Method and system for protecting honey bees from pesticides

Abstract

A method and system for the treatment of honey bees (Apis mellifera), bats, and butterflies protects them from various life threatening conditions, including Colony Collapse Disorder and in particular, provides honey bees with the ability to assimilate and degrade pesticides such as neonicotinoids and fipronil.

Claims

1. A method for providing a honey bee with the ability to assimilate pesticides, comprising, inoculating a honey bee with a culture of pesticide degrading bacteria, wherein the pesticide degrading bacteria include genes whose expression by the pesticide degrading bacteria results in the degradation of the pesticide, said pesticide degrading bacteria being modified to include said genes using a clustered regularly interspaced short palindromic repeats (CRISPR) CRISPR associated protein (Cas) system or using a clustered regularly interspaced short palindromic repeats (CRISPR) from Prevotella and Francisella 1 (Cpf1) nuclease, said pesticide degrading bacteria selected from the group consisting of L. rhamnosus and L. plantarum.

2. The method as set forth in claim 1, wherein said genes express cytochrome P450 enzymes.

3. The method as set forth in claim 1, wherein said genes comprise P450 genes of the CYP6 and CYP3 Glade.

4. The method as set forth in claim 1, wherein said inoculating comprises spraying a honey bee with said pesticide degrading bacteria.

5. The method as set forth in claim 1, wherein said inoculating comprises providing the pesticide degrading bacteria in a sweetened solution.

6. The method as set forth in claim 1, wherein the pesticide comprises a neonicotinoid insecticide.

7. The method as set forth in claim 1, further comprising employing a different CRISPR-Cas9/CRISPR-Cpf1 to ameliorate pathogens in the honey bee gut.

8. The method as set forth in claim 1, wherein said inoculating comprises providing the pesticide degrading bacteria in a sweetened solution or spraying a honey bee with said pesticide degrading bacteria.

9. The method as set forth in claim 6, wherein said genes express cytochrome P450 enzymes.

10. The method as set forth in claim 1, further comprising employing CRISPR-Cas9/CRISPR-Cpf1 to delete antibiotic resistance genes transferred to pathogenic bacteria in the honey bee gut.

11. The method as set forth in claim 2, further comprising employing a different CRISPR-Cas9/CRISPR-Cpf1 to ameliorate pathogens in the honey bee gut.

12. A method for providing a honey bee with the ability to assimilate pesticides, comprising, providing a honey bee with a culture of pesticide degrading bacteria, wherein the pesticide degrading bacteria include genes whose expression by the pesticide degrading bacteria results in the degradation of the pesticide, wherein said step of providing comprises using bacteriophage as a delivery vehicle for said genes, and wherein said pesticide degrading bacteria comprises L. rhamnosus.

13. The method as set forth in claim 12, wherein said genes express cytochrome P450 enzymes.

14. The method as set forth in claim 12, wherein said genes comprise P450 genes of one of the CYP6 and CYP3 clades.

15. The method as set forth in claim 12, wherein said genes are selected from the group consisting of a CYP353D1v2 gene and a SCL3-10 nitrile hydratase beta subunit gene.

16. The method as set forth in claim 12, further comprising employing CRISPR-Cas9/CRISPR-Cpf1 to delete antibiotic resistance genes transferred to pathogenic bacteria in the honey bee gut.

17. The method as set forth in claim 12, further comprising improving honey bee fitness by modifying the honey bee microbiome by using CRISPR-Cas9/CRISPR-Cpf1 to select and modify microbial communities to positively affect honey bee fitness.

18. The method as set forth in claim 12, wherein the pesticide degrading bacteria further comprises bacteria selected from the group consisting of: Lactobacillus spp, Bifidobacterium sp., Gilliamella apicola and Snodgrassella alvi.

19. The method as set forth in claim 16, further comprising employing a different CRISPR-Cas9/CRISPR-Cpf1 to ameliorate pathogens in the honey bee gut.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1 is a depiction of a honeybee, one of the major pollinators that is assisted by employing the method and system of the present invention.

(2) FIG. 2 is a depiction of a Monarch butterfly, which is also a pollinator that is assisted by employing the method and system of the present invention.

(3) FIG. 3 is a depiction of a bat, which is yet another pollinator that is assisted by employing the method and system of the present invention.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE PRESENT INVENTION

(4) Impacts to honey bees from sublethal exposure to imidacloprid in the presence of other stressors have been evaluated in laboratory studies and suggest that pesticides, such as imidacloprid, in combination with pathogens may impact colony health and immune function in honey bees. It is believed that several neonicotinoid insecticides are implicated in the decline of honey bee populations, including the following: imidacloprid. clothianidin, thiamethoxam, and dinotefuran.

(5) To provide necessary and sufficient written disclosure and enablement of the various embodiments of the present invention, the following references are incorporated by reference in their entireties: 20110269119 to Hutchinson, et al.; 20130064796 to Hamdi; 20140212520 to Del Vecchio, et al.; U.S. Pat. No. 9,017,718 to Tan; 20140065218 to Lang et al.; U.S. Pat. Nos. 6,599,883; 8,383,201; 5,158,789; 20070218114 to Sorousch; 20040136923 to Davidson; U.S. Pat. No. 8,999,372 to Davidson; 20090196907 to Bunick; 20090196908 to Lee; 20030124178 to Haley; 20070293587 to Haley; 20100285098 to Haley; 2006-0204591 to Burrell; U.S. Pat. No. 7,087,249 to Burrelll; U.S. Pat. No. 6,210,699 to Acharya; U.S. Pat. No. 8,865,211 to Tzannis; 20140199266 to Park; U.S. Pat. No. 6,599,883 to Romeo; PCT/US2008/080362 to Dussia; 2007-0218114 to Duggan; 2004-0136923 to Davidson; 20110142942 to Schobel; 20040120991 to Gardner et al.; Fuchs et al. U.S. Pat. No. 4,136,162; 20040136923 to Davidson; U.S. Pat. No. 4,163,777 to Mitra; U.S. Pat. No. 5,002,970 to Eby, III; 20040096569 to Barkalow et al.; 20060035008 to Virgallito et al.; 20030031737 to Rosenbloom; U.S. Pat. No. 6,919,373 to Lam et al.; 20050196358 to Georglades et al.; U.S. Pat. No. 3,832,460 to Kosti; 2002002057 to Battey et al.; 20040228804 to Jones, et al.; U.S. Pat. No. 6,054,143 to Jones; U.S. Pat. No. 5,719,196 to Uhari; 20150150792 to Klingman; 20140333003 to Allen; 20140271867 to Myers; 20140356460 to Lutin; 20150038594 to Borges; U.S. Pat. No. 6,139,861 to Friedman; 20150216917 to Jones; 20150361436 to Hitchcock; 20150353901 to Liu; U.S. Pat. No. 9,131,884 to Holmes; 20150064138 to Lu; 20150093473 to Barrangou; 20120027786 to Gupta; 20150166641 to Goodman; 20150352023 to Berg; 20150064138 to Lu; 20150329875 to Gregory; 20150329555 to Liras; 20140199281 to Henn; US20050100559 (proctor and Gamble); 20120142548 to Corsi et al.; U.S. Pat. Nos. 6,287,610, 6,569,474, US20020009520, US20030206995, US20070054008; and U.S. Pat. No. 8,349,313 to Smith; and U.S. Pat. No. 9,011,834 to McKenzie; 20080267933 to Ohlson et al.; 20120058094 to Blasser et al.; 8716327 to Zhao; 20110217368 to Prakash et al.; 20140044734 to Sverdlov et al.; 20140349405 to Sontheimer; 20140377278 to Elinav; 20140045744 to Gordon; 20130259834 to Klaenhammer; 20130157876 to Lynch; 20120276143 to O'Mahony; 20150064138 to Lu; 20090205083 to Gupta et al.; 20150132263 to Liu; and 20140068797 to Doudna; 20140255351 to Berstad et al.; 20150086581 to Li; PCT/US2014/036849; 20160348120 to Esvelt, et al., WO 2013026000 to Bryan and 20180020678 to Scharf et al. and Genomic signatures of honey bee association in an acetic acid symbiont, Smith et. al., bioRxiv preprint (Jul. 11, 2018).

(6) It is believed that honey bees are highly dependent on their hive-mates for acquisition of their normal gut bacteria. Each worker acquires a fully expanded, typical gut community before it leaves the hive. Different colonies may maintain distinct community profiles at the strain level and thus, biological variation among colonies results in part from variation in gut communities. Worker bees develop a characteristic core microbiota within hives. Some Gram-positive members of the core microbiota can be acquired through contact with hive surfaces. Gram-negative species, S. alvi, G. apicola, and F. perrara, appear to be acquired through contact with nurse bees or with fresh feces but not through oral trophallaxis. The eusocial honey bees and bumble bees harbor two specialized gut symbionts, Snodgrassella alvi and Gilliamella apicola, and these microorganisms are specific to bees, with different strains of these bacteria assorting to host species.

(7) Workers initially lack gut bacteria and gain large characteristic communities in the ileum and rectum within 4 to 6 days within hives. The core species of Gram-negative bacteria, Snodgrassella alvi, Gilliamella apicola, and Frischella perrara, are believed to be conveyed via nurses or hindgut material, whereas some Gram-positive species are often transferred through exposure to hive components. G. apicola and S. alvi are mutualistic symbionts with roles in both pathogen defense and nutrition. Their highly restricted distribution and phylogenetic correlation with their hosts are suggestive of a lengthy coevolutionary history with bees and with each other.

(8) Workers possess a consistent set of nine bacterial species that are observed in bees collected worldwide and that dominate their gut communities. Members of the core gut community include Snodgrassella alvi (Betaproteobacteria: Neisseriales) and Gilliamella apicola and Frischella perrara (Gammaproteobacteria: Orbales); three species of Alphaproteobacteria (“Alpha-1,” “Alpha-2.1,” and “Alpha-2.2” and three Gram-positive species (“Bifido,” corresponding to Bifidobacterium asteroides and “Firm-4” and “Firm-5” [both Firmicutes: Lactobacillaceae]. Discrete communities are found in different gut compartments: the crop and midgut contain very few bacteria, whereas hindgut compartments (ileum and rectum) house large communities with characteristic compositional profiles.

(9) Interestingly, queen gut microbiomes do not always reflect those of the workers who tend to them and often lack many of the bacteria that are considered to be “core” to workers. Worker gut microbiotas are relatively consistent across unrelated colony populations and the microbiotas of the related queens are highly variable. Queen bee microbiomes are dominated by enteric bacteria in early life but are comprised primarily of α-proteobacteria at maturity. Bacterial communities in mature queen guts were similar in size to those of mature workers and were characterized by dominant and specific α-proteobacterial strains known to be associated with worker hypopharyngeal glands. It is believed that queen guts are colonized by bacteria from workers' glands.

(10) Workers emerge from the pupal stage without the core gut bacteria and are fully colonized within several days postemergence. Early culture-based studies noted that bees removed from frames as pupae could remain free of gut bacteria through adulthood. During the pupal stage, the shedding of the integument and gut intima bars the carriage of gut microbes from the larval stage to the adult stage. Newly emerged A. mellifera workers (NEWs) are fed via oral trophallaxis by attendant nurse workers, consume bee bread, the fermented pollen food source stored within hives, and have many encounters with adult bees within the hive. Interactions with older bees as well as contact with the comb and bee bread are all potential inoculation routes for young workers.

(11) Honey bees (Apis spp.) and bumble bees (Bombus spp) possess a distinctive gut microbiota dominated by three groups, S. alvi, G. apicola, and Lactobacillus spp., and these form the majority of the gut community. From an evolutionary perspective, specialized gut bacteria represent a unique but ubiquitous form of symbiosis that has thus far escaped close scientific scrutiny. For the eusocial corbiculate bees, there appear to be at least 4 lineages of gut bacteria exhibiting host specificity: S. alvi, G. apicola, Lactobacillus spp, and Bifidobacterium spp. Gilliamella apicola and Snodgrassella alvi are dominant members of the honey bee (Apis spp.) and bumble bee (Bombus spp.) gut microbiota.

(12) Both G. apicola and S. alvi have relatively small genomes with reduced functional capabilities, which is consistent with their being specialized gut symbionts. S. alvi has lost the ability to use carbohydrates for carbon or energy: The glycolysis (Embden-Meyerhof-Parnas), pentose phosphate, and Entner-Doudoroff pathways needed to convert sugars to pyruvate are all missing key enzymes, and thus are predicted to be nonfunctional. This is surprising, considering that the bee diet consists mainly of carbohydrates. Instead, S. alvi possesses transporters for uptake of carboxylates, such as citrate, malate, α-ketoglutarate, and lactate. These can be used directly in the tricarboxylic acid cycle (TCA) cycle or, in the case of lactate, can be converted to pyruvate via lactate dehydrogenase. S. alvi is an obligate aerobe possessing NADH dehydrogenase and cytochrome bo and bd oxidases, but it lacks the TCA cycle enzyme succinyl-CoA synthetase, which catalyzes the interconversion of succinyl-CoA and succinate.

(13) In particular embodiments, the present invention is directed to a system and method used for the biological control of the welfare of bees, and for prophylaxis and treatment of pathological disorders of bees caused by insecticides, and especially neonicotinoids. In certain embodiments, bacteria are modified, preferably via the CRISP R-Cas system, and such bacteria are then provided to honey bees in a fashion such that they can reside in the gut of the honey bee, such bacteria selected from the group consisting of: S. alvi, G. apicola, Lactobacillus spp, and Bifidobacterium spp. Gilliamella apicola and Snodgrassella alvi. The CRISPR-Cas system is employed to enable such modified species to degrade neonicotinoids. CRISPR elements, another widespread system of phage defense, are abundant in G. apicola genomes and may act synergistically with restriction modification systems.

(14) In other embodiments, still other bacteria are introduced into a bee hive environment in a manner such that the bacteria are incorporated into the gut microbiota of at least worker bees or nurse bees, such that the colony can then acquire the ability to assimilate or degrade neonicotinoids that they are exposed to. In this regard, the following bacteria may be employed: Lactobacillus paracasei ssp., Bifidobacterium bifidum, Lactobacillus acidophilus, Lactococcus lactis, Bifidobacterium animalis, Lactobacillus thermophilus, and Bacillus clausii; Lactobacillus plantarum YML001, Lactobacillus plantarum YML004 and Leuconostoc citreum KM20; Ochrobactrum intermedium SCUEC4 strain, wherein the preservation number is CCTCC NO:M2014403; Ochrobactrum intermedium strain LMG3306. Particularly preferred microbes to employ, whether for extraction of their neonicotinoid genes for transplantation into the gut microbes of honey bees, or for the microbes inclusion as a microbe in the gut of honey bees, is a member of the genus ochrobactrum, in the alpha-2 subgroup of the domain Proteobacteria.

(15) Other embodiments of the present invention are directed to modification of the bat gut microbiome so as to enhance the health and survival of bats, e.g. by providing bats with bacteria that can degrade neonicotinoids. In doing so, the adverse effects of WNS can be ameliorated. There are over 190 species within the Phyllostomidae, the New World leaf-nosed bat family. These bats are found from southern USA and northern Mexico to Argentina and are the most ecologically diverse family within the order Chiroptera. They show an evolutionary diversification of dietary strategies from insectivory to diets that include blood, meat from small vertebrates, nectar, fruit and complex omnivorous mixtures. Microbiomes of these bat species include: Gamma-, Alpha-, and Delta-proteobacteria, Tenericutes, Firmicutes, Bacteroidetes, Planctomycetes, Cyanobacteria; Proteobacteria; Gammaproteobacteria; Enterobacteriales; Pasteurella; Deltaproteobacteria; Desulfurellales, Syntrophobacterales, Myxococcales; Rhodospirillales, Rhodobacterales, Rhizobiales, Rickettsiales; Firmicutes; Clostridia; Bacilli; and Cyanobacteria. One striking difference in microbiome composition between plant—(fruit and nectar) vs. animal-eating bats (insectivores and sanguivores) is the great abundance of Crenarchaeota in the later. Overall, archaea are more abundant in the insect and blood eating bats.

(16) The beneficial bacteria on the skin of bats provide vital functions, including processing of skin proteins, freeing fatty acids to reduce invasion of transient microorganisms, and inhibition of pathogenic microorganisms. While some probiotics have been contemplated in the biological control of disease in both aquaculture and agriculture, they have yet to be widely implemented in controlling wildlife disease.

(17) In preferred embodiments of the present invention, bacteria that naturally occur in the bat microbiota are used and even more preferably, certain strains that have been modified to enhance their effectiveness and survival on a bat's skin or gut environment, and/or modified to have particular antibiotic characteristics, are employed. Those that are able to colonize the bat's skin and/or gut are preferred. Augmentation prior to P. destructans exposure is preferably used, but in other embodiments, bacterial augmentation even after exposure to P. destructans can be used to displace such pathogen.

(18) One objective is to employ a strain of bacteria that can effectively persist on bat skin at high enough concentrations to limit P. destructans growth below levels that cause lethal disease.

(19) In certain embodiments, the group of bacteria used comprise Pseudomonas fluorescens, which is known to produce a suite of antifungal compounds that can inhibit many plant fungal pathogens as well as the amphibian fungal pathogens, Batrachochytrium dendrobatidis. Some strains in the P. fluorescens group are also capable of producing mycolysing enzymes that can colonize the mycelia and conidia of fungi rendering them no longer viable. Thus, this bacteria, whether wild type or modified as described herein, is employed in various methods and systems as set forth herein as a biological control agent for reducing infection intensity and increasing survival of bats exposed to P. destructans.

(20) Isolation of such antifungal bacteria can be obtained from the skin of bat species that appear to be better at surviving WNS, and thus, isolates from the skin of E. fuscus, which has lower mortality from WNS compared to other species, is preferably employed. In other embodiments, strains of P. fluorescens (PF3 and PF4) are used.

(21) Administration of effective bacteria that can beneficially assist bats in combating the ill effects of neonicotinoids can be achieved in many ways, including but not limited to spraying colonies of bats with bacterial solutions; providing such bacterial solutions in places where bats frequent in a manner that they will be exposed to the same; purposeful capture and inoculation of members of a colony such that they will be able to then spread the bacteria to other bats in a colony, effectively inoculating the entire colony.

(22) Certain aspects of the present invention are directed to employing genes from the microbe Ochrobactrum intermedium such that bats are able to assimilate and degrade neonicotinoids. Other embodiments employ inoculation of bats with a culture of neonicotinoid degrading bacteria, such as one or more of Ochrobactrum intermedium, Agrobacterium tumefaciens S33, Apergillus oryzae, Pseudomonas putida S16; Arthrobacter nicotinovarans, Microsporum gypseum, Pellicularia filamentosa JTS-208, Pseudomonas sp. 41; Microsporum gypseum; Pseudomonas ZUTSKD; Aspergillus oryzae 112822; and Ochrobactrum intermedium DN2. Preferably wherein the bacteria collection or culture comprises Ochrobactrum intermedium, collection number CGMCC NO. 8839 is used. The invention further provides applications of the Ochrobactrum intermedium to foster degradation of neonicotinoid insecticides by bats while the microbe exists in the gut or skin of the bats, the gut of honey bees and in butterflies.

(23) CRISPR systems may be employed to insert desired genes into the above mentioned (as well as others) microbes that inhabit the bat gut or skin so as to degrade particular insecticides, including neonicotinoids. The bat microbiome enhances host functions, contributing to host health and fitness. One aspect of the present invention is directed to improving bat fitness by modifying the bat microbiome, thus engineering evolved microbiomes with specific effects on the host bat fitness. Thus, by employing host-mediated microbiome selection, one is able to select and modify microbial communities indirectly through the host, thus influencing the bat microbiome and positively affecting bat fitness. The methods that may be used to impose artificial selection on the bat microbiome include various techniques known to those of skill in the art, including CRISPR-Cas and Cpl1 systems. Thus, while particular cultures of particular microbes can be purposefully included into bat populations so as to inhabit their gut or skin microbiome, and by doing so, providing the bats with the ability to degrade neonicotinoids, other embodiments are directed to engineering a modification of the bat gut or skin microbes that do not already possess such neonicotinoid degradation genes.

(24) In certain embodiments, xenobiotic detoxification is employed. In particular embodiments, the conversion of lipid-soluble substances to water-soluble, excretable metabolites is achieved. In a primary detoxification step, a toxin structure is enzymatically altered and rendered unable to interact with lipophilic target sites. Such functionalization is affected primarily by cytochrome P450 monooxygenases (P450) and carboxylesterases (CCE), although other enzymes, including flavin-dependent monooxygenases and cyclooxygenases may also be employed. Further reactions typically involve conjugation of products of the above referenced step to achieve detoxification for solubilization and transport. Glutathione-S-transferases (GST) are the principal enzymes used, although other enzymes may include glycosyltransferases, phosphotransferases, sulfotransferases, aminotransferases, and glycosidases. Nucleophilic compounds can be rendered hydrophilic by UDP-glycosyltransferases. The final stage of detoxification involves transport of conjugates out of cells for excretion. Among the proteins involved in this process are multidrug resistance proteins and other ATP-binding cassette transporters.

(25) In particular embodiments, the present invention is directed to a system and method used for the biological control of the welfare of bats, and for prophylaxis and treatment of pathological disorders of bats caused by insecticides, and especially neonicotinoids. In certain embodiments, bacteria are modified, preferably via the CRISPR-Cas system, and such bacteria are then provided to bats in a fashion such that they can reside in the gut or skin of the bats, such bacteria selected from the group consisting of: Gamma-, Alpha-, and Delta-proteobacteria, Tenericutes, Firmicutes, Bacteroidetes, Planctomycetes, Cyanobacteria; Proteobacteria; Gammaproteobacteria; Enterobacteriales; Pasteurella; Deltaproteobacteria; Desulfurellales, Syntrophobacterales, Myxococcales; Rhodospirillales, Rhodobacterales, Rhizobiales, Rickettsiales; Firmicutes; Clostridia; Bacilli; and Cyanobacteria.

(26) In other embodiments, still other bacteria are introduced into a bat colony environment in a manner such that the bacteria are incorporated into the gut and/or skin microbiota of at least some bats, which can then “infect” other bats with such bacteria, and in so doing, the colony of bats can then acquire the ability to assimilate or degrade neonicotinoids that it is exposed to. In this regard, the following bacteria may be employed: Lactobacillus paracasei ssp., Bifidobacterium bifidum, Lactobacillus acidophilus, Lactococcus lactis, Bifidobacterium animalis, Lactobacillus thermophilus, and Bacillus clausii; Lactobacillus plantarum YML001, Lactobacillus plantarum YML004 and Leuconostoc citreum KM20; Ochrobactrum intermedium SCUEC4 strain, wherein the preservation number is CCTCC NO:M2014403; Ochrobactrum intermedium strain LMG3306. Particularly preferred microbes to employ, whether for extraction of their neonicotinoid genes for transplantation into the gut and/or skin microbes of bats, or for the microbes inclusion as a microbe in the gut or skin of bats, is a member of the genus ochrobactrum, in the alpha-2 subgroup of the domain Proteobacteria.

(27) While not bound by theory, it is believed that still other microbes may be employed in various embodiments of the present invention to address the objective of degrading neonicotinoid-like compounds, such bacteria showing an ability to degrade nicotine. The CRISPR-Cas system is employed to enable such modified species to degrade neonicotinoids. Thus, such system can be used to provide gut or skin bacteria that may grow on the bat skin or gut and can include genes that achieve desired degradation of neonicotinoids via the use of or presence of such genes in nicotine degrading organisms, such as Agrobacterium tumefaciens S33, Apergillus oryzae, Pseudomonas putida S16; Arthrobacter nicotinovarans, Microsporum gypseum, Pellicularia filamentosa JTS-208, Pseudomonas sp. 41; Microsporum gypseum; Pseudomonas ZUTSKD; Aspergillus oryzae 112822; and Ochrobactrum intermedium DN2.

(28) Microbial symbionts are important for host organisms, and insects rely on the communities of microorganisms in their guts for several functions. Hosts have evolved a range of mechanisms to protect themselves against parasites that are a large threat to their fitness. These defenses can extend beyond intrinsic host immunity and incorporate aspects of the environment in which host and parasite interact. Monarch butterfly (Danaus plexippus) larvae actively consume milkweeds (Asclepias spp.) that contain secondary chemical compounds, named cardenolides, which reduce parasite infection and virulence.

(29) Commensalibacter is a genus of acetic acid bacteria and 16S rRNA gene sequences related to the Commensalibacter genus have been recovered from the guts of Drosophila species, honey bees, and bumble bees, as well as from Heliconius erato butterflies. The type strain Commensalibacter intestini A911 was isolated from Drosophila intestines, and the genome sequence of a Commensalibacter symbiont isolated from a monarch butterfly has been reported. Commensalibacter papalotli strain MX01, was isolated from the intestines of an overwintering monarch butterfly. The 2,332,652-bp AT-biased genome of C. papalotli MX01 is the smallest genome for a member of the Acetobacteraceae.

(30) In certain embodiments, Commensalibacter bacteria are modified to render them able to degrade neonicotinoid insecticides and such bacteria are then purposefully provided to the gut biome of monarch butterflies to enable the butterflies to degrade such pesticides, and thus survive and remain viable for reproduction. In a particular embodiment, the genes responsible for the ability to degrade neonicotinoids are derived from the Ochrobactrum intermedium SCUEC4 strain, wherein the preservation number is CCTCC NO:M2014403; and/or Ochrobactrum intermedium strain LMG3306.

(31) CRISPR systems may be employed to insert desired genes into various bacteria that can survive in the gut of the monarch butterfly such that these microbes can degrade particular insecticides, including neonicotinoids.

(32) One aspect of the present invention is directed to improving monarch butterfly fitness by modifying the monarch butterfly microbiome, either by incorporation of select species of bacteria into existing gut microbiomes of the monarch butterfly, or by incorporation of genetic elements into existing bacteria within a monarch butterfly's gut such that the modified microbe is able to degrade neonicotinoids. These engineered microbiomes are purposefully designed to have with specific beneficial effects on the host monarch butterfly fitness. Thus, by employing host-mediated microbiome selection, one is able to select and modify microbial communities indirectly through the host, thus influencing the monarch butterfly microbiome and positively affecting monarch butterfly fitness. The methods that may be used to impose artificial selection on the monarch butterfly microbiome include various techniques known to those of skill in the art, including CRISPR-Cas and Cpl1 systems. Thus, particular cultures of particular microbes can be purposefully included into monarch butterfly populations so as to inhabit their gut microbiome, and by doing so, provide the monarch butterflies with the ability to degrade neonicotinoids. Other embodiments are directed to engineering a modification of the monarch butterfly gut microbes that do not already possess such neonicotinoid degradation genes.

(33) Detoxification gene inventory reduction may reflect an evolutionary history of consuming relatively chemically benign nectar and pollen. Thus, certain embodiments are directed to the development of predictable microbiome-based biocontrol strategies by providing the ability of monarch butterflies to degrade or otherwise assimilate insecticides or other chemical agents, including neonicotinoids. Such a novel biocontrol strategy can not only be used to suppress pathogens, but can also be effectively used to establish microbiomes in a desirable beneficial composition for particular purposes.

(34) In certain embodiments, xenobiotic detoxification is employed to address the problems associated with monarch butterfly health. In particular embodiments, the conversion of lipid-soluble substances to water-soluble, excretable metabolites is achieved. In a primary detoxification step, a toxin structure is enzymatically altered and rendered unable to interact with lipophilic target sites. Such functionalization is affected primarily by cytochrome P450 monooxygenases (P450) and carboxylesterases (CCE), although other enzymes, including flavin-dependent monooxygenases and cyclooxygenases may also be employed. Further reactions typically involve conjugation of products of the above referenced step to achieve detoxification for solubilization and transport. Glutathione-S-transferases (GST) are the principal enzymes used, although other enzymes in insects may include glycosyltransferases, phosphotransferases, sulfotransferases, aminotransferases, and glycosidases. Nucleophilic compounds can be rendered hydrophilic by UDP-glycosyltransferases. The final stage of detoxification involves transport of conjugates out of cells for excretion. Among the proteins involved in this process are multidrug resistance proteins and other ATP-binding cassette transporters.

(35) Any one or more of appropriate bacteria can be modified to express particular genes that have been shown (for example by its inclusion in the bacteria Ochrobactrum intermedium) to degrade neonicotinoids in a manner that preserves monarch butterfly health. One of skill in the art can address compatibility issues with respect to the use of such bacteria and can make modifications thereto to render it tolerable and viable in the gut microbiome of the monarch butterfly. Genetic modification of existing microbes in the monarch butterfly gut can also be performed to render such native bacteria able to produce agents effective in degrading neonicotinoids.

(36) Impacts to monarch butterflies from sublethal exposure to neonicotinoids, such as imidacloprid, especially in the presence of other stressors, is believed to result in severe and significant dysfunctions within and have an adverse impact on monarch colony health and to the immune function in individual monarch butterflies. It is believed that several neonicotinoid insecticides are implicated in the decline of monarch butterfly populations, including the following: imidacloprid. clothianidin, thiamethoxam, and dinotefuran.

(37) In particular embodiments, the present invention is directed to a system and method used for the biological control of the welfare of monarch butterflies, and for prophylaxis and treatment of pathological disorders of monarch butterflies caused by insecticides, and especially neonicotinoids. In certain embodiments, bacteria are modified, preferably via the CRISPR-Cas system, and such bacteria are then provided to monarch butterflies in a fashion such that they can reside in the gut of the monarch butterfly, such bacteria selected from the group consisting of one or more bacteria in six bacterial families: the Acetobacteraceae (Alphaproteobacteria), Moraxellaceae and Enterobacteriaceae (Gammaproteobacteria), Enterococcaceae and Streptococcaceae (Firmicutes), and an unclassified family in the Bacteroidetes phylum. In particular embodiments, the bacteria employed in the present invention include one or more of the following: Commensalibacter, and in particular Commensalibacter intestini A911 and Commensalibacter papalotli strain MX01; Lactobacillus paracasei ssp., Bifidobacterium bifidum, Lactobacillus acidophilus, Lactococcus lactis, Bifidobacterium animalis, Lactobacillus thermophilus, and Bacillus clausii; Lactobacillus plantarum YML001, Lactobacillus plantarum YML004 and Leuconostoc citreum KM20; Ochrobactrum intermedium SCUEC4 strain, wherein the preservation number is CCTCC NO:M2014403; Ochrobactrum intermedium strain LMG3306. Particularly preferred microbes to employ, whether for extraction of their neonicotinoid genes for transplantation into the gut microbes of monarch butterflies, or for the microbes inclusion as a microbe in the gut of monarch butterflies, is a member of the genus ochrobactrum, in the alpha-2 subgroup of the domain Proteobacteria.

(38) Modifying the gut microbiota of monarch butterflies to provide them with the ability to consume chemically defended plants can thus be achieved by varying the monarch butterflies associated microbial communities. Different microbes may be differentially able to detoxify compounds toxic to the monarch or may be differentially resistant to the potential antimicrobial effects of some compounds.

(39) In certain embodiments, the administration of effective bacteria that can beneficially assist monarch butterflies in combating the ill effects of neonicotinoids can be achieved in many ways, including but not limited to spraying colonies of butterflies or caterpillars that will emerge as butterflies with bacterial solutions; providing such bacterial solutions in places where monarch butterflies frequent in a manner that they will be exposed to the same, such as by having bacteria provide in sweetened solutions that monarch butterflies are drawn to; purposeful capture and inoculation of members of a colony such that they will be able to then spread the bacteria to other bats in a colony, effectively inoculating an entire colony of monarch butterflies.

(40) As one of skill in the art of living will appreciate, the reason to preserve nature is not merely within the realm of science, as beauty and literature are rooted in those creatures who share this earth with us. The philosophers of the past recognized this fact. Nietzsche once said: “And to me also, who appreciate life, the butterflies, and soap-bubbles, and whatever is like them amongst us, seem most to enjoy happiness.” Our treasure lies in the beehive of our knowledge. We are perpetually on the way thither, being by nature winged insects and honey gatherers of the mind.” Aristotle chimed in: “As the eyes of bats are to the blaze of day, so is the reason in our soul to the things which are by nature most evident of all.” Karl von Frisch said “Nature has unlimited time in which to travel along tortuous paths to an unknown destination. The mind of man is too feeble to discern whence or whither the path runs and has to be content if it can discern only portions of the track, however small.” So as Nabokov suggested, “do what only a true artist can do . . . pounce upon the forgotten butterfly of revelation.” “Literature and butterflies are the two sweetest passions known to man.”

(41) While specific embodiments and applications of the present invention have been described, it is to be understood that the invention is not limited to the precise configuration and components disclosed herein. Various modifications, changes, and variations which will be apparent to those skilled in the art may be made in the arrangement, operation, and details of the methods and systems of the present invention disclosed herein without departing from the spirit and scope of the invention. Those skilled in the art will appreciate that the conception, upon which this disclosure is based, may readily be utilized as a basis for designing of other methods and systems for carrying out the several purposes of the present invention to instruct and encourage the prevention and treatment of various human diseases. It is important, therefore, that the claims be regarded as including any such equivalent construction insofar as they do not depart from the spirit and scope of the present invention.