicon Valley Clean Water (SVCW) Wastewater Treatment Plant is subjected to centrifugation at 12,000 x g for 10 min, followed by filtration of the supernatant through a 0.5 μπι syringe filter and a 0.2 um syringe filter to isolate a diverse pool of phages. A collection of about 1000 common earwigs (Forficula auricularia) is exposed to the diverse phage pool by misting spray. After 72 hours, surviving earwigs are separated from dead earwigs, and differential metagenomics analysis by shotgun sequencing is performed. In some

embodiments, size exclusion filtration is used to enrich the metagenomics sample in bacteria prior to DNA extraction. In some embodiments, size exclusion filtration is used to enrich the metagenomics sample in viruses prior to DNA extraction. In some embodiments, the pest's feces are subjected to metagenomics analysis before and after treatment with phages. In other embodiments, the Illumina MiSeq Reporter Metagenomics Workflow is used for analysis.

[0115] In some embodiments, the diverse pool of phages used to treat the pest may not possess a high enough titer of useful phages to effectively kill the pest's symbiotic bacteria or to affect a sufficient number of pests to achieve a statistically significant result. It may be necessary, therefore, to perform an "enrichment" step on the phage source by addition of the pest's bacteria followed by an incubation step. In some embodiments, a 50-ml water sample from the Tbilisi River in the Republic of Georgia is mixed with 500 homogenized Formosan termites and incubated at 30 °C for 8 h. The suspension is then subjected to centrifugation at 10,000 x g for 15 min, followed by filtration of the supernatant through a 0.5 μπι syringe filter and a 0.2 μηι syringe filter to isolate an enriched pool of phages. A collection of about 1500 Formosan termites (Coptotermes formosanus) are treated with the supernatant by misting spray. After 48 hours, some of the surviving termites are separated from killed termites and both are subjected to metagenomics analysis. In some embodiments, termites are frozen at -80 °C upon death or shortly thereafter to minimize changes in the microbiome associated with decomposition, and are later thawed and subjected to comparative metagenomics analysis to identify important symbiotic bacteria for which the pest has an obligate or facultative relationship.

[0116| In some embodiments, the pool of phages is obtained from a pest. In some embodiments, the pool of phages is obtained from an extract of a pest infected with phages. In some embodiments, the pool of phages is obtained from the feces or other secretion of the pest. In some embodiments, the pool of phages is collected from the surroundings (such as habitat, water or food source) of the pest.

[0117] The phages identified from the sequencing results can be further isolated from the phage pool. In some embodiments, the inhibitory activity of the phage against each of the symbiotic bacteria is assessed, such as by an in vitro assay and/or by an in vivo assay (i.e., in the pest). Any of the phage isolation or assessment methods described above can be used. A plurality (such as any of 1, 2, 3, 4, 5, 10, 20, 50, or more) of symbiotic bacteria and a plurality (such as any of 1, 2, 3, 4, 5, 10, 20, 50, or more) of phages targeting the symbiotic bacteria may be identified with this in vivo identification method.

Host range expansion

[0118] Phages isolated with the methods described above may be a lytic phage, or a temperate phage. In some embodiments, the phage has a narrow host range. For example, the phage may target no more than about any of 50, 40, 30, 20, 10, 8, 6, 4, 2, or 1 symbiotic bacterium. In some embodiments, the phage is a promiscuous phage with a large host range. For example, the promiscuous phage may target at least about any of 10, 20, 30, 40, 50, or more symbiotic bacterium.

[0119] In some embodiments, the phage is further engineered to expand its host range prior to inclusion in the phage composition. Due to the narrow host range that is typical of naturally occurring phages, it may be necessary to engineer the selected phage such that it is capable of infecting a broader range of host bacteria (i.e. "host range expansion"). Such engineering may be done using genetic techniques that are known to those skilled in the art. In some embodiments, the tail fibers of a phage may be rationally designed and genetically encoded to enable host range expansion. In some embodiments, a tail fiber sequence is obtained from whole-genome sequencing of a pest's bacterial symbiont (the DNA of phages that successfully infected its ancestors is often present in the bacteria's chromosomal DNA) and this sequence is used to modify the tail fiber on a naturally occurring phage. Engineering may also be accomplished using directed evolution of the phage under selection pressure. In some embodiments, the engineering comprises selecting a variant of the phage for the inhibitory activity against a second symbiotic bacterium (or plurality of symbiotic bacteria) of the pest, wherein the phage is initially unable to infect the second symbiotic bacterium (or plurality of symbiotic bacteria) or infects the second symbiotic bacterium (or plurality of symbiotic bacteria) with low efficiency. For example, the phage is initially propagated within its preferred host bacteria, and then gradually "weaned" onto other host bacteria that it was originally unable to infect or to infect with only low efficiency.

[0120] In some embodiments, a lysin is obtained from a phage after host range expansion of the phage to obtain a lysin having a broader substrate.

Phage propagation

[0121] In some embodiments, the phage is further propagated prior to inclusion in the phage composition. In some embodiments, the phage is propagated in a culture of the symbiotic bacterium In some embodiments, the phage is propagated in a culture of a surrogate bacterium In some embodiments, the phage is propagated in a sample rich in bacteria, such as an environmental sample, or a specimen of the pest In some embodiments, the phage is propagated in a pest extract. In some embodiments, the phage is propagated in a live pest. In some embodiments, the phage is propagated in a surrogate host carrying the symbiotic bacterium. The propagated phage may be further extracted, and/or purified prior to inclusion in the phage composition. In some embodiments, the propagated phage is not isolated from the propagation medium (such as the live pest, pest extract, or surrogate host) prior to inclusion in the phage composition.

[0122] When pests are used to propagate phages (i.e. in vivo phage propagation), it is not always ideal to extract the phages by homogenizing the pest, since destruction of the pest is likely to halt phage propagation. Even in the absence of pest homogenization, infection of the pest's important symbiotic bacteria by the phages of interest is likely to lead to a reduction in pest fitness or death. In some cases, it is therefore desirable to use the pests as "continuous- flow reactors," whereby the important symbiotic bacteria within or on the surface of a pest or its surroundings is infected with one or more phages while the pest is intermittently inoculated (i.e. rescued) with the important symbiotic bacteria that enable its continued survival. The phages can then be harvested by non-lethal methods, such as from the pest's feces or other secretions or from collecting infected bacteria in the surroundings. In some embodiments, 300 American cockroaches (Periplaneta americana) are exposed to a phage cocktail that targets Bacteroidetes, Clostridia, and Deltaproteobacteria, important cockroach gut bacteria, by adding the phage cocktail to their mashed potato diet at 10⁸ pfu/g. After 12 hours of feeding, the cockroach diet is switched to a diet of mashed potatoes with 10% fresh cockroach extract (wt%/wt%) from mechanically homogenized non-phage-infected

Periplaneta americana cockroaches, such that the live phage-infected cockroaches are intermittently inoculated with fresh gut bacteria through their diet. Feces from the

cockroaches, which is rich in propagated phages, is collected through a mesh in the cockroach cage that possesses openings that are sufficiently small so that the cockroaches cannot escape but large enough for the fecal droppings to pass. These fecal droppings are then processed in a manner that enriches the phages {e.g., extraction with phage buffer followed by size-exclusion filtration through a 0.2 μπι filter or precipitation with

polyethylene glycol) or renders them non-hazardous to human health without destroying the phages (e.g., radiation, or desiccation and urea treatment).

[0123] In some cases, it may be more desirable to use a "surrogate" organism different than the pest to do in vivo propagation of the phage. For example, if the pest is involved in an obligate relationship with the bacterial host of the phage and the pest is treated with the phage, the pest may die before it can be rescued through inoculation with additional bacterial host. Therefore, a surrogate organism can be used to incubate the bacteria during phage

propagation. In some cases, the substitute organism may already possess as part of its microbiome the host bacteria in which the phage propagates, but in other cases it may be necessary to first inoculate the substitute organism with the bacterial host In some embodiments, the substitute organism belongs to the same genus as the pest. In some embodiments, the substitute organism belongs to a different genus than the pest In some embodiments, the substitute organism is inoculated with the host bacteria through its drinking water. In some embodiments, the host organism secretes the infected host bacteria. Phage composition

[0124] Any of the above methods for isolating a phage in vitro or in vivo can be repeated to obtain a plurality (such as about any of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, or more) of phagic agents (such as phages and/or lysins) against a symbiotic bacterium of the pest. In some embodiments, a plurality (such as at least 2, 3, 4, 5, 6, 10, 15, 20, 50, or more) of symbiotic bacteria of the pest is identified, and one or more (such as at least 2, 3, 4, 5, 6, 10, 15, 20, 50, or more) phagic agents (such as phages and/or lysins) that inhibit each symbiotic bacterium of the pest are obtained. The phage composition prepared using the methods described herein may comprise any number of phagic agents (such as phages and/or lysins), such as at least about any one of 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, or more. Phage compositions comprising one or more phages are also referred herein as "phage cocktails".

[0125] Cocktails are useful because they allow for targeting of a wider host range of bacteria. Furthermore, they allow for each bacterial strain (i.e. subspecies) to be targeted by multiple orthogonal phages, thereby preventing or significantly delaying the onset of resistance. In some embodiments, a cocktail comprises multiple phages targeting one bacterial species. In some embodiments, a cocktail comprises multiple phages targeting multiple bacterial species. In some embodiments, a one-phage "cocktail" is comprised of a single promiscuous phage (i.e. a phage with a large host range) targeting many strains within a species. In some embodiments, the phage cocktail targets bacteria of the genus Wolbachia, and it may be well suited to kill insect pests. In other embodiments, the phage cocktail targets Buchnera aphidicola and Hamiltonella defensa, and it may be suitable to kill aphid pests. In an additional embodiment, the phage cocktail targets Treponema, Bacteroides termitidis, Desulfovibrio, Citrobacter freundii, Enterobacter agglomerans, Enterococcus, and

Laetococcus, and it may show activity against termite pests. In some embodiments, the phage cocktail targets the cyanobacteria Nostoc sp., and it may be suitable to kill lichenized fungi pests. In some embodiments, the phage cocktail targets Aeromonas veronii biovar sobria and a Rikenella-hke bacterium, and it may be effective at killing leeches, such as Hirudo verbana. In some embodiments, the phage cocktail is formulated in a phage buffer (such as 10 mM Tris, pH 7.5, 10 mM MgCl₂, 68 mM NaCl and 1 mM CaCl₂ solution).

[0126] In some embodiments, although a phage cocktail kills the target symbiotic bacteria on or within the pest, this does not ensure that it will kill or degrade the fitness of the pest In embodiments, wherein the pest acts as an obligate symbiont in a mutualistic, commensal, or parasitic relationship with a bacteria, eradication of the bacteria is likely to lead to death of the pest. However, in some cases, the pest may have redundant bacteria that are not targeted by the phage cocktail that may serve the same function as the bacteria that are killed, effectively rescuing the pest from the phage cocktail. It is therefore in some cases important to test the ability of the phage cocktail to kill the pest.

[0127] In some embodiments, the inhibitory activity of the phage composition against the pest is assessed. For example, the phage composition can be applied to the pest in order to assess the inhibitory activity. Methods of application of the phage composition are described in the "Methods of pest control" section. In some embodiments, the inhibitory activity comprises killing at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of a colony of the pest In some embodiments, the inhibitory activity comprises killing the pest within about any of 10 minutes, 20 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 1 day, or 1 week from the time of the application of the phage composition. In some embodiments, the inhibitory activity comprises degrading the fitness of the pest In some embodiments, the fitness is measured by the reproductive rate of the pest. In some embodiments, the phage composition reduces the reproductive rate of the pest by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more. In some embodiments, the fitness is measured by the mobility of the pest. In some embodiments, the phage composition reduces the mobility of the pest by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more. In some embodiments, the fitness is measured by the body weight of the pest. In some embodiments, the phage composition reduces the body weight of the pest by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more. In some embodiments, the fitness is measured by the metabolic rate of the pest In some embodiments, the phage composition reduces the metabolic rate of the pest by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more.

[0128] Based on the inhibitory activity of the phage composition against the pest, in some embodiments, the phage composition is further adjusted. For example, if the phage composition fails to kill or to reduce the fitness of the pest, then the phage composition is adjusted. In some embodiments, the phage composition is adjusted by engineering the phage to expand its host range to obtain an engineered phage, and incorporating the engineered phage in the phage composition. In some embodiments, the phagic agent (such as phage or lysin) is engineered to target redundant symbiotic bacteria of the pest. Methods of expanding the host range of a phage are discussed above in the "Host range expansion" section. In some embodiments, the phage composition is adjusted by including one or more phagic agents (such as phages and/or lysins) that target additional symbiotic bacterium of the pest.

[0129] Suitable concentration of each phage in the phage composition depends on factors such as efficacy, survival rate, transmissibility of the phage, number of distinct phage and/or lysin types in the phage compositions, formulation, and methods of application of the phage composition. In some embodiments, wherein the phage composition is a liquid, the concentration of each phage in the phage composition is at least about any of 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, or more pfu/ml. In some embodiments, wherein the phage composition is a liquid, the concentration of each phage in the phage composition is no more than about any of 10², 10³, 10⁴, 10^s, 10⁶, 10⁷, 10⁸, 10⁹ pfu/ml. In some embodiments, wherein the phage composition is a liquid, the concentration of each phage in the phage composition is any of about 10² to about 10³, about 10³ to about 10⁴, about 10⁴ to about 10^s, 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 10⁸pfu/ml. In some embodiments, wherein the phage composition is a solid, the concentration of each phage in the phage composition is at least about any of 10², 10³, 10⁴, 10^s, 10⁶, 10⁷, 10⁸, 10⁹, or more pfu/g. In some embodiments, wherein the phage composition is a solid, the concentration of each phage in the phage composition is no more than about any of 10², 10³, 10⁴, 10^s, 10⁶, 10⁷, 10⁸, 10⁹ pfu/g. In some embodiments, wherein the phage composition is a solid, the concentration of each phage in the phage composition is any of about 10² to about 10³, about 10³ to about 10⁴, about 10⁴ to about 10⁵, about 10^s 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/g. In some embodiments, wherein the phage composition comprises at least two types of phages, the concentration of each type of the phages can be the same or different For example, in some embodiments, 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.

[0130] Suitable concentration of each lysin in the phage composition depends on factors such as efficacy, stability of the lysin, number of distinct lysin and/or phage types in the phage compositions, formulation, and methods of application of the phage composition. In some embodiments, each lysin in a liquid phage composition is from about 0.1 ng/mL to about 10 mg/mL. In some embodiments, each lysin in a solid phage composition is from about 0.1 ng/g to about 10 mg/g. In some embodiments, wherein the phage composition comprises at least two types of ly sins, the concentration of each type of the lysins can be the same or different

[0131] In some embodiments, the phage composition further comprises a bacteriocin that inhibits a symbiotic bacterium of the pest. In some embodiments, the symbiotic bacterium targeted by the bacteriocin is targeted by the phages in the phage composition. In some embodiments, the symbiotic bacterium targeted by the bacteriocin is not targeted by any phage in the phage composition. In some embodiments, the bacteriocin is a pyocin. In some embodiments, the bacteriocin is engineered. In some embodiments, the bacteriocin is expressed by a bacterial vector. In some embodiments, the phage composition comprises a plurality of bacteriocins, each inhibiting a symbiotic bacterium of the pest.

Exemplary workflows

[0132] Embodiments of the present disclosure include methods for developing

compositions that kill or otherwise reduces the fitness of pests. FIG. 1 shows one exemplary workflow for developing a pest control composition comprising a plurality of phages (also referred herein as "phage cocktail"). In step 1, specimens of the pest are obtained. A metagenomics study is performed on the pest specimen or its immediate environment to identify the bacteria that are present (step 2). Metagenomics study is a powerful sequencing- based technique that allows for the identification of bacteria present within, on the surface of, or around a pest specimen without the requirement for the bacteria to be cultured. The list of bacteria generated from the metagenomics study in step 2 is instructive because it enables proper selection of the culture conditions for obtaining bacterial isolates (step 3). After culturing, clonal isolates of the bacteria are identified in step 4.

[0133] Upon identification, those bacterial isolates that are not already identified in the literature as symbionts through which the pest derives benefit may be screened for the presence of genes implicated in symbiosis through whole-genome sequencing; expression of genes implicated in symbiosis through RNA-seq or microarray; or tested in functional assays (step 5). hi the absence of literature reports or genetic data or functional data suggesting the bacterial isolate may be beneficial to the pest, the isolate is banked for later use (step 6).

[0134] For those bacterial isolates that are likely to provide the pest with enhanced fitness or a survival advantage, screening is performed to identify phages that kill the bacteria (step 7). Once a clonal phage is isolated against the pest's symbiotic bacteria (step 7), the phage is tested for in vivo activity against its bacterial target on or within the pest or in its immediate surroundings. When the phage fails to eliminate the target bacteria on or within the pest, it may be banked for later use (step 8). In the event that the phage successfully kills the target bacteria on or within the pest or its immediate surroundings, the phage is included in a cocktail (step 9).

[0135] Next, the phage cocktail is applied to the pest to test the efficacy of the phage cocktail in killing or degrading the fitness of the pest. If phage-cocktail-treated pests do not die faster than untreated pests of the same age, or the fitness of the phage-cocktail-treated pests is not degraded compared to untreated pests, the workflow is repeated (step 10):

additional bacterial isolates are obtained (step 3) and new phages against them are ultimately added to the cocktail (step 9). Multiple iterations may be required until a suitable phage cocktail is discovered. In cases for which conditions are not amenable to phage stability or delivery, it may be necessary to incorporate the phage cocktail into a delivery or stabilizing vehicle for pest control (step 11).

[0136] FIG. 2 shows an alternative workflow for developing a pest control composition comprising a phage cocktail. Rather than culturing bacteria from the pest to obtain isolates (FIG. 1, step 3), pest-associated bacterial isolates are obtained from a cell bank (FIG. 2, step 12). If the whole-genome sequence of the procured bacterial isolate is not provided, the isolate is sequenced (step 13). If the sequence is not consistent with the putative identity of the isolate, as procured, the isolate is returned or discarded (step 14). However, if the sequence is consistent, phages that kill the bacterial isolate are identified by screening (FIG. 1, step 7). In some embodiments, a phage cocktail is prepared exclusively from commercially obtained bacterial isolates (FIG. 2, step 12). In some embodiments, a phage cocktail is prepared using both bacterial isolates cultured from the pest (FIG. 1, step 3) and

commercially obtained bacterial isolates (FIG. 2, step 12).

Method of preparing a bacteriocin composition

[0137] Further provided are methods of preparing a bacteriocin composition for killing or degrading fitness of a pest, comprising: (a) identifying a symbiotic bacterium in a specimen of the pest; (b) obtaining a bacteriocin that inhibits the symbiotic bacterium; and (c) formulating a composition comprising the bacteriocin to provide the bacteriocin composition. In some embodiments, the method comprises obtaining a plurality of bacteriocins that each inhibits the symbiotic bacterium. In some embodiments, the method comprises identifying a plurality of symbiotic bacteria of the pest, and obtaining one or more bacteriocins that inhibit each symbiotic bacterium. In some embodiments, the bacteriocin is obtained from a natural source. In some embodiments, the bacteriocin is engineered.

[0138] In some embodiments, there is provided a method of preparing a bacteriocin composition for killing or degrading fitness of a pest, comprising: (a) identifying a symbiotic bacterium in a specimen of the pest; and (b) formulating a composition comprising a bacteriocin that inhibits the symbiotic bacterium to provide the bacteriocin composition. In some embodiments, the method comprises formulating a composition comprising a plurality of bacteriocins that each inhibits the symbiotic bacterium. In some embodiments, (he method comprises identifying a plurality of symbiotic bacteria of the pest, and formulating a composition comprising a plurality of bacteriocins, wherein each symbiotic bacterium is inhibited by one or more bacteriocins among the plurality of bacteriocins. In some embodiments, the bacteriocin is obtained from a natural source. In some embodiments, the bacteriocin is engineered.

[0139] In some embodiments, there is provided a method of preparing a bacteriocin composition for killing or degrading fitness of a pest, comprising: (a) identifying a symbiotic bacterium in a specimen of (he pest; and (b) obtaining a composition comprising a bacteriocin that inhibits the symbiotic bacterium to provide the bacteriocin composition. In some embodiments, the method comprises obtaining a composition comprising a plurality of bacteriocins that each inhibits the symbiotic bacterium. In some embodiments, the method comprises identifying a plurality of symbiotic bacteria of the pest, and obtaining a composition comprising a plurality of bacteriocins, wherein each symbiotic bacterium is inhibited by one or more bacteriocins among the plurality of bacteriocins. In some embodiments, the bacteriocin is obtained from a natural source. In some embodiments, the bacteriocin is engineered.

[0140] In some embodiments, (here is provided a method of preparing a bacteriocin composition for killing or degrading fitness of a pest, comprising: (a) identifying a symbiotic bacterium in a specimen of the pest; (b) obtaining a bacteriocin that inhibits the symbiotic bacterium; and (c) formulating a composition comprising the bacteriocin to provide the bacteriocin composition, wherein the symbiotic bacterium is identified by sequencing (such as metagenomics sequencing) the specimen of the pest. In some embodiments, the method comprises obtaining a plurality of bacteriocins that each inhibits the symbiotic bacterium. In some embodiments, the method comprises identifying a plurality of symbiotic bacteria of the pest, and obtaining one or more bacteriocins that inhibit each symbiotic bacterium. In some embodiments, the bacteriocin is obtained from a natural source. In some embodiments, the bacteriocin is engineered.

[0141] In some embodiments, there is provided a method of preparing a bacteriocin composition for killing or degrading fitness of a pest, comprising: (a) identifying a symbiotic bacterium in a specimen of the pest; (b) obtaining a bacteriocin that inhibits the symbiotic bacterium; and (c) formulating a composition comprising the bacteriocin to provide the bacteriocin composition, wherein the symbiotic bacterium is identified by comparative sequencing (such as metagenomics sequencing) of a first specimen of the pest comprising one or more pest individuals affected by an antibacterial treatment (such as applying antibiotics to the pest), and a second specimen of the pest comprising one or more pest individuals unaffected by the antibacterial treatment In some embodiments, the method comprises obtaining a plurality of bacteriocins that each inhibits the symbiotic bacterium. In some embodiments, the method comprises identifying a plurality of symbiotic bacteria of the pest, and obtaining one or more bacteriocins that inhibit each symbiotic bacterium. In some embodiments, the bacteriocin is obtained from a natural source. In some embodiments, (he bacteriocin is engineered.

[0142] In some embodiments, any of the above methods of preparing a bacteriocin composition may further comprise any one or more (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) of the following steps: (i) obtaining a specimen of the pest; (ii) sequencing the specimen of the pest (including comparative sequencing of two or more specimens, such as metagenomics sequencing); (iii) obtaining the symbiotic bacterium (such as from a cell bank, or by isolating the symbiotic bacterium from the specimen); (iv) isolating or culturing the symbiotic bacterium; (v) determining the symbiotic relationship between the symbiotic bacterium and the pest; (vi) isolating a bacteriocin from a natural source; (vii) engineering a naturally- occurring bacteriocin to obtain the bacteriocin targeting the symbiotic bacterium; (viii) assessing the inhibitory activity (such as killing or reduction of fitness) of the bacteriocin against the symbiotic bacterium; and (x) assessing the inhibitory activity (such as lolling or reduction of fitness) of the bacteriocin composition against the pest and adjusting the bacteriocin composition based on the inhibitory activity.

[0143] Thus, in some embodiments, there is provided a method of preparing a bacteriocin composition for killing or degrading fitness of a pest, comprising: (a) sequencing a specimen of the pest to identify a symbiotic bacterium of the pest; (b) obtaining a bacteriocin that inhibits the symbiotic bacterium; and (c) formulating a composition comprising the bacteriocin to provide the bacteriocin composition. In some embodiments, the method comprises obtaining a plurality of bacteriocins that each inhibits the symbiotic bacterium. In some embodiments, the method comprises identifying a plurality of symbiotic bacteria of the pest, and obtaining one or more bacteriocins that inhibits each symbiotic bacterium. In some embodiments, the bacteriocin is obtained from a natural source. In some embodiments, the bacteriocin is engineered.

[0144] In some embodiments, there is provided a method of preparing a bacteriocin composition for killing or degrading fitness of a pest, comprising: (a) sequencing a specimen of the pest to identify a symbiotic bacterium of the pest; (b) isolating the bacteriocin from a natural source; and (c) formulating a composition comprising the bacteriocin to provide the bacteriocin composition. In some embodiments, the method comprises obtaining a plurality of bacteriocins that each inhibits the symbiotic bacterium. In some embodiments, the method comprises identifying a plurality of symbiotic bacteria of the pest, and obtaining one or more bacteriocins that inhibits each symbiotic bacterium.

[0145] In some embodiments, there is provided a method of preparing a bacteriocin composition for killing or degrading fitness of a pest, comprising: (a) sequencing a specimen of the pest to identify a symbiotic bacterium of the pest; (b) engineering a naturally occurring bacteriocin to obtain the bacteriocin that specifically targets the symbiotic bacterium; and (c) formulating a composition comprising the bacteriocin to provide the bacteriocin composition. In some embodiments, the method comprises obtaining a plurality of bacteriocins that each inhibits the symbiotic bacterium, hi some embodiments, the method comprises identifying a plurality of symbiotic bacteria of the pest, and obtaining one or more bacteriocins that inhibits each symbiotic bacterium.

[0146] In some embodiments, there is provided a method of preparing a bacteriocin composition for killing or degrading fitness of a pest, comprising: (a) sequencing a specimen of the pest to identify a symbiotic bacterium of the pest; (b) obtaining a bacteriocin that inhibits the symbiotic bacterium; (c) formulating a composition comprising the bacteriocin to provide the bacteriocin composition; and (d) assessing the inhibitory activity (such as killing or reduction of fitness) of the bacteriocin composition against the pest and adjusting the bacteriocin composition based on the inhibitory activity. In some embodiments, the method comprises obtaining a plurality of bacteriocins that each inhibits the symbiotic bacterium. In some embodiments, the method comprises identifying a plurality of symbiotic bacteria of the pest, and obtaining one or more bacteriocins that inhibits each symbiotic bacterium. In some embodiments, the bacteriocin is obtained from a natural source. In some embodiments, the bacteriocin is engineered.

[0147] In some cases, it may be desirable to use bacteriocins, such as pyocins, instead of phages. Although bacteriocins are proteins rather than viruses, and therefore lack the replication function of phages, they can also be used to kill a host's bacterial symbionts. Bacteriocins may be used in lieu of phages in any of the embodiments of methods of preparing a phage composition or methods of killing or degrading the fitness of a pest described herein for which phage replication is not required to kill a host's bacterial symbionts. For example, bacteriocins can be applied directly to a pest or the environment in which the pest lives.

[0148] Any of the methods described in the "Symbiotic bacteria" section for identifying the symbiotic bacterium can be used in the methods of preparing the bacteriocin composition. Any suitable formulation described in the "Formulation" section may be used in the methods of preparing the bacteriocin composition.

[0149] In some embodiments, the bacteriocin is obtained (such as isolated) from a natural source. In some embodiments, the symbiotic bacterium is present in the natural source. In some embodiments, the natural source is the pest, the pest extract, or the surroundings of the pest. In some embodiments, the bacteriocin is a naturally-occurring bacteriocin. In some embodiments, the bacteriocin is a known bacteriocin targeting the symbiotic bacterium, and is obtained from a commercial source or cloned based on the known sequence of the bacteriocin.

[0150] In some embodiments, the bacteriocin is a high-molecular weight bacteriocin, such as R-type bacteriocins (e.g., pyocins). In some embodiments, the bacteriocin is a low molecular weight polypeptide bacteriocin, such as nisin. In some embodiments, the bacteriocin is naturally produced by Gram-positive bacteria, such as Pseudomonas,

Streptomyces, Bacillus subtilis, Staphylococcus, and lactic acid bacteria (LAB, such as Lactococcus lactis). In some embodiments, the bacteriocin is naturally produced by Gram- negative bacteria, such as Hafnia alvei, Citrobacter freundii, Klebsiella oxytoca, Klebsiella pneumonia, Enterobacter cloacae, and Escherichia coli. In some embodiments, the bacteriocin kills the symbiotic bacterium via cytoplasmic membrane pore formation, cell wall interference, or nuclease activity. Exemplary bacteriocins include, but are not limited to, Class I-IV LAB antibiotics (such as lantibiotics), colicins, microcins, and pyocins. In some embodiments, the bacteriocin is a pyocin. In some embodiments, the pyocin is an R-pyocin, F-pyocin, or S-pyocin. Other naturally occurring and engineered bacteriocins have been described; for example, see Gillor O. etal. Current Pharmaceutical Design, 2005, 11: 1067- 1075, incorporated herein by reference.

[0151] In some embodiments, the bacteriocin is engineered. In some embodiments, a naturally-occurring bacteriocin is engineered such that one or more of its properties are changed relative to the wild-type bacteriocin. Exemplary properties include but are not limited to stability, substrate specificity, binding affinity, immunogenicity, immunotoxicity, and kinetics. For example, site-directed or random mutagenesis of naturally-occurring bacteriocin can be generated to provide bacteriocins with altered properties, such as specificities. In some embodiments, the bacteriocin is obtained by engineering a naturally- occurring bacteriocin to alter the specificity of the naturally-occurring bacteriocin to target the symbiotic bacterium In some embodiments, the bacteriocin is obtained by engineering the tail fiber of a pyocin to obtain the bacteriocin targeting the symbiotic bacterium Methods of engineering bacteriocins are known in the art, for example, see International Patent Application Publication No. WO2007134303, incorporated herein by reference. In some embodiments, the tail fibers on a pyocin are genetically engineered so that it kills bacteria of the genus Wolbachia.

[0152] Any of the above methods for obtaining or engineering a bacteriocin can be repeated to obtain a plurality (such as about any of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) of bacteriocins against a symbiotic bacterium of the pest. In some embodiments, a plurality (such as at least 2, 3, 4, 5, 6, 10, 15, 20, 50, or more) of symbiotic bacteria of the pest is identified, and one or more (such as at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) bacteriocins mat inhibit each symbiotic bacterium of the pest are obtained. The bacteriocin composition prepared using the methods described herein may comprise any number of bacteriocins, such as at least about any one of 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, or more. The term "bacteriocin composition" is used here interchangeably with "bacteriocin cocktail".

[0153] In some embodiments, the inhibitory activity of the bacteriocin composition against the pest is assessed. For example, the bacteriocin composition can be applied to the pest to assess the inhibitory activity. Methods of application of the bacteriocin composition are described in the "Methods of pest control" section. In some embodiments, the inhibitory activity comprises killing at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of a colony of the pest. In some embodiments, the inhibitory activity comprises killing the pest within about any of 10 minutes, 20 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 1 day, or 1 week from the time of the application of the bacteriocin composition. In some embodiments, the inhibitory activity comprises degrading the fitness of the pest. In some embodiments, the fitness is measured by the reproductive rate of the pest. In some embodiments, the bacteriocin composition reduces the reproductive rate of the pest by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more. In some embodiments, the fitness is measured by the mobility of the pest. In some embodiments, the bacteriocin composition reduces the mobility of the pest by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more. In some embodiments, the fitness is measured by the body weight of the pest. In some embodiments, the bacteriocin composition reduces the body weight of the pest by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more. In some embodiments, the fitness is measured by the metabolic rate of the pest. In some embodiments, the bacteriocin composition reduces the metabolic rate of the pest by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more.

[0154] Based on the inhibitory activity of the bacteriocin composition against the pest, in some embodiments, the bacteriocin composition is further adjusted. For example, if the bacteriocin composition fails to kill or to reduce the fitness of the pest, then the bacteriocin composition is adjusted. In some embodiments, the bacteriocin composition is adjusted by engineering the bacteriocin to improve its expression level, stability, and/or efficacy against the symbiotic bacterium, and incorporating the engineered bacteriocin in the bacteriocin composition. In some embodiments, the bacteriocin composition is adjusted by including one or more bacteriocins that target additional symbiotic bacteria of the pest

[0155] In some embodiments, the bacteriocin composition further comprises a phagic agent (such as phage or lysin) that inhibits a symbiotic bacterium of the pest. In some embodiments, the bacteriocin composition further comprises one or more phages. In some embodiments, the bacteriocin composition further comprises one or more lysine. In some embodiments, the symbiotic bacterium targeted by the phagic agent (such as phage or lysin) is targeted by the bacteriocins in the bacteriocin composition. In some embodiments, the symbiotic bacterium targeted by the phagic agent (such as phage or lysin) is not targeted by any bacteriocin in the bacteriocin composition. In some embodiments, the bacteriocin composition comprises a plurality of phagic agents (such as phages and/or lysins), each inhibiting a symbiotic bacterium of the pest.

[0156] Suitable concentration of each bacteriocin in the bacteriocin composition depends on factors such as efficacy, stability of the bacteriocin, number of distinct bacteriocin types in the bacteriocin compositions, formulation, and methods of application of the bacteriocin composition. In some embodiments, each bacteriocin in a liquid bacteriocin composition is from about 0.01 \ig/vdL to about 100 mg/mL. In some embodiments, each bacteriocin in a solid bacteriocin composition is from about 0.01 ug/g to about 100 mg/g. In some embodiments, wherein the bacteriocin composition comprises at least two types of bacteriocins, the concentration of each type of the bacteriocins can be the same or different.

Symbiotic bacteria

[0157] The methods of preparation described above comprise a step of identifying one or more symbiotic bacteria in a specimen of the pest, and the phage composition or the bacteriocin composition prepared by the methods can target (such as inhibit the growth of) the one or more symbiotic bacteria.

[0158] In some embodiments, the symbiotic bacterium is identified by sequencing a specimen of the pest.

[0159] In some embodiments, the symbiotic bacterium is identified by sequencing a specimen of the pest to identify a candidate bacterium associated with the pest, and determining the symbiotic relationship between the candidate bacterium and the pest

[0160] In some embodiments, the symbiotic bacterium is identified by sequencing a specimen of the pest to identify a candidate bacterium associated with the pest, isolating the candidate bacterium from the specimen, and determining the symbiotic relationship between the candidate bacterium and the pest.

[0161] In some embodiments, the symbiotic bacterium is identified by sequencing a specimen or the pest to identify a candidate bacterium associated with the pest, obtaining the candidate bacterium from a cell bank, and determining the symbiotic relationship between the candidate bacterium and the pest.

[0162] In some embodiments, the symbiotic bacterium is identified by sequencing a specimen or the pest to identify a candidate bacterium associated with the pest, obtaining the candidate bacterium (such as by isolating the candidate bacterium from the specimen, or obtaining the candidate bacterium from a cell bank), and identifying a gene of the candidate bacterium implicated in the symbiotic relationship with the pest In some embodiments, the gene is identified by testing the candidate bacterium using a functional assay related to the gene. In some embodiments, the expression level of the gene in the candidate bacterium is Further determined.

[0163] In some embodiments, the symbiotic bacterium is identified by comparative sequencing of a first specimen of the pest comprising one or more pest individuals affected by an antibacterial treatment (such as applying a pool of phages or antibiotics to the pest), and a second specimen of the pest comprising one or more pest individuals unaffected by the antibacterial treatment

[0164] In some embodiments, the symbiotic bacterium is identified by comparative sequencing of a first specimen of the pest comprising one or more pest individuals affected by an antibacterial treatment (such as applying a pool of phages or antibiotics to the pest), and a second specimen of the pest comprising one or more pest individuals unaffected by the antibacterial treatment to identify a candidate bacterium associated with the pest, and determining the symbiotic relationship between the candidate bacterium and the pest

[0165] In some embodiments, the symbiotic bacterium is identified by comparative sequencing of a first specimen of the pest comprising one or more pest individuals affected by an antibacterial treatment (such as applying a pool of phages or antibiotics to the pest), and a second specimen of the pest comprising one or more pest individuals unaffected by the antibacterial treatment to identify a candidate bacterium associated with the pest, isolating the candidate bacterium from the specimen, and detennining the symbiotic relationship between the candidate bacterium and the pest.

[0166] In some embodiments, the symbiotic bacterium is identified by comparative sequencing of a first specimen of the pest comprising one or more pest individuals affected by an antibacterial treatment (such as applying a pool of phages or antibiotics to the pest), and a second specimen of the pest comprising one or more pest individuals unaffected by the antibacterial treatment to identify a candidate bacterium associated with the pest, obtaining the candidate bacterium from a cell bank, and detennining the symbiotic relationship between the candidate bacterium and the pest.

[0167] In some embodiments, the symbiotic bacterium is identified by comparative sequencing a first specimen of the pest comprising one or more pest individuals affected by an antibacterial treatment (such as applying a pool of phages or antibiotics to the pest), and a second specimen of the pest comprising one or more pest individuals unaffected by the antibacterial treatment to identify a candidate bacterium associated with the pest, obtaining the candidate bacterium (such as by isolating the candidate bacterium from the specimen, or obtaining the candidate bacterium from a cell bank), and identifying a gene of the candidate bacterium implicated in the symbiotic relationship with the pest In some embodiments, the gene is identified by testing the candidate bacterium using a functional assay related to the gene. In some embodiments, the expression level of the gene in the candidate bacterium is further determined.

[0168] Any of the methods of preparing a phage composition or a bacteriocin composition described herein may further comprise sequencing a specimen of the pest or comparative sequencing of two or more specimens of the pest to identify to symbiotic bacterium. The sequencing comprises any one or more of the following steps: (i) sequencing a specimen or comparative sequencing of two or more specimen to identify a candidate bacterium associated with the pest; (ii) obtaining the candidate bacterium (such as by isolating the candidate bacterium from the specimen, or obtaining the candidate bacterium from a cell bank); and (iii) determining the symbiotic relationship between the candidate bacterium and the pest. In some embodiments, the step (iii) comprises one or more of the following steps: (1) identifying a gene of the candidate bacterium implicated in the symbiotic relationship with the pest; (2) determining the expression level of the gene in the candidate bacterium; and (3) testing the candidate bacterium using a functional assay related to the gene.

[0169] Any specimen from the pest or in the immediate surrounding of the pest can be used to identify the one or more symbiotic bacteria. In some embodiments, the specimen is the pest. In some embodiments, the specimen is derived from the pest. In some embodiments, the specimen comprises or is derived from pests of a particular developmental stage or of a particular sex. The pest can be obtained from any suitable source. In some embodiments, the pests are obtained from their natural habitat. In other embodiments, they are obtained from locations in which they exist undesirably to humans. In some embodiments, pest specimens are obtained from a supplier. In some embodiments, multiple pests or parts of pests are combined to produce sufficient specimen for genetic extraction and sequencing.

[0170] In some embodiments, two specimens of the pest under different conditions are obtained, each specimen is sequenced, and the sequencing results of the two specimens are compared to identify the symbiotic bacterium. In some embodiments, a plurality of the pest individuals is treated with an antibacterial agent mat kills or degrades the fitness of the pest, and one specimen is obtained from affected pest individuals, and one specimen is obtained from unaffected pest individuals. In some embodiments, wherein the specimens comprise portions of the pest individuals, the same portions are compared for the less-fit or killed pests and the unaffected pests. Any suitable specimens described herein are applicable for use in the comparative sequencing. In some embodiments, the antibacterial agent is a phagic agent (such as phage or lysin), pool of phages, or a phage composition. In some embodiments, the antibacterial agent is a bacteriocin or bacteriocin composition. In some embodiments, the antibacterial agent is an antibiotics or antibiotics composition. In some embodiments, the antibacterial agent comprises one or more phagic agents (such as phages and/or lysins) in the phage composition to be prepared, or one or more bacteriocins in the bacteriocin composition to be prepared. In some embodiments, the antibacterial agent is unrelated to the phage composition or the bacteriocin composition.

[0171] In some embodiments, the specimen is the whole body of the pest. In some embodiments, the entire pest is homogenized and the extracted genetic material is sequenced. In some embodiments, the specimen is a portion of the pest In some embodiments, the pest is dissected and a particular cell type, tissue, organ, or body section is subjected to extraction and sequencing, excluding the rest of the organism. Such methods may be especially suitable in cases for which the symbiotic bacterium is known to be spatially localized on or within a pest. In some embodiments, the specimen is the fruiting body, stomach, or hindgut of the pest.

[0172] In some embodiments, the specimen is inside the pest or on the surface of the pest. In some embodiments, the specimen is the stomach content or feces of the pest. In some embodiments, the specimen is surface bacteria, for example, the pest may also be washed to isolate surface bacteria for study. In some embodiments, the pest is washed to remove the surface bacteria in order to eliminate interference from surface bacteria. In some

embodiments, the pest is washed with an ethanol solution (such as about 70% ethanol, for example, for about three times). In some embodiments, the pest is washed with a bleach solution (such as about 10% bleach, for example, for one time). In some embodiments, the pest is washed with water (such as nanopore water, for example, for about three times). In some embodiments, the pest is washed three times with about 70% ethanol, once with about 10% bleach, and three times with nanopure water.

[0173] In some embodiments, the specimen is derived from the surroundings of the pest. In some embodiments, the specimen is obtained from the habitat of the pest. In some

embodiments, the specimen is obtained from the local environment of the pest, for example, from soil, water, or substrate. In some embodiments, the metagenomics study is performed on the local environment of the pest. Those skilled in the art would recognize that the symbiotic bacteria need not exist on or within the pest, and instead, may exist in its immediate surroundings. For example, symbiotic bacteria for a plant pest may reside in the soil.

[0174] In some embodiments, the specimen is processed by homogenization (such as grinding, digestion, ultrasonication), centrifugation (e.g., to remove sedimentation), and/or size-exclusion filtration etc. to obtain a specimen enriched for the bacteria fraction or bacterial nucleic acids (e.g., DNA or RNA). The specimen may be suspended in any suitable buffer, and genetic material (such as DNA or RNA) is extracted from the specimea In some embodiments, the specimen is resuspended in a sterile lx phosphate-buffered saline. Genetic material can be extracted using any known methods in the art, including using commercially available kits, such as the PowerFood Microbial DNA Isolation Kit (MO BIO Laboratories), PowerSoil DNA Isolation Kit (MO BIO Laboratories), Meta-G-Nome DNA Isolation Kit (Dlumina), SoilMaster DNA Extraction Kit (Illumina), and ExtractMaster Fecal DNA

Extraction Kit (Illumina). DNA libraries may be further prepared from the genetic material using any methods known in the art. For example, in some embodiments, a kit is used for preparing DNA libraries from the genetic material. Suitable commercially available kits for preparing DNA libraries for sequencing analysis include, but are not limited to, Nextera XT DNA Library Preparation Kit (Illumina) and Nextera XT Index Kit (Illumina). In some embodiments, the DNA libraries are subjected to tagmentation prior to shotgun sequencing.

[0175] The specimen or genetic material derived therefrom may be subject to any suitable sequencing analysis in order to identify the symbiotic bacterium or a list of candidate symbiotic bacteria. In some embodiments, the sequencing (including comparative sequencing) is whole genome sequencing of individual bacterial clones from the specimen. In some embodiments, the sequencing (including comparative sequencing) is carried out without culturing the bacterial clones in the specimen. In some embodiments, the sequencing

(including comparative sequencing) is metagenomics sequencing. In some embodiments, the sequencing (including comparative sequencing) is shotgun metagenomics sequencing. In some embodiments, the sequencing (including comparative sequencing) is targeted sequencing of one or more marker genes. In some embodiments, the one or more marker genes are amplified prior to sequencing. In some embodiments, the sequencing (including comparative sequencing) is 16S rRNA or 16S rDNA sequencing. In some embodiments, the 16S rRNA or 16S rDNA is amplified from the specimen for sequencing analysis.

[0176] Any suitable sequencing methods, including, but not limited to, capillary sequencing, PCR-based sequencing, shotgun sequencing, and next-generation sequencing methods, can be used to cany out the sequencing step. Many high-throughput sequencing platforms are available, including, but not limited to MiSeq and NextSeq sequencing platforms (Illumina). Computational pipelines are also known in the art for analyzing the sequencing results. For example, Illumina's BaseSpace cloud computing platform can be used for metagenomics analysis. In some embodiments, the open-source software QIIME is used for 16S metagenomics analysis, and the Greengenes 16S database is used for bacterial identification. Some embodiments use unique molecular identifiers to reduce sequencer error and improve (he accuracy of amplicon-based sequencing. In some embodiments, Amazon Machine Instance versions of algorithms are used.

[0177] The sequencing analysis may provide a list of candidate bacteria, including symbiotic bacteria and non-symbiotic bacteria associated with the pest. In some embodiments, one or more candidate bacteria strains or isolates are obtained. In some embodiments, the candidate bacterium is isolated from the specimen. In some embodiments, said isolating comprises culturing clonal isolates of the candidate bacterium under a culturing condition, and confirming the identity of the cultured clonal isolates. Culture conditions include the nutrient composition, pH, temperature, pressure, atmosphere, and any other specifications required to successfully grow the bacteria of interest Such culture conditions may be aerobic or anaerobic, hi some embodiments, the isolates are cultured at more than about 21% of oxygen. In some embodiments, the isolates are cultured at less than about 21% of oxygen. In some embodiments, the isolates are cultured at the oxygen level normally present in air (such as about 21%). The culturing condition may further comprise different concentrations of C02, nitrogen, and/or other gases, and/or the total pressure may be greater than, less than, or equal to atmospheric pressure. The temperature suitable for culture may include any temperature in which bacteria are known to survive. For example, thermophilic (i.e. heat loving) bacteria are viable up to about 122 °C, while cryophiles (i.e. cold lovers) can exist at about -20 °C. In some embodiments, the culturing conditions necessary to isolate bacteria identified in the sequencing analysis is determined from the literature, such as the ATCC website (atcc.org), Handbook of Microbiological Culture Media, or Handbook of Culture Media for Food and Water Microbiology. In some embodiments, the culture conditions necessary to isolate bacteria identified in the sequencing analysis are determined empirically. In some

embodiments, an extract of the pest is used as a component of the culture media. In some embodiments, the culture media is purchased from a commercially available source. In other embodiments, the culture media is chemically defined. In some embodiments, the culture condition comprises incubation at about 28 °C under anaerobic conditions. In some embodiments, the culture media comprises cellulose.

[0178] In some embodiments, the isolates are further identified. Common techniques for identification of bacterial isolates include, but are not limited to, microarray, polymerase chain reaction (PCR), real-time PCR, matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS), and DNA sequencing. Additional techniques may include bacterial staining, phage typing, and nucleic acid hybridization. In some embodiments, DNA from a bacterial isolate is extracted using the Ultraclean Microbial DNA Extraction Kit (MO BIO Laboratories), tagmented using the Nextera XT DNA Library Preparation Kit (Illumina), and sequenced on a MiSeq instrument (Illumina). Any technique that gives identification of the isolate to at least the genus level (i.e. genus- or species- or strain- levels) is suitable. Any computational tools and databases known in the art may be used, including, but not limited to, Kraken (see, for example, Wood DE & Salzberg SL. Genome Biology 2014, 15:R46), mOTU (see, for example, Sunagawa et al. Nature Methods, 2013, 10: 1196-1199), andNCBI's repository of bacterial genomes. For example, Kraken defines operational taxonomic units and assigns bacterial identification. Some embodiments utilize a database built from NCBI's repository of bacterial genomes to identify the bacterial isolates. In some embodiments, the mOTU algorithm is used to assign bacterial identifications. In some embodiments, de novo assembly of genome of an isolated species is performed. For example, the SPADES algorithm (see, for example, Bankevich A. et al. Journal of Computational Biology, 2012, 19(5): 455-477) is used to perform de novo assembly of bacterial genomes. In some embodiments, de novo assembly is performed on a mixed sample. In some embodiments, unique molecular identifiers are used to reduce sequencer error for shotgun and whole genome sequencing approaches. Bacterial isolates with identities matching the candidate bacteria from the specimen sequencing step can be used for additional analysis and testing, such as to confirm symbiotic relationship between the bacteria and the pest.

[0179] In some embodiments, the candidate symbiotic bacterium is obtained from a cell bank. In some embodiments, the cell bank is ATCC. In some embodiments, the cell bank is the National Collection of Plant Pathogenic Bacteria (UK). In some embodiments, the cell bank is the Belgian Co-ordinated Collections of Micro-organisms (BCCM). In other embodiments, the cell bank is the Deutsche Sammlung von Mikroorganismen und

Zellkulturen (DSMZ, or German Collection of Microorganisms and Cell Cultures). In some embodiments, the cell bank is the Japanese Collection of Research Bioresources (JCRB). [0180] In some embodiments, the candidate symbiotic bacterium obtained for a cell bank is sequenced to confirm the identity of the candidate symbiotic bacterium In some

embodiments, NCBI's Nucleotide BLAST tool is used to confirm the identity of the bacteria by searching a region of the sequenced DNA. In some embodiments, NCBI's Microbial Nucleotide BLAST tool is used to confirm the identity of the bacteria by searching a region of the sequenced DNA. In some embodiments, the Burrows-Wheeler Aligner is used with GATK variant calling to align the sequenced bacterial genome to its putative reference genome. Only bacterial strains with identity matching the candidate symbiotic bacterium from the specimen sequencing step are used for additional analysis and testing, such as to confirm symbiotic relationship between the bacteria and the pest

[0181] In some embodiments, the bacterial isolates or bacterial strains obtained from a cell bank are stored before use. In some embodiments, the isolates are frozen at -80°C in liquid culture media containing 25% glycerol. In some embodiments, the isolates are frozen at - 80°C in commercial freezing media (e.g., Amresco).

[0182] The symbiotic relationship between each of the candidate symbiotic bacteria and the pest need to be confirmed before obtaining a phagic agent (such as phage or lysin) or a bacteriocin targeting the symbiotic bacterium In some embodiments, the symbiotic relationship between a candidate bacterium and the pest is confirmed based on previous publication or database records. In some embodiments, a gene implicated in a symbiotic relationship with the pest is identified from the candidate bacterium in order to confirm the symbiotic relationship. In some embodiments, the gene is identified by sequencing (such as whole-genome sequencing) the candidate symbiotic bacterium In some embodiments, the gene is involved in a metabolic pathway of the pest. For example, in some embodiments, the whole-genome sequence of an isolate from the African cotton stainer Dysdercus fasciatus is screened for bluB-like flavin destructase and fldA genes, which are associated with biosynthesis of vitamin B12. In some embodiments, a gene in the cellulose degradation or other required metabolic pathways (e.g. Fibrobacter succinogenes) is identified from the genome of a candidate symbiotic bacterium to target termites. Further testing related to the gene may be performed to ascertain the symbiotic relationship. In some embodiments, the expression level of the gene is determined in the candidate bacterium. In some embodiments, a functional assay related to the gene is performed on the candidate bacterium. For example, a bacterial isolate from the hindgut of a termite can be tested in a functional assay for cellulase activity. In some embodiments, an isolate from the soil intimately associated with the invasive grass Sorghum halepense, which possesses rhizomes, is tested in a functional assay for nitrogenase activity. In some embodiments, a candidate bacterium expressing one or more genes that are involved in key metabolic pathways of the pest are chosen as the symbiotic bacterium of the pest to be targeted by the phage or bacteriocin composition.

[0183] The methods described above may be used to identify a plurality of symbiotic bacterial isolates (such as strains, species, or genus) of the pest Any number of the symbiotic bacterial isolates may be targeted by the phage or bacteriocin compositions prepared by the methods described herein. In some embodiments, the phage or bacteriocin composition is prepared to target a single symbiotic bacterial isolate. In some embodiments, the phage or bacteriocin composition is prepared to target at least about any of 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 symbiotic bacterial isolates. In some embodiments, the phage or bacteriocin composition is prepared to target any one of about 1 to about 5, about 5 to about 10, about 10 to about 20, about 20 to about 50, about 50 to about 100, about 100 to about 200, about 200 to about 500, about 10 to about 50, about 5 to about 20, or about 10 to about 100 distinct bacterial isolates. Any one or more of the symbiotic bacterial isolates may be an obligate symbiont of the pest, or a facultative symbiont of the pest

[0184] Depending on the identity of the pest, certain symbiotic bacteria may be particularly suitable as targets for preparing the phage or bacteriocin compositions to control the pest In some embodiments, the pest is an animal. In some embodiments, wherein the pest is selected from the group consisting of nematode, spider, mite, and insect, the symbiotic bacterium is Wolbachia. In some embodiments, wherein the pest is a psyllid, the symbiotic bacterium is selected from the group consisting of Carsonella, Wolbachia, Rhizobium, Gordonia, Mycobacterium and Xanthomonas. In some embodiments, wherein the pest is an aphid, the symbiotic bacterium is selected from the group consisting of Buchnera aphidicola and Hamiltonella defense. In some embodiments, wherein the pest is a termite, the symbiotic bacterium is selected from the group consisting of Treponema, Bacteroides, Desulfovibrio, Citrobacter, Enterobacter, Enterococcus, Lactococcus and Methanobrevibacter. In some embodiments, wherein the pest is a leech, the symbiotic bacterium is selected from the group consisting of Aeromonas, Biovar, and a Rikenella-like bacterium. In some embodiments, wherein the pest is a plant (such as a plant with rhizomes), the symbiotic bacterium is selected from the group consisting of Herbaspirillum, Agrobacterium, Xanthomonas, Arthrobacter, Spirillum, and Rhizobium. In some embodiments, wherein the pest is a lichenized fungus, the symbiotic bacterium is Nostoc (such as a cyanobacterium of the Genus Nostoc).

Formulation

[0185] The phage or bacteriocin compositions are formulated either in pure form or together with one or more additional agents (such as excipient, delivery vehicle, carrier, diluent, stabilizer, etc.) to facilitate application of the compositions to the pest. 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 solution, syrup, methylcellulose, methyl- and propylhydroxybenzoates, talc, magnesium stearate and mineral oil.

[0186] Depending on the intended objectives and prevailing circumstances, the phage or bacteriocin composition may be formulated into emulsifiable concentrates, suspension concentrates, directly sprayable or dilutable solutions, coatable pastes, diluted emulsions, spray powders, soluble powders, dispersible powders, wettable powders, dusts, granules, or encapsulations in polymeric substances. In some embodiments, the phage or bacteriocin composition is a liquid. In some embodiments, the phage or bacteriocin composition is a solid. In some embodiments, the phage or bacteriocin composition is an aerosol, such as in a pressurized aerosol can. hi some embodiments, the phage or bacteriocin composition is present in a pest extract. In some embodiments, the phage or bacteriocin composition is present in the waste (such as feces) of the pest. In some embodiments, the phage or bacteriocin composition is present in or on a live pest

[0187] In some embodiments, the method of preparing the phage or bacteriocin

composition comprises incorporating the phage or bacteriocin composition into a delivery vehicle. In some embodiments, the delivery vehicle comprises an excipient. Exemplary excipients include, but are not limited to, solid or liquid carrier materials, solvents, stabilizers, slow-release excipients, colorings, and surface-active substances (surfactants). In some embodiments, the delivery vehicle is a stabilizing vehicle. In some embodiments, the stabilizing vehicle comprises a stabilizing excipient. Exemplary stabilizing excipients include, but are not limited to, epoxidized vegetable oils, antifoaming agents, e.g. silicone oil, preservatives, viscosity regulators, binding agents and tackifiers. In some embodiments of the methods of preparing a phage composition, the stabilizing vehicle is a phage buffer. In some embodiments, the phage composition or the bacteriocin composition is microencapsulated in a polymer bead delivery vehicle. In some embodiments, the stabilizing vehicle protects the phage composition or bacteriocin composition against UV and/or acidic conditions. In some embodiments, the delivery vehicle contains a pH buffer. In some embodiments, the composition is formulated to have a pH in the range of about 4.5 to about 9.0, including for example pH ranges of about any one of 5.0 to about 8.0, about 6.5 to about 7.5, or about 6.5 to about 7.0.

[0188] In some embodiments, the delivery vehicle is the food or water of (he pest. In other embodiments, the delivery vehicle is a food source for the pest. In some embodiments, the delivery vehicle is a food bait for the pest. In some embodiments, the composition is consumed by (he pest In some embodiments, (he composition is delivered by (he pest to a second pest, and consumed by the second pest. In some embodiments, the composition is consumed by the pest or a second pest, and the phage composition or the bacteriophage composition is released to the surrounding of the pest or the second pest via the waste (such as feces) of the pest or the second pest In some embodiments, a bacteriocin is included in food bait intended to be consumed by a pest or carried back to its colony.

[0189] In some embodiments, the delivery vehicle is a bacterial vector. Phagic agents (such as phages and/or lysins) and bacteriocins can be incorporated in a bacterial vector using any suitable cloning methods and reagents well known in (he art, such as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Laboratory, Cold Springs Harbor, New York (1989). "Bacterial vector" as used herein refers to any genetic element, such as plasmids, bacteriophage vectors, transposons, cosmids, and chromosomes, which is capable of replication inside bacterial cells and which is capable of transferring genes between cells. Exemplary bacterial vectors include, but are not limited to, lambda vector system gtl 1, gt WES.tB, Charon 4, and plasmid vectors such as pBR322, pBR325, pACYC177, pACYC184, pUC8, pUC9, pUC18, pUC19, pLG339, pR290, pKC37, pKClOl, SV 40, pBluescript II SK +/- or KS +/- (see "Stratagene Cloning Systems" Catalog (1993) from Stratagene, La Jolla, Calif.), pQE, pIH821, pGEX, pET series (see F.W. Studier et. al., 'Use of T7 RNA Polymerase to Direct Expression of Cloned Genes," Gene Expression Technology Vol. 185 (1990)), and any derivatives thereof. Each bacterial vector may encode one or more phagic agents (such as phages and/or lysins) and/or bacteriocins. In some embodiments, the bacterial vector comprises a phage genome to be expressed and packaged in the target symbiotic bacterium. In some embodiments, (he bacterial vector comprises a nucleic acid molecule encoding a lysin to be expressed in the target symbiotic bacterium or a host bacterium In some embodiments, the lysin is co-expressed with a holin, or the lysin is engineered to have a signal peptide for secretion from the host bacterium. In some embodiments, die bacterial vector comprises a nucleic acid molecule encoding a bacteriocin to be expressed in the target symbiotic bacterium. In some embodiments, the bacterial vector further comprises one or more regulatory elements, such as promoters, termination signals, and transcription and translation elements. In some embodiments, the regulatory sequence is operably linked to a nucleic acid encoding a gene (such as bacteriocin, lysin, or other phage proteins) to be expressed in the target symbiotic bacterium.

[0190] In some embodiments, the bacterial vector is introduced into a bacterial host to be consumed by (he pest or a member in the colony of the pest. In some embodiments, the bacterial host is the target symbiotic bacterium. In some embodiments, the bacterial host is a naturally occurring bacterium of the gut of the pest, or a genetically modified derivative thereof, which can be easily introduced to the pest through ingestion. Exemplary bacterial host for use to carry the bacterial vector include, but are not limited to, Proteobacter, including the genus Pseudomonas; Actinobacter, including Priopionibacterium and

Corynebacterium; Firmicutes, including the any species of the genera Mycoplasma, Bacillus, Streptococcus, Staphylococcus; Fibrobacteres; Spirochaetes, including Treponema and Borrelia; Bacteroides, including the genera Bacteroides and Flavobacterium; and "Termite Group Γ'. Also suitable are any bacteria of the Enterobacteriaceae, including the genus Serratia, including, but not limited to S. marcescens, S. entomophila, S. proteamaculans; any species of Enterobacter, including, but not limited to, E. cloacae, E. aerogenes, E. dissolvens, E. agglomerans, E. hafiiiae; and any species belonging to the following genera: Citrobacter, Escherichia, Klebsiella, Kluyvera, Panotea, Proteus, Salmonella, Xenorhabdus and

Yokenella. In some embodiments, die bacterial vector (or the bacterial host carrying the bacterial vector) is consumed by the pest and transmitted to a second pest In some embodiments, bacteria that are filled with bacteriocins are used to deliver die bacteriocins to the gut of a pest Bacteriocins may be combined with phages in some embodiments.

[0191] The compositions may contain about 0.1% to about 100%, such as any one of about 0.1% to about 95%, about 1% to about 99.9%, about 0.1% to about 10%, about 1% to about 25%, about 10% to about 50%, about 50% to about 99%, or about 0.1% to about 90% of active ingredients (such as phage, lysin or bacteriocin). In some embodiments, the composition comprises at least any of 0.1%, 0.5%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more active ingredients (such as phage, lysin or bacteriocin). In some embodiments, the concentrated agents are preferred as commercial products, the final user normally uses diluted agents, which have a substantially lower concentration of active ingredient.

[0192] Further provided are phage compositions prepared by any of the methods of preparing a phage composition described herein, and bacteriocin compositions prepared by any of the methods of preparing a bacteriocin composition described herein.

[0193] The phage or bacteriocin compositions provided herein kill or otherwise reduce the fitness of pests. In some embodiments, there is provided a phage cocktail which, when administered to a pest, kills one or more of the pest's bacterial symbionts. In some embodiments, there is provided a phage cocktail that contains one or more engineered phages which, when administered to a pest, kills one or more of the pest's bacterial symbionts. In some embodiments, there is provided a phage cocktail which, when administered to a pest or its surroundings, kills one or more bacteria on, within, or in the surroundings of the pest. In some embodiments, there is provided a phage cocktail that contains one or more engineered phages which, when administered to a pest or its surroundings, kills one or more bacteria on, within, or in the surroundings of the pest. In some embodiments, there is provided a phage cocktail which, when administered to a pest or its surroundings, kills one or more bacteria with which the pest has an obligate symbiosis. In some embodiments, there is provided a phage cocktail that contains one or more engineered phages which, when administered to a pest or its surroundings, kills one or more bacteria with which the pest has an obligate symbiosis. In some embodiments, mere is provided a phage cocktail which, when administered to an insect, kills one or more bacteria with which the insect has an obligate symbiosis. In some embodiments, there is provided a phage cocktail which, when administered to a pest or its surroundings, kills one or more bacteria with which the pest has a facultative symbiosis. In some embodiments, there is provided a phage cocktail which, when administered in the vicinity of a pest, kills or significantly degrades the fitness of the pest In some embodiments, mere is provided a phage cocktail which, when added to bait mat is designed to be consumed by the pest, kills or significantly degrades the fitness of the pest. In some embodiments, there is provided a phage cocktail which, when sprayed onto a pest, kills or significantly degrades the fitness of the pest [0194] In some embodiments, there is provided a phage cocktail that degrades the fitness of or kills one or more pests from a group consisting of filarial nematodes, spiders, mites, and insects, comprising lytic or temperate phages that kill one or more bacteria of the genus Wolbachia, wherein the total phage concentration is 10³ pfu/ml or greater in a liquid delivery vehicle, or 10³ pfu/g or greater in a solid delivery vehicle. In some embodiments, there is provided a phage cocktail that degrades the fitness of or kills psyllids, comprising lytic or temperate phages that kill one or more bacteria selected from the group consisting of Carsonella ruddii, Wolbachia, Rhizobium, Gordonia, Mycobacterium, mdXanthomonas, wherein the total phage concentration is 10³ pfu/ml or greater in a liquid delivery vehicle, or 10³ pfu/g or greater in a solid delivery vehicle. In some embodiments, there is provided a phage cocktail that degrades the fitness of or kills aphids, comprising lytic or temperate phages that kill one or more bacteria selected from the group consisting of Buchnera aphidicola and Hamiltonella defensa, wherein the total phage concentration is 10³ pfu/ml or greater in a liquid delivery vehicle, or 10³ pfu/g or greater in a solid delivery vehicle. In some embodiments, there is provided a phage cocktail mat degrades the fitness of or kills termites, comprising lytic or temperate phages that kill one or more bacteria selected from the group consisting of Treponema, Bacteroides, Desulfovibrio, Citrobacter, Enter obacter,

Enterococcus, Lactococcus, mdMethanobrevibacter, wherein the total phage concentration is 10³ pfu/ml or greater in a liquid delivery vehicle, or 10³ pfu/g or greater in a solid delivery vehicle. In some embodiments, there is provided a phage cocktail that degrades the fitness of or kills lichenized fungi, comprising lytic or temperate phages that kill one or more cyanobacteria of the genus Nostoc, wherein the total phage concentration is 10³ pfu/ml or greater in a liquid delivery vehicle, or 10³ pfu/g or greater in a solid delivery vehicle. In some embodiments, there is provided a phage cocktail that degrades the fitness of or kills leeches, comprising lytic or temperate phages that kill one or more bacteria selected from the group consisting of Aeromonas and Rikenella-hke bacteria, wherein the total phage concentration is 10³ pfu/ml or greater in a liquid delivery vehicle, or 10³ pfu/g or greater in a solid delivery vehicle. In some embodiments, there is provided a phage cocktail that degrades the fitness of or kills plants with rhizomes, comprising lytic or temperate phages that kill one or more bacteria selected from the group consisting of Herbaspirillum, Agrobacterium, Xanthomonas, Arthrobacter, Spirillum, and Rhizobium, wherein the total phage concentration is 10³ pfu/ml or greater in a liquid delivery vehicle, or 10³ pfu/g or greater in a solid delivery vehicle. [0195] In some embodiments, there is provided a composition comprising a phage-rich pest extract that degrades the fitness of or kills pests of the same species that are exposed to the composition. In some embodiments, there is provided a composition comprising a phage-rich pest extract that degrades the fitness of or kills pests of a different species that are exposed to the composition. In some embodiments, there is provided a composition comprising a phage cocktail, which when applied to or consumed by a colonizing pest, is carried by the pest to the pest's colony. In some embodiments, there is provided a composition comprising a phage cocktail, which when applied to or consumed by a colonizing pest, is carried by the pest to the pest's colony and is transmitted to one or more of the members of the colony. In some embodiments, there is provided a composition comprising a phage cocktail, which when applied to or consumed by a colonizing pest, is carried by the pest to the pest's colony and is transmitted to one or more of the members of the colony, resulting in the death of one or more of these members. In some embodiments, there is provided a composition comprising a phage cocktail, which when applied to or consumed by a colonizing pest, is carried by a bacterial vector, residing in or on the pest, to the pest's colony. In some embodiments, there is provided a composition comprising a phage cocktail, which when applied to or consumed by a colonizing pest, is carried by a bacterial vector, residing in or on the pest, to the pest's colony and is transmitted to bacteria residing in or on one or more members of the colony. In some embodiments, there is provided a composition comprising a phage cocktail, which when applied to or consumed by a colonizing pest, is carried by a bacterial vector, residing in or on the pest, to the pest's colony and is transmitted to bacteria residing in or on one or more members of the colony, resulting in the death of one or more of these members. In some embodiments, there is provided a composition comprising a phage cocktail and food bait, which is consumed by a colonizing pest and is carried to the pest's colony and excreted through its feces. In some embodiments, there is provided a composition comprising a phage cocktail and food bait, which is consumed by a colonizing pest and is carried to the pest's colony and excreted through its feces, resulting in the death of one or more members of the colony. In some embodiments, there is provided a composition comprising a phage cocktail and food bait, which is carried by a colonizing pest to its colony. In some embodiments, there is provided a composition comprising a phage cocktail and food bait, which is carried by a colonizing pest to its colony and consumed by one or more members of the colony. In some embodiments, there is provided a composition comprising a phage cocktail and food bait, which is carried by a colonizing pest to its colony and consumed by one or more members of the colony, resulting in the death of one or more of these members.

[0196] In some embodiments, there is provided a bacteriocin cocktail which, when administered to a pest, kills one or more of the pest's bacterial symbionts. In some embodiments, there is provided a bacteriocin cocktail that contains one or more engineered bacteriocins which, when administered to a pest, kills one or more of the pest's bacterial symbionts. In some embodiments, there is provided a bacteriocin cocktail which, when administered to a pest or its surroundings, kills one or more bacteria on, within, or in the surroundings of the pest. In some embodiments, there is provided a bacteriocin cocktail that contains one or more bacteriocins which, when administered to a pest or its surroundings, kills one or more bacteria on, within, or in the surroundings of the pest In some embodiments, there is provided a bacteriocin cocktail which, when administered to a pest or its

surroundings, kills one or more bacteria with which the pest has an obligate symbiosis. In some embodiments, there is provided a bacteriocin cocktail that contains one or more bacteriocins which, when administered to a pest or its surroundings, kills one or more bacteria with which the pest has an obligate symbiosis. In some embodiments, there is provided a bacteriocin cocktail which, when administered to an insect, kills one or more bacteria with which the insect has an obligate symbiosis, hi some embodiments, there is provided a bacteriocin cocktail which, when administered to a pest or its surroundings, kills one or more bacteria with which the pest has a facultative symbiosis. In some embodiments, there is provided a bacteriocin cocktail which, when administered in the vicinity of a pest, kills or significantly degrades die fitness of the pest In some embodiments, there is provided a bacteriocin cocktail which, when added to bait that is designed to be consumed by the pest, kills or significantly degrades the fitness of the pest In some embodiments, there is provided a bacteriocin cocktail which, when sprayed onto a pest, kills or significantly degrades the fitness of the pest

[0197] In some embodiments, there is provided a bacteriocin cocktail that degrades the fitness of or kills one or more pests from a group consisting of filarial nematodes, spiders, mites, and insects, comprising lytic or temperate phages that kill one or more bacteria of the genus Wolbachia. In some embodiments, there is provided a bacteriocin cocktail that degrades the fitness of or kills psyllids, comprising lytic or temperate phages that kill one or more bacteria selected from the group consisting of Carsonella ruddii, Wolbachia, Rhizobium, Gordonia, Mycobacterium, and Xanthomonas. In some embodiments, there is provided a bacteriocin cocktail that degrades the fitness of or kills aphids, comprising lytic or temperate phages that kill one or more bacteria selected from the group consisting of Buchnera aphidicola and Hamiltonella defense. In some embodiments, there is provided a bacteriocin cocktail mat degrades the fitness of or kills termites, comprising lytic or temperate phages that kill one or more bacteria selected from the group consisting of Treponema, Bacteroides, Desulfovibrio, Citrobacter, Enterobacter, Enterococcus, Lactococcus, and

Methanobrevibacter. In some embodiments, there is provided a bacteriocin cocktail that degrades the fitness of or kills lichenized fungi, comprising lytic or temperate phages that kill one or more cyanobacteria of the genus Nostoc. In some embodiments, there is provided a bacteriocin cocktail that degrades the fitness of or kills leeches, comprising lytic or temperate phages that kill one or more bacteria selected from the group consisting of Aeromonas and Rikenella-Vke bacteria. In some embodiments, there is provided a bacteriocin cocktail that degrades the fitness of or kills plants with rhizomes, comprising lytic or temperate phages that kill one or more bacteria selected from the group consisting of Herbaspirillum,

Agrobacterium, Xanthomonas, Arthrobacter, Spirillum, and Rhizobium.

[0198] In some embodiments, there is provided a composition comprising a bacteriocin- rich pest extract that degrades the fitness of or kills pests of the same species that are exposed to the composition. In some embodiments, mere is provided a composition comprising a bacteriocin-rich pest extract that degrades the fitness of or kills pests of a different species that are exposed to the composition. In some embodiments, mere is provided a composition comprising a bacteriocin cocktail, which when applied to or consumed by a colonizing pest, is carried by the pest to the pest's colony. In some embodiments, there is provided a composition comprising a bacteriocin cocktail, which when applied to or consumed by a colonizing pest, is carried by the pest to the pest's colony and is transmitted to one or more of the members of the colony. In some embodiments, there is provided a composition comprising a bacteriocin cocktail, which when applied to or consumed by a colonizing pest, is carried by the pest to the pest's colony and is transmitted to one or more of the members of the colony, resulting in the death of one or more of these members, hi some embodiments, there is provided a composition comprising a bacteriocin cocktail, which when applied to or consumed by a colonizing pest, is carried by a bacterial vector, residing in or on the pest, to the pest's colony. In some embodiments, there is provided a composition comprising a bacteriocin cocktail, which when applied to or consumed by a colonizing pest, is carried by a bacterial vector, residing in or on the pest, to the pest's colony and is transmitted to bacteria residing in or on one or more members of the colony. In some embodiments, mere is provided a composition comprising a bacteriocin cocktail, which when applied to or consumed by a colonizing pest, is carried by a bacterial vector, residing in or on the pest, to the pest's colony and is transmitted to bacteria residing in or on one or more members of the colony, resulting in the death of one or more of these members. In some embodiments, there is provided a composition comprising a bacteriocin cocktail and food bait, which is consumed by a colonizing pest and is carried to the pest's colony and excreted through its feces, hi some embodiments, there is provided a composition comprising a bacteriocin cocktail and food bait, which is consumed by a colonizing pest and is carried to the pest's colony and excreted through its feces, resulting in the death of one or more members of the colony. In some embodiments, mere is provided a composition comprising a bacteriocin cocktail and food bait, which is carried by a colonizing pest to its colony. In some embodiments, there is provided a composition comprising a bacteriocin cocktail and food bait, which is carried by a colonizing pest to its colony and consumed by one or more members of the colony. In some

embodiments, there is provided a composition comprising a bacteriocin cocktail and food bait, which is carried by a colonizing pest to its colony and consumed by one or more members of the colony, resulting in the death of one or more of these members.

ΙΠ. Methods of pest control

[0199] The present application further provides a method of killing or degrading the fitness of a pest, comprising applying an effective amount of any one of the phage or bacteriocin compositions described herein.

[0200] Thus, in some embodiments, there is provided a method of killing or degrading fitness of a pest, comprising applying an effective amount of a phage composition, wherein the phage composition is prepared by steps comprising: (a) identifying a symbiotic bacterium in a specimen of the pest; (b) obtaining a phagic agent (such as phage or lysin) mat inhibits the symbiotic bacterium; and (c) formulating a composition comprising the phagic agent (such as phage or lysin) to provide the phage composition. In some embodiments, the phage composition comprises a plurality of phagic agents (such as phages and/or lysins) that each inhibits the symbiotic bacterium. In some embodiments, the phage composition is prepared by identifying a plurality of symbiotic bacteria of the pest, and obtaining one or more phagic agents (such as phages and/or lysins) that inhibit each symbiotic bacterium. In some embodiments, the phagic agent (such as phage or lysin) is obtained from a specimen of the pest, or from an environmental sample. In some embodiments, the phage composition is formulated with a delivery vehicle, such as a bacterial vector, a food bait, or an excipient. In some embodiments, the phage composition is present in a pest extract, a live pest, or in the feces secreted by the pest. In some embodiments, the phage composition is applied directly to the pest. In some embodiments, the phage composition is applied to the surrounding of the pest. In some embodiments, the phage composition is applied to a second pest, wherein the second pest closely associates with the pest (such as being a member of the colony of the pest), and wherein the second pest delivers the phage composition to the pest. In some embodiments, the phage composition is applied via a sprayer.

[0201] In some embodiments, there is provided a method of killing or degrading fitness of a pest, comprising applying an effective amount of a phage composition, wherein the phage composition is prepared by steps comprising: a) identifying a symbiotic bacterium in a specimen of the pest; (b) obtaining a phagic agent (such as phage or lysin) that inhibits the symbiotic bacterium; and (c) formulating a composition comprising the phagic agent (such as phage or lysin) to provide the phage composition, wherein the symbiotic bacterium is identified by sequencing a specimen of the pest. In some embodiments, the phage composition comprises a plurality of phagic agents (such as phages and/or lysins) that each inhibits the symbiotic bacterium. In some embodiments, the phage composition is prepared by identifying a plurality of symbiotic bacteria of the pest, and obtaining one or more phagic agents (such as phages and/or lysins) that inhibit each symbiotic bacterium. In some embodiments, the sequencing is metagenomics sequencing, such as 16S rRNA metagenomics sequencing. In some embodiments, the phagic agent (such as phage or lysin) is obtained from a specimen of the pest, or from an environmental sample. In some embodiments, the phage composition is formulated with a delivery vehicle, such as a bacterial vector, a food bait, or an excipient. In some embodiments, the phage composition is present in a pest extract, a live pest, or in the feces secreted by the pest. In some embodiments, the phage composition is applied directly to the pest. In some embodiments, the phage composition is applied to the surrounding of the pest. In some embodiments, the phage composition is applied to a second pest, wherein the second pest closely associates with the pest (such as being a member of the colony of the pest), and wherein the second pest delivers the phage composition to the pest In some embodiments, the phage composition is applied via a sprayer.

[0202] In some embodiments, there is provided a method of killing or degrading fitness of a pest, comprising applying an effective amount of a phage composition, wherein the phage composition is prepared by steps comprising: (a) identifying a symbiotic bacterium in a specimen of the pest; (b) obtaining a phagic agent (such as phage or lysin) that inhibits the symbiotic bacterium; and (c) formulating a composition comprising the phagic agent (such as phage or lysin) to provide the phage composition, wherein the symbiotic bacterium is identified by comparative sequencing of a first specimen of the pest comprising one or more pest individuals affected by an antibacterial treatment (such as applying a pool of phages to the pest), and a second specimen of the pest comprising one or more pest individuals unaffected by the antibacterial treatment. In some embodiments, the phage composition comprises a plurality of phagic agents (such as phages and/or lysins) that each inhibits the symbiotic bacterium. In some embodiments, the phage composition is prepared by identifying a plurality of symbiotic bacteria of the pest, and obtaining one or more phagic agents (such as phages and/or lysins) that inhibit each symbiotic bacterium. In some embodiments, the comparative sequencing is metagenomics sequencing, such as 16S rRNA metagenomics sequencing. In some embodiments, the phage is identified from results of the comparative sequencing. In some embodiments, the phagic agent (such as phage or lysin) is obtained from the pool of phages. In some embodiments, the phage composition is formulated with a delivery vehicle, such as a bacterial vector, a food bait, or an excipient. In some embodiments, the phage composition is present in a pest extract, a live pest, or in the feces secreted by the pest. In some embodiments, the phage composition is applied directly to the pest In some embodiments, the phage composition is applied to the surrounding of the pest. In some embodiments, the phage composition is applied to a second pest, wherein the second pest closely associates with the pest (such as being a member of the colony of the pest), and wherein the second pest delivers the phage composition to the pest. In some embodiments, the phage composition is applied via a sprayer.

[0203] In some embodiments, there is provided a method of killing or degrading fitness of a pest, comprising applying an effective amount of a phage composition, wherein the phage composition is prepared by steps comprising: (a) sequencing a specimen of the pest to identify a symbiotic bacterium of the pest; (b) isolating a phage that inhibits the symbiotic bacterium from a sample (such as a specimen of the pest, or an environmental sample); optionally (c) engineering the phage to expand the host range of the phage; optionally (d) propagating the phage; (e) formulating a composition comprising the phage to provide the phage composition; and optionally (f) assessing the inhibitory activity (such as killing or reduction of fitness) against the pest and adjusting the phage composition based on the inhibitory activity. In some embodiments, the phage composition comprises a plurality of phages that each inhibits the symbiotic bacterium. In some embodiments, the phage composition is prepared by identifying a plurality of symbiotic bacteria of the pest, and obtaining one or more phages that inhibit each symbiotic bacterium. In some embodiments, the sequencing is metagenomics sequencing, such as 16S rRNA metagenomics sequencing. In some embodiments, the phage composition is formulated with a delivery vehicle, such as a bacterial vector, a food bait, or an excipient In some embodiments, the phage composition is present in a pest extract, a live pest, or in the feces secreted by the pest In some embodiments, the phage composition is applied directly to the pest In some embodiments, the phage composition is applied to the surrounding of the pest. In some embodiments, the phage composition is applied to a second pest, wherein the second pest closely associates with the pest (such as being a member of the colony of the pest), and wherein the second pest delivers the phage composition to the pest. In some embodiments, the phage composition is applied via a sprayer.

[0204] In some embodiments, there is provided a method of killing or degrading fitness of a pest, comprising applying an effective amount of a phage composition, wherein the phage composition is prepared by steps comprising: (a) applying a pool of phages to a plurality of the pest to obtain a first specimen of the pest comprising one or more affected pest individuals and a second specimen comprising one or more unaffected pest individuals; (b) comparatively sequencing the first specimen and the second specimen to identify a symbiotic bacterium from the sequencing results; (c) isolating a phage that inhibits the symbiotic bacterium from the pool of phages; optionally (d) engineering the phage to expand the host range of the phage; optionally (e) propagating the phage; (f) formulating a composition comprising the phage to provide the phage composition; and optionally (g) assessing the inhibitory activity (such as killing or reduction of fitness) against the pest and adjusting the phage composition based on the inhibitory activity. In some embodiments, the phage composition comprises a plurality of phages that each inhibits the symbiotic bacterium. In some embodiments, the phage composition is prepared by identifying a plurality of symbiotic bacteria of the pest, and obtaining one or more phages that inhibit each symbiotic bacterium. In some embodiments, the comparative sequencing is metagenomics sequencing, such as 16S rRNA metagenomics sequencing. In some embodiments, the phage is identified from results of the comparative sequencing. In some embodiments, the phage composition is formulated with a delivery vehicle, such as a bacterial vector, a food bait, or an excipient In some embodiments, the phage composition is present in a pest extract, a live pest, or in the feces secreted by the pest. In some embodiments, the phage composition is applied directly to the pest In some embodiments, the phage composition is applied to the surrounding of the pest. In some embodiments, the phage composition is applied to a second pest, wherein the second pest closely associates with the pest (such as being a member of the colony of the pest), and wherein the second pest delivers the phage composition to the pest In some embodiments, the phage composition is applied via a sprayer.

[0205] In some embodiments, there is provided a method of killing or degrading fitness of a pest, comprising applying an effective amount of a bacteriocin composition, wherein the bacteriocin composition is prepared by steps comprising: (a) identifying a symbiotic bacterium in a specimen of the pest; (b) obtaining a bacteriocin (hat inhibits the symbiotic bacterium; and (c) formulating a composition comprising the bacteriocin to provide the bacteriocin composition. In some embodiments, the bacteriocin is obtained from a natural source. In some embodiments, the bacteriocin is obtained by engineering a naturally- occurring bacteriocin (such as a tail fiber of pyocin) to target the symbiotic bacterium. In some embodiments, the bacteriocin composition comprises a plurality of bacteriocins that each inhibits the symbiotic bacterium. In some embodiments, the bacteriocin composition is prepared by identifying a plurality of symbiotic bacteria of the pest, and obtaining one or more bacteriocins that inhibit each symbiotic bacterium. In some embodiments, the bacteriocin composition is formulated with a delivery vehicle, such as a bacterial vector, a food bait, or an excipient. In some embodiments, the bacteriocin composition is present in a pest extract, a live pest, or in the feces secreted by the pest In some embodiments, the bacteriocin composition is applied directly to the pest. In some embodiments, the bacteriocin composition is applied to the surrounding of the pest. In some embodiments, the bacteriocin composition is applied to a second pest, wherein the second pest closely associates with the pest (such as being a member of the colony of the pest), and wherein the second pest delivers the bacteriocin composition to the pest. In some embodiments, the bacteriocin composition is applied via a sprayer.

[0206] In some embodiments, there is provided a method of killing or degrading fitness of a pest, comprising applying an effective amount of a bacteriocin composition, wherein the bacteriocin composition is prepared by steps comprising: (a) identifying a symbiotic bacterium in a specimen of the pest; (b) obtaining a bacteriocin (hat inhibits the symbiotic bacterium; and (c) formulating a composition comprising (he bacteriocin to provide the bacteriocin composition, wherein the symbiotic bacterium is identified by sequencing a specimen of the pest In some embodiments, the bacteriocin is obtained from a natural source. In some embodiments, the bacteriocin is obtained by engineering a naturally-occurring bacteriocin (such as a tail fiber of pyocin) to target the symbiotic bacterium. In some embodiments, the bacteriocin composition comprises a plurality of bacteriocins that each inhibits the symbiotic bacterium. In some embodiments, the bacteriocin composition is prepared by identifying a plurality of symbiotic bacteria of the pest, and obtaining one or more bacteriocins that inhibit each symbiotic bacterium. In some embodiments, the sequencing is metagenomics sequencing, such as 16S rRNA metagenomics sequencing. In some embodiments, the bacteriocin composition is formulated with a delivery vehicle, such as a bacterial vector, a food bait, or an excipient. In some embodiments, the bacteriocin composition is present in a pest extract, a live pest, or in the feces secreted by the pest. In some embodiments, the bacteriocin composition is applied directly to the pest. In some embodiments, the bacteriocin composition is applied to the surrounding of the pest In some embodiments, the bacteriocin composition is applied to a second pest, wherein the second pest closely associates with the pest (such as being a member of the colony of the pest), and wherein the second pest delivers the bacteriocin composition to the pest In some

embodiments, the bacteriocin composition is applied via a sprayer.

[0207] In some embodiments, there is provided a method of killing or degrading fitness of a pest, comprising applying an effective amount of a bacteriocin composition, wherein the bacteriocin composition is prepared by steps comprising: (a) identifying a symbiotic bacterium in a specimen of the pest; (b) obtaining a bacteriocin that inhibits the symbiotic bacterium; and (c) formulating a composition comprising the bacteriocin to provide the bacteriocin composition, wherein the symbiotic bacterium is identified by comparative sequencing of a first specimen of the pest comprising one or more pest individuals affected by an antibacterial treatment (such as applying antibiotics to the pest), and a second specimen of the pest comprising one or more pest individuals unaffected by the antibacterial treatment In some embodiments, the bacteriocin is obtained from a natural source. In some

embodiments, the bacteriocin is obtained by engineering a naturally-occurring bacteriocin (such as a tail fiber of pyocin) to target the symbiotic bacterium. In some embodiments, the bacteriocin composition comprises a plurality of bacteriocins that each inhibits the symbiotic bacterium. In some embodiments, the bacteriocin composition is prepared by identifying a plurality of symbiotic bacteria of the pest, and obtaining one or more bacteriocins that inhibit each symbiotic bacterium. In some embodiments, the comparative sequencing is metagenomics sequencing, such as 16S rRNA metagenomics sequencing. In some embodiments, the bacteriocin composition is formulated with a delivery vehicle, such as a bacterial vector, a food bait, or an excipient. In some embodiments, the bacteriocin composition is present in a pest extract, a live pest, or in the feces secreted by the pest. In some embodiments, the bacteriocin composition is applied directly to the pest. In some embodiments, the bacteriocin composition is applied to the surrounding of the pest In some embodiments, the bacteriocin composition is applied to a second pest, wherein the second pest closely associates with the pest (such as being a member of the colony of the pest), and wherein the second pest delivers the bacteriocin composition to the pest In some embodiments, the bacteriocin composition is applied via a sprayer.

[0208] The method of killing or degrading the fitness of a pest described herein are applicable to a variety of pests. In some embodiments, the pest is an animal. In some embodiments, the pest is selected from the group consisting of worms (such as nematode, or leech), spider, mite, and insect (such as psyllid, aphid, termite, ant, cockroach). In some embodiments, the pest is a plant, such as a plant with rhizomes. In some embodiments, the pest is a lichenized fungus.

[0209] The phage or bacteriocin composition may be applied to the pest with any suitable methods. In some embodiments, the phage or bacteriocin composition is applied by spraying the pest In some embodiments, the phage or bacteriocin composition is applied to the water in which the pest lives. In some embodiments, the phage or bacteriocin composition is poured onto the soil in which the pest resides. In other embodiments, the phage or bacteriocin composition is aerosolized in the general vicinity of the pest In some embodiments, a kit is used to determine if a particular cocktail eradicates a particular pest.

[0210] Another strategy for killing pests that colonize or associate closely with other members of the same species involves trapping the pest, infecting one or more of its bacterial symbionts using a phage cocktail or bacterial vectors encoding one or more bacteriocins, and releasing the pest, such that it may later interact with and transmit the infection to other pests (i.e., 'Trojan horse strategy"). In some embodiments, this is accomplished by placing the pest into a closed container containing one or more phages and gently shaking the container for a short period of time (i.e. "dusting" the pest). In some embodiments, the pest is sprayed with a liquid composition containing one or more phages or bacterial vectors encoding one or more bacteriocins. In some embodiments, the pest need not be trapped, and can instead be sprayed while the pest is in transit or is otherwise unconfined.

[0211] In some embodiments, (he phage composition or the bacteriocin composition is delivered from a pressurized aerosol can.

[0212] In some embodiments, there is provided a method of killing or otherwise reducing the fitness of a pest, comprising: (a) identifying the pest's bacterial symbionts; (b) isolating phages against the pest's bacterial symbionts; (c) expanding the host range of phages against a pest's bacterial symbionts; and (d) killing or degrading the fitness of a pest by killing one or more of the pest's bacterial symbionts with a phage composition comprising the isolated phages. In some embodiments, the method comprises incorporating the phage composition into a food bait for pest control. In some embodiments, the method comprises aerosolizing the phage composition for pest control. In some embodiments, the method comprises delivering the phage composition (or the isolated phages) to the pest using a bacterial vector.

[0213] In some embodiments, there is provided a method of killing or otherwise reducing the fitness of a pest, comprising: (a) identifying the pest's bacterial symbionts; and (b) killing or degrading the fitness of a pest by killing one or more of the pest's bacterial symbionts with a bacteriocin composition. In some embodiments, the method comprises incorporating the bacteriocin composition into a food bait for pest control. In some embodiments, the method comprises aerosolizing the bacteriocin composition for pest control. In some embodiments, the method comprises delivering the bacteriocin composition to the pest using a bacterial vector.

TV. Kits and articles of manufacture

[0214] The present invention further provides kits, and articles of manufacture (such as products) comprising any of the phage or bacteriocin compositions described herein. The kits and articles of manufacture are for selling or for use to control a pest, such as killing or degrading the fitness of a pest

[0215] In some embodiments, there is provided a kit for killing or degrading fitness of a pest, comprising a phage composition and an instruction, wherein the phage composition is prepared by steps comprising: (a) identifying a symbiotic bacterium in a specimen of the pest; (b) obtaining a phagic agent (such as phage or lysin) that inhibits the symbiotic bacterium; and (c) formulating a composition comprising the phagic agent (such as phage or lysin) to provide the phage composition. In some embodiments, the phage composition comprises a plurality of phagic agents (such as phages and/or lysins) that each inhibits the symbiotic bacterium. In some embodiments, the phage composition is prepared by identifying a plurality of symbiotic bacteria of the pest, and obtaining one or more phagic agents (such as phages and/or lysins) that inhibit each symbiotic bacterium In some embodiments, (he phage composition comprises a plurality of phagic agents (such as phages and/or lysins) that each inhibits the symbiotic bacterium. In some embodiments, the phage composition is prepared by identifying a plurality of symbiotic bacteria of the pest, and obtaining one or more phagic agents (such as phages and/or lysins) that inhibit each symbiotic bacterium. In some embodiments, the phagic agent (such as phage or lysin) is obtained from a specimen of the pest, or from an environmental sample. In some embodiments, the phage composition is formulated with a delivery vehicle, such as a bacterial vector, a food bait, or an excipient. In some embodiments, the phage composition is aerosolized.

[0216] In some embodiments, there is provided a kit for killing or degrading fitness of a pest, comprising a phage composition and an instruction, wherein the phage composition is prepared by steps comprising: (a) identifying a symbiotic bacterium in a specimen of the pest; (b) obtaining a phagic agent (such as phage or lysin) that inhibits the symbiotic bacterium; and (c) formulating a composition comprising the phagic agent (such as phage or lysin) to provide the phage composition, wherein the symbiotic bacterium is identified by sequencing (such as metagenomics sequencing) die specimen of the pest. In some embodiments, the phage composition comprises a plurality of phagic agents (such as phages and/or lysins) that each inhibits the symbiotic bacterium. In some embodiments, the phage composition is prepared by identifying a plurality of symbiotic bacteria of the pest, and obtaining one or more phagic agents (such as phages and/or lysins) that inhibit each symbiotic bacterium In some embodiments, the phagic agent (such as phage or lysin) is obtained from a specimen of the pest, or from an environmental sample. In some embodiments, the phage composition is formulated with a delivery vehicle, such as a bacterial vector, a food bait, or an excipient. In some embodiments, the phage composition is aerosolized.

[0217] n some embodiments, there is provided a kit for killing or degrading fitness of a pest, comprising a phage composition and an instruction, wherein the phage composition is prepared by steps comprising: (a) identifying a symbiotic bacterium in a specimen of the pest; (b) obtaining a phagic agent (such as phage or lysin) that inhibits the symbiotic bacterium; and (c) formulating a composition comprising the phagic agent (such as phage or lysin) to provide the phage composition, wherein the symbiotic bacterium is identified by comparative sequencing (such as metagenomics sequencing) of a first specimen of the pest comprising one or more pest individuals affected by an antibacterial treatment (such as applying a pool of phages to the pest), and a second specimen of the pest comprising one or more pest individuals unaffected by the antibacterial treatment. In some embodiments, the method comprises obtaining a plurality of phagic agents (such as phages and/or lysins) that each inhibits the symbiotic bacterium. In some embodiments, the method comprises identifying a plurality of symbiotic bacteria of the pest, and obtaining one or more phagic agents (such as phages and/or lysins) that inhibit each symbiotic bacterium. In some embodiments, the phage is identified from results of the comparative sequencing. In some embodiments, the phagic agent (such as phage or lysin) is obtained from the pool of phages. In some embodiments, the phage composition is formulated with a delivery vehicle, such as a bacterial vector, a food bait, or an excipient In some embodiments, the phage composition is aerosolized.

[0218] In some embodiments, there is provided a kit for killing or degrading fitness of a pest, comprising a bacteriocin composition and an instruction, wherein the bacteriocin composition is prepared by steps comprising: (a) identifying a symbiotic bacterium in a specimen of the pest; (b) obtaining a bacteriocin that inhibits the symbiotic bacterium; and (c) formulating a composition comprising the bacteriocin to provide the bacteriocin composition. In some embodiments, the bacteriocin is obtained from a natural source. In some

embodiments, the bacteriocin is obtained by engineering a naturally-occurring bacteriocin (such as a tail fiber of pyocin) to target the symbiotic bacterium. In some embodiments, the bacteriocin composition comprises a plurality of bacteriocins that each inhibits the symbiotic bacterium. In some embodiments, the bacteriocin composition is prepared by identifying a plurality of symbiotic bacteria of the pest, and obtaining one or more bacteriocins that inhibit each symbiotic bacterium. In some embodiments, the bacteriocin composition is formulated with a delivery vehicle, such as a bacterial vector, a food bait, or an excipient In some embodiments, the bacteriocin composition is aerosolized.

[0219] In some embodiments, there is provided a kit for killing or degrading fitness of a pest, comprising a bacteriocin composition and an instruction, wherein the bacteriocin composition is prepared by steps comprising: (a) identifying a symbiotic bacterium in a specimen of the pest; (b) obtaining a bacteriocin that inhibits the symbiotic bacterium; and (c) formulating a composition comprising the bacteriocin to provide the bacteriocin composition, wherein the symbiotic bacterium is identified by sequencing (such as metagenomics sequencing) the specimen of the pest. In some embodiments, the bacteriocin is obtained from a natural source. In some embodiments, the bacteriocin is obtained by engineering a naturally-occurring bacteriocin (such as a tail fiber of pyocin) to target the symbiotic bacterium. In some embodiments, the bacteriocin composition comprises a plurality of bacteriocins that each inhibits the symbiotic bacterium. In some embodiments, the bacteriocin composition is prepared by identifying a plurality of symbiotic bacteria of the pest, and obtaining one or more bacteriocins that inhibit each symbiotic bacterium In some embodiments, the bacteriocin composition is formulated with a delivery vehicle, such as a bacterial vector, a food bait, or an excipient. In some embodiments, the bacteriocin composition is aerosolized.

[0220] In some embodiments, there is provided a kit for killing or degrading fitness of a pest, comprising a bacteriocin composition and an instruction, wherein the bacteriocin composition is prepared by steps comprising: (a) identifying a symbiotic bacterium in a specimen of the pest; (b) obtaining a bacteriocin that inhibits the symbiotic bacterium; and (c) formulating a composition comprising the bacteriocin to provide the bacteriocin composition, wherein the symbiotic bacterium is identified by comparative sequencing (such as metagenomics sequencing) of a first specimen of the pest comprising one or more pest individuals affected by an antibacterial treatment (such as applying antibiotics to the pest), and a second specimen of the pest comprising one or more pest individuals unaffected by the antibacterial treatment. In some embodiments, the bacteriocin is obtained from a natural source. In some embodiments, the bacteriocin is obtained by engineering a naturally- occurring bacteriocin (such as a tail fiber of pyocin) to target the symbiotic bacterium. In some embodiments, the bacteriocin composition comprises a plurality of bacteriocins that each inhibits the symbiotic bacterium. In some embodiments, the bacteriocin composition is prepared by identifying a plurality of symbiotic bacteria of the pest, and obtaining one or more bacteriocins that inhibit each symbiotic bacterium. In some embodiments, the bacteriocin composition is formulated with a delivery vehicle, such as a bacterial vector, a food bait, or an excipient. In some embodiments, the bacteriocin composition is aerosolized.

[0221] The kits of the invention are in suitable packaging. Suitable packaging include, but is not limited to, vials, cans (such as pressurized can), bottles, jars, flexible packaging (e.g., Mylar or plastic bags), and the like. Kits may optionally provide additional components such as buffers and interpretative information. The present application thus also provides articles of manufacture, which include vials, cans (such as pressurized can), bottles, jars, flexible packaging, and the like. [0222] The kits and articles of manufacture may contain unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses. For example, kits may be provided that contain sufficient amount of the phage composition or the bacteriocin composition for any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100 or more applications.

[0223] The instructions in the kits contain information generally related to the application of the phage compositions or the bacteriocin compositions, such as the effective amount, frequency, and application routes. The instructions may further contain information related to the storage, and safety information for using the phage compositions or the bacteriocin compositions.

[0224] The phage composition or bacteriocin composition in the kits or articles of manufacture may be concentrated. In some embodiments, the kit further comprises a diluent and instruction for diluting the concentrated phage composition or bacteriocin composition before use.

[0225] The examples below are intended to be purely exemplary of the invention and should therefore not be considered to limit the invention in any way. The following examples and detailed description are offered by way of illustration and not by way of limitation.

EXAMPLES

EXAMPLE 1 - Developing a termite control composition

[0226] This example describes an exemplary experiment designed to develop a termite control composition comprising a phage cocktail that target symbiotic bacteria of termite.

[0227] A sample of termite hindguts is first obtained and subject to 16S rRNA

metagenomics sequencing to describe the microbiome of the termites with the goal of being able to deleteriously affect the microbiome by use of antibacterial agents. Sequencing results are also used to guide bacterial culturing efforts.

[0228] Bacteria from termite guts are directly cultured on a variety of media under aerobic and anaerobic conditions. Isolates from the bacteria culture are obtained, and their identities are determined by sequencing. Termite-associated bacteria which have known roles in the cellulose degradation or other required metabolic pathways (e.g. Fibrobacter succinogenes) for termites are purchased from commercial sources. Such termite-associated bacteria represent candidate symbiotic bacteria of the termites to be targeted by bacteriophages or bacteriocins. [0229] Alternatively, termites are treated with different antibiotics and bacteriocins to observe phenotypic effects on the termites (e.g. length of life, behavioral anomalies, etc.). A sample of termite hindguts after antibiotic or bacteriocin treatment is obtained and subjected to metagenomics sequencing to determine what microbiome population changes have occurred and may be responsible for any observable phenotypic abnormalities. Bacteria species that show such changes in response to the antibiotics or bacteriocin treatment are candidate symbiotic bacteria to be targeted by bacteriophages or bacteriocins.

[0230] To identify phages targeting the candidate symbiotic bacteria, environmental samples containing bacteriophages are obtained, and screened against the symbiotic bacterial isolates for bacteria-killing or inhibition activities. Bacteriophages with specific activities against (he bacterial isolates are further enriched from die environmental samples.

[0231] Once phages are found for any of the symbiotic bacterial strains, termites are treated with these phages and their microbiomes sequenced to observe any resulting dysbiosis. Phenotypic effects on the termites in the phage treatment are assessed. Phage survival and transmissibility through termite treatment are also determined. Phages that result in specific changes in the microbiomes of the termites, effectively kill or reduce the fitness of the termites, and have high survival and transmissibility though termites are selected and included in a phage composition that can be used to control termites.

[0232] Alternatively, bacteriocins that target the symbiotic bacteria strains are obtained, and applied to the termites. The microbiomes of the termites are then sequenced to observe any resulting dysbiosis. Phenotypic effects on the termites in the bacteriocin treatment are assessed. Bacteriocins that result in specific changes in the microbiomes of die termites, and effectively kill or reduce the fitness of the termites are selected and included in a bacteriocin composition that can be used to control termites.

Claims

CLAIMS What is claimed is:

1. A method of preparing a phage composition for killing or degrading fitness of a pest, comprising:

(a) identifying a symbiotic bacterium in a specimen of the pest;

(b) obtaining a phagic agent that inhibits the symbiotic bacterium;

and (c) formulating a composition comprising the phagic agent to provide the phage composition.

2. The method of claim 1, wherein the phagic agent is a lysia

3. The method of claim 1 , wherein the phagic agent is a phage.

4. The method of any one of claims 1-3, wherein the symbiotic bacterium is identified by sequencing the specimen of the pest.

5. The method of any one of claims 1-3, wherein the symbiotic bacterium is identified by comparative sequencing of a first specimen of the pest comprising one or more pest individuals affected by an antibacterial treatment, and a second specimen of the pest comprising one or more pest individuals unaffected by the an antibacterial treatment.

6. The method of claim 5, wherein the antibacterial treatment comprises applying a pool of phages to the one or more pest individuals affected by the antibacterial treatment and the one or more pest individuals unaffected by the antibacterial treatment.

7. The method of claim 5 or claim 6, wherein the pool of phages is present in an

environmental sample.

8. The method of claim 6 or claim 7, wherein the phagic agent is obtained from the pool of phages.

9. The method of any one of claims 4-8, wherein the sequencing or comparative

sequencing is metagenomics sequencing.

10. The method of any one of claims 1-9, wherein the specimen is derived from the pest.

11. The method of any one of claims 1-9, wherein the specimen is derived from die surroundings of the pest.

12. The method of any one of claims 1-11, further comprising isolating the symbiotic bacterium from the specimen.

13. The method of any one of claims 1-8 and 9-12, wherein the phagic agent is obtained from a sample of the pest.

14. The method of any one of claims 1 -8 and 9-12, wherein the phagic agent is obtained from an environmental sample.

15. The method of any one of claims 1-14, further comprising assessing the inhibitory activity of the phagic agent against the symbiotic bacterium.

16. The method of any one of claims 3-15, further comprising engineering the phage to expand the host range of the phage.

17. The method of any one of claims 3-16, further comprising propagating the phage.

18. The method of any one of claims 1-17, further comprising assessing the inhibitory activity of the phage composition against the pest and adjusting the phage composition based on the inhibitory activity.

19. The method of any one of claims 1-18, wherein step (c) comprises incorporating the phage composition into a delivery vehicle.

20. The method of claim 19, wherein the delivery vehicle is a bacterial vector.

21. The method of claim 19, wherein the delivery vehicle is a food bait for the pest.

22. The method of claim 19, wherein the delivery vehicle comprises an excipient

23. The method of any one of claims 1-22, wherein the symbiotic bacterium is an obligate symbiont of the pest

24. The method of any one of claims 1-22, wherein the symbiotic bacterium is a

facultative symbiont of the pest

25. The method of any one of claims 3-24, wherein the phage is a promiscuous phage with a large host range.

26. The method of any one of claims 3-25, wherein the phage is lytic.

27. The method of any one of claims 3-25, wherein the phage is temperate.

28. The method of any one of claims 1-27, wherein the pest is an animal.

29. The method of claim 28, wherein the pest is a termite.

30. The method of claim 29, wherein the symbiotic bacterium is selected from the group consisting of Treponema, Bacteroides, Desulfovibrio, Citrobacter, Enterobacter, Enterococcus, Lactococcus and Methanobrevibacter.

31. The method of any one of claims 1-27, wherein the pest is a plant

32. The method of any one of claims 1-27, wherein the pest is a lichenized fungus.

33. The method of any one of claims 1-32, wherein the method comprises obtaining a plurality of phagic agents that each inhibits the symbiotic bacterium

34. The method of any one of claims 1-33, wherein the method comprises identifying a plurality of symbiotic bacteria of the pest, and obtaining one or more phagic agents that inhibit each symbiotic bacterium

35. A phage composition prepared by any one of the methods of claims 1-34.

36. The phage composition of claim 35, wherein the phage composition is a liquid.

37. The phage composition of claim 36, wherein the phagic agent is a phage, and wherein the concentration of the phage in the phage composition is at least about 10³ pfu/ml.

38. The phage composition of claim 35, wherein the phage composition is a solid.

39. The phage composition of claim 38, wherein the phagic agent is a phage, and wherein the concentration of the phage in the phage composition is at least about 10³ pfti/g.

40. The phage composition of claim 35, wherein the phage composition is an aerosol.

41. The phage composition of any one of claims 35-40, wherein the phage composition is present in a pest extract.

42. The phage composition of any one of claims 35-40, wherein the phage composition is present in a live pest

43. The phage composition of any one of claims 35-40, wherein the phage composition is present in the feces secreted by the pest.

44. The phage composition of any one of claims 35-43, further comprising a bacteriocin that inhibits a symbiotic bacterium of the pest

45. A kit for killing or degrading fitness of a pest, comprising the phage composition of any one of claims 35-44, and an instruction.

46. A method of killing or degrading fitness of a pest comprising applying an effective amount of the phage composition of any one of claims 35-44.

47. The method of claim 46, wherein the phage composition is applied directly to the pest.

48. The method of claim 46, wherein the phage composition is applied to the

surroundings of the pest.

49. The method of claim 46, wherein the phage composition is applied to a second pest, wherein the second pest closely associates with the pest, and wherein the second pest delivers the phage composition to the pest

50. The method of any one of claims 46-49, wherein the phage composition is applied via a sprayer.

51. A method of preparing a bacteriocin composition for killing or degrading fitness of a pest, comprising:

(a) identifying a symbiotic bacterium in a specimen of the pest;

(b) obtaining a bacteriocin that inhibits (he symbiotic bacterium; and

(c) formulating a composition comprising the bacteriocin to provide the bacteriocin composition.

52. The method of claim 51, wherein the symbiotic bacterium is identified by sequencing the specimen of the pest.

53. The method of claim 51, wherein the symbiotic bacterium is identified by

comparative sequencing of a first specimen of the pest comprising one or more pest individuals affected by an antibacterial treatment, and a second specimen of the pest comprising one or more pest individuals unaffected by the antibacterial treatment.

54. The method of claim 52 or claim 53, wherein the sequencing or comparative

sequencing is metagenomics sequencing.

55. The method of any one of claims 51-54, wherein die specimen is derived from the pest.

56. The method of any one of claims 51-54, wherein the specimen is derived from the surroundings of the pest.

57. The method of any one of claims 51-56, further comprising isolating the symbiotic bacterium from the specimen.

58. The method of any one of claims 51-57, wherein the bacteriocin is obtained from a natural source.

59. The method of any one of claims 51-57, wherein the bacteriocin is engineered.

60. The method of any one of claims 51-59, further comprising assessing the inhibitory activity of the bacteriocin composition against the pest and adjusting the bacteriocin composition based on the inhibitory activity.

61. The method of any one of claims 51-60, wherein step (c) comprises incorporating the bacteriocin composition into a delivery vehicle.

62. The method of claim 61, wherein the delivery vehicle is a bacterial vector.

63. The method of claim 61, wherein the delivery vehicle is a food bait for the pest.

64. The method of claim 61, wherein the delivery vehicle comprises an excipient.

65. The method of any one of claims 51-64, wherein the symbiotic bacterium is an

obligate symbiont of the pest.

66. The method of any one of claims 51-64, wherein the symbiotic bacterium is a facultative symbiont of the pest.

67. The method of any one of claims 51-66, wherein the pest is an animal.

68. The method of claim 67, wherein the pest is a termite.

69. The method of claim 68, wherein the symbiotic bacterium is selected from the group consisting of Treponema, Bacteroides, Desulfovibrio, Citrobacter, Enterobacter, Enter -ococcus, Lactococcus and Methanobrevibacter.

70. The method of any one of claims 51-66, wherein the pest is a plant

71. The method of any one of claims 51-66, wherein the pest is a lichenized fungus.

72. The method of any one of claims 51-71, wherein the method comprises obtaining a plurality of bacteriocins that each inhibits the symbiotic bacterium.

73. The method of any one of claims 51-72, wherein the method comprises identifying a plurality of symbiotic bacteria of the pest, and obtaining one or more bacteriocins that inhibit each symbiotic bacterium.

74. A bacteriocin composition prepared by any one of the methods of claims 51-73.

75. The bacteriocin composition of claim 74, wherein the bacteriocin is a pyocin.

76. The bacteriocin composition of claim 74 or claim 75, wherein the bacteriocin

composition is a liquid.

77. The bacteriocin composition of claim 74 or claim 75, wherein the bacteriocin

composition is a solid.

78. The bacteriocin composition of claim 74 or claim 75, wherein the bacteriocin

composition is an aerosol.

79. The bacteriocin composition of any one of claims 74-78, wherein the bacteriocin composition is present in a pest extract.

80. The bacteriocin composition of any one of claims 74-78, wherein the bacteriocin composition is present in a live pest.

81. The bacteriocin composition of any one of claims 74-78, wherein the bacteriocin composition is present in the feces secreted by the pest.

82. The bacteriocin composition of any one of claims 74-81, further comprising a phagic agent that inhibits a symbiotic bacterium of the pest

83. The bacteriocin composition of claim 82, wherein the phagic agent is a phage.

84. The bacteriocin composition of claim 82, wherein the phagic agent is a lysin.

85. The bacteriocin composition of any one of claims 82-84, comprising a plurality of phagic agents, each inhibiting a symbiotic bacterium of the pest.

86. A kit for killing or degrading fitness of a pest, comprising the bacteriocin composition of any one of claims 74-85, and an instruction.

87. A method of killing or degrading fitness of a pest, comprising applying an effective amount of any one of the bacteriocin compositions of claims 74-85.

88. The method of claim 87, wherein the bacteriocin composition is applied directly to the pest.

89. The method of claim 87, wherein the bacteriocin composition is applied to the

surroundings of the pest.

90. The method of claim 87, wherein die bacteriocin composition is applied to a second pest, wherein the second pest closely associates with the pest, and wherein the second pest delivers the bacteriocin composition to the pest.

91. The method of any one of claims 87-90, wherein the bacteriocin composition is

applied via a sprayer.