(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date ;n n /n 19 May 2011 (19.05.2011) 2U11/U57825 Al

(51) International Patent Classification: (74) Agent: LAHRTZ, Fritz; Isenbruck Bosl Horschler LLP, A01K 67/033 (2006.01) C12N 15/63 (2006.01) Prinzregentenstrasse 68, 81675 Munchen (DE). (21) International Application Number: (81) Designated States (unless otherwise indicated, for every PCT/EP20 10/006982 kind of national protection available): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, (22) International Filing Date: CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO, 16 November 2010 (16.1 1.2010) DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, (25) Filing Language: English HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP, KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, (26) Publication Langi English ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, (30) Priority Data: NO, NZ, OM, PE, PG, PH, PL, PT, RO, RS, RU, SC, SD, 10 2009 053 469.5 SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, 16 November 2009 (16.1 1.2009) DE TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. 10 2009 054 265.5 (84) Designated States (unless otherwise indicated, for every 23 November 2009 (23.1 1.2009) DE kind of regional protection available): ARIPO (BW, GH, 10001476.0 12 February 2010 (12.02.2010) EP GM, KE, LR, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, 61/308,143 25 February 2010 (25.02.2010) US ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, (71) Applicant (for all designated States except US): TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, FRAUNHOFER-GESELLSCHAFT ZUR EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, FORDERUNG DER ANGEWANDTEN LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, FORSCHUNG E.V. [DE/DE]; Hansastrasse 27c, 80686 SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, Munchen (DE). GW, ML, MR, NE, SN, TD, TG). (72) Inventors; and Published: (75) Inventors/ Applicants (for US only): RODRIGUES — with international search report (Art. 21(3)) MARKERT DOS SANTOS, Gustavo [DE/DE]; Giebel- — before the expiration of the time limit for amending the str. 29, 70499 Stuttgart (DE). DELAROQUE, Nicolas claims and to be republished in the event of receipt of [FR/DE]; Kobisstr. 2, 043 17 Leipzig (DE). SZARDEN- amendments (Rule 48.2(h)) INGS, Michael [DE/DE]; Oselweg 20, 38302 Wolfen- buttel (DE). ULBERT, Sebastian [DE/DE]; Kochstr. 35, 04275 Leipzig (DE). GIESE, Matthias [DE/DE]; Im Schaffher 24, 69123 Heidelberg (DE).

00 o (54) Title: INDUCTION OF GENE EXPRESSION IN (57) Abstract: The present invention relates to a method for the production of a gene encoded molecule in an in vivo, o wherein a nucleic acid comprising a gene encoding said molecule is fed to the arthropod. Furthermore, the present invention re lates to a method for the delivery of a purified nucleic acid to arthropod cells in vivo, wherein the purified nucleic acid is fed to the arthropod. The present invention relates to methods, uses and solutions to introduce heterologous nucleic acids into , hi 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. Induction of Gene Expression in Arthropods

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 comprising about 80% of all described living species. The most important classes of arthropods are hexapoda (which include insects), (including , mites and scorpions), Crustacea and myriapoda (Brusca and Brusca 2003). An arthropod may be a 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. On the other hand, 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.

It is well-known in the art that cell cultures e.g. SF-9 or SF-21 cell cultures, are widely used in the art and are capable of producing considerable amounts of a gene encoded molecule, in particular a protein. In contrast, 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. There is still a considerable lack of capable techniques to enable a nucleic acid to enter into arthropod cells, which is different to the laborious and time consuming manipulation of germ cell lines.

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.

There is a need for an efficient and simple method for inducing gene expression in arthropods in vivo.

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.

There are numerous disorders that may influence the live of insects, particularly bees, in particular bacterial and viral infectious diseases and various parasites. It is also discussed whether monocultures limit the habitat of the insects and, in this context, reduce diversity of species. Or whether the use of pesticides impairs the immune system of bees so that the animals die. Also, the disputed cultivation of genetically modified plants fell into the focus of criticism again. In spring 2007, it was reported from the U.S.A. that approximately 70 percent of the bees from the east to the west coast disappeared tracelessly, designated as "Colony Collapse Disorder" by the experts. Reason unknown.

However, the infestation by the Varroa mite (Varroa destructor) remains the major verifiable pest of the honey bee. 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.

Most of the higher organisms have developed a specific defense mechanism that protects them from bacteria, viruses but also parasites. Arthropodes do not have this adaptive immune system of the vertebrates. However, they possess an innate unspecific though highly effective immune system that is based on a cascade of differently inducible components. Just recently, for insects, a humoral as well as a cellular immune system has been described that is regulated by various genes. In this context, a background for the following project description is given.

From an economical point of view, the bee is, beside pig and cattle, the third-most important production animal of humans and their pollination activity in agriculture is irreplaceable. In this respect, research and development of biologically safe and highly effective products against the Varroa mite is urgently demanded. As presented in the following, an immunization of the bees against Varroa mites and, in this context, also against Varroa-associated viral pathogenic agents is possible.

Until now, it was found that it is possible to downregulate viruses in bees by orally administered dsRNA constructs (Maori et al., Insect Molecular Biology (2009), 18 (1), 55- 60). The introduction of the 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). However, the manufacture and the handling of dsRNA is difficult as it can be degraded easily.

Further, there is a need for novel insecticides that are easier to handle and that are associated with lesser side effects for humans and the environment. Previous biological insecticides require a viral or animal vector to introduce toxic gene products in certain insects.

Therefore, it was the object of the present invention to provide a method that enables to introduce nucleic acids into insects which method avoids the disadvantages of the state of the art and optionally enables a gene-therapeutic method for insects easily applicable in practice.

In a first aspect, 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.

In the context of the present invention, as shown in the examples, it has been surprisingly found that by feeding a nucleic acid encoding a gene to an arthopod, it is possible to induce expression of said gene in vivo. Furthermore, it has been surprisingly found that the expressed gene product is also found in an ectoparasite living on the arthropod.

Surprisingly, it was found that DNA taken up by insects orally may be read from the transcription/translation apparatus and results in proteins that are encoded by the DNA. In particular, it was found that the gene products that originate from a DNA taken up by an insect can also be detected in parasites that live on these insects. Therewith, a very easy method is created to deliver DNA in insects and insect cells of living entire insects, respectively, for expression. Therewith, on the one hand, 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. Evidently, 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.

Instead of being fed to the arthropod, 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. As well, 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 , aphid, thrips, summer fruit tortrix, potato , cherry fruit fly, Melolontha, European Corn Borer, Rhododendron leafhopper, turnip moth, scale insects, Gypsy moth, mite, (European Grape)Vine moth, Whitefly, Phaenops cyanea, bark- beetle, oak splendor beetle, oak processionary, European green oak moth, Cephalcia abietis, common furniture beetle, Diprion pini, 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, mill moth, old-house borer, Lyctidae, termites and ants. "Heterologous" DNA is understood as a DNA that does not naturally occure in an insect.

In the example section, species from three major orders of insects have been used as model organisms. In view of the close similarity to other arthropods, it is assumed that the results obtained for insects can also be extended to other arthropods.

Throughout the invention, 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.

In a preferred embodiment of the invention, the gene encoded molecule is a protein or a biologically active RNA. In the context of the present invention, the term "biologically active " denotes that the given molecule has a biological function.

Preferably, said biologically active RNA is selected from the group consisting of:

a. tRNA, b. rRNA, c. mRNA, and d. double stranded RNA.

These molecules are known in the art.

Preferably, the RNA is a catalytic RNA capable of catalyzing chemical reactions.

In the most preferred embodiment, the gene encoded molecule is a protein. According to the invention, the term "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.

In a specific embodiment of the present invention, the gene encoded molecule is controlling the rate of the production of other molecules of interest in the arthropod. For example, 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.

According to the invention, 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. Consequently, in the context of the present invention, the term "gene" refers to a DNA or RNA, preferably a DNA encoding said molecule. In this context, 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. Preferably, the nucleic acid is a DNA.

Herein, 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. Such 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. In particular, 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.

Bees are understood as all members of the superfamily "bees and sphecoid wasps" (Apoidea), in particular the "bees" (Apiformes), in particular of the Apidae, in particular of the subfamily Apinae, in particular of the honey bees (Apis), and in particular of the genus Apis mellifera.

An 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. 2 1 (1): 2-4; Henschel A, Buchholz F, Habermann B (2004). "DEQOR: a web-based tool for the design and quality control of siRNAs". Nucleic Acids Res 32 (Web Sever issue): Wl 13-20). In this context, it relates to genes that encode for double-stranded RNA. In particular, the 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 Autographa 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).

Further possible toxic proteins are designated in the book "Biopesticides: Use and Delivery" of Rysanek, P. (Hall, F.R., Menn, J.J. (ed.), Springer). Preferably, 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.

More preferably, 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. Furthermore, it is well-known in the art that viral vectors often tend to influence the systems they are applied to at their own. Thus, in the context of the present invention, a viral vector may influence the insect, in particular the health of the insect and possibly its span of life. Moreover, 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.

Therefore, according to the 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.

According to the invention, 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.

On the other hand, it is also included in the present invention that 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. Furthermore, it is included in the present invention that 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. As used herein, the term "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).

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. These molecules are known in the art (see Sambrook and Russel, 2001).

In particular, 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. In this context, it is preferred to use vectors that are not carrying additional genes such as antibiotic resistance genes, although also such vectors may be used.

Apart from the gene, 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. Such 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.

Furthermore, 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.

Herein, a promoter is understood as a promoter that enables the transcription of DNA sequences in insects. Thereto, 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. In this context, 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. Subsequently, 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. In particular, such promoters are considered that are species specific, i.e., initiate the transcription only in the desired species, genus, family or order of insects.

Furthermore, non-insect promoters are included, such as promoters of viruses that are specific for mammals or monkeys. In particular, 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. Furthermore, the DNAs feasible for the invention may also comprise enhancers. 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 17 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 110-1 15 nucleotides 5' of the beginning of the promoter.

The promoter may be functionally linked with a gene to be expressed.

In principle, the method of the present invention can be applied to all arthropods. In a preferred embodiment of the present invention, the arthropod may be but is not limited to an insect, an , a crustacean and a myriapodum.

In a further preferred embodiment of the present invention, 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. In the most preferred embodiment, the insect is a honey bee Apis mellifera, a mealworm Tenebho molilor, or a Mediterranean field cricket Gryllus bimaculalus.

In another embodiment of the present invention, the arthropod is an arachnid, more preferred a mite or a spider. In the context of the present invention, the nucleic acid is fed to the arthropod. In principle, every feed can be used that is accepted by the arthropod to be fed. This includes any kind material that is consumed orally by the arthropods, independent on whether it is natural feed, agricultural feed or laboratory feed and independent on whether it is consumed naturally or is administered by means of technical devices or is taken up casually. In a preferred embodiment, 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.

As used herein, the term "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. The term "transiently transfected", in contrast, 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.

Furthermore, 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. The production of a pesticide is especially suitable in cases where the arthropod, in particular an insect, is a pest.

Throughout the invention, 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. Additionally, the method of the invention may be used for the production of an arthropod useful in pharmacological screenings. For such screenings the gene product may be the target of drug substances that are preferably tested in a living animal. Most preferably 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.

In particular, 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. As used herein, the term "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.

Similarly, 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.

Furthermore, the method of the invention may be used for protecting the arthropod against pest influences. As used herein, the term "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.

For pest control such method may be employed in that the DNA encodes for RNAi constructs that attack RNAs only occurring in the pest and being vital for the same. Alternatively, 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. Furthermore, it was surprisingly found that also non-insect promoters such as the CMV promoter optionally in connection with the SV40 enhancer enable the transcription of genes in insects.

In particular, the method of the present invention may be used for protecting the arthropod against a parasite or an ectoparasite of the arthropod. In particular, 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. In a preferred embodiment, 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.

In another preferred embodiment, the ectoparasite may be another species of mite, preferably Dermanissus gallinae (Red Poultry Mite, Arachnida, Acari).

In this context, 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.

In further preferred embodiment, 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. Preferably, the arthropode is an insect or a mite. The insect may be Tenebrio molitor. The mite may be Dermanissus gallinae. As used herein, the terms "Tenebrio molitor" and "Tenebrio mollitor" may be understood interschangably.

In another aspect, 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. As shown in the examples, it is possible to induce gene expression simply by feeding a purified nucleic acid to an arthropod. This provides a very simple method of gene delivery.

With respect to this aspect of the invention, all embodiments disclosed above with respect to the nucleic aid, the arthropod, the way of feeding as well as of the use of the method of the invention do also apply.

In a preferred embodiment of said aspect of the invention, said nucleic acid encodes an siRNA or an antisense RNA or is an siR A or antisense RNA. In this case, the siRNA or antisense molecule is produced in the arthropod by feeding the arthropod with the nucleic acid. In this context, the nucleic acid is preferably a plasmid encoding the siRNA and further containing promoter sequences enabling the production of the siRNA. Alternatively, the nucleic acid may be the siRNA or antisense molecule itself. In this case, 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.

The invention is further explained by the following examples and figures, which are intended to illustrate, but not to limit the scope of the present invention.

The invention is further defined by the enclosed claims.

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. In the use, the DNA may be an expression plasmid. In the use, 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. In the use, the promoter may be the CMV promoter. In the use, the insects may be bees. In the use, the diseases may be selected from the group consisting of varroatosis, foulbrood, dysentery, chalkbrood, nosema, and diseases caused by the tracheal mite. In the use, 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. In the use, 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.

Furthermore, the invention relates to a composition for use in a method of the present invention. In a preferred embodiment, the composition is a solution. The solvent may be water.

Further, the invention relates to an aqueous solution containing 50 % per weight of glucose and 0.5 µ µ of DNA, capable for the oral uptake by insects.

The composition may be a solution. In the use, the composition may contain water. In the use, the composition may contain sugar, in particular glucose. In the use, the RNAi may be specific for gene sequences of the Varroa mite.

Further, 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. In the method, the DNA may be an expression plasmid. In the method, 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. In the method, the promoter may be the SV40 promoter or CMV promoter. In the method, the composition may contain water. In the method, the composition may contain sugar, in particular glucose. In the method, the insects may be bees. In the method, 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. In the method, the toxic gene may target vital genes of the insects. The toxic gene may encode for an RNAi. In the method or the use, the duration of administration may be 3-15, 4-12, 6-10 or 8 days.

In the method, 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. In the method, the RNAi may be specific for gene sequences of the Varroa mite.

Further, 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/µl, 0.1-1 g , 0.2-0.8 µ µΐ , 0.3-0.7 µg µl, 0.4-0.6 µg/µl, 0.5 µ µΐ . 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. For administering to bees, 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.

Further, a spraying device for spraying the solution is disclosed. 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. Further, also 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. Brief description of the Figures

Figure 1. pVAX-SV40 plasmid.

Figure 2. pVAX-EGFP-SV40 plasmid

Figure 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).

Figure 5. Muscle and gut preparation of honey bee Apis mellifera which were treated with oral application of DNA-plasmids. These bees were maintained in an incubator at 32°C for 3-4 weeks. The analysis of organs and pictures were made with a Canon Power Shot 66 camera installed in a Carl Zeiss Stemi 2000-C microscope.

Figure 6. Malpighian tubule system of honey bee Apis mellifera was prepared after treatment with oral application of DNA-plasmids and maintained in an incubator at 32°C. The analysis of organs and pictures were made with a Canon Power Shot 66 camera installed in a Carl Zeiss Stemi 2000-C microscope.

Figure 7. Fluorescence analysis. Experiment with EGFP, oral application of pVAX-EGFP- SV40 bee - alimentary organs - malpighian tubule system, lOOx magnification, light exposure 70 ms. Malpighian tubule system of honey bee Apis mellifera 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 70 ms, gamma was 0.6 and gain 1.0X

Figure 8. Experiment without EGFP (negative control), oral application of pVAXl SV40 bee - alimentary organs - malpighian tubule system, lOOx 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 00 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.

Figure 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.

Figure 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.

Figure 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.

Figure 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.

Figure 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. Ι µΐ feces or gut extract was used for the transformation. BL21 cells (30 µΐ) were transformed through electroporation (Voltage 800 V, Capacitance 25 µ , Resistance, 200 Ω), with 1-mm cuvettes and a GenePulser Xcell machine (Bio-Rad).

Figure 17. Sequence of pVAX-EGFP-SV40

Figure 18. Sequence of pVAX-EGFP-SV40-WSSViel

Figure 19. Western blot of a feeding experiment

Figure 20. Western blot of a further feeding experiment

Figure 21. Western blot showing the expression of the ACTX protein (ca. 1 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. Lanes: 1, molecular weight; 2, EGFP-plasmid-fed; 3, HA-control lysate; 4, ACTX-plasmid fed. Position of the ACTX is indicated on the right side.

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. Examples

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.

Abstract

In order to show the capability of the method to feed functional nucleic acid and thereby induce gene expression, 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. The fluorescence was detected in different organs and tissues of the arthropod. In addition, Western blot analysis was performed from samples of the arthropod tissues.

Introduction

It is well-known in the art that insect cell cultures are capable to produce considerable amounts of a gene encoded molecule, in particular a protein. In contrast, 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. There is still a considerable lack of capable techniques to enable a nucleic acid to enter into arthropod cells. 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.

In summary, 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. In the present invention, we introduced a capable method for the use of a nucleic acid in an arthropod in vivo and induce the production of the gene encoded molecule EGFP. 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.

Methods

Antibodies and reagents

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.

Plasmid construction

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). Here, the virus promoter from white spots syndrom virus (Liu et al. 2007) was used to replace the CMV promoter.

DNA-plasmid oral application

The experiments were performed on 10 colonies of honey bee Apis mellifera. Brood combs containing brood in the late pupal stages and 1 day old adults were removed from colonies and maintained 3-4 weeks in an incubator at 32°C (Figure 4). The external parasite mite Varroa destructor was found on the collected bees and was also used for the oral application experiments. In addition, we tested the oral application with two other insect species: the mealworm Tenebrio molitor (coleoptera), a holometabolic insect and in the Mediterranean field cricket Gryllus bimac lat s, a hemimetabolic insect. All insect species were treated with the three plasmids (Figures 1, 2 and 3). Dilution to 500 ng/µΐ plasmid was made with water (for cricket Gryllus bimaculatus) and glucose-water ( 1:1) (for the honey bee Apis mellifera), respectively. The cricket Gryllus bimaculatus was fed with dried feed too. The cricket food was prepared based on the method described by Ibler et al. (Ibler et al., 2009). The crickets were fed with a dried mixture of 2.4 g cricket food + 1 ml [lmg/ml] plasmid. The food for Tenebrio molitor consisted of a dried mixture of 2.4 g meal + 1ml [lmg/ml] plasmid.

Fluorescence microscopy

After the treatment of oral application, different insect tissues were prepared (Figures 5 and 6) under a Carl Zeiss Stemi 2000-C microscope with a Canon Power Shot 66 camera. Intestine and Malpighian tubule system were analyzed by a Leica CTR 4000 fluorescent microscope with a Leica DFC 350 FX camera.

Western blots

In order to confirm the presence of green fluorescent protein western blots from gut and muscle of these insects were performed. 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

To analyze whether the plasmids fed to the insects would leave the animal, we tested for the presence of functional plasmids in the feces. The tests were performed by transformation assays in highly competent E. coli cells. 1 µΐ of feces and intestine cells, respectively, was used for the transformation with electrocompetent BL21 cells. BL21 cells (30 µΐ) were transformed through electroporation with 1-mm cuvettes and a GenePulser Xcell machine (Bio-Rad). After transformation 300 µΐ of SOC medium was added. 100 µΐ of bacteria were spread onto LB plates containing 50 µg µl Kanamycin. The plates were placed at 37°C overnight.

Results

1. Induction of the production of EGFP in the honey bee Apis mellifera EGFP protein could be microscopically detected in the Malphigian tubule system of a honey bee Apis mellifera that had been treated with the oral application of the plasmid pVAX-EGFP-SV40 (Figures 7 and 9). In contrast, a bee that was treated with the negative control, the plasmid pVAX-SV40, did not show any EGFP protein in its Malphigian tubule system (Figure 8). The same result was found in a sample from a muscle of a bee that had been treated with the plasmid pVAX-EGFP-SV40. In the latter case, the EGFP was detected by Western blot analysis (Figure 15, lane 3).

2. Induction of the production of EGFP in the mealworm Tenebrio molitor

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). In contrast, a mealworm that was treated with the negative control, the plasmid pVAX-SV40, did not show any EGFP protein in its intestinal tissue (Figure 11). 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 ).

3. Induction of the production of EGFP in the cricket Gryllus bimaculatus

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).

5. Contamination tests The transformation with electrocompetent cells indicates that the functional plasmid is present in the intestine cells but is absent in the feces (Figure 16). This results indicates that plasmids which are outside of intestine cells were digested and would not spread into nature.

6. Detection Limit

To determine the detection limit of the contamination test, bee feces from untreated bees was spiked with 1 ng of plasmid DNA. In addition, naked DNA was used ( 1 ng, 100 pg, 10 pg). Transformations were performed as described above.

In the transformation experiment with 10 pg of plasmid there were 4 colonies formed. Based on this result it was possible to calculate the number of molecules. The transformation with electrocompetent cells therefore had a detection limit of about 200,000 molecules.

Discussion

In this study, we provide formal experimental evidence that it is possible to induce the production of a gene encoded molecule in a living arthropod by feeding a nucleic acid containing the respective gene. In the presented examples an EGFP protein was expressed in different organs and tissues of different arthropods. The employment of a negative control proved that the induction of gene expression was dependent on the sequence identity of the nucleic acid. Furthermore, it was demonstrated that the employed plasmid was entirely inactivated while passing through the gastrointestinal tract of an arthropod. The latter indicates that a nucleic acid is not spread in nature.

Summarised, 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. In a first step, the nucleic acid may be obtained by standard genetic engineering methods well-known in the art. Subsequently, the plainness of the feeding procedure easily allows the incorporation of the nucleic acid into an arthropod of interest.

Consequently, 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.

Example II

Oral Administration of plasmids to bees (Apis melliferd)

1. Beekeeping Approximately 50 young bees/box were kept in a commercially available plastic box approx. 15x10x12 cm permeable to air in a ventilated incubator (heating cabinet) at approx. 30° Celsius. Prior to the beginning of the experiment, the incubator was disinfected to exclude extrinsic contamination. The average span of live under these laboratory conditions is approx. 3-5 weeks for young bees and, in this context, relates to the natural span of life. Old bees still live at least 2.5 weeks under these conditions. All bees stemmed from beekeepers from Germany and are of European origin.

2. Nutrient Solution/Sugar water The bees were fed with a commercially available sugar solution that was stored at room temperature:

20 g of glucose in

20 ml ofH 2O

3. Plasmids For oral application the following plasmids were used:

3.1 pVAXl EGFP-SV40 (Fig. 18) pVAXl (Invitrogen) + EGFP + SV40 enhancer (with M ) [ 1 µ ΐ] pVAXl EGFP-SV40

3.2 pVAXl-EGFP-WSSViel (Fig. 17) pVAXl (Invitrogen) + EGFP + WSSViel [ 1 µ µΐ] pVAXl-EGFP-WSSViel

3.3 pVAX 1-SV40 (control) pVAXl (Invitrogen) + SV40 enhancer (with Mlul) [ 1 µ ΐ] pVAXl-SV40

4. Oral Application by means of Sugar Water 4.1 pVAXl EGFP-SV40 pVAXl (Invitrogen) + EGFP + SV40 enhancer (with Mlul) [ 1 µg l] pVAXl EGFP-SV40 dilution in a 1.5 ml Eppendorf vial: 600 µΐ of plasmid with 600 µΐ of sugar water solution = 1200 µΐ of nutrient solution with [0.5 g l] plasmid

4.2 pVAXl-EGFP-WSSViel pVAXl (Invitrogen) + EGFP + WSSViel [ 1 µg/ ] pVAXl-EGFP-WSSViel dilution in a .5 ml Eppendorf vial: 600 µΐ of plasmid with 600 µΐ of sugar water solution = 1200 µΐ of nutrient solution with [0.5 g l] plasmid

4.3 pVAXl-SV40 pVAXl (Invitrogen) + SV40 enhancer (with Mlul) [ 1 µg/µl pVAXl-SV40 dilution in a 1.5 ml Eppendorf vial: 600 µΐ of plasmid with 600 µΐ of sugar water solution = 1200 µ of nutrient solution with [0.5 µg/µl plasmid

Dilute plasmids with sugar water give 1200 µΐ of plasmids / sugar water feeding per box (approx. 10 bees) / day

The duration of feeding was between 6 and 10 days.

Fluorescence analysis:

Bees were anesthetized by C0 2 and/or ice Bees and single organs (antenna, compound/complex eye, mouthparts, cerebric, mesothorax, metathorax, first + second pair of wings, esophagus, proventriculus, ventriculus, midgut, hindgut (rectum), malpighian tubule system, feces/excrements) were prepared on preparation plates with Apime Ringer's Solution under the stereomicroscope.

Macroanalysis: analysis of the entire bee and organs for fluorescence: ultraviolet emitter + stereomicroscope analyze bees and organs on bees wax / paraffin preparation plates with Apime Ringer's Solution: Carl Zeiss Stemi 2000-C stereomicroscope - integrated Canon camera (Canon Power Shot 66) Benda ultraviolet emitter type NU-6KL (# 4010705)

Microanalysis: analysis of individual bee organs: fluorescence microscope take individual bee organs of bees wax / paraffin preparation plates (with Apime Ringer's Solution) and prepare: on specimen slide with a drop of DakoCytomation fluorescent mounting medium (code S3023) add cover slip on top, and wait for approx. 3 min again

As can be recognized from 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.7). 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.

Equipment: shutter / lamp Leica CTR 4000 / microscope Leica DMI 4000 B / microscope Leica DFC 350 FX / camera

Exposure time in the fluorescence microscope: 1. for EGFP (green light / fluo L5): 70 s 2. for "normal" (white light / BF): 6 ms

Apis mellifera Ringer's Solution 86 M NaCl corresponds to approx. 5 g/L 5.4 n M KC1 corresponds to approx. 0.4 g/L 3 mM CaCl corresponds to approx. 0.44 g/L p.a. water to 1 liter pH 6.9

Insect preparation plates paraffin: Histosec pastilles, company VWR bees wax: pure, natural, company ROTH Sudan Black, company ROTH

Preparation: Bees wax : paraffin ( 1:1), 60°C 100 g of paraffin + 100 g of bees wax: 1-2 days at 60-70°C + 1-2 ml of Sudan Black (10 mg/ml in EtOH) Subsequently, pour on the Petri dish. Let is cool down.

Preparation of cells as GFP positive control for Western blot: The following cells were obtained in PBS (approximately 200000 cells): BHK21 GFP VeroV76 GFP resuspend the cells with the pipet and transfer in a 1.5 ml Eppi centrifuge 3' 2000 xg RT, discard supernatant (SN) + 250 µΐ of lysis buffer (0.1 % SDS, 50 mM Tris in a. dest.), resuspend and incubate at RT for approximately 1' - freeze at -20°C

For a Western blot, about 50 µΐ of the above cell suspension were diluted + 10 µΐ of 6x loading buffer loaded on the SDS gel (10 µ of cells + 40 µΐ of lx PBS + 10 µΐ of loading buffer).

Expression of EGFP, detection of EGFP in the bee and in Varroa parasiting thereon The bees were treated as in the above examples. The obtained protein extracts were treated as follows: 40 µΐ of protein extract was mixed with 10 µ of mercaptoethanol loading dye SDS buffer and immediately thereafter heated for 5 min at 95°C. Subsequently, 50 µΐ were loaded on an SDS-PAGE gel. A Western blot with a gel of 1.5 mm thickness and a 14 % separation gel (BioRad) was used.

For Western blot of eGFP (27 kDa) from the pVAX-EGFP-WSSViel plasmid a 14 % gel was used. For the detection, the gel was blotted after the flow out of the blue leading band and, subsequently, treated as follows: As first antibody, 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 BHK21 GFP cells served as positive control. Alternatively, VeroV76 GFP cells are feasible.

Bees that received glucose water without DNA addition served as negative control.

It was loaded (Fig. 19): lane sample - 1- 8 µΐ of Page Ruler Plus Prestained Protein Ladder (Fermentas # SM 18 1 1) 2- empty 3- 50 µΐ of Varroa (glucose water) 4- 50 µΐ of Varroa (glucose water) 5- 50 µΐ of Varroa (glucose water) - 6- empty

8- 50 µΐ of bee hemolymph (pVAXl-EGFP-WSSViel) 9- empty 10- 50 µΐ of positive control (40µ1of BH 2 1 GFP cells / PBS + 10 µΐ of 15 % Mercapt. Load. Dye Buffer)

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. Expression of EGFP in bees, detection of EGFP in bee thorax and Varroa Western blot of EGFP of treated bees and Varroa: Used bees were BS-2009.08-MdS-l 1 of Apis mellifera. The DNAs and the controls were administered orally as in the above experiments.

A Western blot with a gel of 1.5 mm thickness and a 14 % separation gel (BioRad) was used.

For Western blot of eGFP (27 kDa) from the pVAX-EGFP-WSSViel plasmid a 14 % gel was used. For the detection, the gel was blotted after the flow out of the blue leading band and, subsequently, treated as follows: As first antibody, 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.

BHK21 GFP cells serve as positive control. Alternatively, VeroV76 GFP cells are feasible.

Bees that received glucose water without DNA addition served as negative control.

Western blot - (BS-2009.09-MdS-06): 1- 8 µΐ of Page Ruler Plus Prestained Protein Ladder (Fermentas # SM 181 1) 2- 50 µΐ of Varroa: 50 x Varroa (glucose water) 3- 50 µΐ of Varroa: 52 x Varroa (pVAXl-EGFP-WSSViel) 4- 50 µΐ of Varroa: 48 x Varroa (pVAXl-EGFP-WSSViel) 5- 50 µ of bee gut: 6 x bee (pVAXl-EGFP-WSSViel) 6- 50 µΐ of bee thorax: 6 x bee (pVAXl-EGFP-WSSViel) 7- 50 µl ofbee gut: 5 x bee (pVAXl-EGFP-WSSViel) 8- 50 µΐ of bee thorax: 5 x bee (pVAXl -EGFP-WSSVie 1) 9- empty 10- 50 µΐ of positive control (BH 2 1 GFP cells)

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). Example HI

Feeding of a DNA plasmid encoding a toxin

DNA plasmids coding for the ACTX toxin from the Australian tunnel web spider were constructed. 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.

For T. mollilor, the feeding was done similar to example 1. 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 µΐ blood, containing 50 µg 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. Survival rates of T. mollitor and D . gallinae was monitored over a period of 5-10 days.

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.

References

EP 815 214 Araujo R.N., Santos A., Pinto F.S., Gontijo N.F., Lehane M.J. and Pereira M.H. (2006).RNA interference of the salivary gland nitrophorin 2 in the triatomine bug Rhodnius prolixus (: Reduviidae) by dsRNA ingestion or injection. Insect Biochem Mol Biol (2006); 36, 683-693. Brusca R.C. & Brusca G.J. (2003) Invertebrates. Sinauer Associates, Sunderland, MA. Henschel A., Buchholz F. and Habermann B. (2004). "DEQOR: a web-based tool for the design and quality control of siRNAs". Nucleic Acids Res 32 (Web Sever issue): W l 13-20. Ibler B., Makert G. R. and Lorenz M. W. (2009) Larval and adult development and organisation of a systemic breeding of the Mediterranean field cricket Gryll s bi ac lat s de Geer, 1773). Zool.Garten 78: 81-101. Liu W. J., Chang Y. S., Wang A. H., Kou G. H. and Lo C. F. (2007) White spot syndrome virus annexes a shrimp STAT to enhance expression of the immediate- early gene iel . J Virol. 8 1: 1461-1471. Maori E., Paldi N., Shafir S., Kalev H., Tsur E., Glick E. and Sela 1. (2009) APV, a bee-affecting virus associated with Colony Collapse Disorder can be silenced by dsRNA ingestion. Insect Molecular Biology (2009), 18 (1), 55-60. McDevitt R., Nisbet A.J. and Huntley J.F. (2006), Ability of a proteinase inhibitor mixture to kill poultry red mite, Dermanyssus gallinae in an in vitro feeding system. Veterinary Parasitology 141, 380-385. Nakagawa Y., Sadilek M., Lehmberg E., Herrmann R., Herrmann R., Moskowitz H., Lee Y.M., Thomas B.A., Shimizu R., Kuroda M., Jones A.D. and Hammock B.D. (1998). Rapid purification and molecular modeling of AalT peptides from venom of Androctonus australis. Arch Insect Biochem Physiol. ;38(2):53-65 Rysanek, P. (Hall, F.R., Menn, J.J. (ed.), "Biopesticides: Use and Delivery", Springer). Sambrook J. and Russel D. W . (2001) Molecular Cloning - Laboratory Manuals, 3rd edition, Cold Spring Harbour Laboratory Press, New York, USA. Tang D.C., Devit M. and Johnston S.A. (1992) Genetic Immunization Is A Simple Method for Eliciting An Immune-Response. Nature 356, 152-1 54. Wahren B . and Liu M. (2005) DNA Vaccines - An Overview. In: DNA Vaccination and Immunotherapy, pp. 1-6. Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany. Wang X.H., Smith R., Fletcher J.I., Wilson H., C.J., Howden M.E.H. and Glenn F. King G.F. (1999), Structure-function studies of ω-atracotoxin, a potent antagonist of insect voltage-gated calcium channels. Eur. J. Biochem. 264, 488-494. Voorhoeve P.M. and Agami R. (2003). Knockdown stands up. Trends Biotechnol. 2 1 (l): 2-4 Claims

1. 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.

2. The method according to claim 1, wherein said gene encoded molecule is a protein or a biologically active RNA.

3. The method of claim 2, wherein said biologically active RNA is selected from the group consisting of:

a. tRNA, b. rRNA, c. rriRNA, and d. double stranded RNA.

4. The method according to any of claims 1 to 3, wherein the gene encoded molecule is a protein.

5. The method according to any of claims 1 to 3, wherein the gene encoded molecule is controlling the rate of the production of other molecules of interest in the anthropode.

6. The method according to any of claims 1 or 5, wherein said nucleic acid is in the form of a purified nucleic acid, is contained in a bacterial cell or virus or is complexed with a transfecting agent.

7. The method according to any of claims 1 to 6, wherein said nucleic acid is 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.

8. The method of any one of claims 1to 7, wherein the nucleic acid is a DNA. . The method according to any of claims 1 to 8, wherein said nucleic acid comprises a promoter active in said arthropod.

10. The method according to any of claims 1 to 9, wherein the arthropod is selected from the group consisting of an insect, an arachnid, a crustacean and a myriapodium.

11. The method according to claim 10, wherein the arthropod is an insect.

12. The method according to claim 11, wherein the insect is a holometabolic insect or a hemimetabolic insect.

13. The method according to claim 11 where the insect is from the order Hymenoptera, Coleoptera or Orthoptera.

14. The method according to any of claims 9 to 12, wherein said insect is a honey bee Apis mellifera, a mealworm Tenebrio molitor, or a Mediterranean field cricket Gryllus bimaculatus.

15. The method according to any of claims 1 to 14, wherein the feed is

a. a liquid feed comprising the nucleic acid;

b. a dry feed mixed with a solution comprising the nucleic acid; or

c. a dry feed comprising the dry nucleic acid.

16. The method according to any of claims 1 to 15, wherein said method is used 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.

17. The method according to claim 15, wherein said method is used to generate a cell culture of transiently transfected or transgenic arthropod cells.

18. The method of any of claims 1 to 17, wherein said method is used for the production of a recombinant protein, especially a pesticide, a regulatory protein or a vaccine antigen. 19. The method of any of claims 1 to 18, wherein said method is used for the production of an arthropod useful in pharmacological screenings.

20. The method of any of claims 1 to 19, wherein said method is used for the production of an arthropod useful for vaccination of insectivores or for protecting insectivores against ectoparasites.

21. The method of any of claims 1 to 19, wherein said method is used for protecting the arthropod against pest influences.

22. The method of any of claims 1 to 19, wherein said method is used for protecting the arthropod against an ectoparasite of the arthropod.

23. The method of claim 22, wherein said ectoparasite is Varroa destructor and said arthropod is a honey bee Apis mellifera.

24. The method according to any one of claims 9 to23, wherein the promoter is functionally linked with a gene to be expressed.

25. The method according to any one of claims 9 to 24, wherein the promoter is the promoter of a virus and the virus is specific for mammalians, or is a promoter that is insect-specific.

26. The method according to claim 25, wherein the promoter is the SV40 promoter or CMV promoter.

27. The method according to claim 26, wherein the promoter is the CMV promoter.

28. The method according to any one of claims 1 to 27, wherein the gene encoding the gene encoded molecule is a therapeutic gene.

29. The method according to any one of claims 1 to 25, wherein the gene encoding the gene encoded molecule is a toxic gene.

30. The method according to claim 1to 29, wherein the toxic gene targets one or more vital genes of the insect. 31. The method according to claim 28, wherein the therapeutic gene targets diseases, wherein the disease is selected from the group consisting of varroatosis, foulbrood, dysentery, chalkbrood, nosema, and diseases caused by the tracheal mite.

32. The method according to any one of claim 1 to 31, wherein the gene encoded molecule is an antisense RNA or an siRNA or encodes for an antisense R A or an siRNA.

33. The method according to any one of claims 1 to 31, wherein the gene encoded molecule is an RNAi construct, or specific or unspecific immune stimulants.

34. The method according to any one of claims 32 or 33, wherein the RNAi construct is specific or unspecific for gene sequences of the Varroa mite.

35. The method according to any of claims 1 to 34, wherein the duration of administration may be 3-15, 4-12, 6-10 or 8 days.

36. The method according to any one of claims 1-35, wherein the gene encoding the gene encoded molecule is administered in a solution.

37. The method according to claim 36, wherein the solution contains water.

38. The method according to any one of claims 36 or 37, wherein the solution contains sugar, in particular glucose.

39. The method according to any one of claims 36 to 38, wherein the solution comprises a sugar and DNA.

40. The method according to any one of claims 36 to 39, wherein the sugar is present in a concentration of from 30-70 % (weight/volume), 40-60 % (weight/volume), 45-55 % (weight/volume or from 50 % (weight/volume) and the DNA is present in a concentration of from 0.1-10 µ µΐ, 0.1-1 µg/ l, 0.2-0.8 µ µΐ, 0.3-0.7 µ µ , 0.4- 0.6 µg/µl or 0.5 µg/µl.

41. The method according to claim 40, wherein the solution is an aqueous solution containing 50 % per weight of glucose and 0.5 µg/µl of DNA, capable for the oral uptake by insects. 42. The method according to any one of claims 1 to 41, wherein said method is for introducing DNA into insect cells, wherein a composition that contains DNA is administered orally to insect(s).

43. The method according to claim 42 for introducing DNA in insect cells, wherein a composition containing DNA is administered orally to insects, and the DNA encodes for a gene product that is toxic for insects.

44. The method according to claim 43 for introducing DNA in insect cells, wherein a composition containing DNA is administered orally to insects, and the DNA encodes for a gene product that is toxic for an arachnid.

45. The method according to claim 44, wherein the arachnid is a mite.

46. The method according to claim 45, wherein the mite is a Varroa mite {Varroa destructor).

47. The method according to claim 45, wherein the mite is a Red Poultry Mite (Dermanissus gallinae).

48. A composition for use in a method according to any one of claims 1to 47.

49. A composition containing DNA for use in a method for the treatment of diseases of insects, wherein the DNA comprises a promoter that is functionally linked with a therapeutic gene, and wherein the administration of the DNA is oral.

50. The composition according to claim 49, wherein the DNA is an expression plasmid.

51. The composition according to any one of claims 49 or 50, wherein the promoter is the promoter of a virus and the virus is specific for mammalians, or comprises a promoter that is insect-specific.

52. The composition according to any of claims 49 to 51, wherein the promoter is the CMV promoter.

53. The composition according to any of claims 49 to 52, wherein the insects are bees. 54. The composition according to any of claims 49 to 53, wherein the diseases are selected from the group consisting of varroatosis, foulbrood, dysentery, chalkbrood, nosema, and diseases caused by the tracheal mite.

55. The composition according to any of claims 49 to 54, wherein the therapeutic gene encodes for an R Ai construct, specific or unspecific immune stimulants.

56. The composition according to claim 55, wherein the RNAi construct is specific for gene sequences of the Varroa mite.

57. The composition according to any one of claims 49 to 56, wherein the duration of administration may be 3-15, 4-12, 6-10 or 8 days.

58. The composition according to any one of claims 49 to 57, wherein the composition is a solution containing sugar and DNA, wherein the sugar is present in a concentration of from 30-70 % (weight/volume), 40-60 % (weight/volume), 45-55 % (weight/volume or from *50 % (weight/volume) and the DNA is present in a concentration of from 0.1-10 µg/µl, 0.1-1 µg/µl, 0.2-0.8 µg/µl, 0.3-0.7 µg/ l, 0.4- 0.6 µg µl or 0.5 µg µl.

59. The composition according to any one of claims 49 to 58, wherein the solvent is water.

60. The composition according to any one of claim 59, wherein the composition is an aqueous solution containing 50 % per weight of glucose and 0.5 µg µl of DNA, capable for the oral uptake by insects.

61. A method for the delivery of a purified nucleic acid to arthropod cells in vivo, wherein the purified nucleic acid is fed to the arthropod.

62. The method according to claim 61, with the features as defined in any of claims 6 to 23.

63. The method according to claim 62, wherein said nucleic acid encodes an siRNA or an antisense RNA or is an siRNA or antisense RNA. 64. The method according to any one of claims 6 1 to 63, wherein the method is for introducing DNA into insect cells, wherein a composition that contains DNA is administered orally to insect(s).

65. The method according to claim 64, wherein the DNA comprises a promoter that is functionally linked with a gene to be expressed.

66. The method according to claim 65, wherein the gene to be expressed is a therapeutic gene.

67. The method according to claim 66, wherein the gene to be expressed is a toxic gene.

68. The method according to any one of claims 64 to 67, wherein the composition is a solution.

69. The method according to any one of claims 64 to 68, wherein the DNA may be an expression plasmid.

70. The method according to any one of claims 64 to 69, wherein the promoter is the promoter of a virus and the virus may be specific for mammalians, or comprise a promoter that is insect-specific.

71. The method according to any one of claims 64 to 70, wherein the promoter is the SV40 promoter or CMV promoter.

72. The method according to any one of claims 64 to 71, wherein the composition contains water.

73. The method according to any one of claims 64 to 72, wherein the composition contains sugar, in particular glucose.

74. The method according to any one of claims 64 to 73, wherein the insects are bees.

75. The method according to any one of claims 64 to 74, wherein the therapeutic gene targets diseases, wherein the disease is selected from the group consisting of varroatosis, foulbrood, dysentery, chalkbrood, nosema, and diseases caused by the tracheal mite. The method according to any one of claims 64 to 75, wherein the toxic gene targ vital genes of the insects.

77. The method according to any one of claims 64 to 76, wherein the toxic gene

78. The method according to any one of claims 64 to 77, wherein the duration of administration may be 3-15, 4-12, 6-10 or 8 days.

79. The method according to any one of claims 64 to 78, wherein the therapeutic gene encodes for an RNAi construct, or specific or unspecific immune stimulants.

80. The method according to any one of claims 64 to 79, wherein the RNAi is designed such that it degrades the RNA sequences of the pathogenic agents that provoke the diseases selected from the group consisting of varroatosis, foulbrood, dysentery, chalkbrood, nosema, and diseases caused by the tracheal mite.

81. The method according to any one of claims 64 to 80, wherein the RNAi is specific for gene sequences of the Varroa mite.

82. The method according to claim 64 for introducing DNA in insect cells, wherein a composition containing DNA is administered orally to insects, and the DNA encodes for a gene product that is toxic for the insects.

83. The method according to claim 64 for introducing DNA in insect cells, wherein a composition containing DNA is adrriinistered orally to insects, and the DNA encodes for a gene product that is toxic for an arachnid.

84. The method according to claim 83, wherein the arachnid is a mite.

85. The method according to claim 84, wherein the mite is a Varroa mite (Varroa destructor).

86. The method according to claim 85, wherein the mite is a Red Poultry Mite (Dermanissus gallinae).

INTERNATIONAL SEARCH REPORT International application No PCT/EP201O/0O6982

A . CLASSIFICATION O F SUBJECT MATTER INV. A01K67/033 C12N15/63 ADD.

According to International Patent Classification (IPC) or to both national classification and IPC

B. FIELDS SEARCHED Minimum documentation searched (classification system followed by classification symbols) A01K C12N

Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched

Electronic data base consulted during the international search (name of data base and, where practical, search terms used)

EPO-Internal BIOSIS, EMBASE, WPI Data

C . DOCUMENTS CONSIDERED TO BE RELEVANT

Category* Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No.

US 6 90 379 A (WOOD H ALAN [US] ) 1,2 ,4, 18 July 2 0 (2000-07-18) 6-12 , 15 , 16, 18, 24,25, 42,48, 61,62, 64,65, 68-70,72 col umn 4 , l i ne 11 l ine 16 col umn 5 , l i ne 18 - l ine 55 col umn 11, l ine 13 l ine 32

Further documents are listed in the continuation of Box C . See patent family annex.

* Special categories of cited documents : "T" later document published after the international filing date or priority date and not in conflict with the application but "A" document defining the general state of the art which is not cited to understand the principle or theory underlying the considered to be of particular relevance invention "E" earlier document but published on or after the international "X" document of particular relevance; the claimed invention filing date cannot be considered novel or cannot be considered to "L" document which may throw doubts on priority claim(s) or involve an inventive step when the document is taken alone which is cited to establish the publication date of another Ύ " document of particular relevance; the claimed invention citation or other special reason (as specified) cannot be considered to involve an inventive step when the Ό " document referring to an oral disclosure, use, exhibition or document is combined with one or more other such docu¬ other means ments, such combination being obvious to a person skilled in the art. "P" document published prior to the international filing date but laterthan the priority date claimed "&" document member of the same patent family

Date of the actual completion of the international search Date of mailing of the international search report

15 March 2011 24/03/2011

Name and mailing address of the ISA/ Authorized officer European Patent Office, P.B. 5818 Patentlaan 2 NL - 2280 HV Rijswijk Tel. (+31-70) 340-2040, Fax: (+31-70) 340-3016 van Heusden, Mi randa INTERNATIONAL SEARCH REPORT International application No PCT/EP201O/0O6982

C(Continuation). DOCUMENTS CONSIDERED TO BE RELEVANT

Category* Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No.

X WO 2005/042753 Al (CHESAPEAKE PERL INC 1,2 ,4, [US] ; JARVIS DONALD [US] ; VAN BEEK NI KOLAI 6-13 , [US] ; F) 12 May 2005 (2005-05-12) 15-19, 24-28, 39-42, 48,61, 62, 64-67, 69-72 Y page 3 , l ine 6 - l ine 11 49-60, page 3 , l ine 29 - page 4 , l i ne 17 73-75, page 14, l ine 3 - l i ne 3 1 79-81, page 40, l ine 30 - page 4 1 , l ine 14 83-86

X LOPEZ M G ET AL: "Trans-complementation 1,2 ,4, of polyhedri n by a stably transformed Sf9 6-12 , 15 , insect cel l l ine al l ows occ baculovi rus 16, 18, occl usion and larval per os infectivi ty" , 24,25, JOURNAL OF BIOTECHNOLOGY, ELSEVI ER SCI ENCE 42,48, PUBLISHERS, AMSTERDAM, NL, 61,62, vol . 145, no. 2 , 64,65, 5 November 2009 (2009-11-05) , pages 68-70,72 199-205 , XP026854245, ISSN: 0168-1656 [retrieved on 2009-11-05] page 202, col umn 2 , paragraph 4

X K0VALEVA ELENA S ET AL: "Recombinant 1,2 ,4, protein producti on i n insect larvae: host 6-12 , 15 , choi ce, t i ssue di stribution , and 16, 18, heterologous gene instabi l i ty" , 24,25, BIOTECHNOLOGY LETTERS, 42,48, vol . 3 1 , no. 3 , March 2009 (2009-03) , 61,62, pages 381-386, XP002590578, 64,65, ISSN: 0141-5492 68-70,72 the whol e document

X SEABAUGH R C ET AL: "Development of a 1,2 ,4,6, Chimeri c Sindbi s Vi rus wi t h Enhanced per 7,9-12, 0s Infection of Aedes aegypti " , 15, 16, VI ROLOGY, ACADEMIC PRESS, ORLANDO, US LNKD- 18,24,25 D0I : 10. 10Q6/VI R0. 1998. 9034, vol . 243, no. 1, 30 March 1998 (1998-03-30) , pages 99-112, XP004445916, ISSN: 0042-6822 page 100, col umn 1, paragraphs 2,3 page 106, col umn 1, paragraphs 2,3

-/-- INTERNATIONAL SEARCH REPORT International application No PCT/EP2Q10/Q06982

C(Continuation). DOCUMENTS CONSIDERED TO BE RELEVANT

Category* Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No.

EP 0 586 892 Al (RES CORP TECHNOLOGI ES INC 1,2 ,4,7 , [US] ) 16 March 1994 (1994-03-16) 8 , 10-12 , 15, 18, 29,30, 35,42, 43,48 page 23 , 1ine 32 - 1ine 5 1

WO 2005/049841 Al (COMMW SCI ENT IND RES 1-3 ,5-7 , ORG [AU] ; BAYER BIOSCI ENCE NV [BE] ; 10-12, WATERHOUSE PE) 2 June 2005 (2005-06-02) 15,24, 29,30, 32,33, 36,37, 42,43, 48, 61-65, 68,72, 76-78,82 page 33 , paragraph 4 page 35 ; examples 2,3

TIAN H0NGGANG ET AL: "Devel opmental 1-3 ,5-8. Control of a Lepidopteran Pest Spodoptera 10-12, exigua by Ingestion of Bacteria Expressi ng 15,24, dsRNA of a Non-Midgut Gene" , 29,30, PL0S ONE, 32,33, vol . 4 , no 7 , E6225, July 2009 (2009-07) , 35,42, pages 1-13 XP002590579 , 43,48 ISSN: 1932-6203 page 10, col umn 1 paragraphs 3 , 4

MEYERING-V0S ET AL: "RNA interference 1-3 ,5-7 , suggests sul faki nins a s sati ety effectors 10-15, i n the cri cket Gryl l us bimaculatus" , 29,30, JOURNAL OF , PERGAM0N 32,33, PRESS, OXFORD, GB LNKD- 35-37, D01 : 1 . 1016/ J . J I NSPHYS . 2007 . 4 . 003 , 48,61-63 vol . 53 , no. 8 , 1 August 2007 (2007-08-01) , pages 840-848, XP022191783, ISSN: 0022-1910 page 841, col umn 2 , paragraph page 842 , col umn 1, paragraph 1 section 3.3 ; page 843; f i gure 3

-/-- INTERNATIONAL SEARCH REPORT International application No PCT/EP2Q10/Q06982

C(Continuation). DOCUMENTS CONSIDERED TO BE RELEVANT

Category* Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No.

HOFFMANN ANDREA ET AL: " Fate of 1,2 ,4, plasmid-bearing, l uci ferase marker gene 6-8, 15, tagged bacteria after feeding t o the soi l 18,42,48 mi croarthropod Onychi urus fimatus (Col lembola) " , FEMS MICROBIOLOGY ECOLOGY, vol . 30, no. 2 , October 1999 (1999-10) , pages 125-135, XP002590599 , ISSN: 0168-6496 * abstract

WO 03/004644 Al (COMMW SCI ENT IND RES ORG 1-3 ,6-8, [AU] ; WHYARD STEVEN [AU] ; CAMERON FIONA 10-16, HELEN) 16 January 2003 (2003-01-16) 20-23, 31-39, 42, 44-48, 61-63 pages 7 , 11,21

MAORI E ET AL: " IAPV, a bee-affecti g 1-3 ,6,7 , v i rus associ ated wi t h Colony Col lapse 10-15, Di sorder can be si lenced by dsRNA 21,32, ingestion" , 33, INSECT MOLECULAR BIOLOGY, BLACKWELL 35-38, SCI ENTI FIC, OXFORD, GB, 48,61-63 vol . 18, no. 1 , 1 February 2009 (2009-02-01) , pages 55- 60, XP002523701 , ISSN: 0962-1075 , D0I : D0I : 10. 1111/J . 1365-2583 . 2009. 00847 . X the whol e document

TEWARS0N N C ET AL: "DETERMINATION OF 49-60, PROTEOLYTIC ACTIVITY I N VARROA-JACOBSONI 73-75, AN ECT0 PARASITIC HEM0PHAG0US MITE OF 79-81, HONEY BEES APIS-SP" , 83-86 APID0L0GI E, vol . 13 , no. 4 , 1982, pages 383-390, XP002627255 , ISSN: 0044-8435 the whol e document

EYRICH U. , RITTER, W. : "Di stribution of a 49-60, systemi c functioning medi cament i n the 73-75, body of the honey bee, Api s mel l i fera" , 79-81, J . APPL. ENT. , 83-86 vol . 109, 1990, pages 15-20, XP002627256, the whol e document

-/-- INTERNATIONAL SEARCH REPORT International application No PCT/EP2Q10/Q06982

C(Continuation). DOCUMENTS CONSIDERED TO BE RELEVANT

Category* Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No.

POPESKOVIC D ET AL: "THE BLOCKADE OF 49-60, HEMOCYANIN FUNCTION OF THE MITE 73-75, VARROA-JACOBSONI AS A SPECI FIC 79-81, PHYSIOLOGICAL BASIS FOR A SYSTEMIC 83-86 TREATMENT OF THE HONEYBEE VARROATOSIS", COMPTES RENDUS DES SEANCES DE LA SOCI ETE DE BIOLOGI E ET DE SES FI LIALES, vol . 180, no. 6 , 1986, pages 663-668, XP009145512 , ISSN: 0037-9026 the whol e document

SHEN MIA0QING ET AL: " Intri cate 49-60, transmi ssion routes and interactions 73-75, between pi corna-l i ke v i ruses (Kashmi r bee 79-81, v i rus and sacbrood v i rus) wi t h the 83-86 honeybee host and the parasi t i c varroa mi t " JOURNAL OF GENERAL VI ROLOGY, vol . 86, no. Part 8 , August 2005 (2005-08) , pages 2281-2289, XP002590581 , ISSN: 0022-1317 the whol e document International application No. INTERNATIONAL SEARCH REPORT PCT/EP2 0 1 0 / 0 0 6 9 8 2

Box No. I Nucleotide and/or amino acid sequence(s) (Continuation of item 1.b of the first sheet)

. With regard to any nucleotide and/or amino acid sequence disclosed in the international application and necessary to the claimed invention, the international search was carried out on the basis of:

a. (means)

on paper

in electronic form

in the international application as filed

together with the international application in electronic form

subsequently to this Authority for the purpose of search

In addition, in the case that more than one version or copy of a sequence listing and/or table relating thereto has been filed □ or furnished, the required statements that the information in the subsequent or additional copies is identical to that in the application as filed or does not go beyond the application as filed, as appropriate, were furnished.

3 . Additional comments:

Form PCT/ISA/21 0 (continuation of first sheet (1)) (July 2009) INTERNATIONAL SEARCH REPORT International application No Information on patent family members PCT/EP201O/0O6982

Patent document Publication Patent family Publication cited in search report date member(s) date

US 6090379 18-07-2000 NONE

WO 2005042753 Al 12·-05-2005 NONE

EP 0586892 Al 16·-03-1994 CA 2101610 Al 08-02-1994 P 3532943 B2 31-05-2004 P 6205681 A 26-07-1994 US 5827684 A 27-10-1998

O 2005049841 Al 02·-06-2005 EP 1687435 Al 09-08-2006 US 2006272049 Al 30-11-2006

WO 03004644 Al 16--01-2003 CA 2455490 Al 16-01-2003 EP 1414959 Al 06-05-2004 P 4330987 B2 16-09-2009 P 2004532653 T 28-10-2004 J P 2009185044 A 20-08-2009 NZ 530969 A 30-09-2005 US 2005095199 Al 05-05-2005