2013 Annual Report
1a.Objectives (from AD-416):
To improve the development and ecological safety of transgenic insects created for improved biological control strategies, and to enhance the functional genomics analysis of pest species providing new resources for novel means of control. By seeking to manage the population size of highly significant invasive pest insects, this project aims to enhance the protection and safety of the Nation's agriculture and food supply. Genetically transformed insects provide significant opportunities to improve the sterile insect technique (SIT) and new strategies based on conditional lethality. A major concern for proposed transgenic insect release programs is the stability of the transgene, and maintenance of their consistent expression. Transgene movement will result in strain breakdown, and may be a prelude to inter-species transgene movement resulting in ecological risks. Random genomic transgene insertions are also problematic due to genomic position effects that influence transgene expression, and insertional mutations that negatively affect host fitness and viability. New vector systems will be created and tested for target-site integrations and vector stabilization. Potential reversion of conditional lethality strains will be assessed in large scale rearing of Drosophila strains.
1b.Approach (from AD-416):
To address transgene instability, a new transposon vector that allows post-integration immobilization by deleting terminal vector sequences required for transposition has been developed and will be tested in three tephritid pest species, Anastrepha suspensa, Anastrepha ludens, and Ceratitis capitata. Random genomic insertions will be addressed by developing a vector system that targets transgene vectors to defined genomic insertion sites based on a recombinase-mediated cassette exchange (RMCE) strategy. RMCE will be tested in the three tephritid species, and a series of stabilized target site strains will identified as optimal sites for future SIT and conditional lethality strains. Potential spontaneous reversion of transgenic conditional lethality strains will be tested in Drosophila by the mass rearing of these strains and selection of surviving individuals under non-permissive (lethal) condition.
This research relates directly to Objective 1. Genetics: Identify developmentally significant genes from whole genome and transcriptome sequencing projects that may be targeted or manipulated in transgenic and nontransgenic insect strains for biological control. Test conditional lethal systems using cell death genes and microRNAs targeted to embryos and vital processes in tephritids and lepidopterans and develop germ-line transformation for the cactus moth and Asian citrus psyllid.
New piggyBac vectors that carry FRT and loxP recombinations sites for genomic targeting have been created, and their insertion by transformation into the Caribbean fruit fly, Anastrepha suspensa, and the Mexican fruit fly, Anastrepha ludens, has been initiated. Recombinase-mediated cassette exchange RMCE) was achieved in the mexfly using a donor plasmid with heterospecific lox sites. RMCE will then be used to both stabilize the piggyBac vector and introduce new genetic marking systems.