1a.Objectives (from AD-416):
Produce and test transgenic Mexican fruit fly (A. ludens) strains that incorporate the tetracycline-suppressible embryonic lethality system developed for medfly. A female-specific component for embryonic lethality will also be developed that should result in male only strains based on female-specific embryonic lethlity.
1b.Approach (from AD-416):
The genetic components for these systems will be first identified and isolated from either A. ludens or A. suspensa. These will include regulatory DNA sequences from embryonic genes such as serendipity, slam, and mdg88; sex-specific intron splicing cassettes isolated from the A. ludens sex-determination gene transformer; and the lethal effector gene, hid. To test the Mediterranean fruit fly embryonic lethality system in Anastrepha species existing driver (srya-tTA) and effector (TRE-hidala5) transgene constructs shown to be functional in the Mediterranean fruit fly will be integrated into a wild A. ludens host strain. Endogenous early and late embryonic genes will be isolated from the Anastrepha species to test their promoters in driver constructs, and efforts will be made to isolate the hid lethal-effector from Anastrepha. Transgenic A. suspensa and A. ludens will be created using the strongest early and late promoter-tra intron-tTA constructs (based on tTA transcription) inserted into DsRed-marked piggyBac vector plasmids having the TRE-tra intron-hidala5 driver and phiC31 attP integrase recombination sites. Transformation experiments will be initiated in A. ludens using transgene vectors yielding the most efficient female-lethality in A. suspensa. If no homozygous line yields 100% female-lethality, the transgenes will be re-mobilized by transposase injection to create new insertion sites that will be tested for more optimal parameters. Optimal homozygous lines will be expanded for large scale sexing, fitness, and mating competitiveness tests in Tapachula and Petapa.
This research relates directly to inhouse project 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.
Transgenic strains have been created in the Mexican fruit fly, Anastrepha ludens, that incorporate fluorescent protein marker genes that allow monitoring of released males, and sperm-specific markers to identify females that have mated with released males. New transgenic strain development has been initiated with vectors that allow female-specific conditional lethality for genetic-sexing in embryos, and these strains are being evaluated.