Biosafety, regulatory aspects and performance assessment of transgenic OX513A strain of Aedes aegypti L. in India

Transcription

1 Biosafety, regulatory aspects and performance assessment of transgenic OX513A strain of Aedes aegypti L. in India Second Annual South Asia Biosafety Conference Colombo, Sri Lanka 15 th / 16 th September, 2014 Gangabishan Bhikulal Investment and Trading Limited At. Dawalwadi, Tq. Badnapur, P.O.Box-76, Jalna , Maharashtra State, India

2 Contents Overview Import of the OX513A RIDL transgenic Ae. aegypti strain in India RIDL Technology Laboratory studies i. Lethal gene expression in homozygous and heterozygous progenies of OX513A strain. ii. iii. Life table comparisons between OX513A strain and wild type strains Mating competitiveness between male adults of OX513A and wild type strains. Conclusions

3 Overview Present scenario of dengue disease incidence and risk zone in India: Today, dengue ranks as the most important mosquito borne viral disease in the world. Everywhere the human and economic cost are staggering Dr Margret Chan Director General World Health Organization Strategies to prevent dengue disease: Protection from being bitten by Aedes mosquitoes Controlling vector mosquito population in the surroundings. High Probability Low Probability GBIT is working in collaboration with OXITEC to drive environmental friendly RIDL technology for the control of Ae. aegypti mosquito, a primary vector responsible for transmitting dengue and chikungunya fever in India. GBIT is a company focused on investing-in and incubating ideas related to human health and agriculture and establishing its related R&D in India. Ref: WHO

4 Contents Overview Import of the OX513A RIDL transgenic Ae. aegypti strain in India RIDL Technology Laboratory studies i. Lethal gene expression in homozygous and heterozygous progenies of OX513A strain. ii. iii. Life table comparisons between OX513A strain and wild type strains Mating competitiveness between male adults of OX513A and wild type strains. Conclusions

5 Import of the OX513A RIDL transgenic Ae. aegypti strain in India Collaboration GBIT collaborated with OXITEC to drive OX513A RIDL technology for the control of dengue Ae. aegypti mosquito in India during year 2008 IBSC constitution Institutional Biosafety Committee (IBSC) for GBIT was constituted for monitoring the research activities as per the IBSC guidelines (IBSC, 2009) setup by Department of Biotechnology (DBT). Application to RCGM Application for importing and conducting studies on OX513A strain was made in the year 2009 to Review Committee for Genetic Manipulation (RCGM) ACL II Laboratory Arthropod Containment Level II (ACL II) laboratory facility (ACL, 2003) was constructed for importing OX513A strain as per the IBSC guidelines & recommendations from RCGM in Import of strain The OX513A strain was imported from Oxitec in the year 2011 for conducting research activities under ACL II facility. Submission of report A detailed report on laboratory assessment of OX513A strain was submitted to RCGM during October 2013 for further evaluation of the strain under open field conditions. Ref: 1. ACL - Arthropod Containment Levels. Vector Borne Zoonot Dis 3: (2003). 2. IBSC Guidelines for Institutional Biosafety Committee, DBT, 2009

6 Arthropod Containment Level II laboratory [Approved by RCGM, 2011] Ref: Arthropod Containment Levels. Vector Borne Zoonot Dis 3: (2003).

7 Contents Overview Import of the OX513A RIDL transgenic Ae. aegypti strain in India RIDL Technology Laboratory studies i. Lethal gene expression in homozygous and heterozygous progenies of OX513A strain. ii. iii. Life table comparisons between OX513A strain and wild type strains Mating competitiveness between male adults of OX513A and wild type strains. Conclusions

8 RIDL Technology Genetic Construct How it works? teto ttav Bi-sex RIDL is based on positive feedback system

9 RIDL Technology Genetic Construct How it works? teto ttav Bi-sex RIDL is based on positive feedback system

10 RIDL Technology RIDL - Release of insects carrying dominant lethal gene Benefits 1. Low-cost scalability 2. Local area control 3. No insect damage 4. Male releases only 5. Built in monitoring systems Challenges 1. Paving the way forwards deregulating the technology LA513A ON-TET LA513A OFF-TET

11 Contents Overview Import of the OX513A RIDL transgenic Ae. aegypti strain in India RIDL Technology Laboratory studies i. Lethal gene expression in homozygous and heterozygous progenies of OX513A strain. ii. iii. Life table comparisons between OX513A strain and wild type strains Mating competitiveness between male adults of OX513A and wild type strains. Conclusions

12 Lethal gene expression in homozygous and heterozygous progenies of OX513A strain. Experimental Design: Heterozygous Eggs: OX513A males X Wild type females Homozygous Eggs: OX513A males X OX513A females Wild Type Eggs: Wild type males X Wild type females (Delhi strain) 1. Heterozygous Eggs: Without Tet (1000 larvae x 4 replica); With Tet-1 µg/ml (1000 x 3) 2. Homozygous Eggs: Without Tet (1000 x 3), with Tet-1 µg/ml (1000x3) & with Tet-30 µg/ml (1000x2) 3. Wild type Eggs: Without Tet (500x2), with Tet-1 µg/ml (500x3) & with Tet-30 µg/ml(500x3) 12

13 Lethal gene expression in homozygous and heterozygous progenies of OX513A strain. Fig 1: Lethal gene expression during developmental stages in heterozygous and homozygous RIDL strain Aedes aegypti reared in the absence of tetracycline Fig 2: Percent adult emergence of heterozygous OX513A strain reared in the absence of tetracycline Stat: Same letters indicate not-significant difference at 0.05 level by one-way ANOVA using Tukey s-b test.

14 Lethal gene expression in homozygous and heterozygous progenies of OX513A strain. Fig 3: Mean / median life span of survived adults reared with / without Tet. Fig 4: Survival curves for male/female adults reared with / without Tet. Stat: Same letters indicate not-significant difference at 0.05 level by one-way ANOVA using Tukey s-b test.

15 Contents Overview Import of the OX513A RIDL transgenic Ae. aegypti strain in India RIDL Technology Laboratory studies i. Lethal gene expression in homozygous and heterozygous progenies of OX513A strain. ii. iii. Life table comparisons between OX513A strain and wild type strains Mating competitiveness between male adults of OX513A and wild type strains. Conclusions

16 Life table comparisons between OX513A strain and wild type strains Experimental Design: Strains: Transgenic: OX513A RIDL strain Wild type: 1) Delhi strain 2) Aurangabad strain Parameters: Development parameters: Larval adult developmental period & adult life span Reproductive parameters Demographic parameters 16

17 Life table comparisons between OX513A strain and wild type strains First instar - adult Female adult life span Male adult life span Stat: Same letters indicate not-significant difference at 0.05 level by one-way ANOVA using Tukey s-b test. Fig 5: Developmental period and male/female life span of transgenic OX513A strain and non-transgenic Delhi and Aurangabad strains of Ae. aegypti Ref: Patil et al. Mating Competitiveness and life-table comparisons between transgenic and Indian wild-type Aedes aegypti L. Pest Manag Sci (2014). (In print)

18 Life table comparisons between OX513A strain and wild type strains Fig 6: Representation of male/female adult survivorship of transgenic OX513A and wild type Delhi and Aurangabad strains of Ae. aegypti Ref: Patil et al. Mating Competitiveness and life-table comparisons between transgenic and Indian wild-type Aedes aegypti L. Pest Manag Sci (2014). (In print)

19 Life table comparisons between OX513A strain and wild type strains Reproductive parameters: Stat: Same letters indicate not-significant difference at 0.05 level by one-way ANOVA using Tukey s-b test. Fig 7: Number of blood meals and eggs laid during life span of female adults of OX513A and wild type strains. Ref: Patil et al. Mating Competitiveness and life-table comparisons between transgenic and Indian wild-type Aedes aegypti L. Pest Manag Sci (2014). (In print)

20 Life table comparisons between OX513A strain and wild type strains Reproductive parameters: Percent hatching Percent adult emergence Proportion of female emergence Stat: Same letters indicate not-significant difference at 0.05 level by one-way ANOVA using Tukey s-b test. Proportion of female adult emergence was same with respect to male adults in each strain by Chi-square test. Fig 8: Percent hatching, percent adult emergence and proportion of adult emergence of OX513A and wild type progenies Ref: Patil et al. Mating Competitiveness and life-table comparisons between transgenic and Indian wild-type Aedes aegypti L. Pest Manag Sci (2014). (In print)

21 Life table comparisons between OX513A strain and wild type strains Developmental parameters: Net Reproductive Rate [R 0 = Ʃ l x m x ] Mean Generation Time [Tc=Ʃ xl x m x / R 0 ] Doubling Time [T d =L n 2 / r m ] Stat: Same letters indicate not-significant difference at 0.05 level by one-way ANOVA using Tukey s-b test. Fig 9: Net Reproductive rate, mean generation time and doubling time for OX513A and wild type strains Ref: Patil et al. Mating Competitiveness and life-table comparisons between transgenic and Indian wild-type Aedes aegypti L. Pest Manag Sci (2014). (In print)

22 Contents Overview Import of the OX513A RIDL transgenic Ae. aegypti strain in India RIDL Technology Laboratory studies i. Lethal gene expression in homozygous and heterozygous progenies of OX513A strain. ii. iii. Life table comparisons between OX513A strain and wild type strains Mating competitiveness between male adults of OX513A and wild type strains. Conclusions

23 Mating competitiveness between male adults of OX513A and wild type strains. Observed Mating Ratio Expected Mating Ratio Chi-square test (X 2 ) p value 122 : 113 : 26 [OX513A : Delhi : Double mating (OX513A & DEL )] 130.5: 130.5: Fig 10: Mating competitiveness between male adults of transgenic OX 513A and wild type Delhi strains with wild type Delhi strain female adults Ref: Patil et al. Mating Competitiveness and life-table comparisons between transgenic and Indian wild-type Aedes aegypti L. Pest Manag Sci (2014). (In print)

24 Contents Overview Import of the OX513A RIDL transgenic Ae. aegypti strain in India RIDL Technology Laboratory studies i. Lethal gene expression in homozygous and heterozygous progenies of OX513A strain. ii. iii. Life table comparisons between OX513A strain and wild type strains Mating competitiveness between male adults of OX513A and wild type strains. Conclusions

25 Conclusions 1. Heterozygous progeny: More than 98% heterozygous progeny collapse when reared in the absence of Tet. And the shorter lifespan observed for OX513A heterozygous emerged adults reared without Tet, indicated an underlying reduction in fitness that would lead to an equivalent or greater proportional reduction in lifespan under harsher conditions. 2. Survival is an important component of vectorial capacity, as the females have to survive at least the extrinsic incubation period, as typically 7-15 days survival is required for Ae. aegypti to have any possibility of transmitting the virus from person to person (Watts et al., 1987) 3. Life Table: Fit in all the analyzed life-table, reproductive as well as demographic parameters in comparison with wild type strains of Ae. aegypti 4. Mating competitiveness: Mating experiments conducted revealed that males had equal potential for mating competitiveness as compared to wild type counterparts (Patil et al., 2014). Present laboratory experimental results supports the OX513A strain to be potential candidate for controlling wild strain population. GBIT has submitted report on laboratory assessment of OX513A strain to RCGM during October 2013 for further evaluation of the strain under open field conditions in India Ref: Watts DM et al. A. Effect of temperature on the vector efficiency of Aedes aegypti for dengue 2 virus. Am. J. Trop. Med. Hyg. 36: (1987).

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