Symposium on Dengue - 2015
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Papers presented at the Symposium on Current Research Activities on Dengue, organized by the Molecular Medicine Unit, on 13th of November 2015
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Item Biology and diversity of Copepods in Sri Lanka and their potential use as predator of Aedes larvae(Moleclar Medicine Unit, Faculty of Medicine, University of Kelaniya, Sri Lanka, 2015) Anukulan, J.W.; Ratnayake, R.M.A.S.; Perera, R.S.M.; Hapukotuwa, R.B.; Iqbal, M.C.M.BACKGROUND: Dengue is a major viral disease in tropical and subtropical countries. Although chemical control of the vector is efficient it causes much damage to the natural environment including destroy of other useful insects. There are many natural predators that prey on mosquito larvae such as copepods, dragonfly, damselfly and larvivorous fish. Among them the use of fish has been practiced extensively to control mosquito larvae. Another potential predator of mosquito larvae is a small aquatic organism called copepod, which is extensively used for biological control in South East Asian countries. We are conducting a preliminary survey on the copepods in Sri Lanka and their ability to prey on larvae of dengue vector Aedes spp. METHODS: Water collected from reservoirs in Kandy (species A), Batticaloa (species B) and Kurunegala (species C) were sieved with a net with 50μm mesh size. The trapped copepods were collected into sampling bottles by washing the net thoroughly. The copepods were observed under a stereo microscope to determine their size. The predation studies were conducted with two ratios: 25 first instar Aedes larvae and 5adult copepods(5:1) and 50 first instar Aedes larvae and 5 adult copepods (10:1). The 5:1 ratio was replicated four times while applying the 10:1 ratio thrice. RESULTS: The body length of the Batticaloa (species B) copepods were larger than the Kandy and Kurunegala copepods. The total mortality of larvae (TM) from the study of 5:1 was species A TM=45, species B, TM=70 and species C, TM=49. The average mortality (AM) was, speciesA, AM=11.25±5.6, species B, AM=17.5±3.1 and species C, AM=12.25±2.5.Mosquito larva predation rate per day by a copepod was, species A 2.25±1.12, species B 3.45±0.62 and species C 2.45±0.5.In the second study with a ratio of 10:1, the total mortality was, species A, TM= 86, species B, TM= 123 and species C, TM= 108. Average mortality was, species A, AM= 28.67±14.17, species B, AM=41±6.24 and species C, AM=39.33±14.53. Mosquito larval predation rate per day by a copepod was, species A, 2.87±1.42, species B, 4.1±0.62 and species C, 3.93±1.45. CONCLUSION: Species B from the Batticaloa, which were with larger in body size showed a better predation ability of Aedes when compared with species A (Kandy) and C (Kurunegala).Item Awareness on dengue and solid waste management among selected school students in Gampaha District of Sri Lanka(Moleclar Medicine Unit, Faculty of Medicine, University of Kelaniya, Sri Lanka, 2015) Radhika, N.M.L.; Gunathilaka, P.A.D.H.N.; Abeyewickreme, W.; Kasturiratne, K.T.A.A.BACKGROUND: Community mobilization is often respected as a powerful tool in ensuring the effective controlling of dengue via promoting effective and sustainable dengue vector control strategies. The current study attempts to investigate the level of awareness on dengue and solid waste management, among selected school students in Gampaha District which is the second highest dengue reported district in Sri Lanka. METHODOLOGY: Awareness programmes on dengue (transmission, vector ecology and preventive measures) and solid waste management were conducted in 10 schools belong to Kelaniya Medical Officer of Health (MOH) area selecting a minimum group of 200 students from each school within the age of 13 - 15 years. Tools such audio-visual presentations, leaflets and videos were used for the awareness programmes. A questionnaire was given to each student prior to the awareness and after the awareness programs as pre and post evaluations respectively in order to assess the general awareness on dengue and solid waste management before and after the awareness programme. One-Way ANOVA, paired t-test and multiple regression techniques were utilized in evaluation of the degree of awareness and key factors affecting the level of awareness, prior and after the conducting the awareness programmes. RESULTS: The degree of initial awareness on dengue and solid waste management of the target school community was recognized to be significantly characterized by the grade of the students and the Education Zonal Category [EZU] (into which their school belongs to) in accordance with One-Way ANOVA (p < 0.05). A significant increment in post awareness of the target school community was recognized due to the conducting of awareness programmes in accordance with the statistics of paired t-test (p < 0.05). Furthermore, based on the multiple linear regression modelling, the post awareness was significantly influenced by the grade of the students and the initial awareness (p < 0.05), regardless of the EZU (p > 0.05). CONCLUSIONS: The awareness of school students on dengue and solid waste management is significantly affected by the grade of the students and EZCs. Awareness programmes are an effective media of raising the awareness of school communities on dengue and solid waste management, while motivating them to actively contribute for the controlling of dengue, via establishing a strong inter sectorial network among schools and other relevant institutions within the Gampaha District.Item Engineering miR-shRNA based molecule to interfere replication of dengue virus in transgenic Aedes aegypti mosquitoes: Bioinformatics approach(Moleclar Medicine Unit, Faculty of Medicine, University of Kelaniya, Sri Lanka, 2015) Ramyasoma, H.P.B.K.D.; Dassanayake, R.S.; Gunawardene, Y.I.N.S.; Kajan, M.; Abeyewickreme, W.BACKGROUND: The genus Flavivirus of the family Flaviviridae includes several vector-borne viruses to which the four serotypes of dengue viruses (DENV-1,-2,-3 and 4) belong to and DENV viruses have a messenger like positive polarity, single-stranded RNA genome approximately 11kb in length which encodes three structural proteins (C-prM-E) and seven Non-Structural proteins (NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5). RNA interference (RNAi) and its properties as a tool has heralded a new era in functional genomics and short double stranded RNAs mediated by RNAi has become a powerful tool for post transcriptional gene silencing. Therefore, this study took the advantage the latter biological phenomenon and designed a multiple miR-shRNA (multi-mir-shRNA) molecule using bioinformatic approach to be effective to block the replication of all dengue serotypes of Sri Lanka. METHODS: Genome sequences of DENV strains belonged to serotypes 1 and 3 isolated from Sri Lanka deposited in GenBank were analyzed for potential sequences for the best siRNA target sites and identified two such sites from DENV 1 and DENV 3 from non structural protein coding sequence of NS5 and structural protein coding sequence of prM consensus regions, respectively. Two more siRNA targets reported from previous study chosen from upstream and downstream of non coding region effective for silencing all DENV serotypes were also used in designing mir-shRNA sequences. The stem region of miR1175 pre-miRNA sequence (miRBase ref: MI0013470) of Aedes aegypi was then replaced with each selected siRNA targets to generate the DENV effective miR-shRNAs and generated miR-shRNAs connected together by placing restriction endonuclease sites between each other to obtain multi-mir-shRNA containing four loop and stem structures. The transgenic gene cassette containing Ae. aegypti carboxypeptidase A promoter, multi-miR-shRNAs and poly adenylation signal of Simian Virus 40 Major Capsid VP1 was then constructed. Expression of this effecter molecule can be achieved by the induction of Ae. aegypti carboxypeptidase A (AeCPA) promoter following blood meal which then ensures activation of RNAi at the time of virus enter into the midgut of mosquito. RESULTS: Folding patterns of the transcript of the designed multi-mir-shRNA cluster were analyzed using online bioinfomatic tool, mfold and the secondary structure of this transcript shown to have optimum endogenous miRNA cleavge/processing with the lowest -ΔG indicating the ability of this design to exert RNAi in mosquito Ae. aegypti. CONCLUSION: Designing multi-miR-shRNA in bioinformatic means an effective way to construct the effector molecule that could exert the maximum RNAi against DENV. However, the effect of design will have to be demonstrated first by transforming to Ae. aegypti mosquitoes and then by evaluating the inhibition of DENV replication in mosquito.Item Maternal transformation of Wolbachia isolated from infected mosquito hosts to Aedes aegypti using micro-injection based procedure: an approach towards integrated dengue vector control(Moleclar Medicine Unit, Faculty of Medicine, University of Kelaniya, Sri Lanka, 2015) Wijegunawardana, N.D.A.D.; Gunawardene, Y.I.N.S.; Chandrasena, T.G.A.N.; Dassanayake, R.S.; Ruanareerate, T.; Kittayapong, P.; Abeyewickreme, W.BACKGROUND: The maternal transformation of Wolbachia (Wb) to Aedes mosquitoes is an approach that has been widely used in Aedes mosquito population control strategies in the integrated dengue vector control efforts. This study details usage of such approach in the maternal transformation of Aedes aegypti using the Wb isolated from infected mosquito hosts using micro-injection based procedure for the first time in Sri Lanka. METHODS: Twenty five ovaries of each adult female Aedes albopictus and Culex quinquifaciatus mosquitoes (14-18 days old) were dissected and Wb was isolated and suspended in 20 μl of 1X Phosphate Buffered Saline (PBS). The isolated samples were kept on ice to maximize the bacteria viability. The exudate was then used in the trans-infection of Ae. aegypti mosquitoes. Infection of Wb into the mosquito Ae. aegypti was carried out by maternal/adult microinjection. To create stable trans-infected lines, the regions near the 3rd leg emerged from the thorax of Ae. aegypti were microinjected to incorporate Wb into the developing germ line and then to facilitate the transmission of Wb to offspring. The volume used for injection based on the swelling of the abdomen, which was monitored throughout the experiment while maintaining the injection pressure, time and number of injecting times. Virgin female Aedes mosquitoes survived after microinjection was fed with 1 ml of 10% sugar solution containing 10,000 units Penicillin/Streptomycin per 100 ml. Survival rate was monitored at 1, 3 and 24 hour/s following microinjection procedure. Survived female mosquitoes resulted from injected line (generation 0 [G0]) was placed in vials with three uninfected male mosquitoes to establish iso-female lines. After egg laying 3 times, G0 females were sacrificed and total DNA was extracted, and PCR was carried out (wsp primers 81F and 691R) using DNA extracted from Ae. aegypti to detect the presence of Wb DNA and its density in the latter mosquito using dilution series of extracted DNA. To select for a stable infection, only offspring from females that came positive for Wb by PCR screening was used as parental stocks. For the samples positive by wsp PCR, strain identification was carried out by performing PCR with 4 different primer sets; wPip, wAlbA, wAlbB and wsp specific for Wb. Females that tested negative for Wb were discarded along with their progeny. The resulting triple infected line will be monitored periodically by PCR to confirm infection status. RESULTS: Wolbachia-triple infected line of Ae. aegypti mosquito was created by using isolated wAlbA+wAlbB+wPip Wolbachia strains. Infection status of newly developed line was monitored by PCR up to G2 and will be continued to G8. Experimental work will be started from G5 to evaluate the use of newly develop Ae. aegypti mosquito line for Wb-based approach under laboratory condition. CONCLUSION: Direct maternal micro-injection technique was used to create Wb infected mosquito line of Ae aegypti, which could be applied in Wb-based dengue vector control activities in Sri Lanka once the line is established.Item Identification of risk factors affecting transmission of dengue in the District of Gampaha, Sri Lanka(Moleclar Medicine Unit, Faculty of Medicine, University of Kelaniya, Sri Lanka, 2015) Withanage, G.P.W.K.; Abeyewickreme, W.; Hapugoda, M.D.INTRODUCTION: Dengue is the most significant mosquito-borne infectious disease in Sri Lanka, causing more than 35,000 cases annually for last five years. The second highest prevalence of dengue was reported from the District of Gampaha for last 10 years. OBJECTIVE: To identify possible risk factors affecting transmission of dengue in selected high risk areas of dengue in the Gampha District. METHODOLOGY: The study was conducted in four high risk Medical Officer of Health (MOH) areas where the annual number of dengue cases greater than 250 during last ten years. In each MOH area, one Grama Niladhari (GN) division with the highest dengue incidence was selected as study areas. In each study area, a cluster of 150, including house-holds, open areas (barren lands, dump yards, and construction sites), abandoned houses, and religious facilities, was selected. A house-hold and entomological surveys were performed in March, 2015 after obtaining an informed written consent. RESULTS: The selected study areas were Akbar town (Mahara MOH), Eriyawatiya (Kelaniya MOH), Kurana (Negombo MOH), and Welikadamulla (Wattala MOH). The size of study human population was 2,544 in 574 house-holds in the study areas and 53.66% (1,365/2,544) were females. The average size of a homestead was 19.3 perches and more than one house was observed in 21.6% (124/574) of land plots. The type of the house-hold was mostly moderate (67.2%-386/574) and the gender of the house-hold head was male in 77.7% (446/574) house-holds. The average number of dwellers in a house-hold was 4 and 7.35% (187/2,544) of the population has previously infected with dengue. Most of house-holds were individual houses with a small garden (98.9%-568/574) and residential function of the house-hold was mainly residential only (83.4%-479/574). The main source of water was piped water (95.1%-546/574), but 39.5% (227/574) using ground-well or tube-well water daily purposes other than drinking and food processing. The most common mosquito preventive measure was bed nets (31.7%-182/574) and 47.6% (273/574) house-holds were using more than one mosquito preventive measures. Collecting tailors of municipal councils is the main solid waste disposal method, while 16.0% (92/574) using burning, burring in pits, composting or open ground due to lack of proper solid waste collection system. Bushes and small trees were the most common vegetation cover of the homesteads (84.5%-485/574) and potential breeding sites were observed in 97.5% (585/600) premises in the study areas. Main mosquito breeding places were plastic and polythene wares, discarded cans and tins, discarded tyres, plant axils, and aluminum and clay pots. The Breteau Index (BI) for Aedes larvae was 4.88 (28/574). Most prominent adult mosquito species in the areas was Aedes albopictus (92.4%-281/304). Dwellers in the study areas have considerable knowledge of the disease and preventive measures, but they reluctant to pursue preventive measures. DISCUSSION: Possible risk factors for transmission of dengue may be crowded conditions, small house-holds and homesteads, poor water and waste management systems, disfavor to pursue preventive measures, and dependence on government vector control programs. Therefore, immense persuade will be required to control dengue in the areas.Item Encapsulation of essential oil blend in β-CD nanoparticles as a repellent for slow release(Moleclar Medicine Unit, Faculty of Medicine, University of Kelaniya, Sri Lanka, 2015) Jayaweera, V.; Eliyapura, A.; Gunasekara, D.; Weerasinghe, L.; Gunathilaka, P.A.D.H.N.; Abeyewickreme, W.; Karunaratne, V.BACKGROUND: Malaria, dengue, and other tropical diseases carried by arthropods such as mosquitoes, ticks and fleas have greatly affected life expectancy, infant mortality, quality of life and work productivity. Essential oils such as citronella, lemon eucalyptus, peppermint, lemon, eucalyptus, lemongrass, geranium, and lavender are known for their mosquito repellency. Due to high volatility of essential oils, these are needed to be reapplied to the skin every 30–60 minutes. Therefore, development of a long lasting mosquito repellent is needed for effective repellent activity. The aim of this experiment is to develop a new blend which encompasses citronella, lemon eucalyptus, lemon and lavender. This oil blend is encapsulated using β-Cyclodextrin (β-CD) to decrease the volatility of the oil blend and increases the long-lasting repellency. METHODS: Citronella, lemon eucalyptus, lemon and lavender were obtained in 15% v/v, 15% v/v, 1% v/v and 1% v/v respectively for a total volume of 100 ml. These oils were mixed with 0.2% w/v of β-Cyclodextrin for encapsulation. The blend was sonicated for 20 min and dissolved in ethanol. This final encapsulated mixture was subjected to bioassays using 110 adult Aedes albopictus mosquitoes against the control. Sitting and biting mosquito behavior patterns were studied against the releasing time. RESULTS: The bioassay results indicated that the encapsulated repellent product is effective against the mosquitos up to 6 hours with zero sitting and biting comparatively to the control. In the 7th hour showed single mosquito sitting with zero biting. CONCLUSION: The encapsulated repellent is effective against the Ae, albopictus mosquitoes for 6 hour time period. Therefore, it is a long-lasting repellent that protect against dengue fever caused by mosquitoes.Item Synthesis of Fe2O3 nanoparticles for the development of a rapid diagnostic test kit for dengue detection(Moleclar Medicine Unit, Faculty of Medicine, University of Kelaniya, Sri Lanka, 2015) Jayarathna, I.P.L.; Gunathilaka, P.A.D.H.N.; Athapaththu, A.M.M.H.; Abeyewickreme, W.BACKGROUND: Hybrid nanoparticles have great potential for biotechnological and biomedical applications. It was recently proposed that biopolymer/co-shell nano-materials could be easily obtained by adapting traditional routes used in pharmaceutical science to design drug delivery system. Most of the case, super-paramagnetic nanoparticles of iron oxides, magnetite (Fe3O4) and maghemite (ᵧ -Fe2O3) have been employed and these interesting magnetic properties are due to finite-size effects and high surface/volume ratio. METHOD: Iron oxide was synthesized by using modified co-precipitation method and resulting particles were characterized using X-ray diffraction (XRD), Transmission Electron Microscopy (TEM) and Diffused Reflectance Fourier Transform-Infrared Spectroscopy (DRIFT-IR). RESULTS: The XRD pattern matches well that of ᵧ -Fe2O3. Six characteristic peaks for ᵧ -Fe2O3 (2θ = 31.7°, 36.7°, 41.1°, 53.4°, 57.0° and 62.6°) marked by their Miller indices [(220), (311), (400), (422), (511) and (440)] were observed for sample. The TEM images reveal that the particles are in 5 – 20 nm range, and well fitted with spaniel cubic structure, but when particles are dry it prefer to agglomerate with neighboring particles to reduce their surface charges. The spectrum of ᵧ -Fe2O3 nanoparticles shows a characteristic broad band at 3410 cm-1 is due to the stretching vibration of H2O molecules. The band corresponding to the bending vibrations of H2O molecules is positioned at 1633 cm-1. Two intense IR bands at 627 and 451 cm-1 are typical for ferrihydrite or ᵧ -Fe2O3 nanoparticles. The spectrum of this sample showed the presence of carbonate groups on the basis of IR bands at 1508, 1340 and 1069 cm-1. The presence of carbonate groups is due to the adsorption of atmospheric carbon dioxide by ᵧ -Fe2O3 nanoparticles. The bands of Fe–O stretching vibrations of ᵧ -Fe2O3 appeared at 627 and 451 cm-1 and the bands at 892 and 796 cm-1 can be assigned to Fe-OH···H bending vibrations. CONCLUSION: Magnetic ᵧ -Fe2O3 nanoparticles were synthesized by the co-precipitation method and this work confirmed that magnetic ᵧ -Fe2O3 nanoparticles are in nano-scale and well matches with spaniel cubic structureItem Designing of immunogenic peptides from Dengue Virus NS1 region for production of monoclonal antibodies as diagnostic intermediates(Molecular Medicine Unit, Faculty of Medicine, University of Kelaniya, Sri Lanka, 2015) Munasinghe, M.M.E.; Chandrasekharan, N.V.; Korbakis, D.; Soosaipillai, A.; Diamandis, E.P.; Athapaththu, A.M.M.H.; Gunathilaka, P.A.D.H.N.; Abeyewickreme, W.BACKGROUND: Small peptide antigens have become an essential tool for antibody production in the recent life science research applications. The immunogenicity of peptide antigens is a critical factor to induce the immune response in order to produce desired antibodies. METHODS: In the current study, we have previously determined four Dengue (DEN) serotype specific peptides, containing 28 Amino Acid (AA) residues were re-designed. The peptides were re-designed considering many factors, for instance, sequence of the Sri Lankan isolates, abundance of Cysteine residues, solubility and the length of the peptide, carrier protein to be used and several other factors such as the N-terminal and C- terminal AAs and multiple AA residues. The peptide sequences were analysed using Antigen Profiler Peptide Tool (Thermo-scientific), Peptide Property Calculator (Genscript) and Swiss-Model (Biozentrum). RESULTS: The protein sequence of the peptides were changed according to the Sri Lankan isolates (AEB98757.1, ACS32038.1, AHG23239.1 and AHN50410.1). Oxidation of Cysteine residues results in significant conformational changes. Replacement of Cysteine with Serine prevents such oxidation reactions and it often retains full biological activity. Generally, peptides with a high number of hydrophobic AA (>50%) may result insoluble peptides. Similarly, to obtain a soluble peptide, it is important to contain at least one charged AA in every five AAs. Hence, the number of hydrophobic residues in the peptides were maintained below 50% and ensured that one out of every five amino acids is charged. The length of the peptide is an important factor as long peptide increases immunogenicity, but also increases the chance for cross-reactivity while a short peptide improves the specificity, but may not be immunogenic. In order to obtain both highly conserved and variable regions among four serotypes, the peptide length was determined as 29 residues. A terminal Cysteine was added to allow peptide conjugation with carrier protein. Keyhole Limpet Hemocyanin was selected as the carrier protein due to its higher immunogenicity. N-terminal Glutamine or Aspargine and C-terminal Proline or Glycine in the sequences were avoided. Finally, the peptides sequences were determined as: DEN1; CPESSDDQRA WNIWEVEDYGFGIFTTNIW,DEN2; CAESPN TNRA WNSLEVEDYGFGVFTTNIW, DEN3;CPESPSASRAWNVWEVEDYGFGVFTTNIW and .DEN4;CSESPNERRAWNSLEVEDYGFGMFTTNIW. CONCLUSION: These peptides have a high potential to be used as peptide antigens for Monoclonal Antibody production.Item Laboratory studies on larvivorous potential of five fish species against Aedes ageypti (Linnaeus) and Ae. albopictus (Skuse)(Molecular Medicine Unit, Faculty of Medicine, University of Kelaniya, Sri Lanka, 2015) Kusumawathie, P.H.D.; Jayasooriya, G.A.J.S.K.; Gunathilaka, P.A.D.H.N.; Hapugoda, M.; Abeyewickreme, W.BACKGROUND: The dengue vectors, namely, Aedes aegypti (Linnaeus) and Ae. albopictus (Skuse) are container breeders, thus, container management is a key element for elimination of vector breeding sites. Use of predator fish is a potential tool for dengue vector control in some container habitats. However, in order to select suitable fish species, larvivorous potential of different fish species needs to be determined. Therefore, the objective of the study was to determine the larvivorous potential of some fresh water fish species against the dengue vectors. METHODOLOGY: Five fish species, namely: Poecilia reticulata, Aplocheilus dayi, Oreochromis niloticus, Rasbora daniconius and Puntius bimaculatus were collected from their natural breeding places during the field surveillance conducted from January-October, 2015. Each fish species was placed in separate fish stock cement tanks at the Regional Office of the Anti- Malaria Campaign in Kandy until they were used for the study. In order to determine the larvivorous potential of the fish, each species was placed in separate enamel trays containing de-chlorinated water and allowed to acclimatize for 30 minutes. A batch of 25 Ae. aegypti and Ae. albopictus larvae was introduced to each tray superlatively and the number of larvae remaining was counted after 24 hours of observation period. Once all the batch of larvae consumes by the fish species, another batch of 25 larvae from respective species was introduced during the observation period. The whole experiment was repeated for 6 times. RESULTS: The mean values of Ae. aegypti larvae consumed by one fish of O. niloticus, P. reticulata, A. dayi, R. daniconius and P. bimaculatus were 97.6, 66.9, 49.3, 62.5 and 47.5 respectively while, one fish of O. niloticus, P. reticulata, A. dayi, R. daniconius and P. bimaculatus consumed 98.4, 62.3, 51.2, 62.5 and 45.4 of mean Ae. albopictus larvae respectively. CONCLUSIONS: All five fish species showed larvivorous activity. However, O. niloticus showed the highest larvivorous potential followed by P. reticulata. Therefore, these two fish species may be considered as a supplementary method to control immature stages of dengue vectors.Item Development of the Sterile Insect Technique (SIT) package for mosquito population control(Molecular Medicine Unit, Faculty of Medicine, University of Kelaniya, Sri Lanka, 2015) Bourtzis, K.Mosquitoes (Diptera: Culicidae) are bloodsucking vector insects of major human pathogens including Plasmodium, dengue and chikungunya which cause infectious diseases resulting to severe morbidity or lethality. According to World Health Organization (WHO), malaria transmitting mosquito species are responsible for more than 600,000 deaths annually, mainly very young children, while more than 2.5 billion people in over 100 countries are at risk due to dengue transmitting mosquitoes. In the absence of efficient, safe and inexpensive drugs and vaccines to combat malaria, dengue and chikungunya, research efforts have focused on the population control of mosquito vector species (mainly Aedes and Anopheles species). In this presentation, I will review recent advances on the development and application of the Sterile Insect Technique (SIT) package for mosquitoes as well as in the field of transgenic and symbiont-based approaches. The pros and cons of each one of these methods, and of their potential combination, will be discussed as well as issues regarding their regulatory approval, stability, sustainability, biosecurity, intellectual property, economic costs and quality control. The results of field trials and future challenges will also be presented.