piggybac- and PhiC31-Mediated Genetic Transformation of the Asian Tiger Mosquito, Aedes albopictus (Skuse)

The Asian tiger mosquito, Aedes albopictus, is a highly invasive mosquito and has spread from South East Asia to Europe, the United States and northern areas of Asia in the past 30 years. Aedes mosquitoes transmit a range of viral diseases, including dengue and chikungunya. Aedes albopictus is generally considered to be somewhat less of a concern in this regard than Aedes aegypti. However a recent mutation in the chikungunya virus dramatically increased its transmission by Aedes albopictus, causing an important outbreak in the Indian Ocean in 2006 that eventually reached Italy in 2007. This highlights the potential importance of this mosquito, which can thrive much further from the Equator than can Aedes aegypti. This paper describes the first genetic engineering of the Asian tiger mosquito. This is an essential step towards the development of genetics-based control methods against this mosquito, and also an invaluable tool for basic research. We describe both transposon-based and site-specific integration methods

Modeling the mutation and reversal of engineered underdominance gene drives

A range of gene drive systems have been proposed that are predicted to increase their frequency and that of associated desirable genetic material even if they confer a fitness cost on individuals carrying them. Engineered underdominance (UD) is such a system and, in one version, is based on the introduction of two independently segregating transgenic constructs each carrying a lethal gene, a suppressor for the lethal at the other locus and a desirable genetic “cargo”. Under this system individuals carrying at least one copy of each construct (or no copies of either) are viable whilst those that possess just one of the transgenic constructs are non-viable. Previous theoretical work has explored various properties of these systems, concluding that they should persist indefinitely in absence of resistance or mutation. Here we study a population genetics model of UD gene drive that relaxes past assumptions by allowing for loss-of-function mutations in each introduced gene. We demonstrate that mutations are likely to cause UD systems to break down, eventually resulting in the elimination of introduced transgenes. We then go on to investigate the potential of releasing “free suppressor” carrying individuals as a new method for reversing UD gene drives and compare this to the release of wild-types; the only previously proposed reversal strategy for UD. This reveals that while free suppressor carrying individuals may represent an inexpensive reversal strategy due to extremely small release requirements, they are not able to return a fully wild-type population as rapidly as the release of wild-types

Population dynamics of engineered underdominance and killer-rescue gene drives in the control of disease vectors

A number of different genetics-based vector control methods have been proposed. Two approaches currently under development in Aedes aegypti mosquitoes are the two-locus engineered underdominance and killer-rescue gene drive systems. Each of these is theoretically capable of increasing in frequency within a population, thus spreading associated desirable genetic traits. Thus they have gained attention for their potential to aid in the fight against various mosquito-vectored diseases. In the case of engineered underdominance, introduced transgenes are theoretically capable of persisting indefinitely (i.e. it is self-sustaining) whilst in the killer-rescue system the rescue component should initially increase in frequency (while the lethal component (killer) is common) before eventually declining (when the killer is rare) and being eliminated (i.e. it is temporally self-limiting). The population genetics of both systems have been explored using discrete generation mathematical models. The effects of various ecological factors on these two systems have also been considered using alternative modelling methodologies. Here we formulate and analyse new mathematical models combining the population dynamics and population genetics of these two classes of gene drive that incorporate ecological factors not previously studied and are simple enough to allow the effects of each to be disentangled. In particular, we focus on the potential effects that may be obtained as a result of differing ecological factors such as strengths of larval competition; numbers of breeding sites; and the relative fitness of transgenic mosquitoes compared with their wild-type counterparts. We also extend our models to consider population dynamics in two demes in order to explore the effects of dispersal between neighbouring populations on the outcome of UD and KR gene drive systems

Modeling the mutation and reversal of engineered underdominance gene drives

A range of gene drive systems have been proposed that are predicted to increase their frequency and that of associated desirable genetic material even if they confer a fitness cost on individuals carrying them. Engineered underdominance (UD) is such a system and, in one version, is based on the introduction of two independently segregating transgenic constructs each carrying a lethal gene, a suppressor for the lethal at the other locus and a desirable genetic “cargo”. Under this system individuals carrying at least one copy of each construct (or no copies of either) are viable whilst those that possess just one of the transgenic constructs are non-viable. Previous theoretical work has explored various properties of these systems, concluding that they should persist indefinitely in absence of resistance or mutation. Here we study a population genetics model of UD gene drive that relaxes past assumptions by allowing for loss-of-function mutations in each introduced gene. We demonstrate that mutations are likely to cause UD systems to break down, eventually resulting in the elimination of introduced transgenes. We then go on to investigate the potential of releasing “free suppressor” carrying individuals as a new method for reversing UD gene drives and compare this to the release of wild-types; the only previously proposed reversal strategy for UD. This reveals that while free suppressor carrying individuals may represent an inexpensive reversal strategy due to extremely small release requirements, they are not able to return a fully wild-type population as rapidly as the release of wild-types

Population dynamics of engineered underdominance and killer-rescue gene drives in the control of disease vectors

A number of different genetics-based vector control methods have been proposed. Two approaches currently under development in Aedes aegypti mosquitoes are the two-locus engineered underdominance and killer-rescue gene drive systems. Each of these is theoretically capable of increasing in frequency within a population, thus spreading associated desirable genetic traits. Thus they have gained attention for their potential to aid in the fight against various mosquito-vectored diseases. In the case of engineered underdominance, introduced transgenes are theoretically capable of persisting indefinitely (i.e. it is self-sustaining) whilst in the killer-rescue system the rescue component should initially increase in frequency (while the lethal component (killer) is common) before eventually declining (when the killer is rare) and being eliminated (i.e. it is temporally self-limiting). The population genetics of both systems have been explored using discrete generation mathematical models. The effects of various ecological factors on these two systems have also been considered using alternative modelling methodologies. Here we formulate and analyse new mathematical models combining the population dynamics and population genetics of these two classes of gene drive that incorporate ecological factors not previously studied and are simple enough to allow the effects of each to be disentangled. In particular, we focus on the potential effects that may be obtained as a result of differing ecological factors such as strengths of larval competition; numbers of breeding sites; and the relative fitness of transgenic mosquitoes compared with their wild-type counterparts. We also extend our models to consider population dynamics in two demes in order to explore the effects of dispersal between neighbouring populations on the outcome of UD and KR gene drive systems

Conditions for success of engineered underdominance gene drive systems

Engineered underdominance is one of a number of different gene drive strategies that have been proposed for the genetic control of insect vectors of disease. Here we model a two-locus engineered underdominance based gene drive system that is based on the concept of mutually suppressing lethals. In such a system two genetic constructs are introduced, each possessing a lethal element and a suppressor of the lethal at the other locus. Specifically, we formulate and analyse a population genetics model of this system to assess when different combinations of release strategies (i.e. single or multiple releases of both sexes or males only) and genetic systems (i.e. bisex lethal or female-specific lethal elements and different strengths of suppressors) will give population replacement or fail to do so. We anticipate that results presented here will inform the future design of engineered underdominance gene drive systems as well as providing a point of reference regarding release strategies for those looking to test such a system. Our discussion is framed in the context of genetic control of insect vectors of disease. One of several serious threats in this context are Aedes aegypti mosquitoes as they are the primary vectors of dengue viruses. However, results are also applicable to Ae. aegypti as vectors of Zika, yellow fever and chikungunya viruses and also to the control of a number of other insect species and thereby of insect-vectored pathogens

Conditions for success of engineered underdominance gene drive systems

Engineered underdominance is one of a number of different gene drive strategies that have been proposed for the genetic control of insect vectors of disease. Here we model a two-locus engineered underdominance based gene drive system that is based on the concept of mutually suppressing lethals. In such a system two genetic constructs are introduced, each possessing a lethal element and a suppressor of the lethal at the other locus. Specifically, we formulate and analyse a population genetics model of this system to assess when different combinations of release strategies (i.e. single or multiple releases of both sexes or males only) and genetic systems (i.e. bisex lethal or female-specific lethal elements and different strengths of suppressors) will give population replacement or fail to do so. We anticipate that results presented here will inform the future design of engineered underdominance gene drive systems as well as providing a point of reference regarding release strategies for those looking to test such a system. Our discussion is framed in the context of genetic control of insect vectors of disease. One of several serious threats in this context are Aedes aegypti mosquitoes as they are the primary vectors of dengue viruses. However, results are also applicable to Ae. aegypti as vectors of Zika, yellow fever and chikungunya viruses and also to the control of a number of other insect species and thereby of insect-vectored pathogens

Genetic Improvements to the Sterile Insect Technique for Agricultural Pests

The sterile insect technique (SIT) relies on area-wide mass-releases of sterile male pest insects, which mate with their wild counterparts and thereby cause a drop in the wild population. In order to improve SIT efficacy or to avoid potential negative effects of such releases, strains of insects have been developed by genetic means. Methods of strain improvement fall into two categories: those generated by classical genetics and those through transgenesis. Here, we describe development and successes of agriculturally important pest insect strains developed through the former, and how transgenic technology is offering a broad spectrum of potential improvements to SIT in a wider range of insects. Also discussed are future prospects and non-technical challenges faced by transgenic technology. The need for environment-friendly pest control methods in agriculture has never been more pressing. SIT and related technologies offer a solution with proven effectiveness

Cross-utilisation of template RNAs by alphavirus replicases

Most alphaviruses (family Togaviridae) including Sindbis virus (SINV) and other human pathogens, are transmitted by arthropods. The first open reading frame in their positive strand RNA genome encodes for the non-structural polyprotein, a precursor to four separate subunits of the replicase. The replicase interacts with cis-acting elements located near the intergenic region and at the ends of the viral RNA genome. A trans-replication assay was developed and used to analyse the template requirements for nine alphavirus replicases. Replicases of alphaviruses of the Semliki Forest virus complex were able to cross-utilize each other’s templates as well as those of outgroup alphaviruses. Templates of outgroup alphaviruses, including SINV and the mosquito-specific Eilat virus, were promiscuous; in contrast, their replicases displayed a limited capacity to use heterologous templates, especially in mosquito cells. The determinants important for efficient replication of template RNA were mapped to the 5' region of the genome. For SINV these include the extreme 5'- end of the genome and sequences corresponding to the first stem-loop structure in the 5' untranslated region. Mutations introduced in these elements drastically reduced infectivity of recombinant SINV genomes. The trans-replicase tools and approaches developed here can be instrumental in studying alphavirus recombination and evolution, but