Advances in area of insect biotechnology including genetic engineering and genomics

Advances in area of insect biotechnology including genetic engineering and genomics

1. Advances in area of insect biotechnology including genetic engineering and genomics Submitted To: Dr. S. S. Shaw Professor Department of Entomology Submitted By: P.KRANTHI Ph.D. Scholar

2. CONTENTS Introduction History Insect biotechnology Methods of genetic transformation Advantages Limitations Future prospects Conclusion

3. Introduction Insects - Abundance group - are significant to human life in numerous ways. There are many beneficial ones and quite a few that are harmful and cause direct or indirect damage to the well-being of human beings. Researchers have been continuously trying to find new ways to mitigate problems of harmful insects like crop pests and also to harness the potential of beneficial ones. In this regard, advances made in genetic engineering have enabled the genetic modification of insects for various purposes .

4. Some of the potential applications of this lie in crop pest management, vector management in public health, production of medically important proteins and genetic improvement of beneficial insects like parasitoids , predators, silk worm and honey bee. The proposed release of genetically engineered insects is evoking serious debate among researchers and environmental groups on safety issues as is happening with transgenic plants and engineered microbes .

5. How it all began? Genetic transformation of insects that involves introduction of DNA from external sources was first tried on a scale-less mutant of the stored-grain pest, Ephestia khuniella , in 1965. Injection of wild-type DNA resulted in the production of adults with wing scales 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. Ex: Mediterranean fruit fly ( Loukeris 1995).

6. What is insect biotechnology ? T he application of biotechnological techniques on insects or their cells to develop products or services for human use. Such products are then applied in agriculture, medicine, and industrial biotechnology . Insect biotechnology has proven to be a useful resource in diverse industries, especially for the production of industrial enzymes, microbial insecticides, insect genes, and many other substances.

7. As a basic research tool, the strategies of insect biotechnology include the sequencing and annotation of insect genomes as well as analyses using comparative genomics. Comparative genomics analyses also make it possible to identify insect specific genes that can be targeted for rational insecticide design.

8. Why Genetically engineered insects?  Benefit public health Enhance agricultural production and Provide new forms of economically useful insects For area-wide control of pests As bioreactors to produce pharma products To develop virus-resistant insect lines To improve disease resistance, pollination attributes in honey bees and high-quality silk production in silk moth ( Gopanathan , 1992)

9. WHAT IS GENOMICS? Genomics is the study of whole genomes of organisms, and incorporates elements from genetics. Genomics uses a combination of recombinant DNA, DNA sequencing methods, and bioinformatics to sequence, assemble, and analyse the structure and function of genomes.

10. Requirements for genetic engineering 1. Gene of interest or exogenous DNA 2. Vector 3. Marker gene 4. Promoter

11. Physical methods 1.Microinjection 2.Biolistics 3.Lipofection 4.Electroporation Methods of genetic transformation Biological methods 1. Vector transformation 2. Sperm mediated transformation 3 . Paratransgenesis

12. 1.Microinjection : Refers to the process of using a micro needle to deliver substances into a living cell. Physical methods

13. 2.Biolistics A genetic engineering technique where particles are accelerated to deliver the genetic material directly into the cell.

14. 3.Lipofection : Lipofection is a lipid-based transfection technology which belongs to biochemical methods including also polymers, dextran and calcium phosphate. Lipofection principle is to associate nucleic acids with cationic lipid formulation.

15. 4.Electroporation An electrical treatment of cells that induces transient pores through which exogenous genetic material can enter the cell.

16. Biological methods I. Vector transformation II. Sperm mediated transformation III. Paratransgenesis

17. 1.VECTOR TRANSFORMATION

18. Transposons T ransposons are generally employed for delivering the gene of interest in insects Mobile pieces of DNA that do not remain fixed at one genomic location but move from one site on a chromosome to another (Liao,2000) Increase their copy number as they move around among chromosomes within individual organisms

19. Transposable elements insects 1. Tc1 elements- Medfly 2. Mariner elements -Fruit fly 3. hAT elements- Drosophila and Helicoverpa 4. Piggy Bac elements- Silkworm

20. For a stable integration of the transgene of interest into the germline of an egg we need two separate plasmids 1.D onor plasmid 2. Helper plasmid

21. 1) Vector/Donor 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 enzyme vector plasmid, carrying the transgene of interest (yellow) and a visible transformation marker, the enhanced green fluorescent protein (EGFP) (green) driven by 3xP3 promoter, within the functional terminal inverted repeats (TIRs)

22. 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 Helper plasmid that codes for a functional transpoase enzyme (violet) with defective TIRs are used

23. VECTOR TRANSFORMATION ( Wimmer , 2003)

24. Selection of Genetically Transformed Insects The genetically engineered insects are selected using either 1.Neomycin phosphotransferase (NPT II) (which confers resistance to neomycin analogues), 2.Organophosphorus dehydrogenase (OPD) (which confers resistance to paraoxan ) 3.T he gene for dieldrin resistance (RDL). 4. Other useful visual markers are the green fluorescent protein (GFP) and its spectral variants .

25. 2. Sperm mediated transformation Factors like low reproductive rates and egg properties prevent DNA introduction The strategy adopted to linearize foreign DNA and introduce it with sperm during the instrumental insemination of virgin queen honey bees. (Robinson,2000)

26. 3. Paratransgenesis (symbiotic control) Paratransgenesis -Frank Richards (1996) The goal of this technique is to control vector-borne diseases. The first step is to identify proteins that prevent the vector species from transmitting the pathogen. The genes coding for these proteins are then introduced into the symbiont, so that they can be expressed in the vector. The final step in the strategy is to introduce these transgenic symbionts into vector populations in the wild. Genes expressed in microbes alter the characteristics of host insects

27. The procedure of insect transformation via transgenic symbionts is depicted

28. The first example of this technique used Rhodnius prolixus which is associated with the symbiont Rhodococcus rhodnii . R. prolixus is an important insect vector of Chagas disease that is caused by Trypanosoma cruzi . The strategy was to engineer R. rhodnii to express proteins such as Cecropin A that are toxic to T. cruzi or that block the transmission of T. cruzi

29. In order to perform paratransgenesis , there are several requirements   The symbiotic bacteria can be grown in vitro easily. They can be genetically modified, such as through transformation with a plasmid containing the desired gene. The engineered symbiont is stable and safe. The association between vector and symbiont cannot be attenuated. Field delivery is easily handled.

30. Advantages of genetically engineered insects In Public health In Agriculture pest 3) In economically useful insects  

31. 1) In public health GM mosquitoes A) Genetically modified malaria causing mosquitoes Mosquitoes are engineered to produce protein that disrupt malarial parasite life cycle within insect Gene (SM1) prevents malarial parasite from penetrating into mosquito mid gut and reaching salivary glands ( Braig and Yan, 2002) Anopheles stephensi is one of the genetically engineered common mosquito species to resist malaria ( Catteruccia , 2003) The GM mosquitoes could be identified by their green fluorescent eyes.

32. B)Genetically modified yellow fever causing mosquitoes Mosquito like Aedes aegypti spread yellow fever GM mosquito to replace these breeds by producing antibacterial protein, limiting its ability to transmit disease (Adelman, 2002)

33. C) Genetically modified African Sleeping sickness causing Tsetse fly It affects more than 50,000 people per year It is caused by Tsetse fly and kissing bug Controlled by paratransgenesis Secondary symbionts like (Wolbachia) that lives inside insect is engineered to kill the trypanosomal parasite (Dale and Welburn , 2001) This parasite passes through insect digestive system. Antitrypansomal gene products are expressed in insect gut making them incapable of transmitting the disease

34. 2 ) In Agriculture pest a) Pink boll worm Sterile insect technique programme (SIT) Protects more than 900,000 acres of cotton Million of male pink boll worm moth were sterilized by irradiation ( Pelloquin , 1999) A lethal gene ( tTA ) is introduced from bacteria. It alters the metabolism of the PBW larvae (Briggs, 2001)

35. B) Transgenic Mediterranean fruit fly Males are sterilized by irradiation prior to release (Lobo, 1999) Sterile males’ mate with feral females hindering female reproduction

36. C) Pierce’s disease Pierce’s disease caused by bacteria It is the lethal infection of grape vines by bacteria- Xyllela fastidiosa ( Bextine , 2004) This bacterium is carried by the vector Glass Winged Sharp Shooter,   Homalodisca vitripennis There is no control measure for this disease Only Controlled by paratransgenesis Anti Xyllela effector proteins (S 1) were isolated and modified to carry in inects against Xyllela (anti-bacterial toxins) (Miller, 2007)

37. D) Transgenic Red flour beetle It is a worldwide pest of stored products Genes responsible for regulating pheromone secretion are mutated ( Daborn , 2002).

38. E) Transgenic predatory mites They feed on Spider Mites, a polyphagous pest. First field trial of a transgenic insect – Transgenic predatory mite Metaseiulus occidentalis , predator of spider mites ( Presnail , 1997)

39. 3) In economically useful insects A) Honey bees Death of honeybees under rearing conditions is mainly because of entomopathogenic diseases and parasites Genetically engineered honeybee with gene coding for hDAF are resistant to diseases, parasites and insecticides (Kimura, 2001)

40. B) Silkworms Genetically modified silkworm produce an industrial and therapeutic proteins like human growth hormone and human collagen (Kadonookuda,1995) Silk glands generally express the introduced L-chain gene and GFP Spider milk (protein) produced from modified silkworm larvae are used to make bullet proof vests, parachutes and artificial ligaments (Lewis,2006)

41. Recent developments in scientific techniques have allowed researchers to create GM silk worms, harnessing the strong protein and other proteins production. One group recently created a GM strain of silkworms that produce human antibodies (Park et al., 2009).

42. Product Source Uses Ref 5.8 kDa honey component of manuka honey Apis spp. For wound repair/healing Tonks et al.2007 Manuka honey, buckwheat honey, honey products of Revamil , propolis Apis spp. Inhibits Gram-positive MRSA, vancomycin-sensitive and vancomycin resistant Enterococci, Streptococcus species isolated from wounds, Gram-negative bacteria associated with wounds such as Pseudomonas aeruginosa , Stenotrophomonas sp and Acinetobacter baumannii Cooper et. al 2011 Examples of potential use of products of insect biotechnology in medicine

43. Quercetin, apigenin, and acacetin Apis spp. Anticancer lymphocytic leukemia Kwakman et al.2012 Bee, wasp, and ant venom products, e.g., melittin Apis spp., Vespula spp., Solenopsis spp., Pachycondyla spp., and Myrmecia s pp. Inhibits or kills cancer cells Szliszka et al.2013 Maggot alloferons Lucilia sericata Antiviral and antitumor activities Oršolić , N. (2012)

44. Cantharidin Blister beetles, Mylabris caragnae , and Mylabris phalerate Treatment of cancers including hepatic, colorectal, melanoma, pancreatic, bladder, breast, and leukemia as well as activities against Plasmodium falciparum and Leishmania major Chernysh et al.2002 Anopheline Anopheles salivary gland Thrombin inhibition and development of anti thrombotics for use as anticoagulants Frank et al.2011

45. Insect cell baculovirus expression vector system

46. Introduced transgenes in insects Insects Gene Character modified A nopheles SM 1 Disease causing ability is destroyed C ulex Defensin   Disease spreading ability is lost Silkworm Spider flagelliform Enhances quality of silk protein   Wolbachia Attacin and Cercopin Infective capacity is lost   Xylella S 1 Disease causing capacity is absent

47. GENOME EDITING DNA is inserted, deleted, mutate or replaced at a particular position in the genome of the organism METHODS: Zinc finger nuclease (ZFN) TALENs - transcription activator-like effector nucleases CRISPR (Clustered regularly interspaced short palindromic repeats) RNAi ( RNA interference )

48. Possible risks in releasing genetically modified insects into the environment may be: Disturbance of ecological balance. Total elimination of a pest species that give rise to another species to fill the vacuum. Viral vectors combining with other wild type viruses and also with the genome of the host. Exchange of transposons between organisms

49. Limitations: 1 . Instability of the introduced genes: Transgenes were reported to get rapidly lost under field conditions (Hoy,2003) 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 2. Horizontal gene transfer Genes are vertically transmitted to progenies and move between individuals of different species in a process known as horizontal gene transfer (by Kidwell, 1992) Seen in insects and bacterial symbionts

50. 3.Poor fitness It is due to the result of introducing transgene thereby making the recipient weaker (Atkinson, 1996) Laboratory rearing is not compatible with competitive behaviour of insect 4. Environmental risks are also associated with the use of genetically modified insects over the open environment. Once they released, they can’t be taken back so lots of investigation and regulation should be done for this research.

51. Future Prospects Transgenic insect approach will help to control harmful insects and create beneficial insects Creation of transgenic insects with increased fitness Biosafety research on transgenic insect has to gain importance in international symposia Risk assessment guidelines require more clarification.

52. Conclusion Genetic engineering of insects for various purposes is at a very nascent stage. The major drawbacks are limited knowledge on molecular genomics of different species, low frequency of transformation, high cost etc. Once these are overcome, genetic engineering of insects would be a job as routine as that of transformation of plants and microbes. Genetically engineered insects offer great scope for crop pest management, which would eventually result in reduced usage of pesticides. In addition, there is a possibility to modify behaviour of insects, improvement of efficiency of parasitoids and predators in biological control, disease and vector control in public health and vector management in plant disease management.

53. It is difficult to predict all of the broader ecological impacts of these changes. It is possible that the elimination or vast reduction in numbers of this organism could have dire consequences for other non-targeted organisms.

54. Progress is impossible without change, and those who cannot change their minds cannot change anything George Bernard shaw