Biosafety training course for Africa related to genetically modified disease vectors
Containment issues during planned field cage trials
- Once transgenic mosquitoes have been created, presuming that they show promise in the laboratory, they will then be studied in outdoor field cages.
- These cages are designed to better understand the effectiveness of GM mosquitoes and any side-effects that could become apparent under more realistic conditions.
- It is impossible to guarantee 100% containment; however the chance of an accidental release can be minimized.
- Care must therefore be taken in the design and implementation of field cage trials.
- These considerations are especially pertinent because:
- the mosquito is a vector of human disease; and
- the transgenes are designed to spread through the mosquito population at faster-than-Mendelian rates of inheritance.
- Laboratory studies.
- Analytic or computer simulation studies.
- Contained field trials.
- Open release trials.
Figure 1: Flow chart describing the phased testing of GM mosquitoes. Column A shows the testing phase, column B shows the steps required for research and development, and Column C shows the field site development pathway.
- First step in phased testing of transgenic mosquitoes.
Evaluate basic characteristics of GM mosquitoes:
- Molecular, genotypic, physiological, behavioral.
- Compare these characteristics to those of wild-type mosquitoes.
- Evaluate the stability of these characteristics over subsequent generations.
Does the anti-pathogen gene work?
- Study effects of the anti-pathogen gene in a laboratory strain of the GM mosquito.
- Then study the effects in mosquitoes collected from the contemplated field trial site.
Does the gene drive system work?
- Evaluate the ability of the gene drive system and anti-pathogen gene to spread through a mixed population of transgenic and wild-type mosquitoes in the laboratory.
Test for unintentional adverse effects of transgenic DNA:
- Increase in vectorial capacity or vector competence for non-target pathogens.
- Increase in blood-feeding.
- Increase in mosquito fitness/longevity.
- Altered population growth rate.
- Increased carrying capacity of environment (e.g. natural TE-generated inversions are associated with aridity tolerance in An. gambiae).
- Decreased susceptibility to control measures (e.g. insecticides).
- Horizontal gene transfer between mosquitoes and other organisms likely to have an undesirable ecological or environmental effect.
- Carry out while laboratory and other biological studies are being performed.
- Help to predict the range of potential effects of a transgenic release.
Incorporate information from all biological studies:
- Parameters measured from laboratory studies.
- Fitness effects of the anti-pathogen gene.
- Properties of the gene drive system.
- Ecology of the local mosquito popualtion.
- Epidemiology of the disease.
- DNA mutation rates and loss of genetic material (leading to loss of anti-pathogen gene function).
Results can contribute to:
- Requirements for an effective disease control strategy.
- Criteria for progressing from the laboratory to cage studies.
- Risk assessment for cage studies and open release.
- Intermediate research stage between the laboratory and an open release.
- Needed to provide a more realistic assessment of the characteristics of GM mosquitoes in the wild.
Figure 2: A mosquito containment facility at the Ifakara Health Research and Development Center in Tanzania.
Requirements before beginning contained field trials:
- Evidence from laboratory and modeling studies that there will be no great harm following an accidental release.
- Reasonable expectation that the results from the trial will provide meaningful insights into the use of GM mosquitoes.
Phases of contained field trials:
- Begin with a field site that is only suitable for mosquitoes at certain times of the year.
- If results are promising and there are no noticeable side-effects, then move to a field site where GM mosquitoes would actually be release.
Before conducting the trial:
- Collect information on mosquito ecology and disease epidemiology at the field site.
- Study the possibility of an accidental release using non-transgenic local mosquitoes.
- Test procedures to detect GM mosquitoes in the wild in the event of an accidental releae.
- Test measures to prevent dispersal of escaped mosquitoes (e.g. vegetation-free zones, traps).
Conducting the trial:
- Begin with an unmixed population of GM mosquitoes.
- Determine the viability and lifespan of GM mosquitoes in the absence of wild-types.
- Frequently monitor inside and outside the cage for:
- GM mosquitoes.
- GM arthropod predators and vertebrate insectivores.
- GM blood hosts.
- Other GM non-target species.
- Reducing the size/scale of the cage trial will reduce the possibility of negative effects.
- Then study a mixture of GM and wild-type mosquitoes.
- Assess interations betwen GM and wild-tpes mosquitoes over several generations.
- Can only perform open release trials after a complete risk assessment has been performed and the efficacy of GM mosquitoes has been ascertained in contained trials.
Phases of open release trials:
- Begin with simple mass release in a small field trial population using transgenic mosquitoes lacking a gene drive system.
- If results are promising and the anti-pathogen gene is effective, then carry out a second release using a less invasive gene drive system (i.e. engineered underdominance) which is not capable of spreading into neighboring populations.
- In the event of unanticipated negative effects, the engineered underdominance constructs and anti-pathogen gene can be removed from the population by mass release of wild-type mosquitoes above a certain threshold.
- Finally, it will be possible to consider releasing the anti-pathogen gene attached to a more invasive gene drive system (e.g. Medea).
- Practices that prevent unplanned or uncontrolled release of organisms into the wild.
- Containment within an enclosed structure.
- Containment procedures familiar from transgenic microbes, plants and plant pests.
- Combine anti-pathogen genes with regulatory elements that are functionally-inactive outside the trial setting.
- Use gene drive systems that are unlikely to spread following an accidental release (e.g. Medea, Wolbachia, engineered underdominance constructs).
- Use GM mosquitoes that are less able to survive or reproduce in the wild.
- Conduct studies in an inhospitable environment to mosquito spread (due to climate or geographical location).
- Use of trap crops, vegetation-free zones or spatial isolation to prevent mosquito spread into neighboring popualtions.
- In general, imagine the possible ways in which containment can be breached.
- Then think about how these conditions can be avoided.
|Possible breaches of containment:
||How they can be avoided:
Structural failure of the cage due to:
- Predictable environmental damage (e.g. wind, rain, UV exposure)
- Unpredictable environmental damage (e.g. tornados, earthquakes, lightning)
- Human damage (e.g. vehicles)
- Animal damage
- Design cage to withhold weather and environmental damage.
- Plan experiments for more environmentally stable times of the year (e.g. not during monsoon season).
- Restrict vehicle and parking access around the facility.
- Plan routine examination of cage integrity.
Accidental release due to:
- Unavoidable accident (e.g. water leak containing eggs, larvae, pupae)
- Preventable accident (e.g. leaving door open due to human error)
- Unapproved change or conscious relaxation of biosecurity protocol
- Inadequate training of personnel.
- Placement of cage to minimize possibility of accidents.
- Write clear and explicit protocols in the languages of all employees.
- Develop safety courses and rehearse procedures regularly.
Deliberate actions leading to a breach:
- Establish good community relations, education and outreach.
- Take local ethical, social and cultural issues into account during site selection.
- Maintain an ongoing commitment before, during and after the trial.
- Install physical barriers and security systems at the site.
- Perform background checks on all potential employees.
Caged GM mosquitoes could mate with wild local mosquitoes through the cage mesh walls.
- Design a cage having two walls of screening.
Possible interactions between caged mosquitoes and non-target organisms:
- Exposure of GM mosquitoes to microbes and nematodes resident in soil or on plants within the cage.
- Entry of small invertebrate organisms from the wild into the cage through cracks, mesh or drains.
- Transfer of organisms into the cage by employees via clothing, footwear, tools or equipment.
- Exposure of organisms brought into the cage as a source of bloodmeal to transgenic material.
- Design transgenic DNA to be non-functional in non-target organisms.
- Design containment facility to prevent entry and exit of small organisms.
- Decontaminate soil, plants, animals and employee clothing before removing them from the cage.
- Prevent interactions between mosquitoes and employees within the cage.
- Treat drainage water from the cage.
Table 1: Possible breaches of containment from field cages and how they can be avoided.
- Risks associated with the potential escape of GM mosquitoes from field cage trials can be minimized through good choices of cage location and design.
- Good policies on surveillance and remediation can help to ameliorate the consequences of an accidental release.
Choice of location:
- Good access to mosquito rearing facility.
- Possibility of safe transport of mosquitoes between the laboratory and field cage.
- Existing infrastructure.
- Community consent.
- Relative isolation from other communities.
- Understanding of local mosquito ecology.
- Stable ground (e.g. not on an earthquake faultline).
- Unlikely to be affected by falling tree branches, fire or flooding.
- For reasonable environmental exposure, it is anticipated that cages will have mesh siding and ceiling.
- Double-wall siding with mesh size 1.2 mm x 1.2 mm (this may not be small enough).
- Mesh should be resistant to ripping, rusting, tearing and stretching.
- Open-mesh roofs are best for simulating a natural environment; but shouldn't be used in regions with torrential rains.
- Ventilation systems should be designed to avoid transfer of mosquitoes and other organisms.
- Use a concrete foundation overlaid with soil.
- Use a double-screen door with air-lock to enter the cage (this will assist the capture of escaped mosquitoes).
Figure 3: A containment room for entry into individual cages designed to enhance protection against escape.
- Survey the outside environment for presence of transgenic material in order to check for inadvertent release.
- Examine cages visually for structural damage due to bird nests, bats, rodents and termites.
- Should have an idea of dispersal range of species being engineered in the event of an escape.
- Collect mosquitoes in the wild using ovitraps and aspirators, then assay for presence of the transgene using PCR.
- Monitor frequencies of target and non-target diseases.
- Regularly check non-target organisms for presence of transgenic DNA.
Prevention of inadvertent release:
- Monitor local weather and environmental conditions.
- Depopulate field cages in the event of an impending storm.
Remediation of inadvertent release:
- Spray inside every surrounding household with insecticide (regularly determine the efficacy of insecticides at the field site).
- Treat water containers and breeding sites with larvicide.
- Continue until GM mosquitoes are no longer detected in the environment for a substantial period of time.
- American Committee of Medical Entomology, 2003 Arthropod Containment Guidelines. American Society of Tropical Medicine and Hygeine, Deerfield, Illinois.
- Benedict, M., P. D’Abbs, S. Dobson, M. Gottlieb, L. Harrington et al., 2008 Guidance for Contained Field Trials of Vector Mosquitoes Engineered to Contain a Gene Drive System: Recommendations of a Scientific Working Group. Vector-Borne and Zoonotic Diseases 8: 127-166.
- Marshall, J. M., 2008 (In press) The effect of gene drive on containment of transgenic mosquitoes. J. Theor. Biol.
- Sinkins, S. P., and F. Gould, 2006 Gene drive systems for insect disease vectors. Nat. Rev. Genet. 7: 427-435.