IMB227.fm Page 605 Tuesday, November 14, 2000 7:17 PM Insect Molecular Biology (2000) 9(6), 605–612 TheBlackwell Science, Ltd piggyBac transposon mediates germ-line transformation in the Oriental fruit fly and closely related elements exist in its genome A. M. Handler1 and S. D. McCombs2 Introduction 1 Center for Medical, Agricultural, and Veterinary Germ-line transformation of nondrosophilid insects has Entomology, Agricultural Research Service, US recently succeeded with four transposon-based vector Department of Agriculture, Gainesville, FL 32608 USA, systems (Loukeris et al., 1995; Jasinskiene et al., 1998; 2Department of Entomology, University of Hawaii, Coates et al., 1998), with the the piggyBac element from the Honolulu, HI 96822, USA cabbage looper moth, Trichoplusia ni, being one of the more widely used to date. PiggyBac has transformed Abstract the Mediterranean (Handler et al., 1998) and Caribbean (Handler & Harrell, 2000) fruit flies, Germ-line transformation of a white eye strain of the Drosophila melanogaster Oriental fruit fly, Bactrocera dorsalis, was achieved (Handler & Harrell, 1999), Bombyx mori (Tamura et al., 2000), with the piggyBac vector, derived from a transposon Tribolium castaneum (Berghammer et al., 1999), and the originally isolated from the cabbage looper moth, pink bollworm (Peloquin et al., 2000). The piggyBac trans- poson was first discovered as the causative agent of few Trichoplusia ni. The vector was marked with the medfly + polyhedra (FP) mutations in a baculovirus that was passed white gene cDNA, and three transgenic lines were through the T. ni cell line TN-368 (Fraser et al., 1983), and identified at a frequency of approximately 2% per fer- it was subsequently found to have originated in the genome tile G . Vector integrations were verified by Southern 0 of the insect species (Cary DNA hybridization, which also revealed the presence et al., 1989). Thus far the trans- of endogenous genomic elements closely related to poson has not been found in any other species nor in any of the insects transformed with the vector, although other piggyBac. Approximately 10–20 elements per genome elements sharing the TTAA insertion site specificity of were evident in several B. dorsalis strains, and sequence analysis of 1.5 kb gene amplification products from two piggyBac have been found in other Lepidoptera (Beames & Summers, 1990). While exhaustive searches for wild strains and the white eye host strain indicated 95% piggyBac have not been made, the existence of piggyBac or related nucleotide and 92% amino acid sequence identity among elements in a wide range of insects would not be unex- resident elements and the T. ni element. PiggyBac was pected given its broad range of vector function in dipterans, not evident by hybridization in other tephritid species, lepidopterans, and a coleopteran. In particular, studies in or insects previously transformed with the transposon. two dipterans, This is the first discovery of piggyBac beyond T. ni, Ceratitis capitata (Handler et al., 1998) and and its existence in a distantly related species has D. melanogaster (Handler & Harrell, 1999), showed that important implications for the practical use of the vector piggyBac transcription and enzymatic activity were auto- and insects transformed with it. nomous, since an unmodified transposase helper catalysed transposition, suggesting that its mobility is not dependent Keywords: piggyBac transposon, germ-line trans- on the T. ni cellular phenotype, or restricted by host specific formation, horizontal transmission, Bactrocera dorsalis, factors in other insects. Tephritidae. A determination of the existence and function of piggyBac in insect species, as well as other organisms, is of particular importance for a vector that may be used for applied purposes. This information provides an indication of the Received 14 June 2000; accepted 10 August 2000. Correspondence: potential stability of the vector in a specific host, as well Dr Alfred Handler, USDA-ARS, 1700 SW 23rd Drive, Gainesville, FL 32608, USA. Tel.: (352) 374 5793; fax: (352) 374 5794; e-mail: as the potential for transmission into nontargeted hosts. [email protected]fl.edu In an effort to apply piggyBac transformation to additional © 2000 Blackwell Science Ltd 605 IMB227.fm Page 606 Tuesday, November 14, 2000 7:17 PM 606 A. M. Handler and S. D. McCombs insect species, we attempted transformation in the Oriental crossed to the we host strain. Of these, 157 were fertile, fruit fly, Bactrocera dorsalis. Bactrocera dorsalis is a highly with three of the G0 matings yielding progeny with pigmented destructive tephritid fruit fly pest whose behaviour and eyes (Fig. 1). One of the lines, Bd1-61, yielded 119 we + population size might be controlled by the molecular mani- putative transformants having red-orange eye colour pheno- pulation of transgenic strains. Furthermore, transformation types, a second line, Bd1-115, yielded five G1 offspring of this species could be tested straightforwardly since a white with yellow eyes and a third line, Bd1-137, yielded nine eye strain was available (McCombs & Saul, 1992) and offspring with pale pink eyes ( Table 2). Putative transformant evidence exists for a close relationship between its white G1 offspring were backcrossed individually, and their G2 eye (we) gene (Xiao, 1997) and that of the medfly (Zwiebel we + offspring and subsequent generations were intermated. et al., 1995), which had previously been tested as a trans- The large number of G1 transformants in the Bd1-61 line, genic marker (Loukeris et al., 1995; Handler et al., 1998). affecting nearly a third of the G1 progeny, is a clustering We report here the transformation of B. dorsalis with the affect that likely represents an early integration into a germ piggyBac vector, and the discovery of nearly identical cell chromosome. piggyBac elements in the host species. Southern hybridization + Results G2 and G3 we flies were subjected to molecular analysis by Southern hybridization, which is typically performed to Transformation experiments determine the number and general integrity of integrations Germ-line transformation of the Oriental fruit fly was tested for particular G1 sublines. For integration number, genomic with a piggyBac vector marked with the medfly white + gene DNA of transformed and nontransformed flies was digested cDNA under hsp70 promoter regulation (Handler et al., 1998), with BglII and probed with a radiolabelled Nsi/Hpa probe and a hsp70-regulated transposase helper (Handler & that spans the piggyBac BglII restriction site (Fig. 2A). This Harrell, 1999). In two experiments, 3742 preblastoderm should result in a 1.6 kb internal vector fragment, and an embryos from a we strain were injected with vector and individual band for each integration that spans the 5′-end helper plasmids at concentrations of 500 µg /ml and 300 µg/ junction site. In addition to the 1.6 kb fragment in putative ml, respectively (Table 1). From these injections, 243 G0 transformant lines, numerous hybridizing fragments were embryos survived to adulthood and were individually back- detected in the transformed as well as the nontransformed Table 1. Transformation experiments Vector:helper G0 lines µ /µ + Expt. g l Eggs injected G0s mated % Fertility G1 progeny with G1 we % Transformed I 500 : 300 2806 205 67 12 299 3 4.5 II 500 : 300 936 38 50 1014 0 0 Figure 1. Eye colour phenotypes of the B. dorsalis strains wild-type (a), white eye (b), and t he Bd[pBCcw] transformant lines Bd1-61 (c), Bd1-115 (d), and Bd1-137 (e). Visible descriptions are given in Table 2. © 2000 Blackwell Science Ltd, Insect Molecular Biology, 9, 605–612 IMB227.fm Page 607 Tuesday, November 14, 2000 7:17 PM piggyBac transposon mediates germ-line transformation in the Oriental fruit fly 607 Table 2. Bd[pBCcw] transformant lines wild and we host strains (autoradiogram exposure times necessary to visualize the 1.6 kb band resulted in over- G line Total G G we + Male Female Phenotype 0 1 1 exposure of the other fragnents). To better determine the Bd1-61 406 119 60 59 red-orange molecular phenotype of these lines, hybridization was Bd1-115 225 5 3 2 yellow performed on the same sample DNA digested with NsiI, Bd1-137 83 9 5 4 pale pink that creates a large internal fragment of 5.6 kb (from the Figure 2. Southern DNA hybridization analysis of Bd[pBCcw] transformant sublines (Bd1-61, 115 and 137), we host strain and wild-type (wt) control samples, and indicated mutant and wild strains of B. dorsalis, B. cucurbitae (B. cuc) and Ceratitis capitata (C. cap). On top is a schematic (not to scale) of the pB[Ccw] vector showing the BglII,NsiI and HindIII restriction sites used to digest genomic DNA, and the 1.4 kb NsiI-HpaI piggyBac and 1.54 kb white vector fragments used as hybridization probes (bars). Above the schematic are the distances used to calculate internal restriction fragment sizes. PiggyBac sequences are in black and hsp70-white marker sequences are in white. For each blot, the restriction enzyme used for digestion and hybridization probe are indicated. DNA size markers are shown to the left of the autoradiograms. Panel (A) shows a BglII digestion hybridized with Nsi/Hpa probe, (B) shows an NsiI digestion hybridized with Nsi/Hpa probe, (C) shows a HindIII digestion hybridized with white probe (arrows indicate resident genomic white sequences), and (D) shows an NsiI digestion hybridized with Nsi/Hpa probe. See Experimental procedures for details. © 2000 Blackwell Science Ltd, Insect Molecular Biology, 9, 605–612 IMB227.fm Page 608 Tuesday, November 14, 2000 7:17 PM 608 A. M. Handler and S. D. McCombs 6.0 kb vector) (Fig. 2B). Using a Nsi/Hpa probe that Nsi/Hpa hybridization was performed on representative hybridizes within this fragment, all the transformed lines DNA samples, digested with NsiI, from B. dorsalis wild, yielded the 5.6 kb vector fragment, which was not detect- mutant, and transformed strains, and DNA from strains from able in the we or wild-type nontransformed lines.