Interplasmid transposition demonstrates piggyBac mobility in vertebrate species

The piggyBac transposon is an extremely versatile helper-dependent vector for gene transfer and germ line transformation in a wide range of invertebrate species. Analyses of genome sequencing databases have identified piggyBac homologues among several sequenced animal genomes, including the human genome. In this report we demonstrate that this insect transposon is capable of transposition in primate cells and embryos of the zebrafish, Danio rerio. piggyBac mobility was demonstrated using an interplasmid transposition assay that has consistently predicted the germ line transformation capabilities of this mobile element in several other species. Both transfected COS-7 primate cells and injected zebrafish embryos supported the helper-dependent movement of tagged piggyBac element between plasmids in the characteristic cut-and-paste, TTAA target-site specific manner. These results validate piggyBac as a valuable tool for genetic analysis of vertebrates.     

Postintegration stability of the silkworm piggyBac transposon

The piggyBac transposon is the most widely used vector for generating transgenic silkworms. The silkworm genome contains multiple piggyBac-like sequences that might influence the genetic stability of transgenic lines. To investigate the postintegration stability of piggyBac in silkworms, we used random insertion of the piggyBac [3 × p3 EGFP afm] vector to generate a W chromosome-linked transgenic silkworm, named W-T. Results of Southern blot and inverse PCR revealed the insertion of a single copy in the W chromosome of W-T at a standard TTAA insertion site. Investigation of 11 successive generations showed that all W-T females were EGFP positive and all males were EGFP negative; PCR revealed that the insertion site was unchanged in W-T offspring. These results suggested that endogenous piggyBac-like elements did not affect the stability of piggyBac inserted into the silkworm genome.     

Molecular evolutionary analysis of the widespread<Emphasis Type="Italic"> piggyBac</Emphasis> transposon family and related "domesticated" sequences

piggyBac is a short inverted-repeat-type DNA transposable element originally isolated from the genome of the moth Trichoplusia ni. It is currently the gene vector of choice for the transformation of various insect species. A few sequences with similarity to piggyBac have previously been identified from organisms such as humans ( Looper), the pufferfish Takifugu rubripes (Pigibaku), Xenopus (Tx), Daphnia (Pokey), and the Oriental fruit fly Bactrocera dorsalis. We have now identified 50 piggyBac-like sequences from publicly available genome sequences and expressed sequence tags (ESTs). This survey allows the first comparative examination of the distinctive piggyBac transposase, suggesting that it might contain a highly divergent DDD domain, comparable to the widespread DDE domain found in many DNA transposases and retroviral integrases which consists of two absolutely conserved aspartic acids separated by about 70 amino acids with a highly conserved glutamic acid about 35 amino acids further away. Many piggyBac-like sequences were found in the genomes of a phylogenetically diverse range of organisms including fungi, plants, insects, crustaceans, urochordates, amphibians, fishes and mammals. Also, several instances of "domestication" of the piggyBac transposase sequence by the host genome for cellular functions were identified. Novel members of the piggyBac family may be useful in genetic engineering of many organisms.Electronic Supplementary Material Supplementary material is available in the online version of this article at     

Formation and loss of large, unstable tandem arrays of the piggyBac transposable element in the yellow fever mosquito, Aedes aegypti

The Class II transposable element, piggyBac, was used to transform the yellow fever mosquito, Aedes aegypti. In two transformed lines only 15–30 of progeny inherited the transgene, with these individuals displaying mosaic expression of the EGFP marker gene. Southern analyses, gene amplification of genomic DNA, and plasmid rescue experiments provided evidence that these lines contained a high copy number of piggyBac transformation constructs and that much of this DNA consisted of both donor and helper plasmids. A detailed analysis of one line showed that the majority of piggyBac sequences were unit-length donor or helper plasmids arranged in a large tandem array that could be lost en masse in a single generation. Despite the presence of a transposase source and many intact donor elements, no conservative (cut and paste) transposition of piggyBac was observed in these lines. These results reveal one possible outcome of uncontrolled and/or unexpected recombination in this mosquito, and support the conclusion that further investigation is necessary before transposable elements such as piggyBac can be used as genetic drive mechanisms to move pathogen-resistance genes into mosquito populations.     

Transposition of the piggyBac element in embryos of Drosophila melanogaster, Aedes aegypti and Trichoplusia ni.

The Lepidopteran transposable element piggyBac is being recognized as a useful vector for genetic engineering in a variety of insect species. This transposon can mediate transformation in the Dipteran species Ceratitis capitata, and can potentially serve as a versatile vector for transformation of a wide variety of insect species. Using a plasmid-based interplasmid transposition assay, we have demonstrated that this transposon, of the short inverted terminal repeat type, is capable of transposition in embryos of three different insect species, Drosophila melanogaster, the yellow fever mosquito Aedes aegypti, and its host of origin, Trichoplusia ni. This assay can confirm the potential utility of piggyBac as a gene transfer tool in a given insect species, and provides an experimental model for assessing molecular mechanisms of transposon movement. Received: 19 November 1998 / Accepted: 1 March 1999     

Post-Integration Silencing of piggyBac Transposable Elements in Aedes aegypti

The piggyBac transposon, originating in the genome of the Lepidoptera Trichoplusia ni, has a broad host range, making it useful for the development of a number of transposon-based functional genomic technologies including gene vectors, enhancer-, gene- and protein-traps. While capable of being used as a vector for the creation of transgenic insects and insect cell lines, piggyBac has very limited mobility once integrated into the genome of the yellow fever mosquito, Aedes aegypti. A transgenic Aedes aegypti cell line (AagPB8) was created containing three integrated piggyBac elements and the remobilization potential of the elements was tested. The integrated piggyBac elements in AagPB8 were transpositionally silent in the presence of functional transposase, which was shown to be capable of catalyzing the movement of plasmid-borne piggyBac elements in the same cells. The structural integrity of one of the integrated elements along with the quality of element-flanking DNA, which is known to influence transposition rates, were tested in D. melanogaster. The element was found to be structurally intact, capable of transposition and excision in the soma and germ-line of Drosophila melanogaster, and in a DNA sequence context highly conducive to element movement in Drosophila melanogaster. These data show that transpositional silencing of integrated piggyBac elements in the genome of Aedes aegypti appears to be a function of higher scale genome organization or perhaps epigenetic factors, and not due to structural defects or suboptimal integration sites.     

Excision of the piggyBac transposable element in vitro is a precise event that is enhanced by the expression of its encoded transposase

The piggyBac Lepidopteran transposable element moves from the cellular genome into infecting baculovirus genomes during passage of the virus in cultured TN-368 cells. We have constructed genetically tagged piggyBac elements that permit analysis of excision when transiently introduced on plasmids into the piggyBac-deficient Spodoptera frugiperda IPLB-SF21AE cell line. Precise excision of the element from these plasmids occurs at a higher frequency in the presence of a helper plasmid that presumably supplies the piggyBac transposase. The results suggest that the piggyBac transposon encodes a protein that functions to facilitate not only insertion, but precise excision as well. This is the first demonstration of piggyBac mobility from plasmid sources in uninfected Lepidopteran cells.     

Host location by the fruit fly parasitoid Diachasmimorpha krausii: role of fruit fly species,life stage and host plant

• 1 Diachasmimorpha krausii is a braconid parasitoid of larval tephritid fruit flies, which feed cryptically within host fruit. At the ovipositor probing stage, the wasp cannot discriminate between hosts that are physiologically suitable or unsuitable for offspring development and must use other cues to locate suitable hosts.
• 2 To identify the cues used by the parasitoid to find suitable hosts, we offered, to free flying wasps, different combinations of three fruit fly species (Bactrocera tryoni, Bactrocera cacuminata, Bactrocera cucumis), different life stages of those flies (adults and larvae) and different host plants (Solanum lycopersicon, Solanum mauritianum, Cucurbita pepo). In the laboratory, the wasp will readily oviposit into larvae of all three flies but successfully develops only in B. tryoni. Bactrocera tryoni commonly infests S. lycopersicon (tomato), rarely S. mauritianum (wild tobacco) but never C. pepo (zucchini). The latter two plant species are common hosts for B. cacuminata and B. cucumis, respectively.
• 3 The parasitoid showed little or no response to uninfested plants of any of the test species. The presence of adult B. tryoni, however, increased parasitoid residency time on uninfested tomato.
• 4 When the three fruit types were all infested with larvae, parasitoid response was strongest to tomato, regardless of whether the larvae were physiologically suitable or unsuitable for offspring development. By contrast, zucchini was rarely visited by the wasp, even when infested with B. tryoni larvae.
• 5 Wild tobacco was infrequently visited when infested with B. cacuminata larvae but was more frequently visited, with greater parasitoid residency time and probing, when adult flies (either B. cacuminata or B. tryoni) were also present.
• 6 We conclude that herbivore‐induced, nonspecific host fruit wound volatiles were the major cue used by foraging D. krausii. Although positive orientation to infested host plants is well known from previous studies on opiine braconids, the failure of the wasp to orientate to some plants even when infested with physiologically suitable larvae, and the secondary role played by adult fruit flies in wasp host searching, are newly‐identified mechanisms that may aid parasitoid host location in environments where both physiologically suitable and unsuitable hosts occur.

     

The ecology of Bactrocera tryoni (Diptera: Tephritidae): what do we know to assist pest management?

The distribution, systematics and ecology of Bactrocera tryoni, the Queensland fruit fly, are reviewed. Bactrocera tryoni is a member of the B. tryoni complex of species, which currently includes four named species, viz. B. tryoni ssp., B. neohumeralis, B. melas and B. aquilonis. The species status of B. melas and B. aquilonis is unclear (they may be junior synonyms of B. tryoni) and their validity, or otherwise, needs to be confirmed as a matter of urgency. While Queensland fruit fly is regarded as a tropical species, it cannot be assumed that its distribution will spread further south under climate change scenarios. Increasing aridity and hot dry summers, as well as more complex, indirect interactions resulting from elevated CO₂, make predicting the future distribution and abundance of B. tryoni difficult. The ecology of B. tryoni is reviewed with respect to current control approaches (with the exception of sterile insect technique (SIT) which is covered in a companion paper). We conclude that there are major gaps in the knowledge required to implement most noninsecticide‐based management approaches. Priority areas for future research include host–plant interactions, protein and cue‐lure foraging and use, spatial dynamics, development of new monitoring tools, investigating the use of natural enemies and better integration of fruit flies into general horticultural IPM systems.     

Identification and characterization of piggyBac-like elements in the genome of domesticated silkworm, Bombyx mori

piggyBac is a short inverted terminal repeat (ITR) transposable element originally discovered in Trichoplusia ni. It is currently the preferred vector of choice for enhancer trapping, gene discovery and identifying gene function in insects and mammals. Many piggyBac-like sequences have been found in the genomes of phylogenetically species from fungi to mammals. We have identified 98 piggyBac-like sequences (BmPBLE1-98) from the genome data of domesticated silkworm (Bombyx mori) and 17 fragments from expressed sequence tags (ESTs). Most of the BmPBLE1-98 probably exist as fossils. A total of 21 BmPBLEs are flanked by ITRs and TTAA host dinucleotides, of which 5 contain a single ORF, implying that they may still be active. Interestingly, 16 BmPBLEs have CAC/GTG not CCC/GGG as the characteristic residues of ITRs, which is a surprising phenomenon first observed in the piggyBac families. Phylogenetic analysis indicates that many BmPBLEs have a close relation to mammals, especially to Homo sapiens, only a few being grouped with the T. ni piggyBac element. In addition, horizontal transfer was probably involved in the evolution of the piggyBac-like elements between B. mori and Daphnia pulicaria. The analysis of the BmPBLEs will contribute to our understanding of the characteristic of the piggyBac family and application of piggyBac in a wide range of insect species.     

Precise marker excision system using an animal‐derived piggyBac transposon in plants

Accurate and effective positive marker excision is indispensable for the introduction of desired mutations into the plant genome via gene targeting (GT) using a positive/negative counter selection system. In mammals, the moth‐derived piggyBac transposon system has been exploited successfully to eliminate a selectable marker from a GT locus without leaving a footprint. Here, we present evidence that the piggyBac transposon also functions in plant cells. To demonstrate the use of the piggyBac transposon for effective marker excision in plants, we designed a transposition assay system that allows the piggyBac transposition to be visualized as emerald luciferase (Eluc) luminescence in rice cells. The Eluc signal derived from piggyBac excision was observed in hyperactive piggyBac transposase‐expressing rice calli. Polymerase chain reaction, Southern blot analyses and sequencing revealed the efficient and precise transposition of piggyBac in these calli. Furthermore, we have demonstrated the excision of a selection marker from a reporter locus in T₀ plants without concomitant re‐integration of the transposon and at a high frequency (44.0% of excision events), even in the absence of negative selection.     

Transposition of the piggyBac element in embryos of Drosophila melanogaster, Aedes aegypti and Trichoplusia ni

The Lepidopteran transposable element piggyBac is being recognized as a useful vector for genetic engineering in a variety of insect species. This transposon can mediate transformation in the Dipteran species Ceratitis capitata, and can potentially serve as a versatile vector for transformation of a wide variety of insect species. Using a plasmid-based interplasmid transposition assay, we have demonstrated that this transposon, of the short inverted terminal repeat type, is capable of transposition in embryos of three different insect species, Drosophila melanogaster, the yellow fever mosquito Aedes aegypti, and its host of origin, Trichoplusia ni. This assay can confirm the potential utility of piggyBac as a gene transfer tool in a given insect species, and provides an experimental model for assessing molecular mechanisms of transposon movement.     

CRISPR/Cas9‐mediated mutagenesis of the white gene in the tephritid pest Bactrocera tryoni

The Queensland fruit fly, Bactrocera tryoni (Froggatt), is a polyphagous horticultural pest in Australia that is capable of causing significant damage to more than 100 different host fruits and vegetables. Chemical applications and ecological control strategies, such as the sterile insect technique (SIT), are commonly used to suppress established populations and eradicate invasive outbreaks following migration. The recently published B. tryoni draft genome provides new opportunities to identify candidate genes for targeted genome modification in order to generate advanced genetic strains for management using sterile insect strategies. Here, we demonstrate CRISPR/Cas‐mediated mutagenesis in B. tryoni through generating a series of frame‐shift mutations in the ATP‐dependent binding cassette transporter, white, causing a classic white‐eye phenotype. This work establishes methods for CRISPR/Cas genome editing in tephritids and demonstrates its potential for developing genetic sexing strains which could be used for SIT‐based pest control.     

Attraction of fruit flies of the genus Bactrocera to colored mimics of host fruit

In tests on feral populations of polyphagous Bactrocera tryoni (Froggatt) adults on host guava trees, both sexes were significantly more attracted to Tangletrap‐coated 50 mm diameter spheres colored blue or white than to similar spheres colored red, orange, yellow, green, or black or to Tangletrap‐coated 50 mm diameter yellow‐green guava fruit. In contrast, in tests on feral populations of oligophagous Bactrocera cacuminata (Hering) on host wild tobacco plants, both sexes were significantly more attracted to Tangletrap‐coated 15 mm diameter spheres colored orange or yellow than to other colors of spheres or to Tangletrap‐coated 15 mm diameter green wild tobacco fruit. Both sexes of both tephritid species were significantly more attracted to blue (in the case of B. tryoni) or orange (in the case of B. cacuminata) 50 mm spheres displayed singly than to blue or orange 15 mm spheres displayed in clusters, even though fruit of wild tobacco plants are borne in clusters. Finally, B. tryoni adults were significantly less attracted to non‐ultraviolet reflecting bluish fruit‐mimicking spheres than to bluish fruit‐mimicking spheres having a slightly enhanced level of ultraviolet reflectance, similar to the reflectance of possible native host fruit of B. tryoni, whose bluish skin color is overlayed with ultraviolet‐reflecting waxy bloom. Responses to fruit visual stimuli found here are discussed relative to responses found in other tephritid species.     

Development and use of a piggyBac‐based jumpstarter system in Drosophila suzukii

Spotted wing drosophila, Drosophila suzukii, is an invasive pest that primarily attacks fresh, soft‐skinned fruit. Although others have reported successful integration of marked piggyBac elements into the D. suzukii genome, with a very respectable transgenesis rate of ～16%, here we take this work a step further by creating D. suzukii jumpstarter strains. These were generated through integration of a fluorescent‐marked Minos element carrying a heat shock protein 70‐driven piggyBac transposase gene. We demonstrate that there is a dramatic increase in transformation rates when germline transformation is performed in a transposase‐expressing background. For example, we achieved transformation rates as high as 80% when microinjecting piggyBac‐based plasmids into embryos derived from one of these D. suzukii jumpstarter strains. We also investigate the effect of insert size on transformation efficiency by testing the ability of the most efficient jumpstarter strain to catalyze integration of differently‐sized piggyBac elements. Finally, we demonstrate the ability of a jumpstarter strain to remobilize an already‐integrated piggyBac element to a new location, demonstrating that our jumpstarter strains could be used in conjunction with a piggyBac‐based donor strain for genome‐wide mutagenesis of D. suzukii.     

Genetic transformation of the codling moth, <Emphasis Type="Italic">Cydia pomonella</Emphasis> L., with <Emphasis Type="Italic">piggyBac</Emphasis> EGFP

Genetic transformation of the codling moth, Cydia pomonella, was accomplished through embryo microinjection with a plasmid-based piggyBac vector containing the enhanced green fluorescent protein (EGFP) gene. Sequencing of the flanking regions around the inserted construct resulted in identification of insect genomic sequences, not plasmid sequences, thus providing evidence that the piggyBac EGFP cassette had integrated into the codling moth genome. EGFP-positive moths were confirmed in the 28th and earlier generations post injection through PCR and Southern blot analyses, indicating heritability of the transgene.     

Temperature effects on “overwintering” phenology of a polyphagous,tropical fruit fly (Tephritidae) at the subtropical/temperate interface

Around the world, several pest tephritids are extending their ranges from warm tropical or Mediterranean climates into cooler temperate regions. The ability to tolerate climatic diversity is uncommon among insects, and understanding the population phenology drivers of such species across different parts of their range will be critical for their management. Here, we determined the role of temperature versus fruit availability on the population phenology of Queensland fruit fly, Bactrocera tryoni. Using a field site located at the subtropical/temperate interface, with host fruits continuously available, we monitored the development times and abundance of B. tryoni, a species which has invaded temperate Australia from the tropics. From fruit samples held at ambient and controlled conditions, the abundance of emerging flies was highly variable among collection dates, but the variance did not reflect the observed changes in temperature. For most samples, the survival rate of flies in a field site was lower than predicted by a day‐degree population model fitted with mean daily field temperatures. The development time of the immature stage in the field was prolonged, presumably due to cooler ambient conditions, but the fitted day‐degree population model consistently over‐predicted estimated development times. Our results indicate that at the subtropical/temperate interface, the decline in B. tryoni populations during winter is only partly driven by temperature and host availability. We classify B. tryoni as a climate generalist, which likely employs physiological as well as behavioural mechanisms to achieve broad climatic tolerance ranges.     

Impact of habitat modification on the distribution and abundance of fruit flies (Diptera: Tephritidae) in Southeast Queensland

Loss of rainforest because of agricultural and urban development may impact the abundance and diversity of species that are rainforest natives. Tropical fruit flies are one group of such organisms indigenous to rainforests. In southeast Queensland, a region subject to rapid urbanization, we assessed the impact of habitat disturbance on the distribution and abundance of native fruit flies. Data on four species (Bactrocera tryoni, Bactrocera neohumeralis, Bactrocera chorista, and Dacus aequalis) were gathered and analyzed over 6 months in three habitat types: suburbia, open sclerophyll forest, and rainforest. We also analyzed the data at a combined "dacine fruit fly" level incorporating all fruit fly species trapped over the period of study (as might occur in a biodiversity assessment): these included the four species already named and Bactrocera melas, Bactrocera bryoniae, Bactrocera newmani, and Dacus absonifacies. Analysis at the species level showed that the polyphagous pest species responded differently to the monophagous species. Bactrocera tryoni, which has more exotic than native hosts, was positively affected by transformation of natural habitat into suburbia whereas B. neohumeralis, which has nearly identical numbers of native and exotic hosts, was found equally across habitat types. Bactrocera chorista and Dacus aequalis, each monophagous on a species-specific rainforest host plant, were most abundant in rainforest. The analysis based on the combined data suggests that replacing rainforest with suburbia has a neutral, or even positive, effect on the abundance of fruit flies as a whole. At the species level, however, it can be seen that this is an erroneous conclusion biased by the abundance of a single pest species. Our discussion raises the issue of analyses at supraspecific levels in biodiversity and impact assessment studies. Received: March 6, 2000 / Accepted: June 19, 2000     

The draft genome of the pest tephritid fruit fly Bactrocera tryoni: resources for the genomic analysis of hybridising species

Background

The tephritid fruit flies include a number of economically important pests of horticulture, with a large accumulated body of research on their biology and control. Amongst the Tephritidae, the genus Bactrocera, containing over 400 species, presents various species groups of potential utility for genetic studies of speciation, behaviour or pest control. In Australia, there exists a triad of closely-related, sympatric Bactrocera species which do not mate in the wild but which, despite distinct morphologies and behaviours, can be force-mated in the laboratory to produce fertile hybrid offspring. To exploit the opportunities offered by genomics, such as the efficient identification of genetic loci central to pest behaviour and to the earliest stages of speciation, investigators require genomic resources for future investigations.

Results

We produced a draft de novo genome assembly of Australia’s major tephritid pest species, Bactrocera tryoni. The male genome (650 -700 Mbp) includes approximately 150Mb of interspersed repetitive DNA sequences and 60Mb of satellite DNA. Assessment using conserved core eukaryotic sequences indicated 98% completeness. Over 16,000 MAKER-derived gene models showed a large degree of overlap with other Dipteran reference genomes. The sequence of the ribosomal RNA transcribed unit was also determined. Unscaffolded assemblies of B. neohumeralis and B. jarvisi were then produced; comparison with B. tryoni showed that the species are more closely related than any Drosophila species pair. The similarity of the genomes was exploited to identify 4924 potentially diagnostic indels between the species, all of which occur in non-coding regions.

Conclusions

This first draft B. tryoni genome resembles other dipteran genomes in terms of size and putative coding sequences. For all three species included in this study, we have identified a comprehensive set of non-redundant repetitive sequences, including the ribosomal RNA unit, and have quantified the major satellite DNA families. These genetic resources will facilitate the further investigations of genetic mechanisms responsible for the behavioural and morphological differences between these three species and other tephritids. We have also shown how whole genome sequence data can be used to generate simple diagnostic tests between very closely-related species where only one of the species is scaffolded.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1153) contains supplementary material, which is available to authorized users.