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WO2011057825A1 - Induction de l'expression génique chez des arthropodes - Google Patents

Induction de l'expression génique chez des arthropodes Download PDF

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Publication number
WO2011057825A1
WO2011057825A1 PCT/EP2010/006982 EP2010006982W WO2011057825A1 WO 2011057825 A1 WO2011057825 A1 WO 2011057825A1 EP 2010006982 W EP2010006982 W EP 2010006982W WO 2011057825 A1 WO2011057825 A1 WO 2011057825A1
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WIPO (PCT)
Prior art keywords
dna
gene
promoter
arthropod
insect
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English (en)
Inventor
Gustavo Rodrigues Markert Dos Santos
Nicolas Delaroque
Michael Szardenings
Sebastian Ulbert
Matthias Giese
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Fraunhofer Gesellschaft zur Foerderung der Angewandten Forschung eV
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Fraunhofer Gesellschaft zur Foerderung der Angewandten Forschung eV
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Priority claimed from DE102009054265A external-priority patent/DE102009054265A1/de
Priority claimed from EP10001476A external-priority patent/EP2356901A1/fr
Application filed by Fraunhofer Gesellschaft zur Foerderung der Angewandten Forschung eV filed Critical Fraunhofer Gesellschaft zur Foerderung der Angewandten Forschung eV
Publication of WO2011057825A1 publication Critical patent/WO2011057825A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/60—New or modified breeds of invertebrates
    • A01K67/61—Genetically modified invertebrates, e.g. transgenic or polyploid
    • A01K67/65—Genetically modified arthropods
    • A01K67/68—Genetically modified insects
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/60—New or modified breeds of invertebrates
    • A01K67/61—Genetically modified invertebrates, e.g. transgenic or polyploid
    • A01K67/65—Genetically modified arthropods
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0075—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the delivery route, e.g. oral, subcutaneous
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/05—Animals modified by non-integrating nucleic acids, e.g. antisense, RNAi, morpholino, episomal vector, for non-therapeutic purpose
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/25—Animals on a special diet
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/70—Invertebrates
    • A01K2227/706—Insects, e.g. Drosophila melanogaster, medfly
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/02—Animal zootechnically ameliorated
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2999/00—Further aspects of viruses or vectors not covered by groups C12N2710/00 - C12N2796/00 or C12N2800/00
    • C12N2999/007—Technological advancements, e.g. new system for producing known virus, cre-lox system for production of transgenic animals

Definitions

  • the present invention relates to the induction of gene expression in arthropods in vivo, especially to the induction of protein expression in insects in vivo. Further, the present invention relates to methods, uses and solutions to introduce heterologous nucleic acids into insects. In particular, the present invention discloses that DNA may be introduced into insects via an oral route, may be transcribed there and may also be retrieved in the sanguivorous (ecto- and endo-)parasites of the insects.
  • Arthropods are a highly diverse group of animals comprising about 80% of all described living animal species.
  • the most important classes of arthropods are hexapoda (which include insects), chelicerata (including spiders, mites and scorpions), Crustacea and myriapoda (Brusca and Brusca 2003).
  • An arthropod may be a pest in agriculture, an ectoparasite on animals, including human, an endoparasite in animals, including human, an epidemiologic vector, a pest destroying property by feeding or contamination or a pest endangering ecological balance.
  • an arthropod may serve as native or agricultural animal feed, as nutrition for humans, as factor to balance an ecosystem or as a source of material susceptible of the industrial utilisation.
  • insect cell cultures e.g. SF-9 or SF-21 cell cultures
  • the production of a gene encoded molecule in an arthropod in vivo is still hampered by the problem to incorporate the required nucleic acid in the cells of said arthropod.
  • Arthropods bear an impermeable barrier, in form of a chitin shield, all over their body surface and are therefore extensively protected from exogenous influences and extensively isolated from the molecular environment. Therefore, the application of a nucleic acid by methods regularly used in the art, such as the use of gene guns (Tang et al 1992), may fail or may be complicated. Furthermore, it is well-known in the art that the use of a nucleic acid, in particular the use of DNA and/or RNA, in vivo does mostly not lead to the desired result. It is well-known that an aforementioned nucleic acid is highly ineffective in in vivo applications in nearly all animal species (Wahren and Liu 2005). It is a common prejudice that this is also true for the use of nucleic acids in living arthropods.
  • Said efficient and simple method for inducing gene expression in arthropods in vivo may be used to treat arthropodes suffering from a disease or disorder. Or, said efficient and simple method for inducing gene expression in arthropods in vivo may be used to prevent a disorder or disease in arthropodes.
  • Varroa mite Varroa destructor
  • the mite pest is accompanied by the infection of various pathogenic agents.
  • Some of the strains of this mite have recently developed some resistance against certain active agents that were still effective until now. For this reason, the alarming situation must be approached in an alternative way.
  • dsRNA constructs are attributed to a particular transmembrane protein in the bee that is known to enable to translocate dsRNA to the interior of a cell (Aronstein et al., Insect Biochem Mol Biol (2006); 36, 683-693).
  • the manufacture and the handling of dsRNA is difficult as it can be degraded easily.
  • the present invention relates to a method for the production of a gene encoded molecule in an arthropod in vivo, wherein a nucleic acid comprising a gene encoding said molecule is fed to the arthropod.
  • a nucleic acid comprising a gene encoding said molecule is fed to the arthropod.
  • the expressed gene product is also found in an ectoparasite living on the arthropod.
  • the gene products that originate from a DNA taken up by an insect can also be detected in parasites that live on these insects.
  • a very easy method is created to deliver DNA in insects and insect cells of living entire insects, respectively, for expression.
  • it is feasible to deliver DNA in insects for therapeutic purposes on the other hand, it is feasible to harm or to kill vermins by DNA that is harmful for them and by gene products that are encoded by this DNA.
  • the DNA upon oral uptake by the insects, the DNA is translocated into the interior of insect cells. Subsequently, the DNA may be read by the transcription apparatus.
  • the nucleic acid may also be administered orally to the arthropod.
  • Therapeutic affects may be achieved in that the delivered DNA encodes for RNAi constructs that specifically degrade the RNA of pathogenic agents.
  • the DNA may encode for immune stimulant molecules that activate or strengthen the innate immune system.
  • Vermins are understood as all insects that provoke harms to agriculture, forestry, stockpiling or in buildings. Vermins are also ("aus") selected from the group consisting of codling moth, aphid, thrips, summer fruit tortrix, potato beetle, cherry fruit fly, Melolontha, European Corn Borer, Rhododendron leafhopper, turnip moth, scale insects, Gypsy moth, Spider mite, (European Grape)Vine moth, Whitefly, Phaenops cyanea, bark- beetle, oak splendor beetle, oak processiffy, European green oak moth, Cephalcia abietis, common furniture beetle, Diprion pini, pine beauty, Bordered White, Pristiphora abietina, black arches, horse chestnut leaf miner, Asian gypsy moth, Trogoxylon impressum, German cockroach, sawtoothed grain beetle, common clothes moth, granary weevils
  • Heterologous DNA is understood as a DNA that does not naturally occure in an insect.
  • the term “gene encoded molecule” refers to all molecules and combinations of molecules that may be generated by gene expression. Particularly, said term includes, but is not limited to a protein, or an RNA. According to the invention, the term “gene encoded molecule” does not include a siRNA or an antisense RNA or DNA.
  • the gene encoded molecule is a protein or a biologically active RNA.
  • biologically active denotes that the given molecule has a biological function.
  • said biologically active RNA is selected from the group consisting of: a. tRNA, b. rRNA,
  • RNA double stranded RNA. These molecules are known in the art.
  • the RNA is a catalytic RNA capable of catalyzing chemical reactions.
  • the gene encoded molecule is a protein.
  • protein includes polypeptides composed of a consecutive sequence of amino acid moieties of all lengths including short peptides, protein domains, high-molecular weight proteins and proteins comprising independent similar or different subunits.
  • the protein may be a protein naturally occurring in arthropods or may be a foreign protein, i.e. a protein which naturally does not occur in arthropods.
  • the gene encoded molecule is controlling the rate of the production of other molecules of interest in the arthropod.
  • the gene encoded molecule may be a protein, e.g. a hormone or a transcription factor, which induces the production of other proteins in the arthropod.
  • the production of the gene encoded molecule is induced by feeding to the arthropod a nucleic acid comprising a gene encoding said gene encoded molecule.
  • the term "gene” refers to a DNA or RNA, preferably a DNA encoding said molecule.
  • the “gene” may be a naturally occurring gene or a part thereof.
  • Said gene is according to the invention comprised in a nucleic acid.
  • This nucleic acid is either a DNA or an RNA, depending on the gene.
  • the nucleic acid is a DNA.
  • DNA is understood as double-stranded DNA that was obtained from a genetic engineering method, for example, by production in monocellular pro- or eukaryotes, such as E. coli or yeast.
  • DNA that is administered orally to insects in the sense of the present invention is in particular not DNA that does originate from the natural feed of the insects.
  • a therapeutic gene is understood as a gene that either encodes for a protein that deploys a therapeutic effect, or that encodes for an RNA that deploys a therapeutic effect in the desired insect.
  • RNA may be an RNAi construct that leads to degradation of the target RNA.
  • the target RNA may belong to an organism that harms the insect.
  • the target RNA may also be part of the insect.
  • DNA is understood as double-stranded deoxyribonucleic acid molecules. These may be linear or circular.
  • DNA may exist as circular plasmid.
  • the plasmids may be amplified in bacteria. The plasmids may be designed in such way that, upon induction, they loose the fractions that are necessary for the amplification in bacteria or other host organisms (minicircle principle, EP 815 214). This would have the advantage that, for example, no antibiotic resistance or "origins of replication" (origins of replication) are introduced in the insects/bees.
  • RNAi construct is understood as a double-stranded RNA that works in accordance with the principle of an interfering RNA. It is known in the state of the art how RNAi constructs have to be designed that degrade target RNA (Voorhoeve et al. (2003). "Knockdown stands up”. Trends Biotechnol.
  • RNAi construct may encode for a dsRNA (double- stranded RNA) against IAPV ("Israeli acute paralysis virus"), as described in the paragraph that is located at the end of the first column and at the beginning of the second column on page 59 of the publication, respectively, of Maori et al. (Insect Molecular Biology (2009); 18 (1 ), 55-60).
  • a toxic gene is understood as a gene that either encodes for a protein deploying a toxic effect in a desire insect, or that encodes for an RNA deploying a toxic effect in a designated insect.
  • Such an RNA may be an RNAi construct that leads to the degradation of a target RNA, for example, an RNA that is vital in the desired insect.
  • Proteins toxic for insects are, particularly, insect toxins produced by insect viruses, spiders, scorpions and insects themselves, for example, the "ecdysteroid UDP-glucosyltransferase” (EGT) from A tographa californica nucleopolyhedrovirus (AcNPV), and the scorpion anti-insect neurotoxin AalT ("Androctonus australis anti-insect toxin", see “Rapid purification and molecular modeling of AalT peptides from venom of Androctonus australis”; Nakagawa Y et al., Arch Insect Biochem Physiol. 1998;38(2):53-65).
  • ETT ecdysteroid UDP-glucosyltransferase
  • AcNPV A tographa californica nucleopolyhedrovirus
  • scorpion anti-insect neurotoxin AalT scorpion anti-insect neurotoxin Aal
  • said nucleic acid is in the form of a purified nucleic acid, is contained in a bacterial cell, or an archeal cell, an eukaryotic cell or virus or virus-like particles or is complexed with a transfecting agent.
  • the DNA may not be included in a bacterial vector or viral vector, in particular not included in a viral vector, more in particular not included in baculoviral or in a Sindbis viral vector.
  • the abnegation of a viral vector may have the advantage that the difficult procedural step(s) relating to molecular cloning of the desired DNA into the viral genome can be dispensed.
  • viral vectors often tend to influence the systems they are applied to at their own.
  • a viral vector may influence the insect, in particular the health of the insect and possibly its span of life.
  • a viral vector may bear the risk of developing increased infectivity upon occurrence of certain mutations.
  • the Sindbis virus is known to cause Sindbis fever, encephalitis in animals including humans. Thus, its use may be avoided in a preferred embodiment of the present invention.
  • a purified nucleic acid or a nucleic acid complexed by non-viral and non-bacterial molecules is preferred, even more preferred a purified nucleic acid.
  • the term "purified nucleic acid” means that the nucleic acid has been purified, e.g. extracted, from the context where it has been produced, preferably a cell or a tissue, more preferably a bacterial cell.
  • Methods for the purification of nucleic acids are known in the art, e.g. from Sambrook and Russel, 2001.
  • the nucleic acid may still be included in the cell where it has been produced, e.g. a bacterial or archeal cell or a eukaryotic cell. This means that the cell itself is fed to the arthropod.
  • the nucleic acid may be contained in a virus (e.g a baculovirus, an adenovirus, a vaccinia virus), a bacteriophage or a virus-like particle.
  • the nucleic acid may further be contained in a transfecting agent.
  • transfecting agent refers to molecular compositions that improve the cellular and systemic uptake of a nucleic acid.
  • Said molecular compositions comprise but are not limited to liposome compositions and complexes and conjugates with fatty acids, synthetic polymers (in particular positively charged or hydrophobic polymers, e.g. polyethylene imine (PE1) and derivatives thereof), polysaccharides (e.g. chitosan), virus-like particles, proteins (in particular DNA- or RNA-interacting proteins and proteins comprising cell- penetrating peptides (CPPs)), protein transduction domains (PTDs) or antimicrobial peptides).
  • synthetic polymers in particular positively charged or hydrophobic polymers, e.g. polyethylene imine (PE1) and derivatives thereof
  • polysaccharides e.g. chitosan
  • virus-like particles e.g. chitosan
  • proteins in
  • Said nucleic acid may preferably be either (i) plasmid DNA, (ii) linear DNA, (iii) circular DNA, (iv) single stranded DNA, (v) RNA, (vi) non natural DNA like molecules or (vii) a hybrid formed out of any of these molecules.
  • plasmid DNA linear DNA
  • iii circular DNA
  • iv single stranded DNA
  • RNA RNA
  • non natural DNA like molecules RNA
  • a hybrid formed out of any of these molecules are known in the art (see Sambrook and Russel, 2001).
  • commercially available plasmids can be used in the method of the invention, wherein the gene of interest is then cloned into said commercially available plasmid.
  • the nucleic acid may contain further nucleic acid sequences, especially regulatory sequences like promoters or enhancers. Promoter and enhancer sequences are known in the art. Since the feeding of the nucleic acid results in the production of the gene encoded molecule, it is particularly preferred that the nucleic acid further contains a promoter active in said arthropod.
  • a promoter may be a promoter which is active in a broad range of cells including e.g. the CMV promoter, or the TK promoter.
  • the promoter may be an arthropod, especially an insect specific promoter.
  • examples include the WSSViel promoter, a virus promoter from white spots syndrome virus which infest arthropods (Liu et al 2007), the B. mori (Bm) actin A3 promoter, the Apis m Actin promoter, and the Drosophila Hsp70 promoter.
  • a promoter is understood as a promoter that enables the transcription of DNA sequences in insects.
  • insect promoters such as the polyhedrin promoter or the "White spot syndrome virus (WSSV) immediate-early promoter one (iel)" (WSSVIE1, see sequence in figure 18) or also promoters are included that are, for instance, responsible for the initiation of the transcription of strongly expressed genes, in particular, housekeeping genes in insects (promoters for GADPH, actin and others).
  • the particular promoter sequence will depend on the insect in which the gene product is to be expressed.
  • a number of DNAs or plasmids could be cloned that present a combination of different promoters having the gene sequence encoding for the desired gene product or a reporter such as GFP.
  • these different promoter/gene product combinations may be screened. For example, then, these combinations may be fed to insects as alimentary additive, i.e., in a solution or as solid matter, as described in the examples.
  • the expression of the gene product or reporter then shows that the promoter is active in the insect, i.e., that it initiates the transcription.
  • such promoters are considered that are species specific, i.e., initiate the transcription only in the desired species, genus, family or order of insects.
  • non-insect promoters are included, such as promoters of viruses that are specific for mammals or monkeys.
  • the promoters of polyoma viruses such as SV40 or the CMV promoter (see the sequence of the CMV promoter in figure 18) are included.
  • Further promoters that are active in insects may be determined by the above- mentioned tests specific for insect promoters.
  • Promoters are functionally linked with those encoding sequences of which they initiate transcription.
  • the encoding sequences may encode for R Ai constructs or proteins.
  • Enhancers may enhance the transcription initiation of a promoter, while they are not directly attached to the sequence of the promoter or the encoding sequence. See the sequence of the SV40 enhancer in figures 1 7 and 18. Enhancers may be located far away or even on another DNA. Preferably, they are located on the same DNA as the promoter and the encoding sequence and they, their 3'end, are 50-150 nucleotides, 75-125 nucleotides, 90-120 nucleotides, or 1 10-1 1 5 nucleotides 5' of the beginning of the promoter.
  • the promoter may be functionally linked with a gene to be expressed.
  • the method of the present invention can be applied to all arthropods.
  • the arthropod may be but is not limited to an insect, an arachnid, a crustacean and a myriapodum.
  • the arthropod is an insect, in more preferred embodiments, the insect is a holometabolic insect or a hemimetabolic insect or the insect is from the order Hymenoptera, Coleoptera or Orthoptera.
  • the insect is a honey bee Apis mellifera, a mealworm Tenebho molilor, or a Mediterranean field cricket Gryll s bimac lalus.
  • the arthropod is an arachnid, more preferred a mite or a spider.
  • the nucleic acid is fed to the arthropod. In principle, every feed can be used that is accepted by the arthropod to be fed.
  • the feed that is used to induce the production of the gene encoded molecules in the arthropods is either a liquid feed comprising the nucleic acid, a dry feed mixed with a solution comprising the nucleic acid or a dry feed comprising the dry nucleic acid or any form of encapsulated nucleic acid (bacteria, archaea, viruses, virus-like particles, eukaryotic cells) in any of these formulations.
  • the method of the invention can be used for all purposes where the production of a gene encoded molecule in an arthropod is suitable and/or desirable. This includes the use of the method of the invention to generate (i) a transfected arthropod or (ii) a transgenic arthropod, where at least part of the arthropod cells are transiently or stably transfected with the nucleic acid.
  • transgenic arthropod refers to an arthropod in which at least in some of the cells the nucleic acid has inserted into the genome in a stable thus irreversible way or to a progeny of said arthropod.
  • transiently transfected refers to a situation where no stable integration of the nucleic acid into the genome has occurred. Consequently, the method of the invention may be used to generate a cell culture of transiently transfected or transgenic arthropod cells, e.g. by isolating the cells from the fed arthropod.
  • the method of the invention may be used for the production of a recombinant protein, especially a pesticide, a regulatory protein or a vaccine antigen.
  • a pesticide especially suitable in cases where the arthropod, in particular an insect, is a pest.
  • the term "vaccine” refers to every gene encoded molecule, in particular a protein, that can provoke a cellular or a humoral immune response in a subject.
  • the arthropod expressing said antigen is eaten by an animal (e.g. small mammals, birds or fish), hence inducing an immune response against said antigen.
  • the method of the invention may be used for the production of an arthropod useful in pharmacological screenings.
  • the gene product may be the target of drug substances that are preferably tested in a living animal.
  • the gene product allows direct monitoring of the actions of insecticides or other agents that change the cellular status. Such monitoring systems, which are well known in the art, can otherwise only be used in certain cases in species accessible to germ line manipulations.
  • the method of the invention may be used for the production of an arthropod useful for vaccination of insectivores or for protecting insectivores against ectoparasites.
  • insectivore refers to all kinds of animals, including human, that feed/eat regularly, sporadically or exceptionally insects or parts thereof, larvae of insects or parts thereof, pupals of insects or part thereof, cocoons of insects or part thereof, eggs of insects or part thereof, imago of insects or part thereof or metabolic products of the insects, larvae, pupal, cocoon or eggs (e.g. silk or other cocoon materials or chitin or chitosan) or products thereof.
  • the arthropod may be a crustacean which is eaten by all kinds of animals, including human, that feed/eat regularly, sporadically or exceptionally Crustacea or parts thereof.
  • the usage of crustaceans similar to insects, opens in addition of the feeding of insects the possibilities to vaccinate fish, which would be extremely useful for commercial fish breeding.
  • the method of the invention may be used for protecting the arthropod against pest influences.
  • pest influences refers to all influences that cause a decrease in the viability or a decrease of the health of the arthropod, in particular but not limited to natural and synthetic toxins and pathogens, in particular viruses, bacteria, moulds, protozoa, other arthropods and other animals, plants and fungi.
  • the DNA encodes for RNAi constructs that attack RNAs only occurring in the pest and being vital for the same.
  • the DNA may encode for proteins that are toxic for the pest.
  • the specificity of the DNA agent may be achieved in that promoters are employed that specifically activate transcription in the vermin only.
  • non-insect promoters such as the CMV promoter optionally in connection with the SV40 enhancer enable the transcription of genes in insects.
  • the method of the present invention may be used for protecting the arthropod against a parasite or an ectoparasite of the arthropod.
  • honey bee Apis mellifera can be protected against the ectoparasite Varroa destructor, as shown in the present examples, because by feeding a honey bee, it is possible to obtain the presence of the gene encoded molecule also in the ectoparasite Varroa destructor.
  • said gene encoded molecule is an acarizide which is harmful to the parasitizing mite Varroa. This is of enormous economic significance, since the damages caused by said ectoparasite are very high. It is estimated that losses due to the weakness and collapse of bee colonies worldwide reach several billion US$ per year.
  • the efficacy of the method of the invention in protecting against parasitic diseases or pest is illustrated in Example III.
  • the ectoparasite may be another species of mite, preferably Dermanissus gallinae (Red Poultry Mite, Arachnida, Acari).
  • the present invention also relates to the use of a nucleic acid comprising a gene encoding a gene encoded molecule for use in a method for protecting an arthropod against pest influences, parasites or ectoparasites or for use in a method for treating an arthropod disease in an arthropod, wherein the nucleic acid is fed to the arthropod or is administered orally. All embodiments described above with respect to the method of the invention also apply to said use of the invention.
  • the gene product encoded by a nucleic acid molecule as used in the context of the present invention preferably by a DNA molecule, that is toxic for an arthropode.
  • the arthropode is an insect or a mite.
  • the insect may be Tenebrio molitor.
  • the mite may be Dermanissus gallinae.
  • the terms "Tenebrio molitor " and "Tenebrio mollitor " may be understood inter schangably.
  • the present invention relates to a method for the delivery of a purified nucleic acid to arthropod cell in vivo, wherein the purified nucleic acid is fed to the arthropod.
  • the purified nucleic acid is fed to the arthropod.
  • said nucleic acid encodes an siRNA or an antisense RNA or is an siR A or antisense RNA.
  • the siRNA or antisense molecule is produced in the arthropod by feeding the arthropod with the nucleic acid.
  • the nucleic acid is preferably a plasmid encoding the siRNA and further containing promoter sequences enabling the production of the siRNA.
  • the nucleic acid may be the siRNA or antisense molecule itself.
  • the siRNA or antisense molecule is preferably protected, e.g. by methylation or by the use of nucleic acid analogues, such as Peptide nucleic acids (PNAs), morpholinos or locked nucleic acids (LNAs), in order to prevent degradation of the molecule.
  • PNAs Peptide nucleic acids
  • LNAs locked nucleic acids
  • Another aspect of the present invention relates to the use of a composition containing DNA for the treatment of diseases of insects, in particular bees, wherein the DNA comprises a promoter that is functionally linked with a therapeutic gene, and wherein the administration of the DNA is oral.
  • the DNA may be an expression plasmid.
  • the promoter may be the promoter of a virus and the virus may be specific for mammalians, or may comprise a promoter that is insect-specific.
  • the promoter may be the CMV promoter.
  • insects may be bees.
  • the diseases may be selected from the group consisting of varroatosis, foulbrood, dysentery, chalkbrood, nosema, and diseases caused by the tracheal mite.
  • the therapeutic gene may encode for an RNAi construct, or specific or unspecific immune stimulants.
  • the RNAi may be designed such that it degrades the RNA sequences of the pathogenic agents that provoke the above-mentioned diseases.
  • the RNAi construct may be specific for gene sequences of the Varroa mite.
  • the invention also relates to a method for introducing DNA into insect cells, wherein a composition that contains DNA is administered orally to insects, and the DNA encodes for a gene product that is toxic for insects.
  • the invention also relates to a method for introducing DNA into insect cells, wherein a composition that contains DNA is administered orally to insects, and the DNA encodes for a gene product that is toxic for an arachnid, in particular wherein the arachnid is a mite, more in particular wherein the mite is a Varroa mite.
  • the invention relates to a composition for use in a method of the present invention.
  • the composition is a solution.
  • the solvent may be water.
  • the invention relates to an aqueous solution containing 50 % per weight of glucose and 0.5 ⁇ g/ ⁇ of DNA, capable for the oral uptake by insects.
  • the composition may be a solution.
  • the composition may contain water.
  • the composition may contain sugar, in particular glucose.
  • RNAi may be specific for gene sequences of the Varroa mite.
  • the invention relates to a method for introducing DNA into insect cells, wherein a composition that contains DNA is administered orally to the insect(s).
  • the DNA may comprise a promoter that is functionally linked with a gene to be expressed, e.g., a therapeutic or toxic gene.
  • the composition may be a solution.
  • the DNA may be an expression plasmid.
  • the promoter may be the promoter of a virus and the virus may be specific for mammalians, or comprise a promoter that is insect-specific.
  • the promoter may be the SV40 promoter or CMV promoter.
  • the composition may contain water.
  • the composition may contain sugar, in particular glucose.
  • the insects may be bees.
  • the therapeutic gene may target diseases, wherein the disease may be selected from the group consisting of varroatosis, foulbrood, dysentery, chalkbrood, nosema, and may be diseases caused by the tracheal mite.
  • the toxic gene may target vital genes of the insects.
  • the toxic gene may encode for an RNAi.
  • the duration of administration may be 3-15, 4-12, 6-10 or 8 days.
  • the therapeutic gene may encode for an RNAi construct, or specific or unspecific immune stimulants.
  • the RNAi may be designed such that it degrades the RNA sequences of the pathogenic agents that provoke the above-mentioned diseases.
  • the RNAi may be specific for gene sequences of the Varroa mite.
  • the invention relates to a solution containing sugar and DNA.
  • the solution may be employed in the above-mentioned methods and use.
  • the sugar may be present in a concentration of from 30-70 % (weight/volume), 40-60 % (weight/volume), 45-55 % (weight/volume or from 50 % (weight/volume).
  • the DNA present in the solution may be in a concentration of from 0.1- 10 ⁇ g/ ⁇ l, 0.1 - 1 ⁇ g/ l, 0.2-0.8 ⁇ , 0.3-0.7 ⁇ g/ ⁇ l, 0.4-0.6 ⁇ g/ ⁇ l, 0.5 ⁇ .
  • the solvent may be water.
  • the solution may be used as feed additive composition for insects, in particular bees.
  • the sugars may be mono-, di- or oligosaccharides and derivatives thereof.
  • the sugars may be such that may attract insects that drink them in solution or such that they can digest.
  • the sugars may be inverted sugars.
  • the sugars may be sucrose, glucose, fructose, maltose. Also combinations of various sugars may be used. Then, the above specifications of concentrations relate to the total sugar concentration.
  • the solution may have a volume of between 15-25 ml or 20 ml.
  • the volume will depend on the size of the beehive or, for other insects, on the amount of insects to which the solution is intended to be administered.
  • the solution will be capable for the administration to insects or bees.
  • the solution will, in particular, contain no substances toxic for insects or bees, other than, possibly, gene products encoded by the contained genes.
  • the DNA may be any DNA described in the above uses and methods.
  • the solution may be provided in a dispenser or in a dish.
  • a spraying device for spraying the solution.
  • the spraying device may comprise a spray nozzle, a suction hose, a container, wherein the container contains the solution, and a pump head for transport of the solution sucked out of the container from the suction hose into the spray nozzle.
  • a spraying device is considered in which the pump head is not necessary as the container is under pressure and, therefore, the solution is pressed into the spray nozzle.
  • FIG 3. pVAX-EGFP-WSSViel plasmid.
  • Figure 4. Brood combs of honey bee Apis mellifera containing brood in the late pupal stages and 1 day old adults were maintained in an incubator at 32°C for several weeks (3-4 weeks).
  • FIG. 7 Fluorescence analysis.
  • the analysis and pictures were made with a Leica DFC 350 FX camera installed in a Leica CTR 4000 fluorescent microscope. The magnification was 100 x, the exposure time was 70 ms, gamma was 0.6 and gain 1.0X;
  • FIG 8. Experiment without EGFP (negative control), oral application of pVAXl SV40 bee - alimentary organs - malpighian tubule system, l OOx magnification, light exposure 70 ms. Malpighian tubule system of negative control honey bee Apis mellifera prepared after oral application of DNA. The analysis and pictures were made with a Leica DFC 350 FX camera installed in a Leica CTR 4000 fluorescent microscope. The magnification was 100 x, the exposure time was 70 ms, gamma was 0.6 and gain 1.OX.
  • Figure 9. Nuclei detection in Malpighian tubule system of honey bee Apis mellifera prepared after treatment with Dapi nucleic acid staining and fluorescent mounting medium. The analysis and pictures were made with a Leica DFC 350 FX camera installed in a Leica CTR 4000 fluorescent microscope. The magnification was 400 x, the exposure time was 70 ms, gamma was 0.6 and gain 1.OX.
  • Figure 10 Intestine of negative control mealworm Tenebrio molitor prepared after treatment with oral application of EGFP-plasmid. The analysis and pictures were made with a Leica DFC 350 FX camera installed in a Leica CTR 4000 fluorescent microscope. The magnification was 100 x, the exposure time was 200 ms, gamma was 0.6 and gain 1.0X.
  • FIG. 11 Intestine of negative control mealworm Tenebrio molitor prepared after treatment with oral application. The analysis and pictures were made with a Leica DFC 350 FX camera installed in a Leica CTR 4000 fluorescent microscope. The magnification was 100 x, the exposure time was 200 ms, gamma was 0.6 and gain l .Ox.
  • FIG. 12 Malpighian tubule system of cricket Gryllus bimaculatus prepared after treatment with oral application of EGFP-plasmid. The analysis and pictures were made with a Leica DFC 350 FX camera installed in a Leica CTR 4000 fluorescent microscope. The magnification was 100 x, the exposure time was 200 ms, gamma was 0.6 and gain 1 .0X.
  • FIG. 13 Malpighian tubule system of negative control cricket Gryllus bimaculatus prepared after treatment with oral application. The analysis and pictures were made with a Leica DFC 350 FX camera installed in a Leica CTR 4000 fluorescent microscope. The magnification was 100 x, the exposure time was 200 ms, gamma was 0.6 and gain 1.OX.
  • FIG. 14 Western blots from Tenebrio molitor performed after oral application of DNA.
  • the lane 1 is the page ruler plus prestained protein ladder.
  • Lane 2 is the negative control (oral application with pVAX-SV40).
  • Lane 3 is empty.
  • Lanes 4 to 6 show EGFP from different animals with a dilution of 1 : 10.
  • Lane 7 shows EGFP from the same sample from lane 6, but with a dilution of 1 :50.
  • Lane 8 and 10 are empty.
  • Lane 9 shows the positive control. All samples were diluted 1 :5 with 15% mercaptoethanol loading dye buffer.
  • FIG 15. Western blots developed from insect cells after oral application of DNA.
  • the lane 1 shows the page ruler plus prestained protein ladder.
  • Lane 2 shows the negative control (oral application with pVAX-SV40).
  • Lane 3 shows EGFP from Apis mellifera oral application and lane 4 from Varroa destructor samples.
  • Lane 5 shows GFP from Tenebrio molitor.
  • Lane 6 shows the positive control. All samples were diluted 1 :5 with 15% mercaptoethanol loading dye buffer.
  • Figure 16. Contamination tests developed with electrocompetent BL21 cells transformation from insect intestine cells and feces extracts after oral application treatment. ⁇ ⁇ feces or gut extract was used for the transformation.
  • BL21 cells (30 ⁇ ) were transformed through electroporation (Voltage 1800 V, Capacitance 25 ⁇ , Resistance, 200 ⁇ ), with 1-mm cuvettes and a GenePulser Xcell machine (Bio-Rad).
  • FIG. 21 Western blot showing the expression of the ACTX protein (ca. 12 kDa, runs as dimer) after feeding of the corresponding DNA-plasmid in T. mollitor.
  • a diagnostic tag (HA-tag) was added to the ACTX gene, to allow the detection via HA-antibodies.
  • Figure 22 Graphic representation of the survival rates after feeding of DNA-plasmids, either coding for ACTX or EGFP to T. mollitor or D. gallinae. Error bars represent the standard deviation.
  • Example I The following example iter alia demonstrates that feeding a nucleic acid to an arthropod is capable to induce the production of gene encoded molecules in said arthropod.
  • a nucleic acid was used in different species of arthropods and the production of the gene encoded molecule enhanced green fluorescent protein (EGFP) was induced in the said arthropods.
  • EGFP enhanced green fluorescent protein
  • the fluorescence was detected in different organs and tissues of the arthropod.
  • Western blot analysis was performed from samples of the arthropod tissues.
  • Arthropods bear an impermeable barrier, in form of a chitin shield, all over their body surface and are therefore extensively protected from exogenous influences and extensively isolated from the molecular environment. Therefore, the application of a nucleic acid by methods regularly used in the art, such as the use of gene guns, may fail or may be complicated. Furthermore, it is well-known in the art that the use of a nucleic acid, in particular the use of DNA and/or RNA, in vivo does mostly not lead to the desired result. It is well-known that an aforementioned nucleic acid is highly ineffective in in vivo applications in nearly all animal species. It is a common prejudice that this is also true for the use of nucleic acids in living arthropods.
  • the problem of the application of a nucleic acid in arthropods to induce the production of a gene encoded molecule existed for a long time and, so far, no capable solution was disclosed in the art, other than laborious and time consuming embryonic manipulation.
  • the nucleic acid DNA encoding for EGFP was fed to the arthropod, whereupon EGFP was induced in different organs of the arthropod. We thereby circumvented the chitin shield barrier.
  • the plasmids used in this invention are based the on pVAXl plasmid purchased from Invitrogen.
  • the colonies of honey bee Apis mellifera were obtained from a beekeeper in Leipzig, Germany.
  • the external parasite mite Varroa destructor was found on the collected bees.
  • the first antibody for Western blotting was purchased from Cell Signaling.
  • the second antibody (the polyclonal goat anti-rabbit immunoglobulins (IgG)/HRP) was obtained from Dako Denmark A/S.
  • Electroporation cuvettes (1 mm) and the GenePulser Xcell machine were supplied by Bio-Rad.
  • the page ruler plus prestained protein ladder was purchased from Fermentas.
  • Three plasmids were constructed ( Figures 1, 2 and 3).
  • the first one (pVAX-SV40) was used as negative control ( Figure 1).
  • This plasmid is based on commercially available pVAXl and was modified by the insertion of a 72bp SV40 enhancer element.
  • the second plasmid pVAX-EGFP-SV40 ( Figure 2) was generated by inserting the coding sequence for EGFP in the MCS of pVAX-SV40, hence downstream of the CMV promoter.
  • the third plasmid was pVAX-EGFP-WSSViel ( Figure 3).
  • the virus promoter from white spots syndrom virus (Liu et al. 2007) was used to replace the CMV promoter.
  • the first antibody for the EGFP detection was diluted 1 :2000 in 5% milk PBS-Tween solution and incorporated for 1.5 h.
  • the second antibody was the polyclonal goat anti-rabbit immunoglobulins (IgG)/HRP. This second antibody was diluted 1 :2000 in 5% milk PBS-Tween solution and incorporated for 1 h. Contamination tests
  • EGFP protein could be microscopically detected in the intestinal tissue of a mealworm Tenebrio molitor that had been treated by the oral application of the plasmid pVAX-EGFP- SV40 ( Figure 10).
  • a mealworm that was treated with the negative control, the plasmid pVAX-SV40 did not show any EGFP protein in its intestinal tissue ( Figure 1 1).
  • the same result was found in a sample from a mealworm that had been treated with the oral application of the plasmids pVAX-EGFP-SV40 and pVAX-SV40.
  • EGFP was only detected when the mealworm was treated with the plasmid pVAX-EGFP-SV40 ( Figure 14 and 15, lane 5). When the mealworm was treated with the negative control pVAX-SV40, no EGFP could be detected ( Figure 14).
  • EGFP protein could be microscopically detected in the Malphigian tubule system of cricket Gryllus bimaculatus that had been treated with the oral application of the plasmid pVAX- EGFP-SV40 ( Figure 12). In contrast, a cricket that was treated with the negative control, the plasmid pVAX-SV40, did not show any EGFP protein in its Malphigian tubule system ( Figure 13). 4. Induction of the production of EGFP in the mite Varroa destructor
  • EGFP protein could be detected in the mite Varroa destructor parasiteizing on a honey bee Apis mellifera that was orally treated with the plasmid pVAX-EGFP-SV40.
  • the detection was performed by Western blot ( Figure 15, lane 4).
  • bee feces from untreated bees was spiked with 1 ng of plasmid DNA.
  • naked DNA was used (1 ng, 100 pg, 10 pg). Transformations were performed as described above.
  • the present invention demonstrates the usability of a method based on feeding of arthropods with feed comprising a nucleic acid comprising a gene encoding a molecule of interest in numerous technical fields.
  • Said method based on feeding may be conducted easily by a person skilled in the art.
  • the nucleic acid may be obtained by standard genetic engineering methods well-known in the art.
  • the plainness of the feeding procedure easily allows the incorporation of the nucleic acid into an arthropod of interest.
  • the present invention represents a capable technical solution to overcome the problem of usability of nucleic acids in living arthropods and inducing production of a molecule of interest therein. Said problem existed for a long time and its solution may allow the use of the presented invention in numerous applications. Furthermore, the prejudice that the in vivo use of nucleic acids administered orally did not result in an induction of the production of molecules of interest was overcome by aforementioned experiments.
  • the bees were fed with a commercially available sugar solution that was stored at room temperature:
  • the duration of feeding was between 6 and 10 days.
  • Fig. 7 and 8 the feeding of the plasmid encoding for GFP leads to the expression of GFP in the organs of the bee.
  • Fig. 8 depicts the feeding of the empty vector, i.e. without the GFP sequence. This result does not only mean that it comes to the expression of the plasmid, but also that the GFP is folded correctly and is, hence, present in the insect natively.
  • 86 mM NaCl corresponds to approx. 5 g/L
  • 5.4 niM KC1 corresponds to approx. 0.4 g/L
  • lysis buffer 0.1 % SDS, 50 mM Tris in a. dest.
  • the obtained protein extracts were treated as follows:
  • GFP antibody (#2555, Cell Signaling) was incubated at a 1 : 1500 dilution in 5 % skimmed milk PBS-Tween (0.5 %) for 1.5 h. After three washing steps with PBS- Tween (0.5 %), the blot was incubated with the second antibody (Polyclonal Goat Anti- Rabbit Immunoglobulins (IgG)/HRP, Dako Denmark A/S - Ref P0448) 1 :2500 in 5 % skimmed milk PBS-Tween (0.5 %) for 1 h.
  • the second antibody Polyclonal Goat Anti- Rabbit Immunoglobulins (IgG)/HRP, Dako Denmark A/S - Ref P0448
  • the BHK21 GFP cells served as positive control.
  • VeroV76 GFP cells are feasible.
  • Varroa glucose
  • GFP As cognizable from the Western blot (Fig. 19), expressed GFP is detectable in the hemolymph of the bees (lane 8).
  • the Varroa mite takes up GFP from the hemolymph via the blood meal (lane 7, at approx. 27.5 kDa).
  • the GFP of the control does not run on the same height as the GFP of the bee as they were expressed in different hosts.
  • the GFP (“green fluorescent protein " ) may be replaced by another (therapeutic or toxic) gene product.
  • GFP antibody (#2555, Cell Signaling) was incubated at a 1 : 1500 dilution in 5 % skimmed milk PBS-Tween (0.5 %) for 1.5 h. After three washing steps with PBS- Tween (0.5 %), the blot was incubated with the second antibody (Polyclonal Goat Anti- Rabbit Immunoglobulins (IgGVHRP, Dako Denmark A/S - Ref P0448) 1 :2500 in 5 % skimmed milk PBS-Tween (0.5 %) for 1 h.
  • the second antibody Polyclonal Goat Anti- Rabbit Immunoglobulins (IgGVHRP, Dako Denmark A/S - Ref P0448) 1 :2500 in 5 % skimmed milk PBS-Tween (0.5 %) for 1 h.
  • BHK21 GFP cells serve as positive control.
  • VeroV76 GFP cells are feasible.
  • Varroa glucose
  • Varroa 52 x Varroa (pVAXl -EGFP-WSSViel)
  • Varroa 48 x Varroa (pVAXl-EGFP-WSSViel )
  • GFP is detectable in Varroa attached to bees that received glucose water with plasmid (lanes 3 and 4). Furthermore, EGFP is detectable in the bee thorax (lanes 6 and 8).
  • Example HI As cognizable from figure 20, GFP is detectable in Varroa attached to bees that received glucose water with plasmid (lanes 3 and 4). Furthermore, EGFP is detectable in the bee thorax (lanes 6 and 8).
  • ACTX is a short protein that specifically blocks Calcium-channels of invertebrates. The toxic effects of the ACTX protein on insects was shown in (Wang et al., 1999).
  • the coding sequence for EGFP in pVAX-EGFP-WSSViel was replaced with the sequence coding for ACTX, according to Wang et al.
  • This plasmid was fed to two arthropods, Tenebrio mollitor (insect) and Dermanissus gallinae (Red Poultry Mite, Arachnida, Acari). Expression of the ACTX protein was verified by western blot ( Figure 21).
  • the EGFP containing plasmid was used as a negative control.
  • D. gallinae was fed in an in vitro-feeding device, according to McDevitt et al. Hereby, mites are placed in a small tube covered with chicken-skin. Chicken blood is placed on top of the skin and mites are allowed to suck the blood through the skin. 10 mites were fed with 250 ⁇ blood, containing 50 ⁇ g plasmid. After 16 hours feeding was stopped, and feeding rates of the mites were determined. Only fed mites (visible via their enlarged body size) were included in the analysis.
  • Results are summarized in Figure 22: Feeding of the ACTX-coding plasmid led to a significant reduction in the survival rate, compared to feeding the EGFP-coding plasmid. T-test values for the differences are p ⁇ 0,002 for T. mollilor. p ⁇ 0,02 for D. gallinae. This example illustrates that arthropods express a gene after feeding the coding plasmid, even if the gene product is a toxic protein, leading to the death of the arthropod, which has implications for applying the present invention in control of parasitic diseases or pests.

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Abstract

La présente invention concerne un procédé pour la production d'une molécule codée par un gène chez un arthropode in vivo, dans lequel un acide nucléique comprenant un gène codant pour ladite molécule est donné dans l'alimentation à l'arthropode. De plus, la présente invention concerne un procédé pour le transfert d'un acide nucléique purifié dans des cellules d'arthropode in vivo, où l'acide nucléique purifié est donné dans l'alimentation à l'arthropode. La présente invention concerne des procédés, utilisations et solutions pour introduire des acides nucléiques hétérologues dans des insectes. En particulier, selon la présente invention, un ADN peut être introduit dans des insectes par voie orale, peut être transcrit dans ceux-ci et peut également être extrait dans les (ecto‑ et endo‑)parasites sanguivores des insectes.
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US9932590B2 (en) 2013-07-02 2018-04-03 The University Court Of The University Of Aberdeen Control of Varroa mite infestation
WO2015153956A1 (fr) * 2014-04-03 2015-10-08 The Board Of Regents Of The Nevada System Of Higher Education On Behalf Of The University Of Nevada, Las Vegas Traitement de la loque américaine provoquée par paenibacillus larvae en utilisant la lysine phagique
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