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United States Department of Agriculture

Agricultural Research Service

Research Project: BIOLOGICALLY-BASED TECHNOLOGIES FOR MANAGEMENT OF CROP INSECT PESTS IN LOCAL AND AREAWIDE PROGRAMS

Location: Insect Behavior and Biocontrol Research Unit

Title: Conditional lethality strains for the biological control of Anastrepha species

Authors
item Schetelig, Marc Florian
item Xavier, Nirmala -
item Handler, Alfred

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: April 20, 2011
Publication Date: N/A

Technical Abstract: Pro-apoptotic cell death genes are promising candidates for biologically-based autocidal control of pest insects as demonstrated by tetracycline (tet)-suppressible systems for conditional embryonic lethality in Drosophila melanogaster (Dm) and the medfly, Ceratitis capitata (Cc). However, for medfly multiple lethal effector lines had to be tested (by mating to promoter-driver lines) to select one hybrid strain that yielded 100% lethality in the absence of tet, which is required for biocontrol using transgenic insects. To optimize lethal effectors for conditional lethality in Anastrepha species, pro-apoptotic genes from the reaper, hid, grim (RHG) family were isolated from the Caribbean fruit fly, A. suspensa (As), and the Mexican fruit fly, A. ludens (Al). RHG proteins are primary regulators of programmed cell death in Drosophila by inhibiting IAP proteins that suppress caspases that effect apoptosis, and their activity at inappropriate times in development typically results in organismal lethality. Using a degenerate PCR approach to conserved domains from the 12 Drosophila species genome project, reaper and hid orthologs were identified that have >50% amino acid sequence identity to their Drosophila homologs, and encode conserved IBM and GH3 motifs that are key for IAP-inhibition and mitochondrial localization. Functional verification of the genes was demonstrated in cell death assays in A. suspensa and heterologous Drosophila S2 embryonic cell cultures, showing that rpr and hid from both species were potent lethal effectors; that As-hid and As-rpr negatively regulated the Drosophila DIAP1 gene to promote apoptosis; and that both genes were cooperative in effecting increased cell death when used together. To test and compare the A. ludens hidAla2 phospho-acceptor variant as a tet-suppressible lethal effector, piggyBac vectors with tet-transactivator (tTA) promoter-driver cassettes (Cc-srya-tTA, As-srya-tTA, and Cc-vas-tTA) and lethal-effector cassettes (TREpDm-hidAla5, TREhs43Dm-hidAla5, and TREhs43Al-hidAla2) were transformed individually into A. suspensa hosts. Dm-hidAla5 effector lines (proven effective in Drosophila and medfly) had low fecundity, and while crosses to driver lines generated high levels of embryonic lethality in the absence of tet, viability was also low with tet. This suggested an unusual low level or leaky expression of Dm-hidAla5 in the absence of tTA-activation in caribfly. In contrast, all Al-hidAla2 effector lines showed normal viability and fecundity, but highly variable (yet reversible) tet-minus lethality rates when crossed to various driver lines. Complete lethality was obtained in one hybrid line from a cross of As-srya-tTA to TREhs43-Al-hidAla2 lines, yielding 96% embryonic and 4% early first larval instar lethality (>1,000 embryos tested). Transgenic lines in A. ludens having the same promoter-driver and lethal-effector transgenes are currently being tested.

Last Modified: 9/1/2014
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