Guoliang Fu

Late-acting dominant lethal genetic systems and mosquito control

BackgroundReduction or elimination of vector populations will tend to reduce or eliminate transmission of vector-borne diseases. One potential method for environmentally-friendly, species-specific population control is the Sterile Insect Technique (SIT). SIT has not been widely used against insect disease vectors such as mosquitoes, in part because of various practical difficulties in rearing, sterilization and distribution. Additionally, vector populations with strong density-dependent effects will tend to be resistant to SIT-based control as the population-reducing effect of induced sterility will tend to be offset by reduced density-dependent mortality.ResultsWe investigated by mathematical modeling the effect of manipulating the stage of development at which death occurs (lethal phase) in an SIT program against a density-dependence-limited insect population. We found late-acting lethality to be considerably more effective than early-acting lethality. No such strains of a vector insect have been described, so as a proof-of-principle we constructed a strain of the principal vector of the dengue and yellow fever viruses, Aedes (Stegomyia) aegypti, with the necessary properties of dominant, repressible, highly penetrant, late-acting lethality.ConclusionConventional SIT induces early-acting (embryonic) lethality, but genetic methods potentially allow the lethal phase to be tailored to the program. For insects with strong density-dependence, we show that lethality after the density-dependent phase would be a considerable improvement over conventional methods. For density-dependent parameters estimated from field data for Aedes aegypti, the critical release ratio for population elimination is modeled to be 27% to 540% greater for early-acting rather than late-acting lethality. Our success in developing a mosquito strain with the key features that the modeling indicated were desirable demonstrates the feasibility of this approach for improved SIT for disease control.

Female-specific flightless phenotype for mosquito control

Dengue and dengue hemorrhagic fever are increasing public health problems with an estimated 50–100 million new infections each year. Aedes aegypti is the major vector of dengue viruses in its range and control of this mosquito would reduce significantly human morbidity and mortality. Present mosquito control methods are not sufficiently effective and new approaches are needed urgently. A “sterile-male-release” strategy based on the release of mosquitoes carrying a conditional dominant lethal gene is an attractive new control methodology. Transgenic strains of Aedes aegypti were engineered to have a repressible female-specific flightless phenotype using either two separate transgenes or a single transgene, based on the use of a female-specific indirect flight muscle promoter from the Aedes aegypti Actin-4 gene. These strains eliminate the need for sterilization by irradiation, permit male-only release (“genetic sexing”), and enable the release of eggs instead of adults. Furthermore, these strains are expected to facilitate area-wide control or elimination of dengue if adopted as part of an integrated pest management strategy.

Successful suppression of a field mosquito population by sustained release of engineered male mosquitoes

Successful suppression of a field mosquito population by sustained release of engineered male mosquitoes

Female-specific insect lethality engineered using alternative splicing.

The Sterile Insect Technique is a species-specific and environmentally friendly method of pest control involving mass release of sterilized insects that reduce the wild population through infertile matings. Insects carrying a female-specific autocidal genetic system offer an attractive alternative to conventional sterilization methods while also eliminating females from the release population. We exploited sex-specific alternative splicing in insects to engineer female-specific autocidal genetic systems in the Mediterranean fruit fly, Ceratitis capitata. These rely on the insertion of cassette exons from the C. capitata transformer gene into a heterologous tetracycline-repressible transactivator such that the transactivator transcript is disrupted in male splice variants but not in the female-specific one. As the key components of these systems function across a broad phylogenetic range, this strategy addresses the paucity of sex-specific expression systems (e.g., early-acting, female-specific promoters) in insects other than Drosophila melanogaster. The approach may have wide applicability for regulating gene expression in other organisms, particularly for combinatorial control with appropriate promoters.

A dominant lethal genetic system for autocidal control of the Mediterranean fruitfly

The Sterile Insect Technique (SIT) used to control insect pests relies on the release of large numbers of radiation-sterilized insects. Irradiation can have a negative impact on the subsequent performance of the released insects and therefore on the cost and effectiveness of a control program. This and other problems associated with current SIT programs could be overcome by the use of recombinant DNA methods and molecular genetics. Here we describe the construction of strains of the Mediterranean fruit fly (medfly) harboring a tetracycline-repressible transactivator (tTA) that causes lethality in early developmental stages of the heterozygous progeny but has little effect on the survival of the parental transgenic tTA insects. We show that these properties should prove advantageous for the implementation of insect pest control programs.

Transposon-free insertions for insect genetic engineering

Methods involving the release of transgenic insects in the field hold great promise for controlling vector-borne diseases and agricultural pests. Insect transformation depends on nonautonomous transposable elements as gene vectors. The resulting insertions are stable in the absence of suitable transposase, however, such absence cannot always be guaranteed. We describe a method for post-integration elimination of all transposon sequences in the pest insect Medfly, Ceratitis capitata. The resulting insertions lack transposon sequences and are therefore impervious to transposase activity.

Transgene-based, female-specific lethality system for genetic sexing of the silkworm, Bombyx mori

Transgene-based genetic sexing methods are being developed for insects of agricultural and public health importance. Male-only rearing has long been sought in sericulture because males show superior economic characteristics, such as better fitness, lower food consumption, and higher silk yield. Here we report the establishment of a transgene-based genetic sexing system for the silkworm, Bombyx mori. We developed a construct in which a positive feedback loop regulated by sex-specific alternative splicing leads to high-level expression of the tetracycline-repressible transactivator in females only. Transgenic animals show female-specific lethality during embryonic and early larval stages, leading to male-only cocoons. This transgene-based female-specific lethal system not only has wide application in sericulture, but also has great potential in lepidopteran pest control.

Field Performance of a Genetically Engineered Strain of Pink Bollworm

Pest insects harm crops, livestock and human health, either directly or by acting as vectors of disease. The Sterile Insect Technique (SIT) – mass-release of sterile insects to mate with, and thereby control, their wild counterparts – has been used successfully for decades to control several pest species, including pink bollworm, a lepidopteran pest of cotton. Although it has been suggested that genetic engineering of pest insects provides potential improvements, there is uncertainty regarding its impact on their field performance. Discrimination between released and wild moths caught in monitoring traps is essential for estimating wild population levels. To address concerns about the reliability of current marking methods, we developed a genetically engineered strain of pink bollworm with a heritable fluorescent marker, to improve discrimination of sterile from wild moths. Here, we report the results of field trials showing that this engineered strain performed well under field conditions. Our data show that attributes critical to SIT in the field – ability to find a mate and to initiate copulation, as well as dispersal and persistence in the release area – were comparable between the genetically engineered strain and a standard strain. To our knowledge, these represent the first open-field experiments with a genetically engineered insect. The results described here provide encouragement for the genetic control of insect pests.

Development of a population suppression strain of the human malaria vector mosquito, Anopheles stephensi

BackgroundTransgenic mosquito strains are being developed to contribute to the control of dengue and malaria transmission. One approach uses genetic manipulation to confer conditional, female-specific dominant lethality phenotypes. Engineering of a female-specific flightless phenotype provides a sexing mechanism essential for male-only mosquito, release approaches that result in population suppression of target vector species.MethodsAn approach that uses a female-specific gene promoter and antibiotic-repressible lethal factor to produce a sex-specific flightless phenotype was adapted to the human malaria vector, Anopheles stephensi. Transposon- and site-specific recombination-mediated technologies were used to generate a number of transgenic An. stephensi lines that when combined through mating produced the phenotype of flight-inhibited females and flight-capable males.ResultsThe data shown here demonstrate the successful engineering of a female-specific flightless phenotype in a malaria vector. The flightless phenotype was repressible by the addition of tetracycline to the larval diet. This conditional phenotype allows the rearing of the strains under routine laboratory conditions. The minimal level of tetracycline that rescues the flightless phenotype is higher than that found as an environmental contaminant in circumstances where there is intensive use of antibiotics.ConclusionsThese studies support the further development of flightless female technology for applications in malaria control programmes that target the vectors.

Use of a regulatory mechanism of sex determination in pest insect control

The sexual development of an insect is defined through a hierarchical control of several sex determining genes. Of these genes, transformer (tra) and doublesex (dsx) are well characterized and functionally conserved, especially dsx. Both genes are regulated at the transcriptional level through sex-specific alternative splicing. Incorporation of a genetically engineered sexspecific splicing module derived from these genes in transgenic systems, such as RIDL (release of insects carrying a dominant lethal), would allow the production of male-only insects for control programmes without any physical intervention.