Strategy for enhanced transgenic strain development for embryonic conditional lethality in suspensa

Marc F. Schetelig1 and Alfred M. Handler

United States Department of , Agriculture Research Service, Center for Medical, Agricultural and Veterinary Entomology, 1700 SW 23rd Drive, Gainesville, FL 32608

Edited by Anthony A. James, University of , Irvine, CA, and approved April 11, 2012 (received for review February 29, 2012)

Here the first reproductive sterility system for the tephritid embryonic lethality systems for these pests as well, and pest, , is presented, based on lethality pri- especially for those 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 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 . 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 , 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 ∣ identity to Ashid was used to isolate the hid ortholog from A. ludens (Alhid). To functionally validate the identity of these he 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 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 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 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 BLASTx search against the National Center for Biotechnology described (12, 13). To determine AlhidAla2 cell death activity Information nr database, the best match was the C. capitata relative to its orthologs, pIE-4 vectors expressing the genes and sry α gene, Ccsryα (9). Comparing the medfly and A. suspensa the pIE-EGFP control were co-transfected into AsE01 cells sry α proteins resulted in a pairwise identity of 60.1% with several (Fig. 1A). Cell death activity was measured as the percent reduc- well-conserved amino acid stretches (Fig. S1). A 2.5 kb upstream tion in the number of surviving green fluorescent cells when pIE- maximal promoter/enhancer region of Assryα (including the 5′ EGFP was co-transfected with a cell death gene vector compared UTR and the entire 5′ upstream flanking sequence (up to the to the control pIE-4. We found that AlhidAla2 has a potent cell next identifiable gene) was then isolated by inverse PCR, fused death function that was greater than the other hid genes and to the tetracycline transactivator (tTA) and subcloned into a variant derivatives tested (Fig. 1A). Relative to 100% cell survival piggyBac vector to generate the promoter-driver component of a in controls, only 1% of AsE01 cells survived when co-transfected Tet-off-based lethality system. The upstream promoter showed with AlhidAla2, which is significantly (P < 0.001) lower than the low conservation when compared to the closest related known 6% survival from unaltered Ashid, as supported by one-way sry α upstream region from medfly. ANOVA statistical analysis (Table S1). This is compared to the In addition, the hid ortholog from the Mexican fruit fly, greater survival after transfection with the Drosophila homologs, A. ludens (Alhid), was isolated by 3′- and 5′-RACE with gene spe- DmhidAla5, and Dmhid, yielding survival rates of 24% and 35%, cific primers designed to the recently identified A. suspensa hid respectively, in the heterologous cell line. The significant differ- SCIENCES APPLIED BIOLOGICAL

Fig. 1. Cell death assays using AsE01 cells. (A) Comparison of cell death activity of pIE-4 overexpression vectors carrying hid orthologs or their phosphomutated variants; (B) Cell death activity of a transgene combination of piggyBac-based driver and effector transformation vectors. In (B), TRE-Dmhid Ala5 and Ala2 Ala5 Ala2 TRE-Alhid represent the effector constructs TREhs43Dmhid and TREhs43Alhid , respectively. Error bars show standard deviations (SD) with the mean percentage of cell survival from three independent experiments shown above.

Schetelig and Handler PNAS ∣ June 12, 2012 ∣ vol. 109 ∣ no. 24 ∣ 9349 Downloaded by guest on October 2, 2021 ences between the wild type and variant hid gene variants resem- D-Assryα-tTA lines to eight effector lines yielded, in several cases, bles earlier results in Drosophila where the phospho-acceptor substantially lower hatch rates. All crosses to the effector line mutated DmhidAla5 promoted greater cell death in developing E-TREp-DmhidAla5 resulted in a hatch rate of 20–57%, with the eyes and in insect cell cultures compared to Dmhid (11, 12). lowest embryonic survival rate in crosses to the strongest To determine if piggyBac transformation vectors carrying the tTA-expressing lines D-Assryα-tTA_F2A (32%) and D-Assryα- driver (D-) and lethal effector (E-) components of the lethality tTA_M1B (20%) (Table S2). Similarly, in crosses to the effector system could be pre-evaluated in cell culture before the more line E-TREhs43-DmhidAla5, the lines D-Assryα-tTA_F2A and laborious generation of transformants for testing in vivo, D-Assryα-tTA_M1B performed best and resulted in 0% and 5% AlhidAla2 was subcloned into the piggyBac transformation vector, survival, respectively (Table 1). Notably, adding tetracycline to pBXL_TREhs43_PUbEGFP. The driver constructs D-Ccsryα- the adult diet did not suppress the lethal effect in all combina- tTA (9) and D-Assryα-tTA were then co-transfected into cells with tions. Instead, lethality was irreversible and, after 10 d, females either E-TREhs43-DmhidAla5 (9) or E-TREhs43-AlhidAla2 failed to oviposit additional . An attempt to generate flies (Fig. 1B) to identify the most effective driver and effector con- homozygous for both driver and effector constructs by inbreeding struct combination. Neither effector construct performed signif- failed, because no double-homozygous flies were observed. One icantly different from the other when co-transfected with either of contributing factor was the highly reduced fecundity of all four the driver constructs. However, in combination with either effec- homozygous E-TREp-DmhidAla5 and E-TREhs43-DmhidAla5 tor construct, D-Ccsryα-tTA reduced cell survival to 31–33% and strains (17–24% :adult ratio compared to 50–72% of other D-Assryα-tTA to 23–24%. Thus, the endogenous Assryα promo- transgenic strains). Taken together, use of the TRE-linked ter/enhancer induced cell death at significantly higher levels DmhidAla5 lethal effector constructs appears to be lethal in (P ¼ 0.002–0.008) than the heterologous C. capitata promoter/ A. suspensa, which was not observed in medfly (9, 10). Since enhancer in the AsE01 cell line. the effect was observed in several lines, where the DmhidAla5 was driven by TREp or TREhs43, but not when TREhs43 was driv- Germline Transformation and Quantitative tTA Expression Analysis. ing AlhidAla2, an effect of the minimal p or hs43 promoter regions The driver (D-Ccsryα-tTA and D-Assryα-tTA) and effector is unlikely. (E-TREp-DmhidAla5 (10), E-TREhs43-DmhidAla5, and Crosses of homozygous D-Assryα-tTA flies to the E-TREhs43- E-TREhs43-AlhidAla2) constructs were then used for germline AlhidAla2 effector line resulted in hatch rates of 4–87%, with the transformation of A. suspensa. For each construct, a maximum of wide variation possibly related to the transgene genomic integra- five independent lines homozygous for the D- or E-transgene tion sites and the level of tTA-expression in the driver lines. The were analyzed. First, the expression of tTA from embryos of each highest lethality in progeny resulted from crosses of homozygous driver line was tested by qPCR. Increases of tTA transcript were D-Assryα-tTA_F2A and D-Assryα-tTA_M1B drivers to the homo- detected for D-Ccsryα-tTA as well as D-Assryα-tTA, with the zygous effector line E-TREhs43-AlhidAla2_F2A, yielding 96% highest tTA expression increase (up to 32-fold) occurring with the embryonic lethality (with 100% L1 lethality) and 79% embryonic endogenous Assryα promoter driving the tTA (Fig. 2). Two of the lethality (100% L3 lethality), respectively (Table S2). Generation five D-Assryα-tTA lines (D-Assryα-tTA_F2A and D-Assryα-tTA_ of a strain (D-F2A/E-F2A) carrying the driver D-Assryα-tTA_- M1B) showed strong relative tTA expression whereas the remain- F2A and the effector E-TREhs43-AlhidAla2_F2A transgenes ing three lines showed only a marginal increase over the control in homozygous condition resulted in 100% embryonic lethality Histone 3 gene (Fig. 2). These differences might be due to geno- when reared on non-Tet diet, which is consistent with the lethal mic positional effects as observed previously in medfly (9). The effect being dosage dependent. In contrast to the DmhidAla5 sole D-Ccsryα-tTA line showed a 12-fold tTA increase, signifi- effector lines, all lines carrying AlhidAla2 showed normal fecund- cantly lower than the strongest D-Assryα-tTA lines. All driver ity when reared on Tet diet (52–58% egg to adult survival). strains carried a red fluorescent PUbDsRed.T3 marker, which was visibly stronger in adults from the strains D-Assryα-tTA_F2A Tet Concentrations for Rearing. Feeding Tet-containing diet to adult and D-Assryα-tTA_M1B, compared to the other lines examined flies is expected to switch off embryonic lethality due to a mater- under epifluorescence microscopy. nal transfer of tetracycline to their progeny. In Drosophila, this Crosses of D-Ccsryα-tTA homozygous flies to eight effector was sufficient to switch off the lethal effect completely (10), lines (Table S2) yielded only moderately decreased larval hatch whereas in medfly an additional low amount of tetracycline or rates between 31 and 64%, on Tet-deficient media, compared to doxycycline (as low as 0.1 μg∕mL) was required in the larval diet the 74% hatch rate of the laboratory-reared A. suspensa wild type (9). Four different adult diet Tet-concentrations (A3, A10, A100, (WT) control strain. In contrast, crosses of five homozygous and A300 μg∕mL) were fed to D-F2A/E-F2A and WT flies, with the laid eggs transferred onto Tet100 larval diet (L100). While both A10 and A100 resulted in viable progeny comparable to other laboratory-reared transgenic strains (51–56% egg to adult survival), A3 and A300 resulted in a highly reduced egg to adult ratio for D-F2A/E-F2A. The WTcontrol flies showed a relatively high number of descendants for A3, A10, and A100 diets (65–69%), while the A300 diet resulted in a highly reduced num- ber of pupae with no descendants. Thus, A3 was not sufficient to

Table 1. Survival rate of embryos transheterozygous for a driver (D) and an effector (E) transgene

E-TREhs43 E-TREhs43 E-TREhs43 E-TREhs43 Dmhid Ala5_ Dmhid Ala5_ Dmhid Ala5_ Alhid Ala2_ Fig. 2. Quantitative PCR on cDNA generated from embryos from D-Ccsryα- F6A, % F7A, % F8A, % F2A, % tTA and D-Assryα-tTA strains. Relative accumulation of tTA transcript D-Assryα-tTA_F2A 0* 11 15 4 (0 L1 survival) (graybars) normalized against AsHis3 transcript using the strain D-Assryα- D-Assryα-tTA_M1B 14 5 22 21 (0 L3 survival) tTA_M9A as a calibrator is shown. Relative values of AsHis3 (black bars) are also calibrated against D-Assryα-tTA_M9A. Error bars show SD, with the mean *Hatch rates of 300 eggs collected from each cross of a homozygous driver fold change from three independent experiments shown above. to a homozygous effector line are shown.

9350 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1203352109 Schetelig and Handler Downloaded by guest on October 2, 2021 efficiently switch off the lethal system in D-F2A/E-F2A, while for the brightest fluorescent driver lines could be used for initial A300 had a negative effect on the number of surviving progeny screening of optimal tTA-expressing lines. in general. In the last step, crosses between pre-validated driver and In a second experiment, D-F2A/E-F2A and WT flies were effector lines provided the final test for embryonic lethality effi- reared on A10 and A100 diet with eggs collected on larval diet ciency, whereupon Tet characteristics for the timing and concen- without tetracycline to determine if the maternal Tet contribution trations required for lethality suppression could be evaluated. was sufficient to suppress lethality in their progeny. As a control, Use of the sole Ccsryα-tTA driver line with any of the effector eggs from both groups were also collected on L100 diet. The eggs lines failed to induce complete lethality. This could be due to from both groups hatched, pupated, and eclosed efficiently the lack of functional sry α promoter conservation between the (Table S3), comparable to controls on L100 diet. Therefore, a tephritid species, similar to the differences between Drosophila maternal source of tetracycline is able to switch off the lethality and medfly sry α (9), or possibly due to genomic positional effects. system effectively in their larval progeny. Toassess the persistence In contrast, two crosses between endogenous Assryα driver lines of tetracycline in the D-F2A/E-F2A strain, double-homozygous to an AlhidAla2 effector line resulted in 100% first instar or third flies were reared on A100 for five days and then placed on adult instar larval lethality, with embryonic lethality of 96% and 79%, diet without Tet. Oviposited eggs were then collected over a one- respectively. week period beginning on day 5. The egg-to-adult ratio decreased In summary, the combination of a tTA driver under endogen- from 51% to 26% over a period of seven days, whereas in medfly ous sry α promoter control and an effector construct utilizing the Ala2 a similar experiment resulted in complete lethality after two days Alhid cell death gene proved to be the most-efficient system (9). This suggests that tetracycline may have enhanced stability in for A. suspensa. The pre-evaluation assays not only indicated that A. suspensa females, possibly due to species differences in gut use of such lines could be highly effective; they allowed a rapid bacterial populations that metabolize the antibiotic. quantitative determination of the specific lines that might be most In a third experiment, the double-homozygous D-F2A/E-F2A effective. Lines exhibiting relatively low tTA expression or induc- strain was reared on non-Tet diet for 8 d after eclosion resulting tion of cellular lethality could be eliminated early in the testing in embryonic lethality. Feeding the adults with A10 or A100 diet process, previous to the generation of transformant individuals on day 8 re-established 48% survival on day 9, indicating that for testing in vivo. The rapid screening of many candidate genes/ lethality in D-F2A/E-F2A was reversible. promoters also allows the identification of the most active ele- ments that are also the least phylogenetically conserved. This Discussion is likely to increase species-specificity for the lethality system, A major bottleneck in creating the safest and most efficient trans- which should enhance the safety of transgenic releases in terms genic insect systems for biologically based control, in a reasonable of potential, though rare, instances of lateral interspecies transfer and cost-effective time period, has been the lack of rapid valida- of transgenes into unintended hosts. tion systems between the initial design of system components and We believe that lethality caused by pro-apoptotic cell death the final test crosses of transformed insects. We demonstrate here genes (e.g., hid, rpr,orgrim), or other well-studied biological that a combination of gene finding, functional analysis in in vitro mechanisms (10), is preferable to systems for which the lethality embryonic cell cultures, and qPCR determination of gene expres- mechanism is unknown, including those dependent on a toxic sion of final transgene constructs can result in the rapid develop- accumulation of the Tet-transcriptional activator (8, 14–17). While ment of transgenic insect strains potentially useful for effective tTA toxicity may be highly effective, it is not species-specific nor is population control of pest species. its precise mode of action known (8). Thus, it is impossible to fully The presented procedure for the development of an embryonic evaluate or anticipate all potential programmatic or environmen- conditional lethality system for an Anastrepha species involved tal risks related to the field-release of this type of transgenic insect three steps: First, newly isolated components (i.e., promoter (18); in particular, are the various issues related to potential sur- drivers and lethal effectors) were tested in an assay that demon- vival of transgenic individuals due to resistance to tTA toxicity, strated that pIE-4 overexpression constructs, as well as expression genetic breakdown of the toxic effect or other inefficiencies of of transgene constructs within final transformation vectors, can the system. be measured quantitatively in AsE01 cells. The results of these Another advantage of the system described here is that the tests reliably provided insights applicable to identifying the transgenic insects can be further altered by site-specific modi- most-promising lethality system components for successful in vivo fications within the vector sequence. This is possible through use in the host species. Importantly, direct quantitative gene phiC31 attP recombination sites placed within the driver and expression and functional assays could be made in the absence effector constructs that have been proven functional in fruit flies of genomic position effects that might confuse relative function. (19–22) and mosquitoes (23, 24). This technology could be used For example, cell death assays showed that the newly isolated for stabilizing the transposon vector, by introducing a tandem SCIENCES endogenous promoter from Assryα performed better than the duplication of one inverted terminal repeat sequence that allows APPLIED BIOLOGICAL medfly Ccsryα promoter, which was consistent with subsequent post-integration deletion of the other terminus, thereby immobi- qPCR analysis in two of the five transgenic lines. The species-spe- lizing remaining transgene sequences. This would enhance stabi- cificity of homologous sryα promoter-driver expression is also lity of the strain and its environmental safety before proceeding beneficial in terms of limiting lethality to A. suspensa and closely from laboratory tests to contained field-trial evaluations. The related species, thereby reducing potential risk to non-target addition of new marking systems, e.g., for sperm identification organisms in the rare event of lateral interspecies transfer. (25–27), or other improvements would be possible using already Second, using only the best-performing components from step evaluated genomic positions instead of fully re-creating and eval- 1, transgenic driver and effector transformant lines were estab- uating strains with new, random genomic integration sites of the lished, with the driver lines further assessed by qPCR for tTA transgenes. transcript levels. Lines with the highest tTA transcript levels A critical consideration for mass rearing Tet-off lethality not only resulted in the highest lethality (when crossed to effector strains is determining the Tet concentration necessary for optimal strains), but also consistently expressed the strongest visible red survival during rearing, and, in particular, the lowest sufficient Tet fluorescence from the PUb-DsRed transgenic marker in adults. concentration necessary to switch the system on or off. In medfly, This might be due to position effects at the vector integration site the amount of Tet was found to be highly dependent on the sys- having a similar positive influence on expression levels of both tem used for generating lethality. The Release of Insects Carrying tTA and DsRed, and, if confirmed by additional tests, selection a Dominant Lethal (RIDL) system based on toxic accumulation

Schetelig and Handler PNAS ∣ June 12, 2012 ∣ vol. 109 ∣ no. 24 ∣ 9351 Downloaded by guest on October 2, 2021 of tTA required 100 μg∕mL Tet to suppress lethality in both vector #1405. #1405 was generated by cloning a SalI-NcoI digested SV40 and adults and larvae (8), whereas the sryα-tTA/TRE-DmhidAla5 an NcoI-EcoRI digested attP220 fragment, amplified with primer pair AH635- AH636 on vector #1215 and AH637-638 on vector pTA-attP (31) into a embryonic lethality system was efficiently suppressed by a 10-fold Ala2 lower concentration of Tet fed to adults, and a 100-fold lower SalI-EcoRI digested pSLfa1180fa vector (30), respectively. The Alhid concentration fed to larvae (9). In A. suspensa, not only did adults sequence is deposited in GenBank (JQ599255). survive on 10 μg∕mL Tet, but their progeny survived without any Transformation Vectors. The driver construct pBXLII_attP220_PUbDsRed.T3_ Tet supplementation. The finding that tetracycline is unnecessary tTA-Assryα (Assryα-tTA; #419) was generated by ligating the FseI/AscI in larval diet for survival is critical since several tons of larval diet digested Assryα-tTA fragment from #1400 in opposite orientation to are typically used daily for mass rearing, and the ability to use a PUbDsRed.T3 in the FseI/AscI site of pBXLII_attP220_PUbDsRed.T3_fa (#1425). Tet-free larval diet greatly improves efficiency, cost savings, and The vector #1425 was created by ligating the hybridized primers AH619- environmental safety. In particular, Tet-free diet can be disposed AH620 in the BglII site of pBXLII_attP220_PUbDsRed.T3 (#1421). The vector of safely without pretreatment, or reused as animal feed (28, 29). #1421 was created by cloning the BsaIcutattP220 fragment, amplified with It will be important to determine if these Tet-characteristics are primer pair AH617-AH618 on pTA-attP (31), in XmaI digested pBXLII_PUbDs- similar in species closely related to A. suspensa. Red.T3 (32). The effector construct pBXLII_PUbEGFP_attP220_SV40-Alhid Ala2-hs43-TRE The three-step procedure we have described and tested Ala2 enabled the generation of an early acting and highly efficient leth- (TREhs43-Alhid ; #1430) was generated by ligating the FseI/AscI Alhid Ala2 ality system in A. suspensa in a relatively short time frame of less digested TREhs43- -SV40_attP220 fragment from M158 in the FseI/ than one year, compared to a development time in medfly of AscI site of pBXLII_PUbEGFP_fa (#1419). The vector #1419 was created by – ligating the hybridized primers AH619-AH620 in the BglII site of pBXLII_ 2.5 3 years (9), despite caribfly having a 25% longer reproductive PUbEGFP.T3 (25). cycle. In medfly, 60 test crosses were necessary to obtain lethal Vectors pBacff attP-sryα2-tTAaPUbDsRedg (Ccsrya-tTA), pBac Ala5 Ala5 lines (9), whereas in caribfly (due to the qPCR evaluation) only ffaattPf TREp-hida PUbEGFPg (TREp-Dmhid ), and pBac Ala5 Ala5 four crosses led to successful lethal lines. The savings in time ffaattPf TREhs43-hida PUbEGFPg (TREhs43-Dmhid ) are as de- and effort afforded by the pre-evaluation strategy can be largely scribed (9). attributed to the elimination of transgenic line development and the reduction of test crosses. Importantly, this procedure should Cell Culture Assays. The overexpression vector pIE4-Alhid Ala2 was created by allow similar conditional lethal strain development in other ligating the NotI-BglII cut Alhid Ala2 fragment from M158 into the pIE4 vector insects controlled with SIT, where cell cultures are available from (Novagen). Cell death assays were then performed in the A. suspensa cell line the same or closely related species, and genetic transformation UFENY-AsE01 as described (12), with the following modifications: For each μ μ μ is possible. These include other tephritids, the closely related well in a 24-well plate, a total of 2 g(1 g plasmid A and 1 g plasmid B) μ mexfly in particular, and several important mosquito and moth was mixed with 4 L lipofectin (Invitrogen) in serum-free media and added to 4.5 × 105 AsE01 cells. The vectors pIE4-Ashid, pIE4-Dmhid, and pIE4- species. Insect species with long generation times and low trans- Ala5 Dmhid are as described (12). formation efficiencies would especially benefit from pre-evalua- tions and promising component selection at an early stage of Germline Transformation. Germline transformation experiments were per- strain development. formed by microinjection of piggyBac constructs (500 ng∕μL) together with the phspBac transposase helper plasmid (200 ng∕μL) into WT embryos (33) Materials and Methods as described (34). G1 offspring were selected by DsRed and EGFP epifluores- α Insect rearing and isolation of the genes Assry and Alhid are described in cence using a Leica MZ FLIII microscope with a Texas Red (ex: 560∕40;em: SI Materials and Methods. The cDNA sequences are deposited in GenBank 610 LP) and a GFP2 filter set (ex: 480∕40; em: 510 LP). Independent homozy- α [accession numbers: JQ599254 (Alhid) and JQ599256 (Assry )] All oligonu- gous strains were established by single pair inbreeding for successive genera- cleotides are shown in Table S4. tions with testing by segregation analysis of transformants outcrossed to wild type flies. ′ Inverse PCR. Inverse PCR was performed to obtain the 5 upstream region of To generate conditional lethality lines, six homozygous driver lines were α μ Assry . First, 1 gofA. suspensa WT genomic DNA was digested with the crossed to eight homozygous effector lines in almost all possible combina- enzyme Sau3AI for 24 h. Restriction fragments were precipitated and self- tions on Tet-containing larval and adult diet. Heterozygous combinations ligated in a volume of 500 μL at 16 °C for 24 h. A PCR using BD Advantage were inbred with progeny visually screened by fluorescent intensity for 2 PCR polymerase (BD Biosciences) was performed on circularized fragments homozygous individuals that were subsequently inbred to generate lethality with the primer pair AH530-AH531, oriented in opposite direction within the lines homozygous for the driver and effector transgene constructs (all crosses 5′UTR/ORF of the gene, using the PCR conditions: 1 min at 95 °C; 6 cycles of performed on Tet-containing larval and adult diet). For screening of double 30 s at 94 °C; 45 s at 66 °C (−2 °C each cycle); 6 min at 68 °C; 25 cycles of 30 s at homozygous lines, the Texas Red and YFP filter set (ex: 500∕20; em: 535∕30) 94 °C; 45 s at 54 °C; 6 min at 68 °C; and 6 min at 68 °C. A resulting 2.5 kb PCR were used. product was cloned into the pCR4 vector (Invitrogen) yielding the clone iAs2600_3 that was sequenced. The upstream sequence of Assryα including the 5´UTR is deposited in GenBank (accession number JQ599257). Quantitative Real-Time PCR. Total RNA was isolated from all embryonic sam- ples (0–24 h collection, including the cellularization stage in A. suspensa), using TRIreagent (Molecular Research Center). The iScriptTM cDNA synthesis Shuttle Vectors. We composed our constructs in the cloning shuttle vector kit (BioRad) and 1 μg RNA were used for cDNA synthesis. Quantitative real- pSLfa1180fa (30). From the shuttle vectors, the constructs can be easily placed in transformation vectors, which carry FseI and AscI sites (fa-sites). pSLaf_ time PCR (qPCR) was performed on approximately 100 ng cDNA using the ™ Assryα-tTA_af (#1400) was created by recombining the 2.5 kb PCR Assryα iQ SYBR Green Supermix (BioRad) in a Chromo4 real-time PCR detector fragment, amplified from iAs2600_3 using primer pair AH562-AH563 into (BioRad). PCR cycling conditions were: 95 °C for 5 min; 45 cycles of 95 °C XbaI cut pSLaf_tTA_af (#1215) (9), using the In-Fusion PCR cloning Kit for 15 s; 60 °C for 10 s; and 72 °C for 10 s with a plate read at the end of each (Clontech). cycle. All reactions were performed on three biological replicates. Gene spe- To generate a mutated version of Alhid lacking two potential MAPK phos- cific primers for tTA (AH621/AH622) and A. suspensa Histone 3 (AsHis3) phorylation sites (Alhid Ala2), the clone M100 containing the complete Alhid (AH439F/AH440R) were designed using Geneious software (Biomatters). ORF in a pCR4 vector was used to generate three fragments by PCR (1 min at Amplified products from randomly selected samples were analyzed on a 2% 95 °C; 30 cycles of 30 s at 94 °C; 30 s at 55 °C; 30 s at 68 °C; and 1 min at 68 °C): A agarose gel, subsequently cloned into pCR4-TOPO vector, and sequenced to 220 bp fragment using primer pair AH664-AH665, a 105 bp fragment using confirm specificity of the tTA and Histone 3 amplification. AH666-AH667, and a 650 bp fragment using AH668-AH669. These fragments Relative accumulation of tTA normalized against AsHis3 was calculated were then recombined into the BglII-NotI digested vector pSL_TREhs43- from the formula 2-ΔΔCt (35), where 2 is the reaction efficiency and ΔΔCt SV40_attP220 (#1403), using the In-Fusion PCR Cloning Kit (Clontech) to is the difference in AsHis3 Ct values between the calibrator (lowest tTA create pSL_TREhs43-Alhid Ala2-SV40_attP220 (M158). #1403 was created by expressing line D-Assryα-tTA_M9A) and the other samples, subtracted from cloning the HindIII-NruI digested TREhs43 fragment, amplified with primer the difference in tTA Ct values between the calibrator (lowest tTA expressing pair AH627-AH628 on vector TREhs43-hid Ala5 (9) in the HindIII-NruI digested line D-Assryα-tTA_M9A) and the other samples.

9352 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1203352109 Schetelig and Handler Downloaded by guest on October 2, 2021 Tetracycline Tests. Backcrosses of transgenic males or females to WT females diet containing 10 or 100 μg∕mL Tet. Eight days post-eclosion eggs were or males were first performed to verify a transgenic/WT progeny ratio of 1∶1, collected and reared on Tet-free larval medium. Eclosion rates were recorded and the same fluorescent marker tissue specificity consistent with single and compared with data from WT A. suspensa flies reared on Tet-free adult vector integrations. Homozygous transgenic lines were then generated by and larval diet. inbreeding and selecting for the strongest red fluorescent flies (for driver lines) or green fluorescent flies (for effector lines). To generate an optimal Statistical Analysis. The program SigmaPlot 11 (Systat Software, Inc.) was used lethality system, homozygous driver lines were crossed to homozygous effec- to perform one-way ANOVA on the different samples. Within these tests, a tor lines, with 300 eggs per cross collected and hatch rate recorded (Table S2). Ala2 normality test was done after Shapiro-Wilk and all pairwise multiple compar- Two crosses (D-Assryα-tTA_F2A x E-TREhs43-Alhid _F2A and D-Assryα-tTA_ M1B x E-TREhs43-Alhid Ala2_F2A) having a reduced hatch rate, larval and ison procedures after Holm-Sidak (Table S1). pupal data were recorded in addition. To determine dosage effects for the lethality system, a double homozygous strain of D-Assryα-tTA_F2A x E- ACKNOWLEDGMENTS. We thank Shelley Olson (funded by TREhs43-Alhid Ala2_F2A was generated by inbreeding on A100/L100 diet and Department of Agriculture (USDA)–Animal and Plant Health Inspection selecting for double homozygous red/green fluorescent flies. These flies were Service–Plant Protection and Quarantine) for excellent technical assistance, then reared on non-Tet diet with the hatch rate of 300 eggs was recorded. Seth Britch for assistance with statistical analysis, and Drs. Nirmala Xavier Starting from larval and adult diet Tet-concentrations of 100 μg∕mL, and Irina Häcker for comments on the manuscript. The project benefitted the minimal Tet concentrations for the line D-F2A/E-F2A was tested with from discussions at International Atomic Energy Agency funded meetings WT as a control. First, flies were reared on four different adult diet Tet con- for the Coordinated Research Project: The Use of Molecular Tools to Improve centrations (A3, A10, A100, A300 μg∕mL), eggs were collected on larval med- the Effectiveness of SIT. Funding is gratefully acknowledged from the USDA– ium containing 100 μg∕mL (L100) and adult eclosion rates were recorded. National Institute of Food and Agriculture–Agriculture and Food Research Second, freshly eclosed flies from the line D-F2A/E-F2A were placed on adult Initiative.

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