insects transgenesis

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20/12/2008 Transgenic Insects 1

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2 Introduction Methods of genetic transfer Markers for insect transgenesis Applications of insect transgenesis Hurdles in insect transgenesis Case studies Future thrust 2 Seminar outline….

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3 Insect pests cause more crop loss Transmission of diseases in human being Evolution of pesticide resistance Dangers to the environment and public health Control of insect pests through genetic engineering techniques gaining importance 3 Introduction

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4 Insects - With newly expressed characteristics New characters – as a result of manipulation of DNA in laboratory Changes - passed on to next generation 4 Genetically modified insects are…

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5 For area-wide control of pests As bioreactors to produce pharma products To develop virus-resistant insect lines To Enhance agricultural production, productivity To Benefit public health To improve disease resistance, pollination attributes in honey bees and high quality silk production in silk moth (Gopanathan, 1992). Genetically modified insects…why?

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6 6 E.F. Knipling, (1937)- Concept of genetic control of insect pest Started with sterilization of Screw worm flies, a serious pest of livestock Experiment on sterile insect resistance management (SIRM) - high sterile to fertile ratio -Achieved by using gamma irradiation, UV rays and mutagens like Ethyl methyl sulphonate Transgenic insect technology is an improvement over sterile insect technology Till now 18 different genera have been manipulated. HISTORY OF GENETICALLY MODIFIED INSECTS

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7 First genetically transformed insect – In 1982 , Rubin and Spradling were successful in transforming Drosophila melanogaster - wild eye colour was restored in a mutant strain After 1995 , the transformation of non-drosophilid arthropods were done in Mediterranean fruit fly (Loukeris). 7

Requirements for gene manipulation:

8 Requirements for gene manipulation Gene of interest or exogenous DNA 2. Vector Marker gene 4. Promoter 8

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9 METHODS OF GENETIC TRANSFORMATION

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10 Physical methods Microinjection Biolistics Lipofection Electroporation Biological methods Transposable elements Sperm mediated transformation Paratransgenesis 10

BIOLOGICAL METHODS OF GENETIC TRANSFER:

11 BIOLOGICAL METHODS OF GENETIC TRANSFER 11 Transposable elements Transposable elements - Mobile pieces of DNA that do not remain fixed at one genomic location , move from one site on a chromosome to another. (Liao, 2000). Increase their copy number as they move around among chromosomes within individual organisms Minos Hermes Mos 1 (mariner) piggyB ac

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12 Transposable element vector systems

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13 Transformation of insects begins by microinjecting embryos with two circular DNA plasmids viz. 1. Vector plasmid Contains a transposable element to be incorporated into host genome under control of a promoter and a marker gene (Handler,1980). Vector plasmid lacks transposase 2. Helper plasmid It catalyzes the excision of the exogenous DNA plus marker gene from the vector plasmid and their insertion into host genome (Rubin and Spradling,1982). Structure of transposable elements and its incorporation into genome is verified by PCR amplification 13

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14 Isolation of desired gene (transgene) Integration of transgene and marker gene with transposable element Excision of transgene and marker gene by transposase Insertion of transgene and marker gene into chromosome PROTOCOL FOR INSECT TRANSFORMATION 14

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15 MARKERS FOR INSECT TRANSGENESIS 15

Universal marker for transgenic insects:

16 Universal marker for transgenic insects Universal marker used to gene transfer in any species is Green Fluorescent Protein (GFP) It is obtained from the jelly fish Aequorea victoria Active in both animal and plant kingdom It requires strong promoter. cause cytotoxicity when expressed at high levels Berghammer et al., (1999) Aequorea victoria

Eye color genes as transformation markers:

17 Eye color genes as transformation markers Production of screening pigments in the compound eye By mutations in these genes, it can be easily distinguishable from wild type Grow well in laboratory cultures Easy detection of transformed individuals E.g. Transformation of the Mediterranean fruit fly, Ceratitis capitata and yellow fever mosquito Aedes aegypti ( Horn et al ., 2002).

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18 Insect transgenesis mediated by fluorescent-marked transposon vectors Contd..

GMI INVOLVED IN CONTROL OF AGRICULTURAL INSECT PESTS:

19 GMI INVOLVED IN CONTROL OF AGRICULTURAL INSECT PESTS 1 . Pink boll worm Million of male pink boll worm moth were sterilized by irradiation in SIT (Pelloquin, 1999) Moths are engineered to contain gene from jelly fish (GFP) + A lethal gene (tTA) is introduced from bacteria (Briggs, 2001) It alters the metabolism of the moth larvae 19

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20 They feed on Spider Mites, a polyphagous pest. First field trial of a transgenic mite - Transgenic predatory mite Metaseiulus occidentalis, predator of spider mites (Presnail, 1997). 20 2. Transgenic predatory mites TransgenicMite

3.Transgenic Red flour beetle:

21 3.Transgenic Red flour beetle It is a worldwide pest of stored products Genes responsible for regulating pheromone secretion are mutated (Daborn, 2002). Specific gene expression is knocked out by RNA interference 21

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22 Transgenic Red flour beetle 22

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23 23 Genetically modified honeybee Death of honeybees under rearing conditions is mainly because of entomopathogens and parasites. Genetically engineered honeybee with gene coding for hDAF are resistant to diseases, parasites and insecticides (Kimura, 2001) Genetic manipulation has improved disease resistance and pollination attributes in honey bees (Rothenbuhler 1979) GMI FOR PRODUCTION OF ECONOMICALLY USEFUL INSECTS

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24 2. Genetically modified silkworm Modified to produce industrial and therapeutic proteins like human growth hormone and human collagen (Kadonookuda,1995) Silk glands generally express the introduced L-chain gene and GFP Cocoons produced from modified insects contain recombinant human collagen. Spider milk (protein) produced from modified silkworm larvae are used to make bullet proof vests, parachutes and artificial ligaments (Lewis,2006) 24 Larvae of transgenic silkworm

RELEASED COMMERCIALLY:

25 RELEASED COMMERCIALLY Predatory mites – In 1997 in US Pink bollworm – In 2001 in Mexico Anopheles mosquito – In 2002 in New Delhi and UP Screw worm fly – Exported from Libya to Kenya and Central America 25

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26 20/12/2008 Transgenic Insects 26 Germ-line transformation of pink bollworm (Lepidoptera : Gelechiidae) mediated by the piggyBac transposable Element. (Peloquin et.al , 2002).

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27 Lepidopteron species are key pests on food and fibre crops world-wide. Field resistance to pesticides presents an increasing challenge to pest control. Manipulation of insect species at the molecular level will become more important to advance our understanding of insect biology and to promote the development of novel field applications. Of immediate use, germ-line transformation can directly contribute to pest control efforts,

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28 28 Experimental procedures Insect strains PBW pupae were received weekly from the PBW Rearing facility Adults were housed at 28 °C and eggs were collected for injection . The vector pB[BmA3EGFP] contains within-piggyBac ends The piggyBac helper plasmid pBacHsp was used to provide a source of transposase

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29 29 2.Microinjection and rearing: Adult females were allowed to oviposit on to glass slides mixture of vector and helper plasmids in buffer was injected at a varied concentrations into the posterior pole G0 embyros were retained on glass slides and allowed to develop at 28°C and 70% relative humidity (RH). Just prior to eclosion (from the eggshell) Single larvae were transferred to a 1.5-ml Eppendorf tube containing approximately 200 mg of PBW diet. (larvae were allowed to develop at 28°C, 70–80% RH) reached the second instar pierce the lid of the tube to permit gas exchange

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30 30 G 0 pupae were separated by sex Adult moths were kept in separate incubators in a egg collection room Mating cages were constructed ,affix white paper towel in interior to collect eggs and provide 6%sucrose solution. G 0 males were provided with three virgin females and G 0 females were provided with two males. G 1 eggs were collected once in three days and transferred to a 50-ml polypropylene screw top tube along with 10–20 g of PBW diet. G 0 were allowed to develop to first larval instar under the same conditions as G 0 s.

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31 31 3. Mating and selection strategy G 1 larvae were examined for EGFP expression under fluorescent stereomicroscope EGFP positive were transferred individually to 1.5-ml Eppendorf tubes. reared to the pupal stage,sexed. Male and female pupae were transferred to separate cages. Add opposite sex of wild-type pupae along with 6% sucrose to each cage. The cage was closed and returned to the incubator. Ten days after emergence oviposition surface was placed on the cage and collect eggs Transfer eggs to polypropylene Fisher 50-ml centrifuge tubes with diet.

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32 32 Adults from the EGFP larvae were always out-crossed to wild-type insects until the introgressive crosses were begun. Screen the larvae for EGFP expression EGFP-positive larvae were selected Backcrossed to the parental wild-type insects. Five generations line 35 EGFP-positive adults to the wild-type parental strain were performed. Maintain lines of EGFP positives by introgression of EGFP-positive insects. Selection of EGFP positive progeny to eliminate wild-type alleles and to build a homozygous line.

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33 33 4. Genomic DNA : Genomic DNA was isolated from PBW larvae selected on the basis of their phenotype, EGFP positiveor wild-type. Concentrations of DNA were determined by spectophotometry. For Southern analysis,

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34 Vector/helper (mg/ml) Injected Developed Fertile adults EGFP Transformation frequency 500/300 800/600 5974 1911 2183 286 86 0 3 0 3.5% 0 Injection and transformation with varied DNA concentrations Results 1.Injection and transformation:

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35 1. Expression of EGFP in third and fourth larval instars of EGFP-transformed line35 compared with wild type PBW larvae. and EGFP-excitation wavelength light . Wild type larvae illuminated under white light

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36 Transformed line 35 larvae illuminated under white light and EGFP-excitation wavelength light

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37 2.Southern blot analysis of DNA extracted from transformed insects of line 35. Hybridization of the probe to EcoRI restricted DNA isolated from EGFP positive individuals of line 35 (lanes1–4, 6,7) phenotypically wild type larvae (lane5), 0.5, 1.0, 2.0, 4.0, 8.0 pg of EcoRI cut vector (lanes 9–13)

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38 3. Western immunoblot of extracted PBW proteins separated on a 15% SDS- PAGE gel, transferred to immobilon P® membrane, probed with a GFP- specific antibody Lanes 1 and 2 contain soluble protein from individual wild type EGFP-negative larvae. Lanes 3 and 4 contain 5mg and 2.5 mg of recombinant EGFP protein respectively .

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39 To use this technology to manipulate genes important for pest control and advance the understanding of the insect order most important to agriculture .

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40 Insect transgenesis and its potential role in agriculture. Alan S. Robinson et al.,

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41 Genetic techniques - Drosophila melanogaster to insect pest species ranging from fruit fly pests of horticulture to mosquito vectors of human disease. Genetic transformation of pest species has proven to be a very important laboratory tool in analyzing gene function and effects on phenotype. The full extension of this technology into the field is yet to be realized A review on the development of transgenic technology in pest insect species and challenges that remain in this applied area of insect genetics and entomology .

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42 Early developments: Early 1970s- Development of gene cloning and in vitro gene splicing techniques in bacteria.. Suggested new possibilities for the use of genetic engineering in many fields of pure and applied biology. In both these areas, insects, either as pests on agricultural crops or as vectors of disease or as insect-transmitted diseases In the public health area still represent a major problem for modern society. 1979 -The application of genetic engineering to solve insect- related problems was first discussed.

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43 1982- Rubin and Spradling were successful in transformation of Drosophila melanogaster using P-element based vectors . It Failure in other insects. because, P-element system depended on the presence of several host factors specific to this species or closely related ones ( Atkinson, 2000). Major problem in early experiments : Lack of efficient transformation markers and Reliance on antibiotic selection to identify putative transformants that proved to be highly ineffective. Overcome: Identification of other transposable elements with a wider host range Isolation of generic transformation markers

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44 Transgenic technology: the current state of the art Genetic transformation of non-drosophilid insects is now possible using four transposon-based gene vectors Mariner, Minos, Hermes and piggy -Bac transposable elements, (Handler, 2001) Used to transform 15 species of insects including Diptera, Hymenoptera, Lepidoptera and Coleoptera The dominant fluorescent protein marker systems, combined with highly conserved gene regulatory sequences, provide reliable methods for detecting, maintaining and recognizing transgenic insects . One agricultural pest, the pink bollworm, has already been registered for testing a fluorescent marking system in a contained release, A malaria vector Anopheles stephensi , has been made partially refractory to an infectious pathogen (Ito et al ., 2002).

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45 Common name Family Species transformed Element References Fruit flies Tephritidae Ceratitis capitata Minos Loukeris et al., 1995 Ceratitis capitata piggyBac Handler et al., 1998 Ceratitis capitata Hermes Michel et al ., 2001 Bactrocera dorsalis piggyBac Handler et al., 2000 Anastrpha suspensa piggyBac Handler et al., 2001 Houseflies Muscidae Musca domestica piggyBac Hediger et al., 2001 Stomoxys calcitrans Hermes O’Brochta et al., 2000 Blowflies Calliphoridae Lucilia cuprina piggyBac Heinrich et al., 2002

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46 Common name Family Species transformed Element References Mosquitoes Culicidae Aedes aegypti Hermes Jasinskiene et al., 1998 Aedesa egypti piggyBac Lobo et al., 2002 Anopheles gambiae piggyBac Grossman et al., 2001 Anopheles stephensi Minos Catterucia et al., 2000 Anopheles stephensi piggyBac Ito et al., 2002 Anopheles albimanus piggyBac Perera et al., 2002 Culex quinquefasciatus hermes Allen et al., 2001

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47 Common name Family / Order Species transformed Element References Saw flies Hymenoptera Athalia rosae piggyBac Sumitani et al .,2003 Silkworm Bombycidae Bombyx mori piggyBac Tamura et al., 2000 Gelechiidmoths Gelechiidae Pectinophora gossypiella piggyBac Peloquin et al .,2000 Darkling beetles Tenebrionidae Tribolium castaneum piggyBac Berghammer et al., 1999 Tribolium castaneum hermes Berghammer et al., 1999

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48 Mobile element interaction with the host genome and their ability to move between species- information is not known… Implementation of transgenic systems Methods to ensure vector stability and strain integrity while maintaining consistent levels of transgene expression. Major areas for application of transgenic technology 1.Sterile insect technique 2.Mass rearing and strain stability 3.Radiation induced sterility

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49 Instability of the introduced genes Transgenes were reported to get rapidly lost under field conditions (Hoy,2000) Experimental release of transgenic predatory mites showed that very few individuals contained the transgene only after three generations while in laboratory strains, it was persistent for over one fifty generations 49 Potential Risks and Research Needs

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50 50 2 . Horizontal gene transfer Difficult to quantify this risk. The whole topic of horizontal gene transfer in insects has received limited scientific attention until relatively recently . It could occur from one insect population to another of the same species, or from one insect species to another, or to other organisms in the environment .

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51 51 3.Gene silencing Multiple mechanisms of transgene silencing occurs in the fruit fly D. melanogaster (Dorer and Henikoff , 1994). it could reduce the effectiveness of transgenic insects after their release in pest management programs. 4.Disruption of ecosystem services The potential effects of transgenic insects on ecosystems is a big topic and difficult to evaluate using laboratory tests (Hoy 2000).

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52 52 5.Current General risk issues Is the transgenic population stable? Has its host or prey range has been altered? Does it have the potential to persist in the environment? Will the transgenic strain will have unintended effects on other species or environmental processes?

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53 53 Creation of transgenic insects with increased fitness Maintenance of stability of genes Risk assessment guidelines require more clarification Overcoming gene silencing problem Prevention of horizontal gene transfer Protocols for evaluating potential risk assessment methods in the permanent releases of transgenic arthropods into the environment Biosafety research on transgenic insect has to gain importance in international symposia FUTURE PROSPECTS

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54 54 Thank U

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