Develop Safe and Efficient Drive Systems for Introgressing Effector Genes into

Mosquito Populations.

Specific Aim 1:  Mosquito densoviruses as tools for population reduction and transduction. Research on AeDNV has the most immediate potential to deliver products for an effective field trial once the field site is selected and the following experiments completed.  Prototype population cage experiments testing the ability of AeDNV to persist, spread and reduce mosquito populations are encouraging in that a relatively low inoculum of virus in a larval rearing site replicates to levels that reduce the mosquito population, and female mosquitoes originally from the site inoculate virus into new sites. 

Specific Aim 2: Synthetic autonomous TEs based on developmentally-regulated genes. Class II transposable elements (TEs) transpose via DNA intermediates in reactions catalyzed by self-encoded transposases that excise and insert (mobilize) the TE through recognition of element-specific terminal inverted repeat (TIR) DNA sequences.  TEs spread by replicative transposition in germ cells in processes that circumvent Mendelian inheritance, and matings between animals with active TEs and those without can result in a majority of the progeny having the TE.  A balance among transposition rates, genetic load and migration results in fixation of the TE in a population  as was observed during the spread of P through D. melanogaster world-wide in a period perhaps as short as 30 years.  Although much remains to be discovered about TE spread, this mobility supports strongly the concept of adapting them as gene drive systems. Transposition is inherently mutagenic and if unregulated could drive to extinction the population in which the TE resides.  However, self-regulation may impede a TE from spreading. We propose to disable the self-regulatory mechanisms while maintaining the ability to control transposition by using the control sequences of genes regulated early in insect development as a basis for engineering a synthetic TE.  We posit that the control sequences of these genes could direct expression of a transposase in germ cells and catalyze the spread of a TE through replicative transposition to all progeny in subsequent generations.  We will characterize a set of genes for their expression profiles and potential ability to donate control sequences to candidate synthetic TEs. Candidate synthetic TEs will be evaluated to determine if they are capable of replicative transposition and for their impact on field-relevant fitness correlates. Linkage of the TE to effector genes developed in the first Project Objective also will be evaluated. Completion of this Specific Aim will demonstrate whether synthetic TEs are viable gene drive mechanisms.

Specific Aim 3: Meiotic Drive mechanisms for introgressing genes. Meiotic drive is a naturally occurring gene drive system in which gametes or embryos not carrying the drive system are killed.  Natural meiotic drive- The best-studied example of meiotic drive is the segregation distorter (SD) system of Drosophila melanogaster.  Males heterozygous for the SD chromosome normally produce in excess of 95-99% SD-carrying sperm.  A comparable system, M^D, is known in Ae. Aegypti, leading to strong sex-ratio distortion (85% male).  Synthetic meiotic drive- Medea (Maternal Effect Dominant Embryonic Arrest) is a natural drive system for which the molecular basis is unknown.  However, the inheritance pattern that confers the drive property is well-known: embryos from heterozygous females are viable only if they inherit the drive system. We expect to obtain data to confirm or reject the potential of each of these approaches to produce a field-useful drive system, and to assess the merits of each of these approaches relative to the other drive systems under development.

Specific Aim 4:  An Underdominance drive mechanisms for introgressing genes.  It has been proposed that a pair of mutually repressing dominant lethals could form the basis of a gene drive system.  Mathematical modeling indicates that this underdominance (UD) system also exhibits useful properties of non-invasiveness, lack of horizontal transfer, re-usability. We expect to obtain data to confirm or reject the potential of this approach to produce a field-useful drive system, and to assess its merits relative to the other drive systems under development.

Specific Aim 5:  Modeling gene drive and dengue transmission. In any system where a large number of biological variables interact to determine the outcome of a process, it is difficult to assess mentally how changes in variables will alter the outcome. Analytic or computer simulation models are helpful in such situations in describing explicitly the system and predicting outcomes. Through workshops, the Network annual meeting, and the Collaborative Web-based Information Portal (Project Initiative), comprehensive models will be developed of the quantitative parameters that affect genetics-based control methods. Completion of this Specific Aim provides tools for analyzing critical population biology issues, and predictions that aid experimental design and policy making. 

Objective 1                          Objective 2                         Objective 3