Patent Application
20210289794
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jul. 17, 2019, is named 51215-012WO2_Sequence_Listing_07.17.19_ST25 and is 60,129 bytes in size.
Plant pests, including insect pests, are pervasive in the human environment.
In a first aspect, the invention comprises a method for decreasing colonization by a bacterium of a gut of a stink bug, the method comprising (a) providing a composition comprising vanillin or an analog thereof; and (b) delivering said composition to an egg from which the stink bug will hatch, whereby colonization by the bacterium within the gut of the stink bug hatched from the egg treated with the composition is decreased relative to a stink bug hatched from an untreated egg.
In some embodiments, the composition is delivered to an egg mass of a stink bug. In some embodiments, the decrease in colonization by the bacterium decreases the fitness of the stink bug, e.g., decreases reproductive ability, survival, rate of development, number of eggs, number of hatched eggs, adult emergence rate, body length, body width, body mass, or cuticle thickness.
In some embodiments, the colonization is in the v4 region of the gut. In some embodiments, colonization by the bacterium of the v4 region of the gut is decreased by at least 5%. In some embodiments, the size of the v4 region of the gut is decreased.
In some embodiments, the stink bug is a Halyomorpha species (e.g., Halyomorpha halys), a Nezara species, an Oebalus species, a Chinavia species, an Euthyrhynchus species, an Euschistus species, an Alcaeorrhynchus species, or a Podisus species.
In some embodiments, the bacterium is an endosymbiont, e.g., an endosymbiont is of the genus Pantoea. In some embodiments, the endosymbiont is Candidatus Pantoea carbekii.
In some embodiments, the composition is a liquid, a solid, an aerosol, a paste, a gel, or a gas composition. In some embodiments, the composition is delivered as a spray. In some embodiments, the composition comprises an agriculturally acceptable carrier. In some embodiments, the composition comprises a wetting solution.
Disclosed herein are compositions and methods for altering the fitness of insects for agriculture or commerce, wherein the composition includes a bacterial colonization-disrupting agent (e.g., an agent (e.g., a lipopolysaccharide (LPS) synthesis inhibitor or a polyhydroxyalkanoate (PHA) synthesis inhibitor) that decreases colonization of a bacteria (e.g., an endosymbiotic bacteria) in the gut of the insect.
In one aspect, provided herein is a method of altering the fitness of an insect comprising delivering to the insect an effective amount of a composition including a bacterial colonization-disrupting agent. In some embodiments, the method includes decreasing the fitness of the insect delivered the bacterial colonization-disrupting agent. Alternatively, in some embodiments, the method includes increasing the fitness of the insect delivered the bacterial colonization-disrupting agent.
In another aspect, provided herein is a method of decreasing bacterial colonization in the gut of an insect including delivering to the insect an effective amount of a composition including a bacterial colonization-disrupting agent.
In some embodiments of the methods herein, the bacterial colonization-disrupting agent is an inhibitor of bacterial metabolism. In some embodiments, the bacterial colonization-disrupting agent is a polyhydroxyalkanoate (PHA) synthesis inhibitor.
In another aspect, provided herein is a method of altering the fitness of an insect including delivering to the insect an effective amount of a composition including a PHA synthesis inhibitor. In some embodiments, the method includes decreasing the fitness of the insect delivered the PHA inhibitor. In some embodiments, the method includes increasing the fitness of the insect delivered the PHA inhibitor.
In some embodiments, the PHA synthesis inhibitor is vanillin or an analog thereof. In some embodiments, the PHA synthesis inhibitor is one or more compounds in Table 1. In some embodiments, the PHA synthesis inhibitor is levulinic acid or an analog thereof. In some embodiments, the PHA synthesis inhibitor is acrylic acid or an analog thereof. In some embodiments, the PHA synthesis inhibitor is 2-bromooctanoic acid or an analog thereof.
In some embodiments of the methods herein, the bacterial colonization-disrupting agent is an inhibitor of cell envelope biogenesis (e.g., biogenesis of the membrane(s) or other structures that surround and protect the bacterial cytoplasm, e.g., cell wall, inner membrane, and outer membrane). In some embodiments of the methods herein, the bacterial colonization-disrupting agent is a lipopolysaccharide (LPS) synthesis inhibitor.
In another aspect, provided herein is a method of altering the fitness of an insect including delivering to the insect an LPS synthesis inhibitor. In some embodiments, the method includes decreasing the fitness of the insect delivered the LPS synthesis inhibitor. In some embodiments, the method includes increasing the fitness of the insect delivered the LPS synthesis inhibitor.
In some embodiments, the LPS synthesis inhibitor is an inhibitor of core oligosaccharide synthesis in the bacteria. In some embodiments, the LPS synthesis inhibitor inhibits an enzyme involved in core oligosaccharide synthesis in the bacteria. In some embodiments, the enzyme has at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to a polypeptide having the amino acid sequence of WaaA, WaaC, WaaF, or WaaG. In some embodiments, the LPS synthesis inhibitor (e.g., the inhibitor of an enzyme involved in LPS synthesis) is a sugar. In some embodiments, the sugar is ADP-2-fluoroheptose (AFH). In some embodiments, the sugar is 2-aryl-5-methyl-4-(5-aryl-furan-2-yl-methylene)-2,4-dihydro-pyrazol-3-ones (DHPO). In some embodiments, the sugar is AFH and DHPO. In some embodiments, the sugar is one or more compounds in Table 7.
In some embodiments, the LPS synthesis inhibitor inhibits expression of a gene involved in core oligosaccharide synthesis in the bacteria. In some embodiments, the gene has at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to a polynucleotide having the nucleotide sequence of waaA, waaC, waaF, or waaG.
In some embodiments, the bacterial colonization-disrupting agent is an inhibitor of bacterial cell wall biogenesis. In some embodiments, the inhibitor of bacterial cell wall biogenesis is an inhibitor of undecaprenyl pyrophosphate phosphatase (UppP), e.g., bacitracin.
In some embodiments, the bacterial colonization-disrupting agent is an inhibitor of flagellar function, e.g., cellulose.
In some embodiments of the methods herein, the insect is a plant pest. In some embodiments, the plant pest is of the order Coleoptera, Diptera, Hemiptera, Lepidoptera, Orthoptera, Thysanoptera, or Acarina. In some embodiments, the insect is a stink bug, bean bug, beetle, weevil, fly, aphid, whitefly, leafhopper, scale, moth, butterfly, grasshopper, cricket, thrip, or mite. In some embodiments, the insect is of the genus Riptortus. In some embodiments, the insect is of the genus Halyomorpha.
In some embodiments of the methods herein, the insect is a vector of an animal pathogen and/or a human pathogen. In some embodiments, the insect is a mosquito, a midge, a louse, a sandfly, a tick, a triatomine bug, a tsetse fly, or flea.
In some embodiments of the methods herein, the bacteria is an endosymbiotic bacteria. In some embodiments, the endosymbiont resides in the gut of the insect. In some embodiments, the bacteria resides in a specialized cell or a specialized organ in the gut of the insect. In some embodiments, the specialized organ is a midgut crypt or a bacteriome. In some embodiments, the specialized cell is a bacteriocyte. In some embodiments, the endosymbiotic bacteria is of the genus Burkholderia. In some embodiments, the endosymbiotic bacteria is of the genus Pantoea.
In some embodiments of the methods herein, the method is effective to decrease the fitness of the insect relative to an untreated insect. In some embodiments, the decrease in fitness of the insect is a decrease (e.g., by about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more than 100%) in reproductive ability, survival, rate of development, number of hatched eggs, adult emergence rate, body length, or weight relative to an untreated insect.
In some embodiments, the method is effective to decrease bacterial colonization (e.g., by about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more than 100%) in the gut of the insect relative to an untreated insect.
In some embodiments, the method is effective to inhibit a physical interaction between the bacteria and the gut of the insect.
In some embodiments of the methods herein, the composition is delivered to the insect to at least one habitat where the insect grows, lives, or reproduces.
In some embodiments of the methods herein, the composition is a liquid, a solid, an aerosol, a paste, a gel, or a gas composition.
In some embodiments of the methods herein, the composition is delivered as an insect comestible composition for ingestion by the insect.
In some embodiments of the methods herein, the composition is delivered to the insect by ingestion, infusion, injection, or spraying. In some embodiments, the composition is delivered to eggs of the insect.
In some embodiments of the methods herein, the composition includes an agriculturally acceptable carrier.
In yet another aspect, provided herein is a modified insect produced by a method including contacting the insect with a composition including a bacterial colonization-disrupting agent in accordance with any of the methods herein.
In a further aspect, provided herein is a screening assay to identify a bacterial colonization-disrupting agent, including the steps of (a) exposing a target insect to one or more agents; and (b) identifying an agent that (i) decreases the fitness of the target insect (e.g., by about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more than 100%), and (ii) inhibits colonization of a bacterium in the gut of the target insect (e.g., by about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more than 100%).
In some embodiments of the assay herein, the decrease in fitness is decreased survival of the target insect. In some embodiments, the decrease in fitness is a decrease in reproductive ability, survival, rate of development, number of hatched eggs, adult emergence rate, body length, or body mass. In some embodiments, the agent is effective to inhibit a physical interaction between the bacteria and the gut of the insect.
In some embodiments of the assay herein, the bacteria is an endosymbiotic bacteria. In some embodiments, the endosymbiotic bacteria resides in the gut of the insect. In some embodiments, the bacteria resides in a specialized cell or a specialized organ in the gut of the insect. In some embodiments, the specialized organ is a midgut crypt or a bacteriome. In some embodiments, the specialized cell is a bacteriocyte. In some embodiments, the bacterium is of the genus Burkholderia. In some embodiments, the bacterium is of the genus Pantoea.
In some embodiments of the assay herein, the bacterial colonization-disrupting agent is a PHA synthesis inhibitor.
In some embodiments of the assay herein, the bacterial colonization-disrupting agent is an LPS synthesis inhibitor.
In some embodiments of the assay herein, the insect is a plant pest. In some embodiments, the plant pest is of the order Coleoptera, Diptera, Hemiptera, Lepidoptera, Orthoptera, Thysanoptera, or Acarina.
In some embodiments of the assay herein, the insect is a vector of an animal pathogen and/or a human pathogen. In some embodiments, the insect is a mosquito, a midge, a louse, a sandfly, a tick, a triatomine bug, a tsetse fly, or flea.
In another aspect, provided herein is a modified insect produced by a method including contacting the insect with a composition including a bacterial colonization-disrupting agent identified by the screening assay herein.
In yet another aspect, provided herein is a method of decreasing the fitness of an insect including delivering to the insect an effective amount of a composition including a bacterial colonization-disrupting agent identified by the screening assay herein.
In a further aspect, provided here in is a composition including a bacterial colonization-disrupting agent and a carrier, wherein the composition is formulated for delivery to an insect, or a habitat thereof.
In some embodiments of the composition herein, the bacterial colonization-disrupting agent is a polyhydroxyalkanoate (PHA) synthesis inhibitor. In some embodiments, the PHA synthesis inhibitor is vanillin or an analog thereof. In some embodiments, the PHA synthesis inhibitor is one or more compounds in Table 1. In some embodiments, the PHA synthesis inhibitor is levulinic acid or an analog thereof. In some embodiments, the PHA synthesis inhibitor is acrylic acid or an analog thereof. In some embodiments, the PHA synthesis inhibitor is 2-bromooctanoic acid or an analog thereof.
In some embodiments of the composition herein, the bacterial colonization-disrupting agent is an inhibitor of bacterial cell envelope biogenesis. In some embodiments, the inhibitor of bacterial cell envelope biogenesis is a lipopolysaccharide (LPS) synthesis inhibitor. In some embodiments, the LPS synthesis inhibitor is an inhibitor of core oligosaccharide synthesis in the bacteria. In some embodiments, the LPS synthesis inhibitor inhibits an enzyme involved in core oligosaccharide synthesis in the bacteria. In some embodiments, the enzyme has at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to a polypeptide having the amino acid sequence of WaaA, WaaC, WaaF, or WaaG. In some embodiments, the LPS synthesis inhibitor (e.g., the inhibitor of an enzyme involved in LPS synthesis) is a sugar. In some embodiments, the sugar is ADP-2-fluoroheptose (AFH). In some embodiments, the sugar is 2-aryl-5-methyl-4-(5-aryl-furan-2-yl-methylene)-2,4-dihydro-pyrazol-3-ones (DHPO). In some embodiments, the sugar is AFH and DHPO. In some embodiments, the sugar is one or more compounds in Table 7.
In some embodiments, the LPS synthesis inhibitor inhibits expression of a gene involved in core oligosaccharide synthesis in the bacteria. In some embodiments, the gene has at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to a polynucleotide having the nucleotide sequence of waaA, waaC, waaF, or waaG.
In some embodiments, the bacterial colonization-disrupting agent is an inhibitor of bacterial cell wall biogenesis. In some embodiments, the inhibitor of bacterial cell wall biogenesis is an inhibitor of undecaprenyl pyrophosphate phosphatase (UppP), e.g., bacitracin.
In some embodiments, the bacterial colonization-disrupting agent is an inhibitor of flagellar function, e.g., cellulose.
In some embodiments of the composition herein, the bacterial colonization-disrupting agent is at least 0.1%, 0.2%, 0.4%, 0.5%, 0.8%, 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the composition. In some embodiments, the carrier is a liquid, a solid, an aerosol, a paste, a gel, or a gas composition. In some embodiments, the carrier is a sugar syrup, corn syrup, or honey. In some embodiments, the carrier is a nanoparticle or lipid membrane.
In some embodiments of the composition herein, the composition is formulated for delivery to the insect, for example, by ingestion, infusion, injection, spraying, smoking, or fogging. In some embodiments, the composition is formulated for delivery to at least one habitat, for example, where the insect grows, lives, reproduces, or feeds. In some embodiments, the composition is formulated for delivery to a plant ingested by the insect. In another aspect, provided herein is a modified plant or part thereof including a bacterial colonization-disrupting agent, wherein the plant or part thereof is ingested by an insect. In some embodiments, the plant is genetically engineered to produce the bacterial colonization-disrupting agent, e.g., by expression from a heterologous genetic construct.
As used herein, the term “bacterial colonization-disrupting agent” refers to an agent that impedes or disrupts colonization of a bacteria in the gut of an insect (e.g., colonization of the surface of the gut or colonization of a cell (e.g., bacteriocyte) or organ (e.g., bacteriome or crypt) therein). For example, the agent may alter properties of the bacteria (e.g., bacterial metabolism or bacterial cell surface), or components thereof, and/or the insect gut, or components thereof, such that the bacteria can no longer adhere, associate with, or propagate in the gut of the insect. Exemplary bacterial colonization-disrupting agents include lipopolysaccharide (LPS) synthesis inhibitors, polyhydroxyalkanoate (PHA) synthesis inhibitors, inhibitors of cell wall biogenesis, and inhibitors of flagellar function.
As used herein, the term “colonizing” refers to persistence of a bacterium in an insect in an amount and for a duration sufficient to establish a population of bacteria in the insect (e.g., insect gut) that persists for the lifespan of the insect. The bacterium, once colonized, may further be vertically transmitted through at least one additional generation, e.g., two or more generations (e.g., life cycles) of the insect.
As used herein, the term “effective amount” refers to an amount of a bacterial colonization-disrupting agent, or composition including said agent sufficient to effect the recited result, e.g., to decrease the fitness of an insect; to reach a target level (e.g., a predetermined or threshold level) of a bacterial colonization-disrupting agent concentration inside a target insect; to reach a target level (e.g., a predetermined or threshold level) of a bacterial colonization-disrupting agent concentration inside a target insect gut; to reach a target level (e.g., a predetermined or threshold level) of a bacterial colonization-disrupting agent concentration inside a target insect bacteriocyte; to reach a target level (e.g., a predetermined or threshold level) of a bacterial colonization-disrupting agent concentration inside a target insect crypt; and/or to decrease colonization of one or more microorganisms (e.g., endosymbiont) in the gut of the target insect.
As used herein “decreasing the fitness of an insect” refers to any unfavorable alteration to insect physiology, or any activity carried out by said insect, as a consequence of administration of a bacterial colonization-disrupting agent, including, but not limited to, any one or more of the following desired effects: (1) decreasing a population of an insect by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (2) decreasing the reproductive rate of an insect by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (3) decreasing the mobility of an insect by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (4) decreasing the body weight of an insect by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (5) decreasing the metabolic rate or activity of an insect by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; or (6) decreasing plant infestation by an insect by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more. A decrease in insect fitness can be determined in comparison to an insect to which the bacterial colonization-disrupting agent has not been administered.
As used herein, the term “fitness” refers to the ability of an insect to survive, grow, and/or to produce surviving offspring. Fitness of an organism may be measured by one or more parameters, including, but not limited to survival, life span, reproductive ability, reproductive rate, reproductive period, number of eggs laid, number of hatched eggs, developmental rate, adult emergence rate, mobility, body size (e.g., body length, body mass, or body width (e.g., pronotal width of a stink bug)), cuticle (exoskeleton) thickness, pigmentation, or metabolic rate.
As used herein, the term “gut” refers to any portion of an insect's gut, including, the foregut, midgut, or hindgut of the insect, and any specialized organ (e.g., crypt or bacteriome) or cell (e.g., bacteriocyte) therein. As used herein, the terms “v1”, “v2”, “v3”, and “v4” refer to morphologically distinct regions of the midgut dissected from an adult hemipteran insect (e.g., a stink bug or a bean bug), which are numbered respectively from anterior to posterior. As used herein, v1 refers to the stomach-like midgut first region; v2 refers to the tubular midgut second region; v3 refers to the expanded sac-like midgut third region; and v4 refers to the midgut fourth region, which contains numerous crypts having lumen that may include symbiotic cells. Bacterial colonization may occur in one, more than one, or all regions of the gut. In some examples, bacterial colonization occurs in the v4 region of the midgut. The v1-v4 regions may also be referred to as m1-m4 (Duron and Noel, Environmental Microbiology Reports, 8(5):715-727).
As used herein, the term “host” refers to an organism (e.g., insect) carrying resident microorganisms (e.g., endogenous microorganisms, endosymbiotic microorganisms (e.g., primary or secondary endosymbionts), commensal organisms, and/or pathogenic microorganisms).
As used herein, “increasing the fitness of an insect” refers to any favorable alteration in insect physiology, phenotype, or any activity of the insect, including, but not limited to, any one or more of the following desired effects: (1) increasing a population of an insect by about 1%, 2%, 3%, 4%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (2) increasing the reproductive rate of an insect by about 1%, 2%, 3%, 4%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (3) increasing the mobility of an insect by about 1%, 2%, 3%, 4%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (4) increasing the body weight of an insect by about 1%, 2%, 3%, 4%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (5) increasing the metabolic rate or activity of an insect by about 1%, 2%, 3%, 4%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (6) increasing pollination (e.g., number of plants pollinated in a given amount of time) by an insect by about 1%, 2%, 3%, 4%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (7) increasing production of insect byproducts (e.g., honey or silk) by about 1%, 2%, 3%, 4%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (8) increasing nutrient content of the insect (e.g., protein, fatty acids, or amino acids) by about 1%, 2%, 3%, 4%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; or (9) increasing insect resistance to pesticides by about 1%, 2%, 3%, 4%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more. An increase in insect fitness can be determined in comparison to a control (e.g., an untreated insect).
The term “insect” or “arthropod” includes any organism belonging to the phylum Arthropoda and to the class Insecta or the class Arachnida, in any stage of development, i.e., immature or adult insects. As used herein, the term “beneficial insect” refers to an insect whose presence confers benefits to agricultural, horticultural, or commercial applications, or whose presence or activity is otherwise desirable.
As used herein, the term “microorganism” refers to bacteria or fungi. Microorganisms may refer to microorganisms resident in an insect (e.g., endogenous microorganisms, endosymbiotic microorganisms (e.g., primary or secondary endosymbionts)) or microorganisms exogenous to the insect, including those that produce bacterial colonization-disrupting agents.
As used herein, the term “peptide,” “protein,” or “polypeptide” encompasses any chain of naturally or non-naturally occurring amino acids (either D- or L-amino acids), regardless of length (e.g., at least 2, 3, 4, 5, 6, 7, 10, 12, 14, 16, 18, 20, 25, 30, 40, 50, 100, or more amino acids), the presence or absence of post-translational modifications (e.g., glycosylation or phosphorylation), or the presence of, e.g., one or more non-amino acyl groups (for example, sugar, lipid, etc.) covalently linked to the peptide, and includes, for example, natural proteins, synthetic, or recombinant polypeptides and peptides, hybrid molecules, peptoids, or peptidomimetics.
As used herein, “percent identity” between two sequences is determined by the BLAST 2.0 algorithm, which is described in Altschul et al. (J. Mol. Biol. 215:403-410, 1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.
As used herein, the term “pest” refers to an insect that causes damage to plants or other organisms, are present where they are not wanted, or otherwise are detrimental to humans, for example, by impacting human agricultural methods or products.
As used herein, the term “plant” refers to whole plants, plant organs, plant tissues, seeds, plant cells, seeds, and progeny of the same. Plant cells include, without limitation, cells from seeds, suspension cultures, embryos, meristematic regions, flowers, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, or microspores. Plant parts include differentiated or undifferentiated tissues including, but not limited to the following: roots, stems, shoots, leaves, pollen, seeds, tumor tissue, and various forms of cells and culture (e.g., single cells, protoplasts, embryos, or callus tissue). The plant tissue may be in a plant or in a plant organ, tissue, or cell culture.
As used herein, the term “symbiont” or “insect symbiont” refers to an intracellular or extracellular microorganism that, upon colonization of an insect, confers fitness benefits to the insect. An “endosymbiont” refers to a microorganism capable of living within an insect cell or organ, such as a bacteriocyte or crypt.
As used herein, the term “untreated insect” or “unmodified insect” refers to an insect, or population thereof, that has not been specifically contacted with or delivered (e.g., in accordance with a method described herein) a bacterial colonization-disrupting agent (e.g., has not been contacted with or delivered a bacterial colonization-disrupting agent at any point in time, or has been assessed at a point in time prior to contact with or delivery of the bacterial colonization-disrupting agent).
Other features and advantages of the invention will be apparent from the following Detailed Description and the Claims.
The figures are meant to be illustrative of one or more features, aspects, or embodiments of the invention and are not intended to be limiting.
Provided herein are methods and compositions including bacterial colonization-disrupting agents useful for decreasing or preventing bacterial colonization in the gut of insects. The integrity of the gut microbiota is important for insect fitness. A number of insects have evolved to be obligatorily dependent on bacterial symbionts, including intracellular symbionts (e.g., endosymbionts). Many of these bacteria reside in the gut of insects, and in some cases, the insect harbors such bacteria in specialized cells (bacteriocytes) or organs (bacteriomes or crypts). By impeding colonization of bacteria in the insect gut or in the specialized organs or cells therein, the present methods and compositions can be used to decrease the fitness of a variety of insects, such as insects that are considered pests in agricultural or commercial industries or otherwise insects harmful to humans or animals (e.g., insect vectors of disease).
A variety of bacterial colonization-disrupting agents are useful in the present methods. The methods and compositions described herein are based, in part, on the examples which illustrate how different agents, for example, lipopolysaccharide (LPS) synthesis inhibitors, polyhydroxyalkanoate (PHA) synthesis inhibitors, inhibitors of cell wall biogenesis, or inhibitors of flagellar function, can be used to decrease colonization of symbiotic microorganisms in insect hosts (e.g., endosymbiotic Burkholderia in bean bugs or Candidatus Pantoea carbekii in stink bugs) to decrease the fitness of these hosts. Screening methods are also provided herein for identifying additional bacterial colonization-disrupting agents.
Provided herein are methods of altering the fitness (e.g., decreasing the fitness or increasing the fitness) of an insect by delivering to the insect a composition including a bacterial colonization-disrupting agent. Examples of insects that can be targeted by the present methods, fitness benefits that can be conferred by the present methods, and methods for delivering the bacterial colonization-disrupting agent to insects are further described, below.
i. Insects
The bacterial colonization-disrupting agents herein can be applied to a variety of insects. For example, the insect may be an agricultural pest. Pests include insects that cause damage to plants or other organisms, or otherwise are detrimental to humans, for example, human agricultural methods or products.
In some instances, the insect is of the order: Acari, Araneae, Anoplura, Coleoptera, Collembola, Dermaptera, Dictyoptera, Diplura, Diptera (e.g., spotted-wing Drosophila), Embioptera, Ephemeroptera, Grylloblatodea, Hemiptera (e.g., aphids, Greenhous whitefly), Homoptera, Hymenoptera, Isoptera, Lepidoptera, Mallophaga, Mecoptera, Neuroptera, Odonata, Orthoptera, Phasmida, Plecoptera, Protura, Psocoptera, Siphonaptera, Siphunculata, Thysanura, Strepsiptera, Thysanoptera, Trichoptera, or Zoraptera.
In some instances, the insect is of the class Arachnida, for example, Acarus spp., Aceria sheldoni, Aculops spp., Aculus spp., Amblyomma spp., Amphitetranychus viennensis, Argas spp., Boophilus spp., Brevipalpus spp., Bryobia graminum, Bryobia praetiosa, Centruroides spp., Chorioptes spp., Dermanyssus gaffinae, Dermatophagoides pteronyssinus, Dermatophagoides farinae, Dermacentor spp., Eotetranychus spp., Epitrimerus gyri, Eutetranychus spp., Eriophyes spp., Glycyphagus domesticus, Halotydeus destructor, Hemitarsonemus spp., Hyalomma spp., Ixodes spp., Latrodectus spp., Loxosceles spp., Metatetranychus spp., Neutrombicula autumnalis, Nuphersa spp., Oligonychus spp., Ornithodorus spp., Ornithonyssus spp., Panonychus spp., Phyllocoptruta oleivora, Polyphagotarsonemus latus, Psoroptes spp., Rhipicephalus spp., Rhizoglyphus spp., Sarcoptes spp., Scorpio maurus, Steneotarsonemus spp., Steneotarsonemus spinki, Tarsonemus spp., Tetranychus spp., Trombicula alfreddugesi, Vaejovis spp., or Vasates lycopersici.
In some instances, the insect is of the class Chilopoda, for example, Geophilus spp. or Scutigera spp.
In some instances, the insect is of the order Collembola, for example, Onychiurus armatus.
In some instances, the insect is of the class Diplopoda, for example, Blaniulus guttulatus;
In some instances, the insect is of the class Insecta, e.g. from the order Blattodea, for example, Blattella asahinai, Blattella germanica, Blatta orientalis, Leucophaea maderae, Panchlora spp., Parcoblatta spp., Periplaneta spp., or Supella longipalpa.
In some instances, the insect is of the order Coleoptera, for example, Acalymma vittatum, Acanthoscelides obtectus, Adoretus spp., Agelastica alni, Agriotes spp., Alphitobius diaperinus, Amphimallon solstitialis, Anobium punctatum, Anoplophora spp., Anthonomus spp., Anthrenus spp., Apion spp., Apogonia spp., Atomaria spp., Attagenus spp., Bruchidius obtectus, Bruchus spp., Cassida spp., Cerotoma trifurcata, Ceutorrhynchus spp., Chaetocnema spp., Cleonus mendicus, Conoderus spp., Cosmopolites spp., Costelytra zealandica, Ctenicera spp., Curculio spp., Cryptolestes ferrugineus, Cryptorhynchus lapathi, Cylindrocopturus spp., Dermestes spp., Diabrotica spp. (e.g., corn rootworm), Dichocrocis spp., Dicladispa armigera, Diloboderus spp., Epilachna spp., Epitrix spp., Faustinus spp., Gibbium psylloides, Gnathocerus cornutus, Hellula undalis, Heteronychus arator, Heteronyx spp., Hylamorpha elegans, Hylotrupes bajulus, Hypera postica, Hypomeces squamosus, Hypothenemus spp., Lachnosterna consanguinea, Lasioderma serricorne, Latheticus oryzae, Lathridius spp., Lema spp., Leptinotarsa decemlineata, Leucoptera spp., Lissorhoptrus oryzophilus, Lixus spp., Luperodes spp., Lyctus spp., Megascelis spp., Melanotus spp., Meligethes aeneus, Melolontha spp., Migdolus spp., Monochamus spp., Naupactus xanthographus, Necrobia spp., Niptus hololeucus, Oryctes rhinoceros, Oryzaephilus surinamensis, Oryzaphagus oryzae, Otiorrhynchus spp., Oxycetonia jucunda, Phaedon cochleariae, Phyllophaga spp., Phyllophaga helleri, Phyllotreta spp., Popillia japonica, Premnotrypes spp., Prostephanus truncatus, Psyffiodes spp., Ptinus spp., Rhizobius ventralis, Rhizopertha dominica, Sitophilus spp., Sitophilus oryzae, Sphenophorus spp., Stegobium paniceum, Sternechus spp., Symphyletes spp., Tanymecus spp., Tenebrio molitor, Tenebrioides mauretanicus, Tribolium spp., Trogoderma spp., Tychius spp., Xylotrechus spp., or Zabrus spp.;
In some instances, the insect is of the order Diptera, for example, Aedes spp., Agromyza spp., Anastrepha spp., Anopheles spp., Asphondylia spp., Bactrocera spp., Bibio hortulanus, Calliphora erythrocephala, Calliphora vicina, Ceratitis capitata, Chironomus spp., Chrysomyia spp., Chrysops spp., Chrysozona pluvialis, Cochliomyia spp., Contarinia spp., Cordylobia anthropophaga, Cricotopus sylvestris, Culex spp., Culicoides spp., Culiseta spp., Cuterebra spp., Dacus oleae, Dasyneura spp., Delia spp., Dermatobia hominis, Drosophila spp., Echinocnemus spp., Fannia spp., Gasterophilus spp., Glossina spp., Haematopota spp., Hydrellia spp., Hydrellia griseola, Hylemya spp., Hippobosca spp., Hypoderma spp., Liriomyza spp., Lucilia spp., Lutzomyia spp., Mansonia spp., Musca spp. (e.g., Musca domestica), Oestrus spp., Oscinella frit, Paratanytarsus spp., Paralauterborniella subcincta, Pegomyia spp., Phlebotomus spp., Phorbia spp., Phormia spp., Piophila casei, Prodiplosis spp., Psila rosae, Rhagoletis spp., Sarcophaga spp., Simulium spp., Stomoxys spp., Tabanus spp., Tetanops spp., or Tipula spp.
In some instances, the insect is of the order Heteroptera, for example, Alydidae, Anasa tristis, Antestiopsis spp., Boisea spp., Blissus spp., Calocoris spp., Campylomma livida, Cavelerius spp., Cimex spp., Collaria spp., Creontiades dilutus, Dasynus piperis, Dichelops furcatus, Diconocoris hewetti, Dysdercus spp., Euschistus spp., Eurygasterspp., Heliopeltis spp., Horcias nobilellus, Leptocorisa spp., Leptocorisa varicornis, Leptoglossus phyllopus, Lygus spp., Macropes excavatus, Miridae, Monalonion atratum, Nezara spp., Oebalus spp., Pentatomidae, Piesma quadrata, Piezodorus spp., Psallus spp., Pseudacysta persea, Rhodnius spp., Sahlbergella singularis, Scaptocoris castanea, Scotinophora spp., Stephanitis nashi, Tibraca spp., or Triatoma spp.
In some instances, the insect is of the order Homoptera, for example, Acizzia acaciaebaileyanae, Acizzia dodonaeae, Acizzia uncatoides, Acrida turrita, Acyrthosipon spp., Acrogonia spp., Aeneolamia spp., Agonoscena spp., Aleyrodes proletella, Aleurolobus barodensis, Aleurothrixus floccosus, Allocaridara malayensis, Amrasca spp., Anuraphis cardui, Aonidiella spp., Aphanostigma pini, Aphis spp. (e.g., Apis gossypii), Arboridia apicalis, Arytainilla spp., Aspidiella spp., Aspidiotus spp., Atanus spp., Aulacorthum solani, Bemisia tabaci, Blastopsylla occidentalis, Boreioglycaspis melaleucae, Brachycaudus helichrysi, Brachycolus spp., Brevicoryne brassicae, Cacopsylla spp., Calligypona marginata, Carneocephala fulgida, Ceratovacuna lanigera, Cercopidae, Ceroplastes spp., Chaetosiphon fragaefolii, Chionaspis tegalensis, Chlorita Chondracris rosea, Chromaphis juglandicola, Chrysomphalus ficus, Cicadulina mbila, Coccomytilus haffi, Coccus spp., Cryptomyzus ribis, Cryptoneossa spp., Ctenarytaina spp., Dalbulus spp., Dialeurodes citri, Diaphorina citri, Diaspis spp., Drosicha spp., Dysaphis spp., Dysmicoccus spp., Empoasca spp., Eriosoma spp., Erythroneura spp., Eucalyptolyma spp., Euphyllura spp., Euscelis bilobatus, Ferrisia spp., Geococcus coffeae, Glycaspis spp., Heteropsylla cubana, Heteropsylla spinulosa, Homalodisca coagulata, Homalodisca vitripennis, Hyalopterus arundinis, Icerya spp., Idiocerus spp., Idioscopus spp., Laodelphax striatellus, Lecanium spp., Lepidosaphes spp., Lipaphis erysimi, Macrosiphum spp., Macrosteles facifrons, Mahanarva spp., Melanaphis sacchari, Metcalfiella spp., Metopolophium dirhodum, Monellia costalis, Monelliopsis pecanis, Myzus spp., Nasonovia ribisnigri, Nephotettix spp., Nettigoniclla spectra, Nilaparvata lugens, Oncometopia spp., Orthezia praelonga, Oxya chinensis, Pachypsylla spp., Parabemisia myricae, Paratrioza spp., Parlatoria spp., Pemphigus spp., Peregrinus maidis, Phenacoccus spp., Phloeomyzus passerinii, Phorodon humuli, Phylloxera spp., Pinnaspis aspidistrae, Planococcus spp., Prosopidopsylla flava, Protopulvinaria pyriformis, Pseudaulacaspis pentagona, Pseudococcus spp., Psyllopsis spp., Psylla spp., Pteromalus spp., Pyrilla spp., Quadraspidiotus spp., Quesada gigas, Rastrococcus spp., Rhopalosiphum spp., Saissetia spp., Scaphoideus titanus, Schizaphis graminum, Selenaspidus articulatus, Sogata spp., Sogatella furcifera, Sogatodes spp., Stictocephala festina, Siphoninus phillyreae, Tenalaphara malayensis, Tetragonocephela spp., Tinocallis caryaefoliae, Tomaspis spp., Toxoptera spp., Trialeurodes vaporariorum, Trioza spp., Typhlocyba spp., Unaspis spp., Viteus vitifolii, or Zygina spp.
In some instances, the insect is of the order Hymenoptera, for example, Acromyrmex spp., Athalia spp., Atta spp., Diprion spp., Hoplocampa spp., Lasius spp., Monomorium pharaonis, Sirex spp., Solenopsis invicta, Tapinoma spp., Urocerus spp., Vespa spp., or Xeris spp.
In some instances, the insect is of the order Isopoda, for example, Armadillidium vulgare, Oniscus asellus, or Porcellio scaber.
In some instances, the insect is of the order Isoptera, for example, Coptotermes spp., Cornitermes cumulans, Cryptotermes spp., Incisitermes spp., Microtermes obesi, Odontotermes spp., or Reticulitermes spp.
In some instances, the insect is of the order Lepidoptera, for example, Achroia grisella, Acronicta major, Adoxophyes spp., Aedia leucomelas, Agrotis spp., Alabama spp., Amyelois transitella, Anarsia spp., Anticarsia spp., Argyroploce spp., Barathra brassicae, Borbo cinnara, Bucculatrix thurberiella, Bupalus piniarius, Busseola spp., Cacoecia spp., Caloptilia theivora, Capua reticulana, Carpocapsa pomonella, Carposina niponensis, Cheimatobia brumata, Chilo spp., Choristoneura spp., Clysia ambiguella, Cnaphalocerus spp., Cnaphalocrocis medinalis, Cnephasia spp., Conopomorpha spp., Conotrachelus spp., Copitarsia spp., Cydia spp., Dalaca noctuides, Diaphania spp., Diatraea saccharalis, Earias spp., Ecdytolopha aurantium, Elasmopalpus lignosellus, Eldana saccharina, Ephestia spp., Epinotia spp., Epiphyas postvittana, Etiella spp., Eulia spp., Eupoecilia ambiguella, Euproctis spp., Euxoa spp., Feltia spp., Galleria mellonella, Gracillaria spp., Grapholitha spp., Hedylepta spp., Helicoverpa spp., Heliothis spp., Hofmannophila pseudospretella, Homoeosoma spp., Homona spp., Hyponomeuta padella, Kakivoria flavofasciata, Laphygma spp., Laspeyresia molesta, Leucinodes orbonalis, Leucoptera spp., Lithocolletis spp., Lithophane antennata, Lobesia spp., Loxagrotis albicosta, Lymantria spp., Lyonetia spp., Malacosoma neustria, Maruca testulalis, Mamstra brassicae, Melanitis leda, Mocis spp., Monopis obviella, Mythimna separata, Nemapogon cloacellus, Nymphula spp., Oiketicus spp., Oria spp., Orthaga spp., Ostrinia spp., Oulema oryzae, Panolis flammea, Parnara spp., Pectinophora spp., Perileucoptera spp., Phthorimaea spp., Phyllocnistis citrella, Phyllonorycter spp., Pieris spp., Platynota stultana, Plodia interpunctella, Plusia spp., Plutella xylostella, Prays spp., Prodenia spp., Protoparce spp., Pseudaletia spp., Pseudaletia unipuncta, Pseudoplusia includens, Pyrausta nubilalis, Rachiplusia nu, Schoenobius spp., Scirpophaga spp., Scirpophaga innotata, Scotia segetum, Sesamia spp., Sesamia inferens, Sparganothis spp., Spodoptera spp., Spodoptera praefica, Stathmopoda spp., Stomopteryx subsecivella, Synanthedon spp., Tecia solanivora, Thermesia gemmatalis, Tinea cloacella, Tinea pellionella, Tineola bisselliella, Tortrix spp., Trichophaga tapetzella, Trichoplusia spp., Tryporyza incertulas, Tuta absoluta, or Virachola spp.
In some instances, the insect is of the order Orthoptera or Saltatoria, for example, Acheta domesticus, Dichroplus spp., Gryllotalpa spp., Hieroglyphus spp., Locusta spp., Melanoplus spp., or Schistocerca gregaria.
In some instances, the insect is of the order Phthiraptera, for example, Damalinia spp., Haematopinus spp., Linognathus spp., Pediculus spp., Ptirus pubis, or Trichodectes spp.
In some instances, the insect is of the order Psocoptera for example Lepinatus spp., or Liposcelis spp.
In some instances, the insect is of the order Siphonaptera, for example, Ceratophyllus spp., Ctenocephalides spp., Pulex irritans, Tunga penetrans, or Xenopsylla cheopsis.
In some instances, the insect is of the order Thysanoptera, for example, Anaphothrips obscurus, Baliothrips biformis, Drepanothrips reuteri, Enneothrips flavens, Frankliniella spp., Heliothrips spp., Hercinothrips femoralis, Rhipiphorothrips cruentatus, Scirtothrips spp., Taeniothrips cardamomi, or Thrips spp.
In some instances, the insect is of the order Zygentoma (=Thysanura), for example, Ctenolepisma spp., Lepisma saccharina, Lepismodes inquilinus, or Thermobia domestica.
In some instances, the insect is of the class Symphyla, for example, Scutigerella spp.
In some instances, the insect is a mite, including but not limited to, Tarsonemid mites, such as Phytonemus pallidus, Polyphagotarsonemus latus, Tarsonemus bilobatus, or the like; Eupodid mites, such as Penthaleus erythrocephalus, Penthaleus major, or the like; Spider mites, such as Oligonychus shinkajii, Panonychus citri, Panonychus mori, Panonychus ulmi, Tetranychus kanzawai, Tetranychus urticae, or the like; Eriophyid mites, such as Acaphylla theavagrans, Aceria tulipae, Aculops lycopersici, Aculops pelekassi, Aculus schlechtendali, Eriophyes chibaensis, Phyllocoptruta oleivora, or the like; Acarid mites, such as Rhizoglyphus robini, Tyrophagus putrescentiae, Tyrophagus similis, or the like; Bee brood mites, such as Varroa jacobsoni, Varroa destructor or the like; Ixodides, such as Boophilus microplus, Rhipicephalus sanguineus, Haemaphysalis longicornis, Haemophysalis flava, Haemophysalis campanulata, Ixodes ovatus, Ixodes persulcatus, Amblyomma spp., Dermacentor spp., or the like; Cheyletidae, such as Cheyletiella yasguri, Cheyletiella blakei, or the like; Demodicidae, such as Demodex canis, Demodex cati, or the like; Psoroptidae, such as Psoroptes ovis, or the like; Scarcoptidae, such as Sarcoptes scabiei, Notoedres cati, Knemidocoptes spp., or the like. In certain instances, the insect is a bean bug (e.g., a Riptortus species, e.g., Riptortus pedestris).
In some instances, the insect is a stink bug, e.g., a member of the Pentatomidae, e.g., a Halyomorpha species (e.g., Halyomorpha halys (St{dot over (a)}l)), a Nezara species (e.g., Nezara viridula), an Oebalus species (e.g., Oebalus pugnax), a Chinavia species (e.g., Chinavia hilaris), an Euthyrhynchus species (e.g., Euthyrhynchus floridanus), an Euschistus species (e.g., Euschistus servus), an Alcaeorrhynchus species (e.g., Alcaeorrhynchus grandis), or a Podisus species. In certain instances, the stink bug is the brown marmorated stink bug (Halyomorpha halys (St{dot over (a)}l)).
The methods and compositions provided herein may also be used with any insect host that is considered a vector for a pathogen that is capable of causing disease in animals.
For example, the insect host may include, but is not limited to those with piercing-sucking mouthparts, as found in Hemiptera and some Hymenoptera and Diptera such as mosquitoes, bees, wasps, midges, lice, tsetse fly, fleas and ants, as well as members of the Arachnidae such as ticks and mites; order, class or family of Acarina (ticks and mites) e.g. representatives of the families Argasidae, Dermanyssidae, Ixodidae, Psoroptidae or Sarcoptidae and representatives of the species Amblyomma spp., Anocenton spp., Argas spp., Boophilus spp., Cheyletiella spp., Chorioptes spp., Demodex spp., Dermacentor spp., Denmanyssus spp., Haemophysalis spp., Hyalomma spp., Ixodes spp., Lynxacarus spp., Mesostigmata spp., Notoednes spp., Ornithodoros spp., Ornithonyssus spp., Otobius spp., otodectes spp., Pneumonyssus spp., Psoroptes spp., Rhipicephalus spp., Sancoptes spp., or Trombicula spp.; Anoplura (sucking and biting lice) e.g. representatives of the species Bovicola spp., Haematopinus spp., Linognathus spp., Menopon spp., Pediculus spp., Pemphigus spp., Phylloxera spp., or Solenopotes spp.; Diptera (flies) e.g. representatives of the species Aedes spp., Anopheles spp., Calliphora spp., Chrysomyia spp., Chrysops spp., Cochliomyia spp., Cw/ex spp., Culicoides spp., Cuterebra spp., Dermatobia spp., Gastrophilus spp., Glossina spp., Haematobia spp., Haematopota spp., Hippobosca spp., Hypoderma spp., Lucilia spp., Lyperosia spp., Melophagus spp., Oestrus spp., Phaenicia spp., Phlebotomus spp., Phormia spp., Acari (sarcoptic mange) e.g., Sarcoptidae spp., Sarcophaga spp., Simulium spp., Stomoxys spp., Tabanus spp., Tannia spp. or Zzpu/alpha spp.; Mallophaga (biting lice) e.g. representatives of the species Damalina spp., Felicola spp., Heterodoxus spp. or Trichodectes spp.; or Siphonaptera (wingless insects) e.g. representatives of the species Ceratophyllus spp., Xenopsylla spp; Cimicidae (true bugs) e.g. representatives of the species Cimex spp., Tritominae spp., Rhodinius spp., or Triatoma spp.
In some instances, the insect is a blood-sucking insect from the order Diptera (e.g., suborder Nematocera, e.g., family Colicidae). In some instances, the insect is from the subfamilies Culicinae, Corethrinae, Ceratopogonidae, or Simuliidae. In some instances, the insect is of a Culex spp., Theobaldia spp., Aedes spp., Anopheles spp., Aedes spp., Forciponiyia spp., Culicoides spp., or Helea spp. In certain instances, the insect is a mosquito. In certain instances, the insect is a tick. In certain instances, the insect is a mite. In certain instances, the insect is a biting louse.
Alternatively, the insect may be a beneficial insect, such as a plant pollinator, a natural competitor of a pest, or a producer of useful substances for humans or animals. The term beneficial insect refers to an insect that confers a benefit (e.g., economical and/or ecological) to humans, animals, an ecosystem, and/or the environment. For example, the insect may be an insect that is involved in the production of a commercial product, including, but not limited to, insects cultivated to produce food (e.g., honey from honey bees, e.g., Apis mellifera), materials (such as silk from Bombyx mori), and/or substances (e.g., lac from Laccifer lacca or pigments from Dactylopius coccus and Cynipidae). In some instances, the insect may be harvested, or one or more parts of the insect may be harvested, and processed for use in the manufacture of a consumable product, including any product safe for human or animal consumption (e.g., ingestion). Additionally, the insect may include insects that are used in agricultural applications, including insects that aid in the pollination of crops, spreading seeds, or pest control. Further, in some instances, the insect may be an insect that is useful for waste disposal and/or organic recycling (e.g., earthworms, termites, or Diptera larvae). The insect may be one that has its native (i.e., unaltered) microbiota. Alternatively, the insect may be one that has received probiotic compositions prior to or during delivery of the bacterial colonization-disrupting agent.
In some instances, the insect may be harvested and distributed in a whole form (e.g., as the whole, unprocessed insect) as a consumable product. In some instances, the whole harvested insect is processed (e.g., ground up) and distributed as a consumable product. Alternatively, one or more parts of the insect (e.g., one or more body parts or one or more substances) may be extracted from the insect for use in the manufacture of a consumable product. In some instances, the insect may be a moth, butterfly, fly, cricket, grasshopper, locust, spider, or beetle. In some instances, an insect species is selected based upon their natural nutritional profile or nutrient content. Examples of nutrients include vitamins, carbohydrates, amino acids, polypeptides, or fatty acids.
In some instances, the insect produces a useable product (e.g., honey, silk, beeswax, or shellac). In some instances, the insect is a bee. Exemplary bee genera include, but are not limited to Apis, Bombus, Trigona, and Osmia. In some instances, the bee is a honeybee (e.g., an insect belonging to the genus Apis). In some instances, the honeybee is the species Apis mellifera (the European or Western honey bee), Apis cerana (the Asiatic, Eastern, or Himalayan honey bee), Apis dorsata (the “giant” honey bee), Apis florea (the “red dwarf” honey bee), Apis andreniformis (the “black dwarf” honey bee), or Apis nigrocincta. In some instances, the insect is a silkworm. The silkworm may be a species in the family Bombycidae or Saturniidae. In some instances, the silkworm is Bombyx mori In some instances, the insect is a lac bug. The lac bug may be a species in the family Kerriidae. In some instances, the lac bug is Kerria lacca.
In some instances, the insect aids in pollination of a plant (e.g., bees, beetles, wasps, flies, butterflies, or moths). In some examples, the insect aiding in pollination of a plant is beetle. In some instances, the beetle is a species in the family Buprestidae, Cantharidae, Cerambycidae, Chrysomelidae, Cleridae, Coccinellidae, Elateridae, Melandryidae, Meloidae, Melyridae, Mordellidae, Nitidulidae, Oedemeridae, Scarabaeidae, or Staphyllinidae. In some instances, the insect aiding in pollination of a plant is a butterfly or moth (e.g., Lepidoptera). In some instances, the butterfly or moth is a species in the family Geometridae, Hesperiidae, Lycaenidae, Noctuidae, Nymphalidae, Papilionidae, Pieridae, or Sphingidae. In some instances, the insect aiding in pollination of a plant is a fly (e.g., Diptera). In some instances, the fly is in the family Anthomyiidae, Bibionidae, Bombyliidae, Calliphoridae, Cecidomiidae, Certopogonidae, Chrionomidae, Conopidae, Culicidae, Dolichopodidae, Empididae, Ephydridae, Lonchopteridae, Muscidae, Mycetophilidae, Phoridae, Simuliidae, Stratiomyidae, or Syrphidae. In some instances, the insect aiding in pollination is an ant (e.g., Formicidae), sawfly (e.g., Tenthredinidae), or wasp (e.g., Sphecidae or Vespidae). In some instances, the insect aiding in pollination of a plant is a bee. In some instances, the bee is in the family Andrenidae, Apidae, Colletidae, Halictidae, or Megachilidae.
In some instances, the insect aids in pest control. For example, the insect aiding in pest control may be a species belonging to the family Braconidae (e.g., parasitoid wasps), Carabidae (e.g., ground beetles), Chrysopidae (e.g., green lacewings), Coccinellidae (e.g., ladybugs), Hemerobiidae (e.g., brown lacewings), Ichneumonidae (e.g., ichneumon wasps), Lampyridae (e.g., fireflies), Mantidae (e.g., praying mantises), Myrmeleontidae (e.g., antilions), Odonata (e.g., dragonflies and damselflies), or Syrphidae (e.g., hoverfly). In other instances, the insect aiding in pest control is an insect that competes with an insect that is considered a pest (e.g., an agricultural pest). For example, the Mediterranean fruit fly, Ceratitis capitata is a common pest of fruits and vegetables worldwide. One way to control C. captitata is to release the sterilized male insect into the environment to compete with wild males to mate the females. In these instances, the insect may be a sterilized male belonging to a species that is typically considered a pest.
In some instances, the insect aids in degradation of waste or organic material. In some examples, the insect aiding in degradation of waste or organic material belongs to Coleoptera or Diptera. In some instances, the insect belonging to Diptera is in the family Calliphoridae, Curtonotidae, Drosophilidae, Fanniidae, Heleomyzidae, Milichiidae, Muscidae, Phoridae, Psychodidae, Scatopsidae, Sepsidae, Sphaeroceridae, Stratiomyidae, Syrphidae, Tephritidae, or Ulidiidae. In some instances, the insect belonging to Coleoptera is in the family Carabidae, Hydrophilidae, Phalacaridae, Ptiliidae, or Staphylinidae.
In particular instances, the bacterial colonization-disrupting agents disclosed herein may be used to increase the fitness of a honeybee.
ii. Decreasing Insect Fitness
In instances where the bacterial colonization-disrupting agent disrupts colonization of bacteria beneficial to an insect, the present methods are effective to decrease the fitness of the insect. For example, a bacterial colonization-disrupting agent as described herein can be contacted with an insect in an amount and for a time sufficient to: (a) reach a target level (e.g., a predetermined or threshold level) of concentration inside a target insect (e.g., inside the gut, or a cell (e.g., bacteriocyte) or organ (e.g., bacteriome or crypt) therein); and (b) decrease the fitness of the target insect. The decrease in insect fitness may manifest as a deterioration or decline in the physiology of the insect (e.g., as measured by survival) as a consequence of administration of the bacterial colonization-disrupting agent. The fitness of the insect may be measured by one or more parameters, including, but not limited to, reproductive rate, lifespan, mobility, fecundity, body weight, metabolic rate or activity, or survival in comparison to an insect to which the bacterial colonization-disrupting agent has not been administered.
For example, the methods or compositions provided herein may be effective to decrease the overall health of the insect or to decrease the overall survival of the insect. In some instances, the decreased survival of the insect is about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more than 100% relative to a reference level (e.g., a level found in an insect that does not receive a bacterial colonization-disrupting agent). In some instances, the methods and compositions are effective to decrease insect reproduction (e.g., reproductive rate) in comparison to an insect to which the bacterial colonization-disrupting agent has not been administered. In some instances, the methods and compositions are effective to decrease other physiological parameters, such as mobility, body weight, life span, fecundity, or metabolic rate, by about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or greater than 100% relative to a reference level (e.g., a level found in an insect that does not receive a bacterial colonization-disrupting agent).
In some instances, the decrease in insect fitness may manifest as a decrease in the production of one or more nutrients in the insect (e.g., vitamins, carbohydrates, amino acids, or polypeptides) in comparison to an insect to which the bacterial colonization-disrupting agent has not been administered. In some instances, the methods or compositions provided herein may be effective to decrease the production of nutrients in the insect (e.g., vitamins, carbohydrates, amino acids, or polypeptides) by about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or greater than 100% relative to a reference level (e.g., a level found in an insect that does not receive a bacterial colonization-disrupting agent). In some instances, the methods or compositions provided herein may decrease nutrients in the insect by decreasing the production of nutrients by one or more microorganisms (e.g., endosymbiont) in the insect in comparison to an insect to which the bacterial colonization-disrupting agent has not been administered.
In some instances, the decrease in insect fitness may manifest as an increase in the insect's sensitivity to a pesticidal agent and/or a decrease in the insect's resistance to a pesticidal agent in comparison to an insect to which the bacterial colonization-disrupting agent has not been administered. In some instances, the methods or compositions provided herein may be effective to increase the insect's sensitivity to a pesticidal agent by about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or greater than 100% relative to a reference level (e.g., a level found in an insect that does not receive a bacterial colonization-disrupting agent). The pesticidal agent may be any pesticidal agent known in the art, including insecticidal agents. In some instances, the methods or compositions provided herein may increase the insect's sensitivity to a pesticidal agent by decreasing the insect's ability to metabolize or degrade the pesticidal agent into usable substrates.
In some instances, the decrease in insect fitness may manifest as an increase in the insect's sensitivity to an allelochemical agent and/or a decrease in the insect's resistance to an allelochemical agent in comparison to an insect to which the bacterial colonization-disrupting agent has not been administered. In some instances, the methods or compositions provided herein may be effective to decrease the insect's resistance to an allelochemical agent by about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or greater than 100% relative to a reference level (e.g., a level found in an insect that does not receive a bacterial colonization-disrupting agent). In some instances, the allelochemical agent is caffeine, soyacystatin N, monoterpenes, diterpene acids, or phenolic compounds. In some instances, the methods or compositions provided herein may increase the insect's sensitivity to an allelochemical agent by decreasing the insect's ability to metabolize or degrade the allelochemical agent into usable substrates in comparison to an insect to which the bacterial colonization-disrupting agent has not been administered.
In some instances, the methods or compositions provided herein may be effective to decease the insect's resistance to parasites or pathogens (e.g., fungal, bacterial, or viral pathogens or parasites) in comparison to an insect to which the bacterial colonization-disrupting agent has not been administered. In some instances, the methods or compositions provided herein may be effective to decrease the insect's resistance to a pathogen or parasite (e.g., fungal, bacterial, or viral pathogens; or parasitic mites) by about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or greater than 100% relative to a reference level (e.g., a level found in an insect that does not receive a bacterial colonization-disrupting agent).
In some instances, the decrease in insect fitness may manifest as other fitness disadvantages, such as decreased tolerance to certain environmental factors (e.g., a high or low temperature tolerance), decreased ability to survive in certain habitats, or a decreased ability to sustain a certain diet in comparison to an insect to which the bacterial colonization-disrupting agent has not been administered. In some instances, the methods or compositions provided herein may be effective to decrease insect fitness in any plurality of ways described herein. Further, the bacterial colonization-disrupting agent may decrease insect fitness in any number of insect classes, orders, families, genera, or species (e.g., 1 insect species, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 200, 250, 500, or more insect species). In some instances, the bacterial colonization-disrupting agent acts on a single insect class, order, family, genus, or species. Insect fitness may be evaluated using any standard methods in the art. In some instances, insect fitness may be evaluated by assessing an individual insect. Alternatively, insect fitness may be evaluated by assessing an insect population.
iii. Increasing Insect Fitness
In instances where the bacterial colonization-disrupting agent disrupts colonization of bacteria harmful to an insect (e.g., pathogenic bacteria), the present methods are effective to confer a variety of fitness benefits to insects. For example, the increase in insect fitness may manifest as an improvement in the physiology of the insect (e.g., improved health or survival, or increased nutritional profile) as a consequence of administration of the bacterial colonization-disrupting agent. The fitness of the insect may be measured by one or more parameters, including, but not limited to, reproductive rate, lifespan, mobility, fecundity, body weight, nutritional profile, metabolic rate or activity, or survival in comparison to an insect to which the bacterial colonization-disrupting agent has not been administered. In some instances, the bacterial colonization-disrupting agent may increase the fitness of the insect in a transient manner. Alternatively, the bacterial colonization-disrupting agent may increase the fitness of the insect for the duration of the insect's lifespan.
For example, the methods or compositions provided herein may be effective to improve the overall health of the insect or to improve the overall survival of the insect in comparison to an insect to which the bacterial colonization-disrupting agent has not been administered. In some instances, the improved survival of the insect is about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or greater than 100% greater relative to a reference level (e.g., a level found in an insect that does not receive a bacterial colonization-disrupting agent).
In some instances, the methods and compositions are effective to increase insect reproduction (e.g., reproductive rate) in comparison to an insect to which the bacterial colonization-disrupting agent has not been administered. In some instances, the methods and compositions are effective to increase other physiological parameters, such as mobility, body weight, life span, fecundity, or metabolic rate, by about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or greater than 100% relative to a reference level (e.g., a level found in an insect that does not receive a bacterial colonization-disrupting agent).
In some instances, the increase in insect fitness may manifest as an increased production of a product generated by said insect in comparison to an insect to which the bacterial colonization-disrupting agent has not been administered. In some instances, the methods or compositions provided herein may be effective to increase the production of a product generated by the insect, as described herein (e.g., honey, beeswax, beebread, propolis, silk, or lac), by about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or greater than 100% relative to a reference level (e.g., a level found in an insect that does not receive a bacterial colonization-disrupting agent).
For example, the methods or compositions provided herein may be effective to improve the nutritional profile of the insect or to improve the overall nutrient content (e.g., vitamin, carbohydrate, amino acid, polypeptide, or fatty acid content) of the insect in comparison to an insect to which the bacterial colonization-disrupting agent has not been administered. In some instances, the improved nutritional profile or nutrient content of the insect is about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or greater than 100% greater relative to a reference level (e.g., a level found in an insect that does not receive a bacterial colonization-disrupting agent).
In some instances, the increase in insect fitness may manifest as an increase in the frequency or efficacy of a desired activity carried out by the insect (e.g., pollination, predation on pests, seed spreading, or breakdown of waste or organic material) in comparison to an insect to which the bacterial colonization-disrupting agent has not been administered. In some instances, the methods or compositions provided herein may be effective to increase the frequency or efficacy of a desired activity carried out by the insect (e.g., pollination, predation on pests, seed spreading, or breakdown of waste or organic material) by about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or greater than 100% relative to a reference level (e.g., a level found in an insect that does not receive a bacterial colonization-disrupting agent).
In some instances, the increase in insect fitness may manifest as an increase in the production of one or more nutrients in the insect (e.g., vitamins, carbohydrates, amino acids, or polypeptides) in comparison to an insect to which the bacterial colonization-disrupting agent has not been administered. In some instances, the methods or compositions provided herein may be effective to increase the production of nutrients in the insect (e.g., vitamins, carbohydrates, amino acids, or polypeptides) by about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or greater than 100% relative to a reference level (e.g., a level found in an insect that does not receive a bacterial colonization-disrupting agent). In some instances, the methods or compositions provided herein may increase nutrients in the insect by increasing the production of nutrients by one or more microorganisms (e.g., endosymbiont) in the insect.
In some instances, the increase in insect fitness may manifest as a decrease in the insect's sensitivity to a pesticidal agent and/or an increase in the insect's resistance to a pesticidal agent in comparison to an insect to which the bacterial colonization-disrupting agent has not been administered. In some instances, the methods or compositions provided herein may be effective to decrease the insect's sensitivity to a pesticidal agent by about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or greater than 100% relative to a reference level (e.g., a level found in an insect that does not receive a bacterial colonization-disrupting agent). In some instances, the insect's sensitivity to the pesticidal agent is altered by administering a bacterial colonization-disrupting agent that degrades a pesticidal agent (e.g., pesticidal-degrading bacteria, e.g., a neonicotinoid-degrading bacteria or an organophosphorus insecticide-degrading bacteria). The pesticidal agent may be any pesticidal agent known in the art, including insecticidal agents. In some instances, the pesticidal agent is a neonicotinoid (e.g., imidacloprid) or an organophosphorus insecticide (e.g., a phosphorothioate, e.g., fenitrothion). In some instances, the methods or compositions provided herein may decrease the insect's sensitivity to a pesticidal agent by increasing the insect's ability to metabolize or degrade the pesticidal agent into usable substrates.
In some instances, the increase in insect fitness may manifest as a decrease in the insect's sensitivity to an allelochemical agent and/or an increase in the insect's resistance to an allelochemical agent in comparison to an insect to which the bacterial colonization-disrupting agent has not been administered. In some instances, the methods or compositions provided herein may be effective to increase the insect's resistance to an allelochemical agent by about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or greater than 100% relative to a reference level (e.g., a level found in an insect that does not receive a bacterial colonization-disrupting agent). In some instances, the allelochemical agent is caffeine, soyacystatin N, monoterpenes, diterpene acids, or phenolic compounds. In some instances, the methods or compositions provided herein may decrease the insect's sensitivity to an allelochemical agent by increasing the insect's ability to metabolize or degrade the allelochemical agent into usable substrates.
In some instances, the methods or compositions provided herein may be effective to increase the insect's resistance to parasites or pathogens (e.g., fungal, bacterial, or viral pathogens; or parasitic mites (e.g., Varroa destructor mite in honeybees)) in comparison to an insect to which the bacterial colonization-disrupting agent has not been administered. In some instances, the methods or compositions provided herein may be effective to increase the insect's resistance to a pathogen or parasite (e.g., fungal, bacterial, or viral pathogens; or parasitic mites (e.g., Varroa destructor mite in honeybees)) by about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or greater than 100% relative to a reference level (e.g., a level found in an insect that does not receive a bacterial colonization-disrupting agent).
In some instances, the increase in insect fitness may manifest as other fitness advantages, such as improved tolerance to certain environmental factors (e.g., a high or low temperature tolerance), improved ability to survive in certain habitats, or an improved ability to sustain a certain diet (e.g., an improved ability to metabolize soy vs corn) in comparison to an insect to which the bacterial colonization-disrupting agent has not been administered. In some instances, the methods or compositions provided herein may be effective to increase insect fitness in any plurality of ways described herein. Further, the bacterial colonization-disrupting agent may increase insect fitness in any number of insect classes, orders, families, genera, or species (e.g., 1 insect species, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 200, 250, 500, or more insect species). In some instances, the bacterial colonization-disrupting agent acts on a single insect class, order, family, genus, or species.
In some embodiments of the methods herein, the method is effective to increase the fitness of the insect relative to an untreated insect. In some embodiments, the increase in fitness is an increase in survival, life span, reproductive ability, reproductive rate, reproductive period, number of eggs laid, number of hatched eggs, developmental rate, adult emergence rate, mobility, body size (e.g., body length, body mass, or body width (e.g., pronotal width of a stink bug)), cuticle (exoskeleton) thickness, pigmentation, or metabolic rate of the insect relative to an untreated insect. In some embodiments, the increase in fitness is an increase in vitellogenin protein in the insect relative to an untreated insect. In some embodiments, the increase in fitness is an increase in vitellogenin gene expression in the insect relative to an untreated insect.
Insect fitness may be evaluated using any standard methods in the art. In some instances, insect fitness may be evaluated by assessing an individual insect. Alternatively, insect fitness may be evaluated by assessing an insect population. For example, an increase in insect fitness may manifest as an increase in successful competition against other insects, thereby leading to an increase in the size of the insect population.
iv. Insects in Agriculture
By decreasing the fitness of insects, such as agricultural pests (e.g., stink bugs or bean bugs), that are harmful to plants, or increasing the fitness of beneficial insects (e.g., pollinating insects, e.g., bees) the bacterial colonization-disrupting agents provided herein may be effective to promote the growth of plants that are typically harmed by said insects. The bacterial colonization-disrupting agent may be delivered to the plant using any of the formulations and delivery methods described herein, in an amount and for a duration effective to decrease insect fitness and thereby benefit the plant, e.g., increase crop growth, increase crop yield, decrease pest infestation, and/or decrease damage to plants. This may or may not involve direct application of the bacterial colonization-disrupting agent to the plant. For example, in instances where the primary insect habitat is different than the region of plant growth, the bacterial colonization-disrupting agent may be applied to either the primary insect habitat, the plants of interest, or a combination of both.
In some instances, the plant may be an agricultural food crop, such as a cereal, grain, legume, fruit, or vegetable crop, or a non-food crop, e.g., grasses, flowering plants, cotton, hay, hemp. The compositions described herein may be delivered to the crop any time prior to or after harvesting the cereal, grain, legume, fruit, vegetable, or other crop. Crop yield is a measurement often used for crop plants and is normally measured in metric tons per hectare (or kilograms per hectare). Crop yield can also refer to the actual seed generation from the plant. In some instances, the bacterial colonization-disrupting agent may be effective to increase crop yield (e.g., increase metric tons of cereal, grain, legume, fruit, or vegetable per hectare and/or increase seed generation) by about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more in comparison to a reference level (e.g., a crop to which the bacterial colonization-disrupting agent has not been administered).
In some instances, the plant (e.g., crop) may be at risk of developing a pest infestation (e.g., by an insect) or may have already developed a pest infestation. The methods and compositions described herein may be used to reduce or prevent pest infestation in such crops by reducing the fitness of insects that infest the plants. In some instances, the bacterial colonization-disrupting agent may be effective to reduce crop infestation (e.g., reduce the number of plants infested, reduce the pest population size, reduce damage to plants) by about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more in comparison to a reference level (e.g., a crop to which the bacterial colonization-disrupting agent has not been administered). In other instances, the bacterial colonization-disrupting agent may be effective to prevent or reduce the likelihood of crop infestation by about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more in comparison to a reference level (e.g., a crop to which the bacterial colonization-disrupting agent has not been administered).
Any suitable plant tissues may benefit from the compositions and methods described herein, including, but not limited to, somatic embryos, pollen, leaves, stems, calli, stolons, microtubers, and shoots. The methods described herein may include treatment of angiosperm and gymnosperm plants such as acacia, alfalfa, apple, apricot, artichoke, ash tree, asparagus, avocado, banana, barley, beans, beet, birch, beech, blackberry, blueberry, broccoli, brussels sprouts, cabbage, canola, cantaloupe, carrot, cassava, cauliflower, cedar, a cereal, celery, chestnut, cherry, Chinese cabbage, citrus, clemintine, clover, coffee, corn, cotton, conifers, cowpea, cucumber, cypress, eggplant, elm, endive, eucalyptus, fava beans, fennel, figs, fir, fruit and nut trees, geranium, grape, grapefruit, groundnuts, ground cherry, gum hemlock, hemp, hickory, kale, kiwifruit, kohlrabi, larch, lettuce, leek, lemon, lime, locust, pine, maidenhair, maize, mango, maple, melon, millet, mushroom, mustard, nuts, oak, oats, okra, onion, orange, an ornamental plant or flower or tree, papaya, palm, parsley, parsnip, pea, peach, peanut, pear, peat, pepper, persimmon, pigeon pea, pine, pineapple, plantain, plum, pomegranate, potato, pumpkin, radicchio, radish, rapeseed, raspberry, rice, rye, sorghum, sallow, soybean, spinach, spruce, squash, strawberry, sugarbeet, sugarcane, sunflower, sweet potato, sweet corn, tangerine, tea, tobacco, tomato, trees, triticale, turf grasses, turnips, a vine, walnut, watercress, watermelon, wheat, yams, yew, and zucchini.
v. Insects as Disease Vectors
By decreasing the fitness of host insects that carry animal pathogens, the bacterial colonization-disrupting agents provided herein are effective to reduce the spread of vector-borne diseases. The bacterial colonization-disrupting agent may be delivered to the insects using any of the formulations and delivery methods described herein, in an amount and for a duration effective to reduce transmission of the disease, e.g., reduce vertical or horizontal transmission between vectors and/or reduce transmission to animals. For example, the bacterial colonization-disrupting agent described herein may reduce vertical or horizontal transmission of a vector-borne pathogen by about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more in comparison to a host organism to which the bacterial colonization-disrupting agent has not been administered. As an another example, the bacterial colonization-disrupting agent described herein may reduce vectorial competence of an insect vector by about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more in comparison to a host organism to which the bacterial colonization-disrupting agent has not been administered.
Non-limiting examples of diseases that may be controlled by the compositions and methods provided herein include diseases caused by Togaviridae viruses (e.g., Chikungunya, Ross River fever, Mayaro, Onyon-nyong fever, Sindbis fever, Eastern equine enchephalomyeltis, Wesetern equine encephalomyelitis, Venezualan equine encephalomyelitis, or Barmah forest); diseases caused by Flavivirdae viruses (e.g., Dengue fever, Yellow fever, Kyasanur Forest disease, Omsk haemorrhagic fever, Japaenese encephalitis, Murray Valley encephalitis, Rocio, St. Louis encephalitis, West Nile encephalitis, or Tick-borne encephalitis); diseases caused by Bunyaviridae viruses (e.g., Sandly fever, Rift Valley fever, La Crosse encephalitis, California encephalitis, Crimean-Congo haemorrhagic fever, or Oropouche fever); disease caused by Rhabdoviridae viruses (e.g., Vesicular stomatitis); disease caused by Orbiviridae (e.g., Bluetongue); diseases caused by bacteria (e.g., Plague, Tularaemia, Q fever, Rocky Mountain spotted fever, Murine typhus, Boutonneuse fever, Queensland tick typhus, Siberian tick typhus, Scrub typhus, Relapsing fever, or Lyme disease); or diseases caused by protozoa (e.g., Malaria, African trypanosomiasis, Nagana, Chagas disease, Leishmaniasis, Piroplasmosis, Bancroftian filariasis, or Brugian filariasis).
vi. Application Methods
An insect described herein can be exposed to a composition including the bacterial colonization-disruption agent herein in any suitable manner that permits delivering or administering the composition to the insect or to an egg or egg mass from which the insect will hatch. The bacterial colonization-disrupting agent may be delivered either alone or in combination with other active or inactive substances and may be applied by, for example, spraying, injection (e.g., microinjection), through plants, pouring, dipping, in the form of concentrated liquids, gels, solutions, suspensions, sprays, powders, pellets, briquettes, bricks and the like, formulated to deliver an effective concentration of the bacterial colonization-disrupting agent. Amounts and locations for application of the compositions described herein are generally determined by the habitat of the insect, the lifecycle stage at which the insect can be targeted by the bacterial colonization-disrupting agent, the site where the application is to be made, and the physical and functional characteristics of the bacterial colonization-disrupting agent.
In some instances, the composition is sprayed directly onto a plant e.g., crops, by e.g., backpack spraying, aerial spraying, crop spraying/dusting etc. In instances where the bacterial colonization-disrupting agent is delivered to a plant, the plant receiving the bacterial colonization-disrupting agent may be at any stage of plant growth. For example, formulated bacterial colonization-disrupting agents can be applied as a seed-coating or root treatment in early stages of plant growth or as a total plant treatment at later stages of the crop cycle. In some instances, the bacterial colonization-disrupting agent may be applied as a topical agent to a plant. In some instances, the composition is sprayed or applied onto an egg or an egg mass from which the insect will hatch.
Further, the bacterial colonization-disrupting agent may be applied (e.g., in the soil in which a plant grows, or in the water that is used to water the plant) as a systemic agent that is absorbed and distributed through the tissues of a plant. In some instances, plants or food organisms may be genetically transformed to express the bacterial colonization-disrupting agent. For example, in some instances, the bacterial colonization-disrupting agent is delivered in a modified plant for ingestion by the insect. Alternatively, the bacterial colonization-disrupting agent may be delivered in an attenuated bacteria or modified bacteria for ingestion by the insect.
Delayed or continuous release can also be accomplished by coating the bacterial colonization-disrupting agent or a composition with the bacterial colonization-disrupting agent(s) with a dissolvable or bioerodable coating layer, such as gelatin, which coating dissolves or erodes in the environment of use, to then make the bacterial colonization-disrupting agent available, or by dispersing the agent in a dissolvable or erodable matrix. Such continuous release and/or dispensing means devices may be advantageously employed to consistently maintain an effective concentration of one or more of the bacterial colonization-disrupting agents described herein.
In some instances, the bacterial colonization-disrupting agent may be recommended for field application as an amount of agent per hectare (g/ha or kg/ha) or the amount of active ingredient (e.g., bacterial colonization-disrupting agent) per hectare (kg a.i./ha or g a.i./ha). Bacterial colonization-disrupting agents of the invention can be applied at a variety of amounts per hectare, for example at about 0.0001, 0.001, 0.005, 0.01, 0.1, 1, 2, 10, 100, 1,000, 2,000, 5,000 (or any range between about 0.0001 and 5,000) kg/ha. For example, about 0.0001 to about 0.01, about 0.01 to about 10, about 10 to about 1,000, about 1,000 to about 5,000 kg/ha.
In some instances where the bacterial colonization-disrupting agent is delivered to an insect or an egg or egg mass produced by the insect, the insect, egg, or egg mass can be simply “soaked” or “sprayed” with a solution including the bacterial colonization-disrupting agent. In other instances, the bacterial colonization-disrupting agents may be administered to the insect by oral ingestion, but may also be administered by means which permit penetration through the cuticle or penetration of the insect's respiratory system. For example, the bacterial colonization-disrupting agent can be linked to a food component (e.g., comestible) of the insect for ease of delivery and/or in order to increase uptake of the bacterial colonization-disrupting agent by the insect. Methods for oral introduction include, for example, directly mixing a bacterial colonization-disrupting agent with the insect's food, spraying the bacterial colonization-disrupting agent in the insect's habitat or field, as well as engineered approaches in which a species that is used as food is engineered to express a bacterial colonization-disrupting agent, then fed to the insect to be affected. In some instances, for example, the bacterial colonization-disrupting agent can be incorporated into, or overlaid on the top of, the insect's diet. For example, the bacterial colonization-disrupting agent can be sprayed onto a field of crops which an insect inhabits.
The bacterial colonization-disrupting agent can also be incorporated into the medium in which the insect grows, lives, reproduces, feeds, or infests. For example, a bacterial colonization-disrupting agent can be incorporated into a food container, feeding station, protective wrapping, or a hive. For some applications the bacterial colonization-disrupting agent may be bound to a solid support for application in powder form or in a trap or feeding station. As an example, for applications where the composition is to be used in a trap or as bait for a particular insect, the compositions may also be bound to a solid support or encapsulated in a time-release material.
A variety of bacterial colonization-disrupting agents may be used in accordance with the present methods. Bacterial colonization-disrupting agents can be differentiated either by their chemical composition, or by their physiological functions. For example, the agent may alter properties of the bacteria (e.g., bacterial metabolism or bacterial cell surface) and/or the insect gut, such that the bacteria can no longer adhere, associate with, or propagate in the gut of the insect. Exemplary bacterial colonization-disrupting agents and methods of screening for such agents are further described, below. Colonization of the insect (e.g., colonization of a bacteriome of the insect, the gut of the insect, or the v4 region of the gut of the insect) may be decreased by between 1% and 100%, e.g., decreased by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or decreased by 100%.
The size (e.g., area or mass) of a cell, organ, region, or tissue of the insect that may be colonized by a bacterium (e.g., a bacteriocyte or a v4 region of the gut) may be decreased as a result of treatment with a colonization-disrupting agent, e.g., decreased by between 1% and 100%, e.g., decreased by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or decreased by 100%. In some examples, the size of the cell, organ, region, or tissue of the insect that may be colonized (e.g., a bacteriocyte or a v4 region of the gut) is used as a measure of colonization; for example, a smaller size of the cell, organ, region, or tissue may indicate a greater decrease in colonization.
i. Classes of Bacterial Colonization-Disrupting Agents
In some instances, the bacterial colonization-disrupting agent alters (e.g., inhibits) bacterial metabolism. Bacteria residing in the gut of an insect depend on production of certain nutrients to thrive in the insect, or a cell or organ therein. For example, polyhydroxyalkanoate (PHA) is a linear polyester that is synthesized and used as storage compounds of carbon and energy sources. Generally, the biosynthesis of PHA granules is promoted when bacteria face stressful environments, such as nutrition-deficient conditions. As described in Example 1, synthesis of PHA is one exemplary bacterial metabolic pathway that can be targeted to disrupt bacterial colonization of the insect gut (e.g., such as colonization of the gut of Riptortus pedestris by Burkholderia).
Accordingly, in some instances, the bacterial colonization-disrupting agent is a PHA synthesis inhibitor. PHA granules are mainly synthesized from acetyl coenzyme A (acetyl-CoA) by three different enzymes, such as the products of phaA (ketothiolase), phaB (acetyl-CoA reductase), and phaC (PHA synthase). The surfaces of PHA granules are surrounded by various proteins, such as PhaP (a surface protein of PHA granules; phasin), PhaR (a negative regulator of PhaP), and PhaZ (PHA depolymerase). In some instances, the bacterial colonization-disrupting agent is an inhibitor of a gene involved in PHA biosynthesis, such as phaA, phaB, phaC, phaP, phaR, or phaZ gene expression. In other instances, the bacterial colonization-disrupting agent in binds a protein involved in PHA biosynthesis, such as PhaA, PhaB, PhaC, PhaP, PhaR, or PhaZ. In certain instances the PHA synthesis inhibitor is vanillin or an analog thereof (Table 1; Table 2). In other instances, the PHA synthesis inhibitor is levulinic acid or an analog thereof, e.g, an analog as provided in Table 4; acrylic acid or an analog thereof, e.g, an analog as provided in Table 5; or 2-bromooctanoic acid (2BA) or an analog thereof, e.g, an analog as provided in Table 6. In still other instances, the PHA synthesis inhibitor is furfural, 2,3-butanedione, 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), or 4-pentenoic acid.
In some instances, the bacterial colonization-disrupting agent alters properties of the surface of the bacterial cell by, for example, targeting the biogenesis of the bacterial cell envelope (e.g., biogenesis of the membrane(s) or other structures that surround and protect the bacterial cytoplasm, e.g., cell wall, inner membrane, and outer membrane). The cell envelope represents the outermost layers of the bacterial cell and, in general, functions in the protection of the cell, communication with the environment, maintenance of cellular shape, stability, and rigidity of the cell, as well as allowing appropriate metabolism, growth, division, and colonization of the bacteria. Accordingly, in some instances, the bacterial colonization-disrupting agent targets genes or proteins required for the biosynthesis of molecules important for the integrity of the cell envelope, including the biosynthesis carbohydrate-containing macromolecules such as lipopolysaccharides (LPSs), peptidoglycan, lipoteichoic acids, teichoic acids, capsule polysaccharides, and lipoarabinomannan.
For example, LPS represents a major component of the outer leaflet of the outer membrane, and is composed of three domains: lipid A, core oligosaccharide (OS) and O-specific polysaccharide (or O antigen). As described in Examples 2 and 3, LPS biosynthesis (e.g., core oligosaccharide synthesis, e.g., L-Heptoses synthesis), is one exemplary cell envelope biogenesis pathway that can be targeted to disrupt bacterial colonization of the insect gut (e.g., disrupt colonization of the endosymbiont Burkholderia in the gut of Riptortus pedestris (Example 2) or disrupt colonization of the endosymbiont Candidatus Pantoea carbekii in the gut of Halyomorpha halys (Example 3)).
Accordingly, in some instances, the bacterial colonization-disrupting agent is an LPS synthesis inhibitor. In some instances, the LPS synthesis inhibitor is an inhibitor of core oligosaccharide synthesis in the bacteria. For example, the LPS synthesis inhibitor may inhibit an enzyme involved in core oligosaccharide synthesis in the bacteria, such as WaaA, WaaC, WaaF, or WaaG, or an enzyme. In some instances, the LPS synthesis inhibitor inhibits an enzyme having at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to a polypeptide having the amino acid sequence of WaaA, WaaC, WaaF, or WaaG. In some instances, the LPS synthesis inhibitor inhibits expression of a gene involved in core oligosaccharide synthesis in the bacteria, such as waaA, waaC, waaF, or waaG. In some instances, the LPS synthesis inhibitor inhibits expression of a gene having at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to a polynucleotide having the nucleotide sequence of waaA, waaC, waaF, or waaG. Exemplary LPS synthesis inhibitors are provided in Table 3.
In certain instances, the LPS synthesis inhibitor (e.g., core oligosaccharide synthesis inhibitor, e.g., L-Heptoses synthesis inhibitor) is a sugar. For example, the sugar may be ADP-2-fluoroheptose (AFH). Alternatively, the sugar may be 2-aryl-5-methyl-4-(5-aryl-furan-2-yl-methylene)-2,4-dihydro-pyrazol-3-ones (DHPO). In some instances, the sugar is AFH and DHPO. In some instances, the sugar is a structural analog of ADP-beta-L-glycero-D-manno-heptopyranose. For example, the sugar may be one or more compounds in Table 7. In some instances, the sugar is ADP-2-deoxy-2-fluoro-heptose. In some instances, the LPS inhibitor is a fullerene hexa-adducts bearing 12 copies of peripheral sugars displaying the mannopyranose core structure of bacterial l,d-heptoside.
In another example, undecaprenyl-pyrophosphate (UPP) is a 55-carbon polyisoprenoid lipid carrier that is required to transport peptidoglycan precursors across the cell membrane during bacterial peptidoglycan synthesis. Undecaprenyl pyrophosphate phosphatases (Upp-Pases, e.g., UppP or bcrC) are required for the synthesis and recycling of UPP. Accordingly, in some instances, the bacterial colonization-disrupting agent is an inhibitor of a Upp-Pase, e.g., a UppP inhibitor. In some instances, the UppP inhibitor is bacitracin, tripropeptin C (TPPC), a lipophilic hydroxyalkyl phosphonic acid, or a series of benzoic acids and phenylphosphonic acids.
In some instances, the bacterial colonization-disrupting agent alters the motility of the bacterial cell by, for example, targeting the function (e.g., rotation) of flagella. Accordingly, in some instances, the bacterial colonization-disrupting agent is a flagellar function inhibitor. In some instances, the flagellar function inhibitor is cellulose.
The bacterial colonization-disrupting agent may be used in a composition containing a single agent or may be used in a composition containing a mixture of different bacterial colonization-disrupting agents. The composition including the bacterial colonization-disrupting agent may include any number or type of bacterial colonization-disrupting agents, such as at least about any one of 1 bacterial colonization-disrupting agent, 2, 3, 4, 5, 10, 15, 20, or more bacterial colonization-disrupting agents.
The bacterial colonization-disrupting agent may be formulated in a composition for any of the uses described herein. A suitable concentration of each bacterial colonization-disrupting agent in the composition depends on factors such as efficacy, stability of the bacterial colonization-disrupting agent, number of distinct bacterial colonization-disrupting agents, the formulation, and methods of application of the composition. Exemplary formulations and compositions including bacterial colonization-disrupting agents are described in the section entitled “Formulations and Compositions.”
ii. Screening Methods to Identify Bacterial Colonization-Disrupting Agents
Included herein is a screening assay for identifying bacterial colonization-disrupting agents that are effective to inhibit colonization of bacteria in an insect and thereby decrease the insect's fitness. The screening assay involves identifying a bacterial colonization-disrupting agent by (a) exposing a target insect to one or more agents; and (b) identifying an agent that (i) decreases the fitness of the target insect, and (ii) inhibits colonization of a bacterium in the gut of the target insect.
Host fitness may be measured by any parameters described herein, including, but not limited to, measurements of reproductive rate, lifespan, mobility, fecundity, body weight, metabolic rate or activity, or survival in comparison to an insect to which the candidate agent has not been administered. The decrease in fitness may be compared to a predetermined threshold or a reference level. For example, the decrease in fitness (e.g., overall survival) may be a decrease of about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more than 100% relative to a reference level (e.g., untreated insect).
Inhibition of bacterial colonization can be measured by a number of methods known in the art, including in vitro or in vivo assays. Changes to colonization of bacteria in the insect as a consequence of the agent may be determined by methods including, but not limited to, polymerase chain reaction (PCR), quantitative PCR, real-time PCR, flow cytometry, microarray, fluorescence microscopy, transmission electron microscopy, fluorescence in situ hybridization (e.g., FISH), and DNA sequencing. The decrease in colonization may be compared to a predetermined threshold or a reference level. For example, the decrease in colonization may be a decrease of about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more than 100% relative to a reference level (e.g., untreated bacteria).
The bacteria targeted by the bacterial colonization-disrupting agent described herein may include any bacteria resident in the gut of the host, or a cell or organ therein, including, but not limited to, any bacteria described herein. Bacteria resident in the host may include, for example, symbiotic (e.g., endosymbiotic microorganisms that provide beneficial nutrients or enzymes to the host) pathogenic bacteria, or commensal microorganisms. An endosymbiotic microorganism may be a primary endosymbiont or a secondary endosymbiont. A symbiotic bacteria may be an obligate symbiont of the host or a facultative symbiont of the host.
Microorganisms resident in the host may be acquired by any mode of transmission, including vertical, horizontal, or multiple origins of transmission. Transmission modes of insect symbionts includes environmental determination, coprophagy, smearing of brood cell or egg surface, social acquisition, capsule transmission or infection via jelly-like secretions. Some symbionts, like gut symbionts, are horizontally acquired from the environment in each generation. For example, the bean bug, Riptortus pedestris (Hemiptera: Alydidae), harbors a specific gut symbiont of the genus Burkholderia, which is acquired orally from the environment by second-instar nymphs. Bean bugs have a specialized symbiotic organ (crypts) in a posterior midgut fourth region (M4) to host the symbionts.
Exemplary bacteria that may be targeted in accordance with the methods and compositions provided herein, include, but are not limited to, Xenorhabdus spp, Photorhabdus spp, Candidatus spp, Pantoea spp, Buchnera spp, Blattabacterium spp, Baumania spp, Wigglesworthia spp, Wolbachia spp, Rickettsia spp, Orientia spp, Sodalis spp, Burkholderia spp, Cupriavidus spp, Frankia spp, Snirhizobium spp, Streptococcus spp, Wolinella spp, Xylella spp (e.g., Xylella fastidiosa), Erwinia spp, Agrobacterium spp, Bacillus spp, Commensalibacter spp. (e.g., Commensalibacter intestine), Paenibacillus spp, Streptomyces spp, Micrococcus spp, Corynebacterium spp, Acetobacter spp (e.g., Acetobacter pomorum), Cyanobacteria spp, Salmonella spp, Rhodococcus spp, Pseudomonas spp, Lactobacillus spp (e.g., Lactobacillus plantarum), Lysobacter spp., Herbaspirillum spp., Enterococcus spp, Gluconobacter spp. (e.g., Gluconobacter morbifer), Alcaligenes spp, Hamiltonella spp., Klebsiella spp, Paenibacillus spp, Serratia spp., Arthrobacter spp, Azotobacter spp., Corynebacterium spp, Brevibacterium spp, Regiella spp. (e.g., Regiellan insecticola), Thermus spp, Pseudomonas spp, Clostridium spp, Mortierella spp. (e.g., Mortierella elongata), or Escherichia spp.
Non-limiting examples of bacteria that may be targeted by the methods and compositions provided herein are shown in Table 8. In some instances, the 16S rRNA sequence of the bacteria targeted by the bacterial colonization-disrupting agent has at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, 99.9%, or 100% identity with a sequence listed in Table 8.
Carsonella ruddii
Candidatus Portiera
aleyrodidarum BT-B
Buchnera aphidicola str.
pisum)
Buchnera aphidicola str.
graminum)
Buchnera aphidicola str.
Buchnera aphidicola BCc
Buchnera aphidicola
Buchnera aphidicola str.
Buchnera aphidicola str.
kondoi)
Buchnera aphidicola str.
ambrosiae)
Buchnera aphidicola
Candidatus Annandia
pinicola
Moranella endobia
Ishikawaella capsulata
Baumannia
cicadellinicola
Sodalis-like bacterium
Rhopalus
sapporensis
Candidatus Hartigia
pinicola
Wigglesworthia
glossinidia
Tremblaya phenacola
Candidatus Tremblaya
princeps
Nasuia deltocephalinicola
Candidatus Zinderia
insecticola CARI
Profftella armatura
Diaphorina citri,
Diceroprocta
semicincta
Wolbachia strain wPip
Culex
quinquefasciatus
Candidatus Uzinura
diaspidicola
Sulcia muelleri
Symbiotaphrina buchneri
Stegobium
paniceum
Symbiotaphrina kochii
Lasioderma
serricome
Burkholderia strain SFA1
Riptortus
pedestris
Burkholderia strain KM-A
Riptortus
pedestris
Burkholderia strain KM-G
Riptortus
pedestris
The compositions described herein may be formulated either in pure form (e.g., the composition contains only the bacterial colonization-disrupting agent) or together with one or more additional agents (such as excipient, delivery vehicle, carrier, diluent, stabilizer, etc.) to facilitate application or delivery of the compositions. 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. The composition may include a wetting solution (e.g., a non-ionic wetting solution), e.g., SilWet®.
To allow ease of application, handling, transportation, storage, and maximum activity, the bacterial colonization-disrupting agent can be formulated with other substances. The bacterial colonization-disrupting agent can be formulated into, for example, baits, concentrated emulsions, dusts, emulsifiable concentrates, fumigants, gels, granules, microencapsulations, seed treatments, suspension concentrates, suspoemulsions, tablets, water soluble liquids, water dispersible granules or dry flowables, wettable powders, and ultra-low volume solutions.
The bacterial colonization-disrupting agent can be applied as aqueous suspensions or emulsions prepared from concentrated formulations of such agents. Such water-soluble, water-suspendable, or emulsifiable formulations are either solids, usually known as wettable powders, or water dispersible granules, or liquids usually known as emulsifiable concentrates, or aqueous suspensions. Wettable powders, which may be compacted to form water dispersible granules, comprise an intimate mixture of the bacterial colonization-disrupting agent, a carrier, and surfactants. The carrier is usually selected from among the attapulgite clays, the montmorillonite clays, the diatomaceous earths, or the purified silicates.
Effective surfactants, including from about 0.5% to about 10% of the wettable powder, are found among sulfonated lignins, condensed naphthalenesulfonates, naphthalenesulfonates, alkylbenzenesulfonates, alkyl sulfates, and non-ionic surfactants such as ethylene oxide adducts of alkyl phenols.
Emulsifiable concentrates can comprise a suitable concentration of a bacterial colonization-disrupting agent, such as from about 50 to about 500 grams per liter of liquid dissolved in a carrier that is either a water miscible solvent or a mixture of water-immiscible organic solvent and emulsifiers. Useful organic solvents include aromatics, especially xylenes and petroleum fractions, especially the high-boiling naphthalenic and olefinic portions of petroleum such as heavy aromatic naphtha. Other organic solvents may also be used, such as the terpenic solvents including rosin derivatives, aliphatic ketones such as cyclohexanone, and complex alcohols such as 2-ethoxyethanol. Suitable emulsifiers for emulsifiable concentrates are selected from conventional anionic and non-ionic surfactants.
Aqueous suspensions comprise suspensions of a water-insoluble bacterial colonization-disrupting agent dispersed in an aqueous carrier at a concentration in the range from about 5% to about 50% by weight. Suspensions are prepared by finely grinding the active agent and vigorously mixing it into a carrier comprised of water and surfactants. Ingredients, such as inorganic salts and synthetic or natural gums may also be added, to increase the density and viscosity of the aqueous carrier.
The bacterial colonization-disrupting agent may also be applied as granular compositions that are particularly useful for applications to the soil. Granular compositions can contain, for example, from about 0.5% to about 10% by weight of the bacterial colonization-disrupting agent, dispersed in a carrier that comprises clay or a similar substance. Such compositions are usually prepared by dissolving the formulation in a suitable solvent and applying it to a granular carrier which has been pre-formed to the appropriate particle size, in the range of from about 0.5 to about 3 mm. Such compositions may also be formulated by making a dough or paste of the carrier and compound and crushing and drying to obtain the desired granular particle size.
Dusts containing the present compositions are prepared by intimately mixing the bacterial colonization-disrupting agent in powdered form with a suitable dusty agricultural carrier, such as kaolin clay, ground volcanic rock, and the like. Dusts can suitably contain from about 1% to about 10% of the packets. They can be applied as a seed dressing or as a foliage application with a dust blower machine.
It is equally practical to apply the present formulation in the form of a solution in an appropriate organic solvent, usually petroleum oil, such as the spray oils, which are widely used in agricultural chemistry.
The bacterial colonization-disrupting agent can also be applied in the form of an aerosol composition. In such compositions the packets are dissolved or dispersed in a carrier, which is a pressure-generating propellant mixture. The aerosol composition is packaged in a container from which the mixture is dispensed through an atomizing valve.
Another embodiment is an oil-in-water emulsion, wherein the emulsion comprises oily globules which are each provided with a lamellar liquid crystal coating and are dispersed in an aqueous phase, wherein each oily globule comprises at least one compound which is agriculturally active, and is individually coated with a monolamellar or oligolamellar layer including: (1) at least one non-ionic lipophilic surface-active agent, (2) at least one non-ionic hydrophilic surface-active agent and (3) at least one ionic surface-active agent, wherein the globules having a mean particle diameter of less than 800 nanometers. Further information on the embodiment is disclosed in U.S. patent publication 20070027034 published Feb. 1, 2007. For ease of use, this embodiment will be referred to as “OIWE.”
Additionally, generally, when the molecules disclosed above are used in a formulation, such formulation can also contain other components. These components include, but are not limited to, (this is a non-exhaustive and non-mutually exclusive list) wetters, spreaders, stickers, penetrants, buffers, sequestering agents, drift reduction agents, compatibility agents, anti-foam agents, cleaning agents, and emulsifiers. A few components are described forthwith.
A wetting agent is a substance that when added to a liquid increases the spreading or penetration power of the liquid by reducing the interfacial tension between the liquid and the surface on which it is spreading. Wetting agents are used for two main functions in agrochemical formulations: during processing and manufacture to increase the rate of wetting of powders in water to make concentrates for soluble liquids or suspension concentrates; and during mixing of a product with water in a spray tank to reduce the wetting time of wettable powders and to improve the penetration of water into water-dispersible granules. Examples of wetting agents used in wettable powder, suspension concentrate, and water-dispersible granule formulations are: sodium lauryl sulfate; sodium dioctyl sulfosuccinate; alkyl phenol ethoxylates; and aliphatic alcohol ethoxylates.
A dispersing agent is a substance which adsorbs onto the surface of particles and helps to preserve the state of dispersion of the particles and prevents them from reaggregating. Dispersing agents are added to agrochemical formulations to facilitate dispersion and suspension during manufacture, and to ensure the particles redisperse into water in a spray tank. They are widely used in wettable powders, suspension concentrates and water-dispersible granules. Surfactants that are used as dispersing agents have the ability to adsorb strongly onto a particle surface and provide a charged or steric barrier to reaggregation of particles. The most commonly used surfactants are anionic, non-ionic, or mixtures of the two types. For wettable powder formulations, the most common dispersing agents are sodium lignosulfonates. For suspension concentrates, very good adsorption and stabilization are obtained using polyelectrolytes, such as sodium naphthalene sulfonate formaldehyde condensates. Tristyrylphenol ethoxylate phosphate esters are also used. Non-ionics such as alkylarylethylene oxide condensates and EO-PO block copolymers are sometimes combined with anionics as dispersing agents for suspension concentrates. In recent years, new types of very high molecular weight polymeric surfactants have been developed as dispersing agents. These have very long hydrophobic ‘backbones’ and a large number of ethylene oxide chains forming the ‘teeth’ of a ‘comb’ surfactant. These high molecular weight polymers can give very good long-term stability to suspension concentrates because the hydrophobic backbones have many anchoring points onto the particle surfaces. Examples of dispersing agents used in agrochemical formulations are: sodium lignosulfonates; sodium naphthalene sulfonate formaldehyde condensates; tristyrylphenol ethoxylate phosphate esters; aliphatic alcohol ethoxylates; alkyl ethoxylates; EO-PO (ethylene oxide-propylene oxide) block copolymers; and graft copolymers.
An emulsifying agent is a substance which stabilizes a suspension of droplets of one liquid phase in another liquid phase. Without the emulsifying agent the two liquids would separate into two immiscible liquid phases. The most commonly used emulsifier blends contain alkylphenol or aliphatic alcohol with twelve or more ethylene oxide units and the oil-soluble calcium salt of dodecylbenzenesulfonic acid. A range of hydrophile-lipophile balance (“HLB”) values from 8 to 18 will normally provide good stable emulsions. Emulsion stability can sometimes be improved by the addition of a small amount of an EO-PO block copolymer surfactant.
A solubilizing agent is a surfactant which will form micelles in water at concentrations above the critical micelle concentration. The micelles are then able to dissolve or solubilize water-insoluble materials inside the hydrophobic part of the micelle. The types of surfactants usually used for solubilization are non-ionics, sorbitan monooleates, sorbitan monooleate ethoxylates, and methyl oleate esters.
Surfactants are sometimes used, either alone or with other additives such as mineral or vegetable oils as adjuvants to spray-tank mixes to improve the biological performance of the bacterial colonization-disrupting agent on the target. The types of surfactants used for bioenhancement depend generally on the nature and mode of action of the bacterial colonization-disrupting agent. However, they are often non-ionics such as: alkyl ethoxylates; linear aliphatic alcohol ethoxylates; aliphatic amine ethoxylates.
A carrier or diluent in an agricultural formulation is a material added to the bacterial colonization-disrupting agent to give a product of the required strength. Carriers are usually materials with high absorptive capacities, while diluents are usually materials with low absorptive capacities. Carriers and diluents are used in the formulation of dusts, wettable powders, granules, and water-dispersible granules.
Organic solvents are used mainly in the formulation of emulsifiable concentrates, oil-in-water emulsions, suspoemulsions, and ultra low volume formulations, and to a lesser extent, granular formulations. Sometimes mixtures of solvents are used. The first main groups of solvents are aliphatic paraffinic oils such as kerosene or refined paraffins. The second main group (and the most common) comprises the aromatic solvents such as xylene and higher molecular weight fractions of C9 and C10 aromatic solvents. Chlorinated hydrocarbons are useful as cosolvents to prevent crystallization of the bacterial colonization-disrupting agent when the formulation is emulsified into water. Alcohols are sometimes used as cosolvents to increase solvent power. Other solvents may include vegetable oils, seed oils, and esters of vegetable and seed oils.
Thickeners or gelling agents are used mainly in the formulation of suspension concentrates, emulsions, and suspoemulsions to modify the rheology or flow properties of the liquid and to prevent separation and settling of the dispersed particles or droplets. Thickening, gelling, and anti-settling agents generally fall into two categories, namely water-insoluble particulates and water-soluble polymers. It is possible to produce suspension concentrate formulations using clays and silicas. Examples of these types of materials, include, but are not limited to, montmorillonite, bentonite, magnesium aluminum silicate, and attapulgite. Water-soluble polysaccharides have been used as thickening-gelling agents for many years. The types of polysaccharides most commonly used are natural extracts of seeds and seaweeds or are synthetic derivatives of cellulose. Examples of these types of materials include, but are not limited to, guar gum; locust bean gum; carrageenam; alginates; methyl cellulose; sodium carboxymethyl cellulose (SCMC); hydroxyethyl cellulose (HEC). Other types of anti-settling agents are based on modified starches, polyacrylates, polyvinyl alcohol, and polyethylene oxide. Another good anti-settling agent is xanthan gum.
Microorganisms can cause spoilage of formulated products. Therefore preservation agents are used to eliminate or reduce their effect. Examples of such agents include, but are not limited to: propionic acid and its sodium salt; sorbic acid and its sodium or potassium salts; benzoic acid and its sodium salt; p-hydroxybenzoic acid sodium salt; methyl p-hydroxybenzoate; and 1,2-benzisothiazolin-3-one (BIT).
The presence of surfactants often causes water-based formulations to foam during mixing operations in production and in application through a spray tank. In order to reduce the tendency to foam, anti-foam agents are often added either during the production stage or before filling into bottles. Generally, there are two types of anti-foam agents, namely silicones and non-silicones. Silicones are usually aqueous emulsions of dimethyl polysiloxane, while the non-silicone anti-foam agents are water-insoluble oils, such as octanol and nonanol, or silica. In both cases, the function of the anti-foam agent is to displace the surfactant from the air-water interface.
“Green” agents (e.g., adjuvants, surfactants, solvents) can reduce the overall environmental footprint of crop protection formulations. Green agents are biodegradable and generally derived from natural and/or sustainable sources, e.g., plant and animal sources. Specific examples are: vegetable oils, seed oils, and esters thereof, also alkoxylated alkyl polyglucosides.
In some instances, the bacterial colonization-disrupting agent can be freeze-dried or lyophilized. See U.S. Pat. No. 4,311,712. The bacterial colonization-disrupting agent can later be reconstituted on contact with water or another liquid. Other components can be added to the lyophilized or reconstituted, for example, other agricultural agents, agriculturally acceptable carriers, or other materials in accordance with the formulations described herein.
Other optional features of the composition include carriers or delivery vehicles that protect the bacterial colonization-disrupting agent against UV and/or acidic conditions. In some instances, the delivery vehicle contains a pH buffer. In some instances, the composition is formulated to have a pH in the range of about 4.5 to about 9.0, including for example pH ranges of 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.
In some instances, the composition includes a bait. The bait can be in any suitable form, such as a solid, paste, pellet or powdered form. The bait can also be carried away by the insect back to a population of said insect (e.g., a colony or hive). The bait can then act as a food source for other members of the colony, thus providing an effective amount of a bacterial colonization-disrupting agent for a large number of insects and potentially an entire insect colony.
The baits can be provided in a suitable “housing” or “trap.” Such housings and traps are commercially available and existing traps can be adapted to include the compositions described herein. The housing or trap can be box-shaped for example, and can be provided in pre-formed condition or can be formed of foldable cardboard for example. Suitable materials for a housing or trap include plastics and cardboard, particularly corrugated cardboard. The inside surfaces of the traps can be lined with a sticky substance in order to restrict movement of the insect once inside the trap. The housing or trap can contain a suitable trough inside which can hold the bait in place. A trap is distinguished from a housing because the insect cannot readily leave a trap following entry, whereas a housing acts as a “feeding station” which provides the insect with a preferred environment in which they can feed and feel safe from predators.
In some instances, the composition includes an attractant (e.g., a chemoattractant). The attractant may attract an adult insect or immature insect (e.g., larva) to the vicinity of the composition. Attractants include pheromones, a chemical that is secreted by an animal, especially an insect, which influences the behavior or development of others of the same species. Other attractants include sugar and protein hydrolysate syrups, yeasts, and rotting meat. Attractants also can be combined with an active ingredient and sprayed onto foliage or other items in the treatment area.
Various attractants are known which influence insect behavior as an insect's search for food, oviposition or mating sites, or mates. Attractants useful in the methods and compositions described herein include, for example, eugenol, phenethyl propionate, ethyl dimethylisobutyl-cyclopropane carboxylate, propyl benszodioxancarboxylate, cis-7,8-epoxy-2-methyloctadecane, trans-8,trans-0-dodecadienol, cis-9-tetradecenal (with cis-11-hexadecenal), trans-11-tetradecenal, cis-11-hexadecenal, (Z)-11,12-hexadecadienal, cis-7-dodecenyl acetate, cis-8-dodecenyul acetate, cis-9-dodecenyl acetate, cis-9-tetradecenyl acetate, cis-11-tetradecenyl acetate, trans-11-tetradecenyl acetate (with cis-11), cis-9,trans-11-tetradecadienyl acetate (with cis-9,trans-12), cis-9,trans-12-tetradecadienyl acetate, cis-7,cis-11-hexadecadienyl acetate (with cis-7,trans-11), cis-3,cis-13-octadecadienyl acetate, trans-3,cis-13-octadecadienyl acetate, anethole and isoamyl salicylate. Additionally, means other than chemoattractants may also be used to attract insects, including lights in various wavelengths or colors.
The bacterial colonization-disrupting agent can also be incorporated into the medium in which the insect grows, lives, reproduces, feeds, or infests. For example, a bacterial colonization-disrupting agent can be incorporated into a food container, feeding station, protective wrapping, or a hive. For some applications the bacterial colonization-disrupting agent may be bound to a solid support for application in powder form or in a trap or feeding station. As an example, for applications where the composition is to be used in a trap or as bait for a particular insect, the compositions may also be bound to a solid support or encapsulated in a time-release material. In some instances, the bacterial colonization-disrupting agent is formulated in a fog, smoke, or other treatment suitable for application to an insect habitat.
In formulations and in the use forms prepared from these formulations, the bacterial colonization-disrupting agent may be in a mixture with other agricultural agents or otherwise applied in coincidence with other agricultural agents, such as pesticidal agents (e.g., insecticides, antihelminthics, sterilants, acaricides, nematicides, molluscicides, or fungicides), attractants, plant growth-regulating substances, pollen, sucrose, fertilizers, plant growth regulators, safeners, semiochemicals, or herbicides.
For further information on agricultural formulations, see “Chemistry and Technology of Agrochemical Formulations” edited by D. A. Knowles, copyright 1998 by Kluwer Academic Publishers. Also see “Insecticides in Agriculture and Environment—Retrospects and Prospects” by A. S. Perry, I. Yamamoto, I. Ishaaya, and R. Perry, copyright 1998 by Springer-Verlag.
The following is an example of the methods of the invention. It is understood that various other embodiments may be practiced, given the general description provided above.
This Example demonstrates the disruption of colonization of the gut symbiont Burkholderia in a hemipteran insect, the bean bug (Riptortus pedestris), to descrease insect fitness through the administration of polyhydroxyalkanoate (PHA) synthesis inhibitors. The bean bug R. pedestris (Hemiptera: Heteroptera: Coreoidea) is a notorious pest of leguminous crops, such as soy-bean and cowpea.
Experimental Design:
Insect Rearing and Burkholderia Infection
The R. pedestris bean bugs are reared in the insect incubator at 28° C. under a long-day condition of 16 h light and 8 h dark. Briefly, nymphs are reared in clean plastic containers supplied with soybean seeds and distilled water containing 0.05% ascorbic acid (DWA). The plastic containers are cleaned every day, and the soybean seeds and DWA are changed with fresh ones every 2 days. When the insects emerge as adults, they are transferred to larger plastic containers with soybean seeds and DWA. In addition, cotton pads are attached to the walls of the plastic containers for egg laying. Eggs are collected every day and transferred to new cages for hatching. When newborn nymphs molt to second-instar nymphs, DWA containing 107 cells/ml cultured Burkholderia is provided for the colonization of Burkholderia in a small petri dish. Burkholderia symbiont used is a rifampicin-resistant (Rfr) spontaneous mutant strain RPE75.
Administration of Burkholderia cultured with PHA synthesis inhibitor vanillin A PHA synthesis inhibitor, vanillin is purchased from Sigma-Aldrich (Cat no. V1104-2G). The working concentration of vanillin made in the YG medium is 1 g/ml. The symbiont strain is grown to an early log phase in YG medium (containing rifampicin at 50 ug/ml) on a gyratory shaker (150 rpm) at 30° C. For the positive control Burkholderia is cultured in YG medium only. Colony-forming unit (CFU) values are estimated by plating the culture media on YG agar plates containing adequate antibiotics. Symbiont cells are harvested by centrifuging the culture media, suspended in DWA and adjusted to 104 CFU/mL in DWA.
Immediately after first instar nymphs molt to the second instar, DWA is removed from the rearing containers so that the nymphs are kept without drinking water overnight. Then, DWA containing 104 CFU/mL symbiont cells is supplied to the rearing containers for 24 h, which the second instar nymphs can immediately exploit to acquire the Burkholderia symbionts cultured with PHA synthase inhibitors or the positive control Burkholderia cultured in YG medium only. Then, the symbiont-containing DWA is replaced by symbiont-free DWA, and the nymphs are reared to adulthood.
Direct Feeding of PHA Synthesis Inhibitor Vanillin to R. pedestris
The vanillin working solution (1 g/ml) is made from the stock solution into the distilled water. The vanillin working solution is dispensed into a feeding tube and put into the plastic rearing container for bean bug feeding. Immediately after first instar nymphs molt to the second instar, DWA is removed from the rearing containers so that the nymphs are kept without drinking water overnight. The following day, the vanillin solution along with 104 CFU/mL symbiont cells is supplied to the rearing containers for 24 h, which the second instar nymphs can immediately exploit to acquire the PHA synthase inhibitor vanillin and Burkholderia symbionts. The positive controls are the nymphs fed with 104 CFU/mL symbiont cells only. Then, the symbiont-containing DWA is replaced by DWA, and the nymphs are reared to adulthood.
Quantification of Burkholderia Colonized in R. pedestris Midgut by qPCR
Quantitative PCR (qCPR) is performed using iTaq SYBR green (Biorad) and Applied Biosystems QuantStudio 7 Flex QPCR system (Thermo Fisher) with primers BSdnaA-F and BSdnaA-R targeting a 0.15 kb region of the dnaA gene of the Burkholderia symbiont as described in (Kikuchi et al. 2011; Kikuchi and Fukatsu, 2014). Total DNA is extracted from M4 and M4B parts by using the Blood & Cell Culture DNA Mini Kit (Qiagen, Cat number 13323). and the extracted DNA is eluted in 200 μL water. Each of the PCR mixtures contains 10 μL in volume. qPCR is performed using a qPCR amplification ramp of 1.6 degrees C./s and the following conditions: 1) 95° C. for 10 minutes, 2) 95° C. for 15 seconds, 3) 60° C. for 30 seconds, 4) repeat steps 2-3 40×, 5) 95° C. for 15 seconds, 6) 60° C. for 1 minute, 7) ramp change to 0.15 degrees C./s, 8) 95° C. for 1 second. A standard curve for the dnaA gene is generated with standard samples of the target PCR fragment amplified with the primers BSdnaA-F and BSdnaA-R. qPCR data is analyzed using analytical software (Thermo Fisher Scientific, QuantStudio Design and Analysis).
Measurement of R. pedestris Fitness
The survival rates after administration of Burkholderia cultured with PHA synthase inhibitor vanillin or direct feeding of vanillin to the second-instar nymphs and both positive controls are estimated every day until 25 days after hatching by counting the dead insects. The adult emergence rate is estimated by counting the newly molted adult insects from the late-fifth-instar nymphs. To measure the body length and weight, adult insects (3 days after molting) are sacrificed by submerging in acetone for 5 min and are dried completely in a 70° C. oven for 30 min. Soybean seeds are not supplied to insects 24 h before sacrificing to exclude the weight of the soybean.
In comparison with the positive controls of R. pedestris fed with Burkholderia cultured in YG medium only and direct feeding of Burkholderia only, the titers of Burkholderia in the midgut of R. pedestris offspring are expected to be reduced by either administration of Burkholderia cultured with vanillin or direct feeding of vanillin to R. pedestris.
This example demonstrate the disruption of Burkholderia colonization in a hemipteran model, the bean bug, Riptortus pedestris to decrease fitness in the insect through the administration of sugar analogs, ADP-2-fluoroheptose (AFH) and 2-aryl-5-methyl-4-(5-aryl-furan-2-yl-methylene)-2,4-dihydro-pyrazol-3-ones (DHPO).
Experimental Design:
Insect Rearing and Burkholderia Infection
The R. pedestris bean bugs are reared in the insect incubator at 28° C. under a long-day condition of 16 h light and 8 h dark. Briefly, nymphs are reared in clean plastic containers supplied with soybean seeds and distilled water containing 0.05% ascorbic acid (DWA). The plastic containers are cleaned every day, and the soybean seeds and DWA are changed with fresh ones every 2 days. When the insects emerge as adults, they are transferred to larger plastic containers with soybean seeds and DWA. In addition, cotton pads are attached to the walls of the plastic containers for egg laying. Eggs are collected every day and transferred to new cages for hatching. When newborn nymphs molted to second-instar nymphs, DWA containing 107 cells/ml cultured Burkholderia is provided for the colonization of Burkholderia in a small petri dish. Burkholderia symbiont is a rifampicin-resistant (Rfr) spontaneous mutant strain RPE75.
Administration of Burkholderia Cultured with Sugar Analogs
Two sugar analogs, ADP-2-fluoroheptose (AFH) (Dohi et al., 2008, Chemistry 14, 9530-9539) and 2-aryl-5-methyl-4-(5-aryl-furan-2-yl-methylene)-2,4-dihydro-pyrazol-3-ones (DHPO) (Moreau et al., 2008. Bioorg. Med. Chem. Lett. 18, 4022-4026) inhibiting L-Heptoses synthesis are synthesized by CRO. The working concentration of AHF and DHPO made in the YG medium is 1 g/ml. The symbiont strain is grown to an early log phase in YG medium (containing rifampicin at 50 ug/ml) on a gyratory shaker (150 rpm) at 30° C. The positive control of Burkholderia is cultured in YG medium only. Colony-forming unit (CFU) values are estimated by plating the culture media on YG agar plates containing adequate antibiotics. Symbiont cells are harvested by centrifuging the culture media, suspended in DWA and adjusted to 104 CFU/mL in DWA.
Immediately after first instar nymphs molt to the second instar, DWA is removed from the rearing containers so that the nymphs are kept without drinking water overnight. Then, DWA containing 104 CFU/mL symbiont cells is supplied to the rearing containers for 24 h, which the second instar nymphs can immediately exploit to acquire the Burkholderia symbionts cultured with AHF or DHPO or the positive control Burkholderia cultured in YG medium only. Then, the symbiont-containing DWA is replaced by symbiont-free DWA, and the nymphs are reared to adulthood.
Direct Feeding of Sugar Analogs to R. pedestris
Two sugar analogs, AFH and DHPO (Moreau et al., 2008. Bioorg. Med. Chem. Lett. 18, 4022-4026) inhibiting L-Heptoses synthesis are synthesized by CRO. The working solutions (1 g/ml) for AFH and DHPO are made from the stock into the distilled water. The two sugar analogs working solution are dispensed into the feeding tube and put into the plastic rearing container for bean bug feeding. Immediately after first instar nymphs molted to the second instar, DWA is removed from the rearing containers so that the nymphs are kept without drinking water overnight. The following day, the vanillin solution along with 104 CFU/mL symbiont cells is supplied to the rearing containers for 24 h, which the second instar nymphs immediately exploited, leading to the acquisition of AFH or DHPO and Burkholderia symbionts. The positive control is the nymphs fed with 104 CFU/mL symbiont cells only. Then, the symbiont-containing DWA is replaced by DWA, and the nymphs are reared to adulthood.
Quantification of Burkholderia Colonized in R. pedestris Midgut by qPCR
Quantitative PCR (qCPR) is performed using iTaq SYBR green (Biorad) and Applied Biosystems QuantStudio 7 Flex QPCR system (Thermo Fisher) with primers BSdnaA-F and BSdnaA-R targeting a 0.15 kb region of the dnaA gene of the Burkholderia symbiont as described (Kikuchi et al. 2011; Kikuchi and Fukatsu, 2014). Total DNA is extracted from M4 and M4B parts by using the Blood & Cell Culture DNA Mini Kit (Qiagen, Cat number 13323). and the extracted DNA is eluted in 200 μL water. Each of the PCR mixtures contains 10 μL in volume. qPCR is performed using a qPCR amplification ramp of 1.6 degrees C./s and the following conditions: 1) 95° C. for 10 minutes, 2) 95° C. for 15 seconds, 3) 60° C. for 30 seconds, 4) repeat steps 2-3 40×, 5) 95° C. for 15 seconds, 6) 60° C. for 1 minute, 7) ramp change to 0.15 degrees C./s, 8) 95° C. for 1 second. A standard curve for the dnaA gene is generated with standard samples of the target PCR fragment amplified with the primers BSdnaA-F and BSdnaA-R. qPCR data is analyzed using analytic (Thermo Fisher Scientific, QuantStudio Design and Analysis) software.
Measurement of R. pedestris Fitness
The survival rates after administration of Burkholderia cultured with AFH or DHPO or direct feeding of AFH or DHPO to the second-instar nymphs and both positive controls are estimated every day until 25 days after hatching by counting the dead insects. The adult emergence rate is estimated by counting the newly molted adult insects from the late-fifth-instar nymphs. To measure the body length and weight, adult insects (3 days after molting) are sacrificed by submerging in acetone for 5 min and are dried completely in a 70° C. oven for 30 min. Finally, all fitness parameters are recorded. Soybean seeds are not supplied to insects 24 h before sacrificing to exclude the weight of the soybean.
In comparison with the positive controls of R. pedestris fed with Burkholderia cultured in YG medium only and direct feeding of Burkholderia only, the titers of Burkholderia in the midgut of R. pedestris offspring are expected to be reduced by either administration of Burkholderia cultured with two sugar analogs, AFH and DHPO, or direct feeding of AFH and DHPO to R. pedestris.
This example describes disruption of the gut symbiont Candidatus Pantoea carbekii colonization in the hemipteran brown marmorated stink bug, Halyomorpha halys (St{dot over (a)}l) to decrease insect fitness by administration of sugar analogs.
Experimental Design:
Identification of Genes Required for Synthesizing Core Oligosaccharides of Candidatus Pantoea Carbekii
By searching the Candidatus Pantoea carbekii genome (AB012554.1) in Genbank, four genes for synthesizing core oligosaccharide have been identified (Table 9). The identification of these four genes suggests the P. carbekii synthesizes the core oligosaccharide on its cell surface. In addition, these four genes share high similarity to the genes in the core oligosaccharide pathways of the gut symbiont Burkholderia from the bean bug, Riptortus pedestris.
Candidatus Pantoea carbekii in the brown marmorated
Candidatus Pantoea carbekii
Halyomorpha halys Lab Colony Rearing and Maintenance
Halyomorpha halys no-diapause lab colony is originally from Phillip Alampi Beneficial Insect Laboratory, New Jersey Department of Agriculture, and is maintained in rearing cages (299 cm cube with 24 by 24 mesh, BioQuip Products, Rancho Dominguez, Calif.) in the laboratory. They are held in a growth chamber (28° C., 60-70% relative humidity, and a photoperiod of 16:8 [L:D] h) and are provided diet that included green beans and egg based artificial diet. A green bean plant and Euonymus japonicus plant are provided in the cage for H. halys oviposition and resting, respectively.
Administration Sugar Analogs by Spraying on Egg Masses of Halyomorpha halys
The working concentrations AHF and DHPO are 100 μg/ml in water. A total of 30 egg masses on leaf disks are removed from the colony in a single day during peak egg production. There are two sugar analogs, AHF and DHPO treatments and a water-sprayed negative control, each containing 10 egg masses are setup in a petri dish. Ten egg masses are laid face-up in each of the deep petri dish (15 mm×100 mm). AHF, DHPO or the water (negative control) are applied on the egg masses (1 ML per petri dish) using a Master Airbrush Brand Compressor Model C-16-B Black Mini Airbrush Air Compressor. The compressor is cleaned with ethanol before, after, and between treatments. The liquid is feed through the compressor using a quarter inch tube. A new tube is used for each treatment.
Measurement of Halyomorpha halys Fitness and Fecundity
The sprayed egg masses are reared under the same conditions as in the laboratory colony rearing section above. The number of hatched eggs is recorded for each egg mass and then averaged over all masses per replicate. The newly hatched nymphs in each container are reared to determine the number surviving to the second instar. The survival rate of the nymphs at each stadium is estimated every day until 25 days after hatching by counting the dead insects. The adult emergence rate is estimated by counting the newly molted adult insects from the late-fifth-instar nymphs. To measure the body length and weight, adult insects (3 days after molting) are sacrificed by submerging in acetone for 5 min and dried completely in a 70° C. oven for 30 min. Finally, all fitness parameters are recorded. Green beans are not supplied to insects 24 h before sacrificing to exclude the weight of the diet.
Quantification of Candidatus Pantoea Carbekii Titers by qPCR
Total DNA is extracted from M4 and M4B parts of midgut by using the Blood & Cell Culture DNA Mini Kit (Qiagen, Cat number 13323). and the extracted DNA is eluted in 200 μL water. Quantitative PCR (qCPR) is performed using iTaq SYBR green (Biorad) and Applied Biosystems QuantStudio 7 Flex QPCR system (Thermo Fisher) with primers (forward: GCATATAAAGATTTTACTCTTTAGGTGGC (SEQ ID NO: 5) and reverse: CTCGAAAGCACCAATCCATTTCT (SEQ ID NO: 6)) (Bansal et al. 2014). Two control primers for stink bug mitochondrial DNA are used (forward: CGAATCCCATTGTTTGTGTG (SEQ ID NO: 7) and reverse: AGGGTCTCCTCCTCCTGATG (SEQ ID NO: 8) (Bansal et al. 2014). Each of the PCR mixtures contains 10 μL in volume. qPCR is performed using a qPCR amplification ramp of 1.6 degrees C./s and the following conditions: 1) 95° C. for 10 minutes, 2) 95° C. for 15 seconds, 3) 60° C. for 30 seconds, 4) repeat steps 2-3 40×, 5) 95° C. for 15 seconds, 6) 60° C. for 1 minute, 7) ramp change to 0.15 degrees C./s, 8) 95° C. for 1 second. qPCR data is analyzed using analytic (Thermo Fisher Scientific, QuantStudio Design and Analysis) software.
In comparison with the negative control offspring hatched from the eggs sprayed with water only, the titers of P. carbekii in the midgut of H. halys offspring are expected to be reduced by spraying on egg masses with two sugar analogs, AFH and DHPO.
In comparison with the negative control offspring hatched from the eggs sprayed with water only, the fitness and fecundity of H. halys offspring are expected to be reduced by spraying on egg masses with two sugar analogs, AFH and DHPO.
Together, the data described in these Examples are expected to demonstrate the ability to kill and decrease the development, reproductive ability, longevity, and/or endogenous bacterial populations, e.g., fitness, of several hemipterans by treating them with colonization disrupting agents using multiple delivery methods.
Below are provided examples demonstrating that reducing the bacterial symbionts Candidatus Pantoea carbekii (hereafter referred to as “P. carbekii”) and Burkholderia in their respective hemipteran insect hosts, the brown marmorated stink bug (Halyomorpha halys (St{dot over (a)}l)) and the bean bug (Riptortus pedestris), reduces the fitness of each insect.
This example demonstrates that disruption of colonization of the bacterial symbiont Candidatus Pantoea carbekii (hereafter referred to as “P. carbekii”) in a hemipteran insect host, the brown marmorated stink bug Halyomorpha halys (St{dot over (a)}l), reduces the fitness of the host. Developmental stages of H. halys are shown in
Experimental Design:
Halyomorpha halys Lab Colony Rearing and Maintenance
A Halyomorpha halys no-diapause lab colony was obtained from the Phillip Alampi Beneficial Insect Rearing Laboratory (BIRL), State of New Jersey Department of Agriculture. Following receipt from the BIRL, the lab colony was maintained in Thermo Fisher Scientific environmental incubators (24° C., ambient humidity, and 16:8 [L:D] photoperiod). Adult cages were fed fresh green beans and a seed mixture of peanuts, sunflower seeds, and buckwheat; green beans were changed every other day and the seed mixture was changed weekly. Egg clutches were collected daily from colony cages and placed into hatching containers (all egg clutches into a single container) that contained only 5 mL water tubes (stuffed with cotton). Upon hatching, nymphs were provided diet pellets (ad libitum) that contained split pea, almonds, buckwheat, sunflower seeds, wheat germ, ascorbic acid, and Wesson's salt.
Treatment of Eggs to Remove Symbionts
Four- to five-day-old H. halys egg clutches were submerged in absolute (˜95%) ethanol for 5 minutes, then submerged in 8% sodium hypochlorite (extra strength bleach) for 45 seconds, and were finally gently rinsed with purified water before being placed on a paper towel to dry. Control eggs were left untreated. To confirm the efficacy of the treatments, DNA was extracted from a subset of treated vs. control nymphs at the 2nd, 3rd and 4th instars and was screened for the P. carbekii symbionts using qPCR, as described below. P. carbekii abundance was reduced in the treated group (
Quantification of P. carbekii Titers by RT-qPCR
Total RNA was extracted from nymphs using total RNA isolation and purification kits (both from Thermo Fisher Scientific), and the extracted RNA was eluted in 100 μL water. Quantitative reverse transcription PCR (RT-qCPR) was performed using RT-qPCR kits (Thermo Fisher Scientific) with primers targeting the P. carbekii DNAK gene (forward primer sequence: TGCAGAAATTTGTGGCGGTG (SEQ ID NO: 1); reverse primer sequence: CGTTGCCTCAGAAAACGGTG (SEQ ID NO: 2)). Primers for the stink bug 60S rRNA gene (forward primer sequence: AACAGGCAAGCTGCTATCTC (SEQ ID NO: 3) and reverse primer sequence: CTGTCCCTTGGTGGTTCTTT (SEQ ID NO: 4)) were used to normalize the bacterial quantities. Each of the PCR mixtures was 10 μL in volume. RT-qPCR was performed using a PCR amplification ramp of 1.6° C./s and the following conditions: 1) 48° C. for 30 min; 2) 95° C. for 10 minutes; 3) 95° C. for 15 seconds; 4) 55° C. for 30 seconds; 5) repeat steps 3-4 40×, 6); 95° C. for 15 seconds; 7) 55° C. for 1 minute; 8) ramp change to 0.15° C./s; 9) 95° C. for 1 second. RT-qPCR data was analyzed using analytic software (Thermo Fisher Scientific).
Set-Up of Replicates and Data Collection
After egg treatment, eggs were allowed to hatch and develop to the second instar (larvae only require drinking water during this period). For each replicate, ten second instars from each treatment were placed in plastic cages containing a paper towel, water tube, and a green bean; water tubes were changed weekly, and green beans were changed every other day. Total replicates were 28 for the control treatment and 23 for the bleach/ethanol treatment. The number of survivors and the number of insects in each instar were recorded for each replicate daily. Symbiont removal increased the average amount of time between successive developmental instars compared to the control group (
Once nymphs reached adulthood, adults from each treatment group were respectively pooled into large colony cages where the number of adults (male and female), egg masses, and eggs per mass were counted daily.
The average number of eggs in each egg mass was significantly lower for females reared from ethanol-treated and bleached eggs compared to controls (
Guts were dissected from H. halys individuals of the same age from the bleach/ethanol treatment group or the control group. Gut health was observed to be inferior and the symbiont-containing v4 region of the gut was degenerated in the bleach/ethanol treatment group (
Insect size and coloring were observed to differ between H. halys individuals of the same age from the bleach/ethanol treatment group or the control group (
The width of the pronotum (standard fitness measure for stinkbugs) was measured for all males and females for comparison. Pronotal width was significantly reduced in male and female individuals hatched from bleached eggs (
This example demonstrates the disruption of colonization of the gut symbiont P. carbekii in the brown marmorated stink bug (Halyomorpha halys (St{dot over (a)}l)) through the administration of polyhydroxyalkanoate (PHA) synthesis inhibitors.
Experimental Design:
The polyhydroxyalkanoate (PHA) synthesis inhibitors used were vanillin, levulinic acid, acrylic acid (AA), and 2-bromooctanoic acid (2BA).
Halyomorpha halys Lab Colony Rearing and Maintenance
A Halyomorpha halys no-diapause lab colony was reared as described in Example 4. Egg clutches were collected daily from colony cages and placed into hatching containers (5 egg clutches per container, maximum) that contained 30 mL water tubes (stuffed with cotton), fresh green beans, and a seed mixture of peanuts.
Administration of PHA Synthesis Inhibitors by Egg Mass Treatment:
Working concentrations of the PHA synthesis inhibitors (vanillin, levulinic acid, acrylic acid (AA), and 2-bromooctanoic acid (2BA)) were made up to 100 μg/ml in water. The solutions of PHA inhibitors were spiked with a non-ionic wetting solution to a final concentration of 0.025% to increase the wettability and spreading of the agents on the eggs. As a negative control, no agents were added to the 0.025% non-ionic wetting solution, and as a positive control, 100 μg/ml of the antibiotic rifamycin S was used. Egg masses on leaf disks were removed from the colony in a single day during peak egg production. Each egg mass was then placed in a container with a paper towel at the bottom, and a tube of water plugged with cotton was provided as a water source for the hatchlings. 100 μl of the agent was pipetted onto the eggs to wet them completely. This allowed the agent to interact directly with the bacteria before the bacteria was able to colonize the host. Once the 1st instar hatchlings molted to the 2nd instar stage, food was provided in the form of 500 mg diet pellets (see “Halyomorpha halys lab colony rearing and maintenance” above). Late 2nd instars were collected and frozen to extract DNA to assay for the symbiont levels, as described in Example 4.
Results
The levels of P. carbekii were significantly lower in the positive control (Rifamycin S) compared to the negative (water) control. All the four PHA inhibitors (vanillin, levulinic acid, acrylic acid (AA), and 2-bromooctanoic acid (2BA)) used caused a reduction in the symbiont levels per host relative to the water control (
Based on the results from Example 4 showing decreased fitness in H. halys having reduced colonization by P. carbekii, the lower levels of the symbionts may lead to decreased fitness in the PHA inhibitor-treated insects. PHA synthase inhibitors are taken as being useful in the invention.
This example demonstrates the disruption of colonization of the gut symbiont P. carbekii in the brown marmorated stink bug, Halyomorpha halys (St{dot over (a)}l), through the administration of a UppP inhibitor, bacitracin.
Experimental Design
Halyomorpha halys Lab Colony Rearing and Maintenance
A Halyomorpha halys no-diapause lab colony was reared as described in Example 4. Egg clutches were collected daily from colony cages and placed into hatching containers (5 egg clutches per container, maximum) that contained 30 mL water tubes (stuffed with cotton), fresh green beans, and a seed mixture of peanuts.
Administration of a UppP Inhibitor by Egg Mass Treatment
A working concentration of bacitracin was made up to 100 μg/ml in water and spiked with Silwet® L-77 to a final concentration of 0.025% to increase the wettability and spreading of the agents on the eggs. As a negative control, no agents were added to the wetting solution, and as a positive control, 100 μg/ml of the antibiotic rifamycin S was used. Egg masses on leaf disks were removed from the colony in a single day during peak egg production. Each egg mass was then placed in a container with a paper towel at the bottom, and a tube of water plugged with cotton was provided as a water source for the hatchlings. 100 μl of the agent was pipetted onto the eggs to wet them completely. This allowed the agent to interact directly with the bacteria before the bacteria was able to colonize the host. Once the 1st instar hatchlings molted to the 2nd instar stage, food was provided in the form of 500 mg pellets of the artificial diet described above. The same food was provided to the 2nd instars until they molted to the 3rd instar stage. The third instars were collected and frozen to extract DNA to assay for the symbiont levels, as described in Example 4.
Results
The levels of P. carbekii were significantly lower in the positive control (Rifamycin S) and the bacitracin treatment group compared to the negative control (
This example demonstrates the disruption of colonization of the gut symbiont P. carbekii in a hemipteran insect host, the brown marmorated stink bug (Halyomorpha halys (St{dot over (a)}l)), through the administration of a flagellar function inhibitor, cellulose.
Experimental Design
Halyomorpha halys Lab Colony Rearing and Maintenance
A Halyomorpha halys no-diapause lab colony was reared as described in Example 4. Egg clutches were collected daily from colony cages and placed into hatching containers (5 egg clutches per container, maximum) that contained 30 mL water tubes (stuffed with cotton), fresh green beans, and a seed mixture of peanuts.
Administration of Flagellar Function Inhibitors by Egg Mass Treatment
A working concentration of cellulose was made up to 100 μg/ml in water. The solution of cellulose was spiked with a non-ionic wetting solution to a final concentration of 0.025% to increase the wettability and spreading of the agent on the eggs. As a negative control, no agents were added to the wetting solution, and as a positive control, 100 μg/ml of the antibiotic rifamycin S was used. Egg masses on leaf disks were removed from the colony in a single day during peak egg production. Each egg mass was then placed in a container with a paper towel at the bottom, and a tube of water plugged with cotton was provided as a water source for the hatchlings. 100 μl of the agent was pipetted onto the eggs to wet them completely. This allowed the agent to interact directly with the bacteria even before the bacteria is able to colonize the host. Once the 1st instar hatchlings molted to 2nd instar stage, food was provided in the form of 500 mg pellets of the artificial diet described above. The same food was provided to the 2nd instars until they molted to the 3rd instar stage. The third instars were collected and frozen to extract DNA to assay for the symbiont levels, as described in Example 4.
Results
The levels of P. carbekii were significantly lower in the positive control (Rifamycin S) and in the cellulose treatment group compared to the negative control (
This example describes disruption of the gut symbiont P. carbekii colonization in the hemipteran brown marmorated stink bug, Halyomorpha halys (St{dot over (a)}l) by administration of sugar analogs. This example is provided to evaluate the ability of sugar analogs to kill and decrease the development, reproductive ability, longevity and endogenous bacterial populations, e.g., fitness, of a hemipteran insect.
Experimental Design
Identification of Genes Required for Synthesizing Core Oligosaccharides of P. carbekii
By searching the P. carbekii genome (AB012554.1) in Genbank, four genes for synthesizing core oligosaccharide have been identified (Table 11). The identification of these four genes suggests that P. carbekii could synthesize the core oligosaccharide on its cell surface. These data provide the grounds for disrupting P. carbekii colonization in Halyomorpha halys by inhibiting the core oligosaccharide synthesis process via administration of sugar analogs.
Candidatus Pantoea carbekii in the brown
Candidatus Pantoea carbekii
Halyomorpha halys Lab Colony Rearing and Maintenance
A Halyomorpha halys no-diapause lab colony is obtained as described in Example 4. Following receipt from the BIRL, the insects are held in a growth chamber as described above and are provided a pellet diet. A green bean plant and a Euonymus japonicus plant are provided in the cage for H. halys oviposition and resting, respectively.
Administration of Sugar Analogs by Spraying on Egg Masses of Halyomorpha halys
Two sugar analogs, ADP-2-fluoroheptose (AFH) (Dohi et al., Chemistry, 14(31): 9530-9539, 2008) and 2-aryl-5-methyl-4-(5-aryl-furan-2-yl-methylene)-2,4-dihydro-pyrazol-3-ones (DHPO) (Moreau et al., Bioorg. Med. Chem. Lett., 18(14): 4022-4026, 2008), which inhibit the function of WaaC (heptosyl transferase), are synthesized by a Contract Research Organization (CRO).
The working concentrations of AFH and DHPO are 100 μg/ml in water. A total of 30 egg masses on leaf disks are removed from the colony in a single day during peak egg production. Ten egg masses are laid face-up in each well of a deep petri dish (15 mm×100 mm). AFH, DHPO or water (negative control) are applied on the egg masses (1 mL per petri dish) using a Master Airbrush Brand Compressor Model C-16-B Black Mini Airbrush Air Compressor. The compressor is cleaned with ethanol before, after, and between treatments. The liquid is fed through the compressor using a quarter inch tube. A new tube is used for each treatment.
Measurement of Halyomorpha halys Fitness and Fecundity
The sprayed egg masses are reared under the conditions described above. The number of hatched eggs is recorded for each egg mass and then averaged over all masses per replicate. The newly hatched nymphs in each container are reared to determine the number surviving to the second instar. The survival rate of the nymphs at each stadium is estimated every day until 25 days after hatching by counting the dead insects. The adult emergence rate is estimated by counting the newly molted adult insects from the late-fifth-instar nymphs. To measure the body length and weight, adult insects (3 days after molting) are sacrificed by submerging in acetone for 5 min and dried completely in a 70° C. oven for 30 min. Finally, all fitness parameters are recorded. Green beans are not supplied to insects 24 h before sacrificing to exclude the weight of the diet.
Quantification of P. carbekii Titers by RT-qPCR
Total RNA is extracted from nymphs using RNA isolation and purification kits (both from Thermo Fisher Scientific), and the extracted RNA is eluted in 100 μL water. Quantitative reverse transcription PCR (RT-qCPR) is performed using a RT-qPCR kit (Thermo Fisher Scientific) with primers targeting the P. carbekii DNAKgene (forward primer sequence: TGCAGAAATTTGTGGCGGTG (SEQ ID NO: 1); reverse primer sequence: CGTTGCCTCAGAAAACGGTG (SEQ ID NO: 2)). Primers for the stink bug 60S rRNA gene (forward primer sequence: AACAGGCAAGCTGCTATCTC (SEQ ID NO: 3) and reverse primer sequence: CTGTCCCTTGGTGGTTCTTT (SEQ ID NO: 4)) are used to normalize the bacterial quantities. Each of the PCR mixtures is 10 μL in volume. RT-qPCR is performed using a PCR amplification ramp of 1.6° C./s and the following conditions: 1) 48° C. for 30 min; 2) 95° C. for 10 minutes; 3) 95° C. for 15 seconds; 4) 55° C. for 30 seconds; 5) repeat steps 3-4 40×, 6); 95° C. for 15 seconds; 7) 55° C. for 1 minute; 8) ramp change to 0.15° C./s; 9) 95° C. for 1 second. RT-qPCR data is analyzed using analytic software (Thermo Fisher Scientific).
Sugar analogs reducing the fitness or fecundity or both of H. halys offspring, in view of appropriate controls, are taken as useful in the invention.
This example demonstrates the disruption of colonization of the gut symbiont Burkholderia in a hemipteran insect, the bean bug (Riptortus pedestris) through the administration of the polyhydroxyalkanoate (PHA) synthesis inhibitor vanillin or a vanillin analog. This example is provided to evaluate the ability of this disruption to cause a fitness decrease in the insect.
The bean bug R. pedestris (Hemiptera: Heteroptera: Coreoidea) is a notorious pest of leguminous crops, such as soy-bean and cowpea. R. pedestris harbors a specific gut symbiont of the genus Burkholderia, which is acquired orally from the environment by second-instar nymphs. Bean bugs have a specialized symbiotic organ (crypts) in a posterior midgut fourth region (M4) to host the symbionts.
Experimental Design:
Insect Rearing and Burkholderia Infection
The R. pedestris bean bugs are reared in an insect incubator at 28° C. under a long-day condition of 16 h light and 8 h dark. Briefly, nymphs are reared in clean plastic containers supplied with soybean seeds and distilled water containing 0.05% ascorbic acid (DWA). The plastic containers are cleaned every day, and the soybean seeds and DWA are changed with fresh ones every 2 days. When the insects emerge as adults, they are transferred to larger plastic containers with soybean seeds and DWA. In addition, cotton pads are attached to the walls of the plastic containers for egg laying. Eggs are collected every day and transferred to new cages for hatching. When newborn nymphs molt to second-instar nymphs, DWA containing 107 cells/ml cultured Burkholderia is provided in a small petri dish for the colonization by Burkholderia of the bean bugs. The Burkholderia symbiont used is the rifampicin-resistant (Rfr) spontaneous mutant strain RPE75 (provided by Dr. Takema Fukatsu, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Center, Tsukuba, Japan).
Administration of Burkholderia Cultured with PHA Synthesis Inhibitor Vanillin
A PHA synthesis inhibitor, vanillin, is purchased from Sigma-Aldrich (Cat. no. V1104-2G). A working concentration of vanillin is made at 1 g/ml in YG medium (0.5% yeast extract, 0.4% glucose, and 0.1% NaCl). The symbiont strain is grown to an early log phase in YG medium (containing rifampicin at 50 μg/ml) on a gyratory shaker (150 rpm) at 30° C. For the positive control, Burkholderia is cultured in YG medium only. Colony-forming unit (CFU) values are estimated by plating the culture media on YG agar plates containing adequate antibiotics. Symbiont cells are harvested by centrifuging the culture media, suspended in DWA, and adjusted to 104 CFU/mL in DWA.
Immediately after first instar nymphs molt to the second instar, DWA is removed from the rearing containers so that the nymphs are kept without drinking water overnight. Then, DWA containing 104 CFU/mL symbiont cells is supplied to the rearing containers for 24 h, which the second instar nymphs can exploit to acquire the Burkholderia symbionts cultured with PHA synthase inhibitors or the positive control Burkholderia cultured in YG medium only. Then, the symbiont-containing DWA is replaced by symbiont-free DWA, and the nymphs are reared to adulthood.
Direct Feeding of PHA Synthesis Inhibitor Vanillin to R. pedestris
Immediately after first instar nymphs molt to the second instar, DWA is removed from the rearing containers so that the nymphs are kept without drinking water overnight. The following day, the vanillin solution (1 mg/ml) along with 104 CFU/mL symbiont cells is supplied to the rearing containers for 24 h, which the second instar nymphs can exploit to acquire the PHA synthase inhibitor vanillin and Burkholderia symbionts. Positive controls are nymphs fed with 104 CFU/mL symbiont cells only. Then, the symbiont-containing DWA is replaced by DWA, and the nymphs are reared to adulthood.
Quantification of Burkholderia Colonized in R. pedestris Midgut by qPCR
Quantitative PCR (qPCR) is performed using qPCR kits (Thermo Fisher) with primers BSdnaA-F and BSdnaA-R targeting a 0.15 kb region of the dnaA gene of the Burkholderia symbiont, as described in (Kikuchi et al., Applied and Environmental Microbiology, 77: 4075-4081, 2011; Kikuchi and Fukatsu, Molecular Ecology, 23: 1445-1456, 2014). Total DNA is extracted from the M4 and M4B parts of the midgut by using Blood & Cell Culture DNA Mini Kit (Qiagen, Cat number 13323). and the extracted DNA is eluted in 200 μL water. Each of the PCR mixtures contains 10 μL in volume. qPCR is performed using a qPCR amplification ramp of 1.6° C./s and the following conditions: 1) 95° C. for 10 minutes; 2) 95° C. for 15 seconds; 3) 60° C. for 30 seconds; 4) repeat steps 2-3 40×; 5) 95° C. for 15 seconds; 6) 60° C. for 1 minute; 7) ramp change to 0.15° C./s; 8) 95° C. for 1 second. A standard curve for the dnaA gene is generated with standard samples of the target PCR fragment amplified with the primers BSdnaA-F and BSdnaA-R. qPCR data is analyzed using analytical software (Thermo Fisher Scientific).
Measurement of R. pedestris Fitness
The survival rates after administration of Burkholderia cultured with PHA synthase inhibitor vanillin or direct feeding of vanillin to the second-instar nymphs and both positive controls are estimated every day until 25 days after hatching by counting the dead insects. The adult emergence rate is estimated by counting the newly molted adult insects from the late-fifth-instar nymphs. To measure the body length and weight, adult insects (3 days after molting) are sacrificed by submerging in acetone for 5 min and are dried completely in a 70° C. oven for 30 min. Soybean seeds are not supplied to insects 24 h before sacrificing to exclude the weight of the soybean.
Vanillin or analogs thereof which reduce titers of Burkholderia in R. pedestris offspring, in view of appropriate controls, are taken as useful in the invention.
This example demonstrates the disruption of Burkholderia colonization in a hemipteran model, the bean bug (Riptortus pedestris) through the administration of the sugar analogs ADP-2-fluoroheptose (AFH) and 2-aryl-5-methyl-4-(5-aryl-furan-2-yl-methylene)-2,4-dihydro-pyrazol-3-ones (DHPO). This example is provided to evaluate the ability of this disruption to cause a fitness decrease in the insect.
Experimental Design
Insect Rearing and Burkholderia Infection
R. pedestris is reared as described in Example 6.
Administration of Burkholderia Cultured with Sugar Analogs
Two sugar analogs, ADP-2-fluoroheptose (AFH) (Dohi et al., Chemistry, 14(31): 9530-9539, 2008) and 2-aryl-5-methyl-4-(5-aryl-furan-2-yl-methylene)-2,4-dihydro-pyrazol-3-ones (DHPO) (Moreau et al., Bioorg. Med. Chem. Lett., 18(14): 4022-4026, 2008), which inhibit the function of WaaC (heptosyl transferase), are synthesized by a Contract Research Organization (CRO).
The working concentration of AFH and DHPO made in the YG medium is 1 g/ml. The symbiont strain is grown to an early log phase in YG medium (containing rifampicin at 50 μg/ml) on a gyratory shaker (150 rpm) at 30° C. The positive control of Burkholderia is cultured in YG medium only. Colony-forming unit (CFU) values are estimated by plating the culture media on YG agar plates containing adequate antibiotics. Symbiont cells are harvested by centrifuging the culture media, suspended in DWA, and adjusted to 104 CFU/mL in DWA.
Immediately after first instar nymphs molt to the second instar, DWA is removed from the rearing containers so that the nymphs are kept without drinking water overnight. Then, DWA containing 104 CFU/mL symbiont cells is supplied to the rearing containers for 24 h, which the second instar nymphs can exploit to acquire the Burkholderia symbionts cultured with AFH or DHPO or the positive control Burkholderia cultured in YG medium only. Then, the symbiont-containing DWA is replaced by symbiont-free DWA, and the nymphs are reared to adulthood.
Direct Feeding of Sugar Analogs to R. pedestris
AFH and DHPO are synthesized by CRO. The working solutions (1 g/ml) for AFH and DHPO are made from the stock into the distilled water. The two sugar analog working solutions are dispensed into the feeding tube and put into the plastic rearing container for bean bug feeding. Immediately after first instar nymphs molt to the second instar, DWA is removed from the rearing containers so that the nymphs are kept without drinking water overnight. The following day, the AFH and DHPO solution along with 104 CFU/mL symbiont cells is supplied to the rearing containers for 24 h, which the second instar nymphs can exploit, leading to the acquisition of AFH or DHPO and Burkholderia symbionts. The positive control is nymphs fed with 104 CFU/mL symbiont cells only. Then, the symbiont-containing DWA is replaced by DWA, and the nymphs are reared to adulthood.
Quantification of Burkholderia Colonized in R. pedestris Midgut by qPCR
Quantitative PCR (qCPR) is performed as described in Example 6.
Measurement of R. pedestris Fitness
The survival rates after administration of Burkholderia cultured with AFH or DHPO or direct feeding of AFH or DHPO to the second-instar nymphs and both positive controls are estimated every day until 25 days after hatching by counting the dead insects. The adult emergence rate is estimated by counting the newly molted adult insects from the late-fifth-instar nymphs. To measure the body length and weight, adult insects (3 days after molting) are sacrificed by submerging in acetone for 5 min and are dried completely in a 70° C. oven for 30 min. Finally, all fitness parameters are recorded. Soybean seeds are not supplied to insects 24 h before sacrificing to exclude the weight of the soybean.
Sugar analogs reducing titers of Burkholderia in R. pedestris offspring, in view of appropriate controls, are taken as useful in the invention.
Some embodiments of the invention are within the following numbered paragraphs.
1. A method of decreasing the fitness of an insect comprising delivering to the insect an effective amount of a composition comprising a bacterial colonization-disrupting agent.
2. A method of inhibiting bacterial colonization in the gut of an insect comprising delivering to the insect an effective amount of a composition comprising a bacterial colonization-disrupting agent.
3. The method of paragraph 2, wherein the method is effective to increase the fitness of the insect relative to an untreated insect.
4. The method of any one of paragraphs 1-3, wherein the bacterial colonization-disrupting agent is a polyhydroxyalkanoate (PHA) synthesis inhibitor.
5. A method of decreasing the fitness of an insect comprising delivering to the insect an effective amount of a composition comprising a PHA synthesis inhibitor.
6. The method of paragraph 4 or 5, wherein the PHA synthesis inhibitor is vanillin.
7. The method of paragraph 4 or 5, wherein the PHA synthesis inhibitor is one or more compounds in Table 1.
8. The method of paragraph 4 or 5, wherein the PHA synthesis inhibitor is levulinic acid.
9. The method of paragraph 4 or 5, wherein the PHA synthesis inhibitor is acrylic acid.
10. The method of paragraph 4 or 5, wherein the PHA synthesis inhibitor is 2-bromooctanoic acid.
11. The method of any one of paragraphs 1-3, wherein the bacterial colonization-disrupting agent is an inhibitor of bacterial cell envelope biogenesis.
12. The method of paragraph 11, wherein the inhibitor of bacterial cell envelope biogenesis is a lipopolysaccharide (LPS) synthesis inhibitor.
13. A method of decreasing the fitness of an insect comprising delivering to the insect an effective amount of a composition comprising an LPS synthesis inhibitor.
14. The method of paragraph 12 or 13, wherein the LPS synthesis inhibitor is an inhibitor of core oligosaccharide synthesis in the bacteria.
15. The method of paragraph 14, wherein the LPS synthesis inhibitor inhibits an enzyme involved in core oligosaccharide synthesis in the bacteria.
16. The method of paragraph 15, wherein the enzyme has at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to a polypeptide having the amino acid sequence of WaaA, WaaC, WaaF, or WaaG.
17. The method of any one of paragraphs 12-16, wherein the LPS synthesis inhibitor is a sugar.
18. The method of paragraph 17, wherein the sugar is ADP-2-fluoroheptose (AFH).
19. The method of paragraph 17, wherein the sugar is 2-aryl-5-methyl-4-(5-aryl-furan-2-yl-methylene)-2,4-dihydro-pyrazol-3-ones (DHPO).
20. The method of paragraph 17, wherein the sugar is AFH and DHPO.
21. The method of paragraph 17, wherein the sugar is one or more compounds in Table 7.
22. The method of paragraph 14, wherein the LPS synthesis inhibitor inhibits expression of a gene involved in core oligosaccharide synthesis in the bacteria.
23. The method of paragraph 22, wherein the gene has at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to a polynucleotide having the nucleotide sequence of waaA, waaC, waaF, or waaG.
24. The method of any one of paragraphs 1-3, wherein the bacterial colonization-disrupting agent is an inhibitor of bacterial cell wall biogenesis.
25. The method of paragraph 24, wherein the inhibitor of bacterial cell wall biogenesis is an inhibitor of undecaprenyl pyrophosphate phosphatase (UppP).
26. The method of paragraph 25, wherein the inhibitor of UppP is bacitracin.
27. The method of any one of paragraphs 1-3, wherein the bacterial colonization-disrupting agent is an inhibitor of flagellar function.
28. The method of paragraph 27, wherein the inhibitor of flagellar function is cellulose.
29. The method of any one of paragraphs 1-28, wherein the insect is a plant pest.
30. The method of paragraph 29, wherein the plant pest is of the order Coleoptera, Diptera, Hemiptera, Lepidoptera, Orthoptera, Thysanoptera, or Acarina.
31. The method of paragraph 30, wherein the insect is a stink bug, bean bug, beetle, weevil, fly, aphid, whitefly, leafhopper, scale, moth, butterfly, grasshopper, cricket, thrip, or mite.
32. The method of paragraph 31, wherein the insect is of the genus Riptortus.
33. The method of paragraph 32, wherein the insect is of the genus Halyomorpha.
34. The method of any one of paragraphs 1-33, wherein the insect is a vector of an animal pathogen and/or a human pathogen.
35. The method of paragraph 34, wherein the insect is a mosquito, a midge, a louse, a sandfly, a tick, a triatomine bug, a tsetse fly, or flea.
36. The method of any one of paragraphs 1-35, wherein the bacteria is an endosymbiotic bacteria.
37. The method of paragraph 36, wherein the endosymbiont resides in the gut of the insect.
38. The method of paragraph 37, wherein the bacteria resides in a specialized cell or a specialized organ in the gut of the insect.
39. The method of paragraph 38, wherein the specialized organ is a midgut crypt or a bacteriome.
40. The method of paragraph 38, wherein the specialized cell is a bacteriocyte.
41. The method of any one of paragraphs 36-40, wherein the endosymbiotic bacteria is of the genus
Burkholderia.
42. The method of any one of paragraphs 36-40, wherein the endosymbiotic bacteria is of the genus Pantoea.
43. The method of any one of paragraphs 1, 2, and 4-41, wherein the method is effective to decrease the fitness of the insect relative to an untreated insect.
44. The method of paragraph 43, wherein the decrease in fitness of the insect is a decrease in reproductive ability, survival, rate of development, number of hatched eggs, adult emergence rate, body length, or weight.
45. The method of any one of paragraphs 1-44, wherein the method is effective to decrease bacterial colonization in the gut of the insect relative to an untreated insect.
46. The method of any one of paragraphs 1-45, wherein the composition is delivered to the insect to at least one habitat where the insect grows, lives, or reproduces.
47. The method of any one of paragraphs 1-46, wherein the composition is a liquid, a solid, an aerosol, a paste, a gel, or a gas composition.
48. The method of any one of paragraphs 1-47, wherein the composition is delivered as an insect comestible composition for ingestion by the insect.
49. The method of any one of paragraphs 1-48, wherein the composition is delivered to eggs of the insect.
50. The method of any one of paragraphs 1-49, wherein the composition is delivered to the insect by ingestion, infusion, injection, or spraying.
51. The method of any one of paragraphs 1-50, wherein the composition comprises an agriculturally acceptable carrier.
52. A modified insect produced by a method comprising contacting the insect with a composition comprising a bacterial colonization-disrupting agent in accordance with the methods of any one of paragraphs 1-51.
53. A screening assay to identify a bacterial colonization-disrupting agent, comprising the steps of
(a) exposing a target insect to one or more agents; and
(b) identifying an agent that:
54. The assay of paragraph 53, wherein the decrease in fitness is decreased survival of the target insect.
55. The assay of paragraph 53, wherein the decrease in fitness is a decrease in reproductive ability, survival, rate of development, number of hatched eggs, adult emergence rate, body length, or body mass.
56. The assay of any one of paragraphs 53-55, wherein the bacteria is an endosymbiotic bacteria.
57. The assay of paragraph 56, wherein the endosymbiotic bacteria resides in the gut of the insect.
58. The assay of paragraph 57, wherein the bacteria resides in a specialized cell or a specialized organ in the gut of the insect.
59. The assay of paragraph 58, wherein the specialized organ is a midgut crypt or a bacteriome.
60. The assay of paragraph 58, wherein the specialized cell is a bacteriocyte.
61. The assay of any one of paragraphs 53-58, wherein the bacterium is of the genus Burkholderia.
62. The assay of any one of paragraphs 53-60, wherein the bacterium is of the genus Pantoea.
63. The assay of any one of paragraphs 53-62, wherein the insect is a plant pest.
64. The assay of paragraph 63, wherein the plant pest is of the order Coleoptera, Diptera, Hemiptera, Lepidoptera, Orthoptera, Thysanoptera, or Acarina.
65. The assay of any one of paragraphs 53-62, wherein the insect is a vector of an animal pathogen and/or a human pathogen.
66. The assay of paragraph 65, wherein the insect is a mosquito, a midge, a louse, a sandfly, a tick, a triatomine bug, a tsetse fly, or flea.
67. A modified insect produced by a method comprising contacting the insect with a composition comprising a bacterial colonization-disrupting agent identified by the screening assay of any one of paragraphs 53-66.
68. A method of decreasing the fitness of an insect comprising delivering to the insect an effective amount of a composition comprising a bacterial colonization-disrupting agent identified by the screening assay of any one of paragraphs 53-66.
69. A composition comprising a bacterial colonization-disrupting agent and a carrier, wherein the composition is formulated for delivery to an insect, or a habitat thereof.
70. The composition of paragraph 69, wherein the bacterial colonization-disrupting agent is a polyhydroxyalkanoate (PHA) synthesis inhibitor.
71. The composition of paragraph 70, wherein the PHA synthesis inhibitor is vanillin.
72. The composition of paragraph 70, wherein the PHA synthesis inhibitor is one or more compounds in Table 1.
73. The composition of paragraph 70, wherein the PHA synthesis inhibitor is levulinic acid.
74. The composition of paragraph 70, wherein the PHA synthesis inhibitor is acrylic acid.
75. The composition of paragraph 70, wherein the PHA synthesis inhibitor is 2-bromooctanoic acid.
76. The composition of paragraph 69, wherein the bacterial colonization-disrupting agent is an inhibitor of bacterial cell envelope biogenesis.
77. The composition of paragraph 76, wherein the inhibitor of bacterial cell envelope biogenesis is a lipopolysaccharide (LPS) synthesis inhibitor.
78. The composition of paragraph 77, wherein the LPS synthesis inhibitor is an inhibitor of core oligosaccharide synthesis in the bacteria.
79. The composition of paragraph 77 or 78, wherein the LPS synthesis inhibitor inhibits an enzyme involved in core oligosaccharide synthesis in the bacteria.
80. The composition of paragraph 79, wherein the enzyme has at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to a polypeptide having the amino acid sequence of WaaA, WaaC, WaaF, or WaaG.
81. The composition of any one of paragraphs 78-80, wherein the LPS synthesis inhibitor is a sugar.
82. The composition of paragraph 81, wherein the sugar is ADP-2-fluoroheptose (AFH).
83. The composition of paragraph 81, wherein the sugar is 2-aryl-5-methyl-4-(5-aryl-furan-2-yl-methylene)-2,4-dihydro-pyrazol-3-ones (DHPO).
84. The composition of paragraph 81, wherein the sugar is AFH and DHPO.
85. The composition of paragraph 81, wherein the sugar is one or more compounds in Table 7.
86. The composition of paragraph 77 or 78, wherein the LPS synthesis inhibitor inhibits expression of a gene involved in core oligosaccharide synthesis in the bacteria.
87. The composition of paragraph 86, wherein the gene has at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to a polynucleotide having the nucleotide sequence of waaA, waaC, waaF, or waaG.
88. The composition of paragraph 69, wherein the bacterial colonization-disrupting agent is an inhibitor of bacterial cell wall biogenesis.
89. The composition of paragraph 88, wherein the inhibitor of bacterial cell wall biogenesis is an inhibitor of UppP.
90. The composition of paragraph 89, wherein the inhibitor of UppP is bacitracin.
91. The composition of paragraph 69, wherein the bacterial colonization-disrupting agent is an inhibitor of flagellar function.
92. The composition of paragraph 91, wherein the inhibitor of flagellar function is cellulose.
93. The composition of any one of paragraphs 69-92, wherein the bacterial colonization-disrupting agent is at least 0.1%, 0.2%, 0.4%, 0.5%, 0.8%, 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the composition.
94. The composition of any one of paragraphs 69-93, wherein the carrier is a liquid, a solid, an aerosol, a paste, a gel, or a gas composition.
95. The composition of any one of paragraphs 69-93, wherein the carrier is a sugar syrup, corn syrup, or honey.
96. The composition of any one of paragraphs 69-93, wherein the carrier is a nanoparticle or lipid membrane.
97. The composition of any one of paragraphs 69-96, wherein the composition is formulated for delivery to the insect by ingestion, infusion, injection, spraying, smoking, or fogging.
98. The composition of any one of paragraphs 69-97, wherein the composition is formulated for delivery to at least one habitat where the insect grows, lives, reproduces, or feeds.
99. The composition of any one of paragraphs 69-98, wherein the composition is formulated for delivery to a plant ingested by the insect.
100. A method for decreasing colonization by a bacterium of a gut of a stink bug, the method comprising:
101. The method of paragraph 100, wherein the composition is delivered to an egg mass of a stink bug.
102. The method of paragraph 100, wherein the decrease in colonization by the bacterium decreases the fitness of the stink bug.
103. The method of paragraph 102, wherein the decrease in the fitness of the stink bug is a decrease in reproductive ability, survival, rate of development, number of eggs, number of hatched eggs, adult emergence rate, body length, body width, body mass, or cuticle thickness.
104. The method of paragraph 100, wherein the colonization is in the v4 region of the gut.
105. The method of paragraph 104, wherein colonization by the bacterium of the v4 region of the gut is decreased by at least 10%.
106. The method of paragraph 104, wherein the size of the v4 region of the gut is decreased.
107. The method of paragraph 100, wherein the stink bug is a Halyomorpha species.
108. The method of paragraph 107, wherein the stink bug is Halyomorpha halys.
109. The method of paragraph 100, wherein the bacterium is an endosymbiont.
110. The method of paragraph 109, wherein the endosymbiont is of the genus Pantoea.
111. The method of paragraph 110, wherein the endosymbiont is Candidatus Pantoea carbekii.
112. The method of paragraph 100, wherein the composition is a liquid, a solid, an aerosol, a paste, a gel, or a gas composition.
113. The method of paragraph 100, wherein the composition is delivered as a spray.
114. The method of paragraph 100, wherein the composition comprises an agriculturally acceptable carrier.
115. The method of paragraph 100, wherein the composition comprises a wetting solution.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated in their entirety by reference.
This application claims benefit of U.S. Provisional Application No. 62/703,304, filed Jul. 25, 2018, the contents of which are incorporated herein by reference in their entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US19/43354 | 7/25/2019 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62703304 | Jul 2018 | US |
