Method and System for Protecting Monarch Butterflies from Pesticides

Abstract

A method and system for the treatment of Monarch butterflies (Danaus plexippus Kluk (Lepidoptera: Nymphalidae) to protect them from various life-threatening conditions, including the negative effects of various pesticides, provides Monarch butterflies with the ability to assimilate and degrade pesticides such as neonicotinoids and fipronil. Certain embodiments involve the inoculation of flowers by honey bees with desired bacteria that are able to degrade pesticides, such that when Monarch butterflies visit such flowers, they are exposed to such bacteria, transforming the microbiome of the Monarch butterflies so that pesticides can be degraded, thus enhancing the health of the Monarch butterflies.

Claims

1. A method for providing a Monarch butterfly with the ability to assimilate pesticides, comprising, inoculating a Monarch butterfly 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 clade.

4. The method as set forth in claim 1, wherein said inoculating comprises spraying a Monarch butterfly 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 Monarch butterfly gut.

8. A method for providing a Monarch butterfly with the ability to assimilate pesticides, comprising, inoculating a Monarch butterfly 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 selected from the group consisting of L. rhamnosus and L. plantarum.

9. The method as set forth in claim 8, wherein said inoculating comprises one of providing the pesticide degrading bacteria in a sweetened solution, spraying a Monarch butterfly with said pesticide degrading bacteria, and having a honey bee inoculate a flower with the pesticide degrading bacteria.

10. The method as set forth in claim 8, wherein said pesticide degrading bacteria is 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, and wherein said genes express cytochrome P450 enzymes.

11. The method as set forth in claim 8, further comprising employing CRISPR-Cas9/CRISPR-Cpf1 to delete antibiotic resistance genes transferred to pathogenic bacteria in the Monarch butterfly gut.

12. The method as set forth in claim 8, further comprising employing a different CRISPR-Cas9/CRISPR-Cpf1 to ameliorate pathogens in the Monarch butterfly gut.

13. A method for providing a Monarch butterfly with the ability to assimilate pesticides, comprising, providing a Monarch butterfly 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 selected from the group consisting of L. rhamnosus, Commensalibacter intestine, Commensalibacter papalotli, Lactobacillus paracasei, Bifidobacterium bifidum, Lactobacillus acidophilus, Lactococcus lactis, Bifidobacterium animalis, Lactobacillus thermophilus, Bacillus clausii; Lactobacillus plantarum, Leuconostoc citreum and Ochrobactrum intermedium.

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

15. The method as set forth in claim 13, wherein said genes comprise P450 genes of one of the CYP6 and CYP3 clades.

16. The method as set forth in claim 13, wherein said genes are selected from the group consisting of a CYP353D1v2 gene and a SCL3-10 nitrile hydratase beta subunit gene.

17. The method as set forth in claim 13, further comprising employing CRISPR-Cas9/CRISPR-Cpf1 to delete antibiotic resistance genes transferred to pathogenic bacteria in the Monarch butterfly gut.

18. The method as set forth in claim 13, further comprising improving Monarch butterfly fitness by modifying a Monarch butterfly microbiome by using CRISPR-Cas9/CRISPR-Cpf1 to select and modify microbial communities to positively affect Monarch butterfly fitness.

19. The method as set forth in claim 13, wherein said pesticide degrading bacteria comprises L. rhamnosus.

20. The method as set forth in claim 13, further comprising employing a different CRISPR-Cas9/CRISPR-Cpf1 to ameliorate pathogens in the Monarch butterfly gut.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0037] FIG. 1 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.

[0038] FIG. 2 shows the transformation of the monarch caterpillar from its chrysalis stage to emerge as a mature butterfly.

[0039] FIG. 3 shows a Monarch caterpillar feeding on milkweed.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE PRESENT INVENTION

[0040] It is believed that several neonicotinoid insecticides are implicated in the decline of Monarch butterfly populations, including the following: imidacloprid. clothianidin, thiamethoxam, and dinotefuran.

[0041] 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 Duggan; 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; 20040120991to 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.; U.S. Pat. No. 8,716,327 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).

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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. 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. 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.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] 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 (Gamma proteobacteria), 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.

[0051] 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.

[0052] 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.

[0053] 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.”

[0054] 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.