Strategy for enhanced transgenic strain development for embryonic conditional lethality in Anastrepha suspensa Marc F. Schetelig1 and Alfred M. Handler United States Department of Agriculture, Agriculture Research Service, Center for Medical, Agricultural and Veterinary Entomology, 1700 SW 23rd Drive, Gainesville, FL 32608 Edited by Anthony A. James, University of California, Irvine, CA, and approved April 11, 2012 (received for review February 29, 2012) Here the first reproductive sterility system for the tephritid fruit embryonic lethality systems for these insect pests as well, and fly pest, Anastrepha suspensa, is presented, based on lethality pri- especially for those species amenable to SIT control and insect marily limited to embryos heterozygous for a conditional lethal transformation. A caveat, however, is that the embryonic lethality transgene combination. This tetracycline (Tet)-suppressible system systems tested required a species-specific sryα promoter to drive Ala5 uses a driver construct having the promoter from the newly iso- the tTA, and while the phosphomutated Drosophila hid lated embryo-specific A. suspensa serendipity α gene linked to the lethal effector (11) resulted in complete embryonic lethality in Tet-transactivator. This was used to drive expression of a phospho- both D. melanogaster (10) and in C. capitata (9), the latter study mutated variant of the pro-apoptotic cell death gene, hid,from suggested that these systems are most effective using genes for A. ludens, that was isolated, based on its identity to A. suspensa promoters and lethal effectors endogenous to the respective host hid. The Alhid Ala2 variant was shown to have the highest cell death species. This specificity has the advantage of limiting the lethal activity in an in vitro A. suspensa cell death assay compared to the system primarily to the host species, which is an important con- orthologous genes Ashid, Dmhid, and the variant Dmhid Ala5. These sideration for risk assessment of released transgenic insects. On cell death assays also allowed a determination of the most-efficient the other hand, isolating and validating the genes and transgene driver-effector cassette combinations for use in A. suspensa trans- constructs required can be a lengthy and time-consuming process, formants, resulting in two hybrid strains exhibiting 100% lethality. as occurred in the previous studies, and would be more so for One strain was 96% lethal in embryos in the absence of tetracy- species difficult to transform and having long reproductive cycles. cline, with none surviving past the first larval instar, which is critical With the desire to test the Tet-off embryonic lethality system in for pests that are most damaging in late-larval stages. We demon- another tephritid species and to make the evaluation process for strate that the isolation and in vitro validation of species-specific species-specific system components more efficient, we initiated promoters and lethal effector genes can greatly improve the effi- development of a lethality system for Anastrepha species using ciency of creating high-performance conditional lethality strains qPCR and in vitro functional assays. First, homologs to the that may be extended to other insect pest species. Drosophila pro-apoptotic cell death genes, hid and reaper (rpr), were previously isolated from A. suspensa (12), and sequence apoptosis ∣ insect pest management ∣ sterile insect technique identity to Ashid was used to isolate the hid ortholog from A. ludens (Alhid). To functionally validate the identity of these he Caribbean fruit fly (caribfly), Anastrepha suspensa, and the genes, a cell death assay was developed using embryonic cell lines TMexican fruit fly (mexfly), A. ludens, are established model derived from a homologous (A. suspensa) and heterologous organisms for the genera Anastrepha, which are economically (D. melanogaster) species (12). This assay was used to assess the important insect pests (1–4). An effective biologically based con- relative capacity of these genes to induce cell death in A. suspensa cells, in addition to creating and testing a phosphomutated ver- trol system for these, and other tephritid pests, is the sterile insect Ala2 technique (SIT) that relies on the field release en masse of males sion of Alhid (Alhid ). A similar variant of D. melanogaster hid sterilized by irradiation, resulting in progeny that are zygotic was found to decrease the natural ability of hid down-regulation lethal (5, 6). In the process of developing improved methods for by phosphorylation (11) and was effective in the lethality systems SIT programs for the Mediterranean fruit fly (medfly), Ceratitis tested in Drosophila and medfly. capitata, two transgenic approaches have been taken based on Second, to avoid lengthy in vivo testing of the various embryo- tetracycline-suppressibilty of lethality in the progeny of released nic promoter driver and lethal effector components requiring males, using the Tet-off gene expression system (7–9). One system transformant lines for each possible combination, we used cell uses self-regulated accumulation of the tetracycline-controlled death assays to test these components to find the optimal com- transactivator (tTA) that is toxic at high concentrations. The lim- bination. Transgenic flies carrying these systems were then eval- itation of this system, however, is that unlike typical SIT, lethality uated by qPCR, that confirmed in vivo the trend of lethal occurs in late larval or pupal progeny after damage to host plants intensity first observed in in vitro cell death assays. The presented has been sustained (8). The other system, however, uses embryo- evaluation strategy improves the rapid selection of optimal nic-specific serendipity α (sryα) promoters to drive tTA expression, vectors from a large number of potential candidates and reduces which then activates expression of the Drosophila hidAla5 pro- apoptotic lethal gene. Significantly, transgenic hybrid strains were Author contributions: M.F.S. and A.M.H. designed research; M.F.S. performed research; created for both Drosophila melanogaster and medfly in which M.F.S. contributed new reagents/analytic tools; M.F.S. and A.M.H. analyzed data; and complete embryonic lethality occurred in the progeny of paren- M.F.S. and A.M.H. wrote the paper. tals reared on media lacking tetracycline, thereby eliminating pest The authors declare no conflict of interest. individuals previous to the damaging larval stages (9, 10). This article is a PNAS Direct Submission. The ability to conditionally regulate embryonic lethality in Data deposition: The sequences reported in this paper have been deposited in the insects is important, since many plant pests are most harmful GenBank database (accession nos. JQ599254–JQ599257). as larvae, including fruit flies and moths, among others, as well 1To whom correspondence should be addressed. E-mail: [email protected]. as serious animal pests, such as the New World screwworm. It This article contains supporting information online at www.pnas.org/lookup/suppl/ would therefore be highly beneficial to develop similar Tet-off doi:10.1073/pnas.1203352109/-/DCSupplemental. 9348–9353 ∣ PNAS ∣ June 12, 2012 ∣ vol. 109 ∣ no. 24 www.pnas.org/cgi/doi/10.1073/pnas.1203352109 Downloaded by guest on October 2, 2021 the lengthy and labor-intensive studies required for the genera- gene (Ashid) (12). These two Hid homologs are identical except tion and functional analysis of transgenic flies. For A. suspensa, for a single amino acid substitution (Fig. S2), indicating a very this resulted in the development of two effective lethality strains high level of conservation that surpasses the similarity of hid in less than one year, relative to the several years taken to develop in D. melanogaster and its closest known relative, D. simulans, effective strains for Drosophila and medfly (both having shorter having eight substitutions between them. Studies in Drosophila, reproductive cycles). Notably, one of the caribfly strains achieved where hid phosphorylation sites were mutated to hidAla3 and 100% lethality in the first larval instar, which is essential for larval hidAla5, resulted in improved cell death activity (11). Therefore, pests. It is expected that similar protocols will be applicable to Alhid was analyzed in-silico for possible MAPK phosphorylation many other pest species, allowing the rapid creation of new sites, according to definitions in Drosophila (11), revealing two strains for biologically based population control systems. potential sites at positions 85 (PASP) and 116 (PRTP) that were subsequently mutated to PAAP and PRAP, respectively, to create Results the variant AlhidAla2. Isolation of Early Embryonic and Cell Death Genes. In a first step, the embryonic cellularization-specifically expressed gene, serendipity Evaluation of Alhid Ala2 and the Promoter Region of Assry α in α (sryα), was isolated from A. suspensa. The complete ORF and its A. suspensa Cell Assays. To validate the cell death activity of UTRs were isolated by 3′- and 5′-RACE PCR with gene-specific AlhidAla2, it was first cloned into the overexpression vector primers designed on an in-silico isolated putative Assryα fragment pIE-4 and compared to pIE-4 vectors containing Ashid, from an A. suspensa transcriptome. The gene consists of a 377 bp DmhidAla5, and Dmhid (Fig. 1) (12). All cell death assays were 5′UTR, a 1,941 bp open reading frame, and a 459 bp 3′UTR. In a performed in A. suspensa AsE01 embryonic cells, as previously