Selection and Establishment of an Appropriate Field Site for Genetic Control Trials.

Specific Aim 1: Selection and establishment of field sites. One site will be selected from as many as six geographically isolated locations. Four oceanic islands and one ecological island have been identified already, and a sixth site could be added if it meets initial criteria. Site isolation is important as a secondary barrier to the spread of transgenes should mosquitoes escape from the primary containment of the laboratory or outdoor cages. The selection process includes visits to all candidate sites to gather information relevant to site selection criteria, deliberation among relevant project participants (PI, CRs, and NRs), selection of the best location, and establishing the infrastructure and protocols for an effective field station.  Completion of this Specific Aim provides the most appropriate site for Ae. aegypti field studies and large outdoor cage experiments.

Specific Aim 2: Develop strategies to involve community participation in genetics-based methods and analyze ethical, legal and social issues (ELSI).  Community engagement and consultation with local persons at candidate field sites as well as with persons responsible for regulating research using transgenic insects, will be combined with limited aspects of capacity-building to enable appropriate mechanisms of informed consent and community consent for field trials.  Developing the most appropriate biosafety approval procedures to meet legal requirements of NIH and partner countries and institutions for the expected social science and eventual biological/medical trials requires a dialogue with the target communities. The two-way process of community engagement is designed to enhance the local communities understanding of the scientific, cultural, ethical and spiritual aspects of the proposed research, and possible future options for disease control, and to also ensure that the control strategies have regard for local and national values.

Specific Aim 3: Construct and develop procedural protocols for large outdoor cages that will be used to assess genetically modified Ae. aegypti. This effort involves the construction and evaluation of large, ACL2-level outdoor cages at the field station.  Greenhouses (Filclair Corp, France) provide stability, containment, and security for research in a semi-field setting that simulates natural environmental conditions while maintaining stringent containment requirements. These units have double-mesh siding, foundation posts placed in a concrete footing 1 m deep into the soil, and posts, arches, and gutters fit together with a one-piece forged post head with entry through a 2 x 4 m double-door air-lock. Large Outdoor Enclosures: Five units will be constructed each containing two separate experimental enclosures measuring 20 m x 28 m.  A 4 m x 38 m corridor constitutes a barrier to mosquito escape and connects the two enclosures in each unit.  Because Ae. aegypti is endophilic, houses built with local materials in the local style will be placed inside each enclosure. Two houses will be placed in each enclosure so mosquitoes can move between them. Appropriate resting places (clothing on hangers) and local vegetation will be placed indoors and outdoors, respectively.  Water storage containers will provide oviposition sites for female Ae. aegypti, and other relevant features will be placed inside and outside of houses in order to simulate the natural environment of local Ae. aegypti.  Open-mesh roofs may prove problematic (torrential rains may damage or wash-out containers) and should we forgo their use, oviposition sites will be replenished with collected rain water following the local rain pattern. Selection of the location where outdoor enclosures will be constructed will be based on discussions among local collaborators; project participants (PI, CRs, and NRs); and the appropriate regulatory, public health, and government bodies.  At this time mosquitoes will be trapped from the local population in order to establish a genetically-diverse laboratory strain (GDLS) derived from the field site which can be used in initial laboratory studies comparing wild-type mosquitoes with modified ones. 

Specific Aim 4:  Characterize local vector population using mark-release-recapture (MRR).  Accurate monitoring of dispersal rates and migration distances of mosquitoes is needed to track introduction, persistence, and spread of novel alleles.  MRR will be used to characterize free-ranging local mosquitoes to develop monitoring procedures.  MRR will be used to determine survival, dispersal within and among study communities/houses, size of target populations and patterns of oviposition. Twenty-five fluorescently-marked adult blood-fed mosquitoes will be released in each of 20 adjacent houses (n = 500 per site), and color marks will designate the release house.  Adults will be collected by aspiration and immature mosquitoes (different larval instars and pupae) collected every week for at least four months from houses within a 1 km radius of the release houses. This schedule allows the team to collect from each site a day for four consecutive days, and then make collections monitoring for escape on the fifth day. 

Specific Aim 5: Mating behavior comparisons of wild-type and modified Ae. aegypti.  Genetically-modified mosquitoes will not replace or reduce wild vector populations unless they compete successfully for mates with their wild type counterparts. Mating behavior of local and genetically-modified mosquitoes under semi-field conditions (field studies on local mosquitoes) will be assessed in the laboratory for key components of male mating behavior. 

Specific Aim 6: Interactions of genetically-modified and wild-type mosquitoes in laboratory and field cages.  Interactions among genetically-modified and wild-type mosquitoes will influence the success of a genetics-based control method. Cage experiments will determine the effects on net fitness of specific genotypes and phenotypes in transgene-bearing mosquitoes. These experiments require introgression of the desired genes into a genetically-diverse laboratory strain (GDLS) derived from the field site (Specific Aim 3) that are produced and tested by the Cage Trial Initiative (Resources and Facilities). Phase I and II trials screen and eliminate transgenic strains that are not competitive for further evaluation. Phase I trials assess the viability in laboratory cages of a GDLS-transgene strain in direct competition with the parental GDLS. Phase II trials assess the same strain in competition with field site (FS) mosquitoes. Phase III studies are identical to Phase II except that the experiments take place in the large outdoor cages. In some cases, Phase II may be bypassed and a strain moved directly to Phase III trials. This incremental evaluation process has a built-in emphasis on safety and ethics and will result in the identification of appropriate strains of modified mosquitoes that can be used in genetics-based control methods. 

Objective 1                          Objective 2                         Objective 3