Genomic targeting by recombinase-mediated cassette exchange in the spotted wing drosophila, Drosophila suzukii.

Drosophila suzukii is a significant pest of stone and small fruits. The genome of this species has been sequenced and manipulated by transposon-mediated transformation and CRISPR/Cas9 gene editing. These technologies open a variety of possibilities for functional genomics and genetic modifications that might improve biologically based population control strategies. Both of these approaches, however, would benefit from genome targeting that would avoid position effects and insertional mutations associated with random transposon vector insertions, and the limited DNA fragment insertion size allowed by gene editing. Here, we describe an efficient recombinase-mediated cassette exchange (RMCE) system for D. suzukii in which heterospecific lox recombination sites were integrated into the genome by transposon-mediated transformation and subsequently targeted for double recombination by a donor vector in the presence of Cre recombinase. Three loxN/lox2272 landing site lines have previously been created in D. suzukii, and quantitative PCR determined that polyubiquitin-regulated enhanced green fluorescent protein expression is least susceptible to position effect suppression in the 443_M26m1 line. We presume that RMCE target sites may also be inserted more specifically into the genome by homology-directed repair gene editing, thereby avoiding position effects and mutations, while eliminating restrictions on the size of donor constructs for subsequent insertion.

Tetracycline-suppressible female lethality and sterility in the Mexican fruit fly, Anastrepha ludens.

The sterile insect technique (SIT) involves the mass release of sterile males to suppress insect pest populations. SIT has been improved for larval pests by the development of strains for female-specific tetracycline-suppressible (Tet-off) embryonic lethal systems for male-only populations. Here we describe the extension of this approach to the Mexican fruit fly, Anastrepha ludens, using a Tet-off driver construct with the Tet-transactivator (tTA) under embryo-specific Anastrepha suspensa serendipity α (As-sry-α) promoter regulation. In the absence of tetracycline, tTA acts upon a Tet-response element linked to the pro-apoptotic cell death gene lethal effector, head involuation defective (hid), from A. ludens (Alhid(Ala2) ) that contains a sex-specific intron splicing cassette, resulting in female-specific expression of the lethal effector. Parental adults double-homozygous for the driver/effector vectors were expected to yield male-only progeny when reared on Tet-free diet, but a complete lack of oviposited eggs resulted for each of the three strains tested. Ovary dissection revealed nonvitellogenic oocytes in all strains that was reversible by feeding females tetracycline for 5 days after eclosion, resulting in male-only adults in one strain. Presumably the sry-α promoter exhibits prezygotic maternal expression as well as zygotic embryonic expression in A. ludens, resulting in a Tet-off sterility effect in addition to female-specific lethality.

Not all GMOs are crop plants: non-plant GMO applications in agriculture.

Since tools of modern biotechnology have become available, the most commonly applied and often discussed genetically modified organisms are genetically modified crop plants, although genetic engineering is also being used successfully in organisms other than plants, including bacteria, fungi, insects, and viruses. Many of these organisms, as with crop plants, are being engineered for applications in agriculture, to control plant insect pests or diseases. This paper reviews the genetically modified non-plant organisms that have been the subject of permit approvals for environmental release by the United States Department of Agriculture/Animal and Plant Health Inspection Service since the US began regulating genetically modified organisms. This is an indication of the breadth and progress of research in the area of non-plant genetically modified organisms. This review includes three examples of promising research on non-plant genetically modified organisms for application in agriculture: (1) insects for insect pest control using improved vector systems; (2) fungal pathogens of insects to control insect pests; and (3) virus for use as transient-expression vectors for disease control in plants.

Germ-line transformation of the Queensland fruit fly, Bactrocera tryoni, using a piggyBac vector in the presence of endogenous piggyBac elements.

We report the heritable germ-line transformation of the Queensland fruit fly, Bactrocera tryoni, using a piggyBac vector marked with either the fluorescent protein DsRed or EGFP. A transformation frequency of 5-10% was obtained. Inheritance of the transgenes has remained stable over more than 15 generations despite the presence of endogenous piggyBac sequences in the B. tryoni genome. The sequence of insertion sites shows the usual canonical pattern of piggyBac integraton into TTAA target sites. An investigation of endogenous piggyBac elements in the B. tryoni genome reveals the presence of sequences almost identical to those reported recently for the B. dorsalis complex of fruit flies and two noctuid moths, suggesting a common origin of piggyBac sequences in these species. The availability of transformation protocols for B. tryoni has the potential to deliver improvements in the performance of the Sterile Insect Technique for this pest species.

Characterization of the proteasomebeta2 subunit gene and its mutant allele in the tephritid fruit fly pest, Anastrepha suspensa.

In Drosophila melanogaster the beta2 proteasome subunit gene, Prosbeta2, was first identified as a dominant temperature sensitive mutant, DTS-7, that causes pupal lethality at 29 degrees C but allows survival to adulthood at 25 degrees C. To explore the use of proteasome mutations for a conditional lethal system in insect pests, we identified and isolated the beta2 subunit gene of the 20S proteasome from the Caribbean fruit fly, Anastrepha suspensa. The caribfly ortholog AsProsbeta2 was isolated from pupal cDNA by 5' and 3' RACE. The AsProsbeta2 protein has high amino acid sequence similarity to predicted insect Prosbeta2 subunits and homologs from yeast and mammals, and it contains the well conserved amino acids that confer catalytic activity and substrate specificity. AsProsbeta2 is a single copy gene and its RNA accumulates throughout all developmental stages of the caribfly. For functional studies a point mutation, analogous to the Prosbeta2(1) mutation in D. melanogaster, was introduced into AsProsbeta2 to create an aberrant protein with a Gly170Arg substitution. Consistent with the DTS-7 mutation, transgenic insects carrying the mutant allele undergo normal metamorphosis at the permissive temperature (25 degrees C) but at the non-permissive temperature (29 degrees C) they exhibit effective pupal lethality. This is the first report of a functional characterization of a Prosbeta2 cognate based on the creation of a dominant temperature-sensitive mutation. This type of temperature-dependent lethality could be used for biological control, where transgenic insects are reared to adulthood at 25 degrees C or lower and then released into the field where ambient temperatures averaging 29 degrees C or greater cause lethality in their progeny.

Development and utilization of transgenic New World screwworm, Cochliomyia hominivorax.

The New World screwworm (NWS), Cochliomyia hominivorax (Coquerel) (Diptera: Calliphoridae), was the first insect to be effectively controlled using the sterile insect technique (SIT). Recent efforts to improve SIT control of this species have centred on the development of genetically transformed strains using the piggyBac transposon vector system. Eight transgenic strains were produced incorporating an enhanced green fluorescent protein (EGFP) marker gene under polyubiquitin regulation that has the potential for use as a genetic marking system for released males. The transgenic strains were genetically and phenotypically characterized, including for life fitness parameters and mating competitiveness. These characteristics were unique for each strain and thus some strains were deemed suitable for incorporation into SIT eradication programmes. The strain with the best attributes is designated 'CLAY'. Four of the strains, including CLAY, have been successfully cryopreserved so that their original characteristics should be unchanged when further evaluation is required. With the demonstration of efficient germ-line transformation in NWS, allowing production of fit and competitive transformants, it is now possible to consider further transgenic strain development to improve SIT that are currently being tested in other dipteran species. This includes strains that allow genetic marking with fluorescent proteins, genetic sexing by female lethality, male-specific fluorescent sorting and male sterility by testis-specific lethality. The SIT may also be improved upon by new strategies resulting in lethality of offspring of released insects using conditional lethal systems based upon temperature-dependent or dietary tetracycline regulation of lethal gene expression. Both the creation of new NWS transgenic strains and the ecological safety of their release will be enhanced by new vector systems that allow specific genomic targeting of vector constructs and their subsequent immobilization, ensuring transgene and strain stability.

Highly similar piggyBac elements in Bactrocera that share a common lineage with elements in noctuid moths.

The piggyBac IFP2 transposable element, originally discovered in a Trichoplusia ni cell line, also exists as nearly identical elements in other noctuid lepidopterans, and in several species of the tephritid genus Bactrocera. To further define the distribution of piggyBacs in Bactrocera, and compare their relationship to sequences found in Lepidoptera, a survey by PCR amplification was performed in a range of Bactrocera species. Highly similar piggyBac sequences were found in all B. dorsalis complex species tested, as well as in species in the B. zonata and B. frauenfeldi complexes. All nucleotide sequences had > 94% identity to corresponding sequences in the T. ni IFP2 element, and > 88% identity among the sequences. Conserved primers did not amplify any distantly related sequences that have been found by computational searches in a wider range of insect and non-insect species. Notably, 55 nucleotide substitutions relative to IFP2 were common to all the Bactrocera sequences, 44 of which exist in piggyBacs previously sequenced from moths, with 17 resulting in amino acid substitutions. These piggyBac elements, that apparently traversed orders by horizontal transfer, probably arose from a lineage separate from IFP2 and the other known elements in T. ni. Implications for the presence of nearly identical piggyBacs, in widely distributed insects, to the applied use of piggyBac vectors are discussed.

piggyBac internal sequences are necessary for efficient transformation of target genomes.

A previously reported piggyBac minimal sequence cartridge, which is capable of efficient transposition in embryo interplasmid transposition assays, failed to produce transformants at a significant frequency in Drosophila melanogaster compared with full-length or less extensive internal deletion constructs. We have re-examined the importance of these internal domain (ID) sequences for germline transformation using a PCR strategy that effectively adds increasing lengths of ID sequences to each terminus. A series of these piggyBac ID synthetic deletion plasmids containing the 3xP3-ECFP marker gene are compared for germline transformation of D. melanogaster. Our analyses identify a minimal sequence configuration that is sufficient for movement of piggyBac vectored sequences from plasmids into the insect genome. Southern hybridizations confirm the presence of the piggyBac transposon sequences, and insertion site analyses confirm these integrations target TTAA sites. The results verify that ID sequences adjacent to the 5' and 3' terminal repeat domains are crucial for effective germline transformation with piggyBac even though they are not required for excision or interplasmid transposition. Using this information we reconstructed an inverted repeat cartridge, ITR1.1k, and a minimal piggyBac transposon vector, pXL-BacII-ECFP, each of which contains these identified ID sequences in addition to the terminal repeat configuration previously described as essential for mobility. We confirm in independent experiments that these new minimal constructs yield transformation frequencies similar to the control piggyBac vector. Sequencing analyses of our constructs verify the position and the source of a point mutation within the 3' internal repeat sequence of our vectors that has no apparent effect on transformation efficiency.

piggyBac transformation of the New World screwworm, Cochliomyia hominivorax, produces multiple distinct mutant strains.

Sterile insect technique (SIT) programs are designed to eradicate pest species by releasing mass-reared, sterile insects into an infested area. The first major implementation of SIT was the New World Screwworm Eradication Program, which successfully eliminated the New World screwworm (NWS), Cochliomyia hominivorax (Coquerel) (Diptera: Calliphoridae), from the Continental US, Mexico and much of Central America. Ionizing radiation is currently used for sterilization, but transgenic insect techniques could replace this method, providing a safer, more cost-effective alternative. Genetic transformation methods have been demonstrated in NWS, and verified by Southern blot hybridization, PCR and sequencing of element insertion junctions. A lethal insertional mutation and enhancer detection-like phenotypic expression variations are presented and discussed. In addition to supporting the eradication efforts, transformation methods offer potential means to identify genes and examine gene function in NWS.

Germ-line transformation of the South American malaria vector, Anopheles albimanus, with a piggyBac/EGFP transposon vector is routine and highly efficient.

Stable and efficient germ-line transformation was achieved in the South American malaria vector, Anopheles albimanus, using a piggyBac vector marked with an enhanced green fluorescent protein gene regulated by the Drosophila melanogaster polyubiquitin promoter. Transgenic mosquitoes were identified from four independent experiments at frequencies ranging from 20 to 43% per fertile G0. Fluorescence was observable throughout the body of larvae and pupae, and abdominal segments of adults. Transgenic lines analysed by Southern hybridization had one to six germ-line integrations, with most lines having three or more integrations. Hybridized transposon vector fragments and insertion site sequences were consistent with precise piggyBac-mediated integrations, although this was not verified for all lines. The piggyBac/PUbnlsEGFP vector appears to be a robust transformation system for this anopheline species, in contrast to the use of a piggyBac vector in An. gambiae. Further tests are needed to determine if differences in anopheline transformation efficiency are due to the marker systems or to organismal or cellular factors specific to the species.

Polyubiquitin-regulated DsRed marker for transgenic insects.

Genetic transformation of most insect systems requires dominant-acting markers that do not depend on reverting a mutant phenotype in a host strain, andfor this purpose GFP has proven to be useful in several insect orders. However, detection of multiple transgenes and reporters for gene expression requires the development of new visible markers that can be unambiguously detected when co-expressed with GFP The DsRed fluorescentprotein has spectral characteristics that are most distinct from GFP and GFP variants, and we have explored the use of DsRed as a selectable marker for piggyBac transformation in Drosophila melanogaster and its use as a reporter when co-expressed with GFP. Transformants marked with polyubiquitin-regulated DsRed1 were detected throughout development at a relatively high frequency, and they exhibited brighter fluorescence than transformants marked with EGFP. The use of a Texas Red filter set eliminated detection of EGFP fluorescence and autofluorescence, and DsRed expressedfrom a reporter construct could be unambiguously detected when co-expressed with EGFP DsRed should prove to be a highly efficient marker system for the selection of transformant insects and as a reporter in gene expression studies.

A current perspective on insect gene transformation.

The genetic transformation of non-drosophilid insects is now possible with several systems, with germ-line transformation reported in published and unpublished accounts for about 12 species using four different transposon vectors. For some of these species, transformation can now be considered routine. Other vector systems include viruses and bacterial symbionts that have demonstrated utility in species and applications requiring transient expression, and for some, the potential exists for genomic integration. Many of these findings are quite recent, presenting a dramatic turning point in our ability to study and manipulate agriculturally and medically important insects. This review discusses these findings from the perspective of all the contributions that has made this technology a reality, the research that has yet to be done for its safe and efficient use in a broader range of species, and an overview of the available methodology to effectively utilize these systems.

Transformation of the Caribbean fruit fly, Anastrepha suspensa, with a piggyBac vector marked with polyubiquitin-regulated GFP.

Germ-line transformation was achieved in the Caribbean fruit fly, Anastrepha suspensa, using a piggyBac vector marked with an enhanced green fluorescent protein gene regulated by the Drosophila melanogaster polyubiquitin promoter. Four transgenic G(0) lines were selected exhibiting unambiguous GFP expression. Southern hybridization indicated the presence of one to four integrations in each of the transgenic lines with two integrations verified as piggyBac-mediated by sequencing their insertion sites. Fluorescence was detectable throughout development, and in adults was most intense from the thoracic flight muscle. Although adult cuticle quenched fluorescence, GFP was routinely detectable in the thorax. A quantitative spectrofluorometric assay was developed for GFP fluorescence that indicated differing levels of fluorescence among the transgenic lines, suggesting some level of position effect variegation/suppression. These results are encouraging for the use of this marker system in insect species not amenable to mutation-based visible markers. Together with the piggyBac vector, a transformation system is presented that has the potential to be universally applicable in insect species.

The piggyBac transposon mediates germ-line transformation in the Oriental fruit fly and closely related elements exist in its genome.

Germ-line transformation of a white eye strain of the Oriental fruit fly, Bactrocera dorsalis, was achieved with the piggyBac vector, derived from a transposon originally isolated from the cabbage looper moth, Trichoplusia ni. The vector was marked with the medfly white+ gene cDNA, and three transgenic lines were identified at a frequency of approximately 2% per fertile G0. Vector integrations were verified by Southern DNA hybridization, which also revealed the presence of endogenous genomic elements closely related to piggyBac. Approximately 10-20 elements per genome were evident in several B. dorsalis strains, and sequence analysis of 1.5 kb gene amplification products from two wild strains and the white eye host strain indicated 95% nucleotide and 92% amino acid sequence identity among resident elements and the T. ni element. PiggyBac was not evident by hybridization in other tephritid species, or insects previously transformed with the transposon. This is the first discovery of piggyBac beyond T. ni, and its existence in a distantly related species has important implications for the practical use of the vector and insects transformed with it.

Germline transformation of Drosophila melanogaster with the piggyBac transposon vector.

Germline transformation of Drosophila melanogaster was attempted with the piggyBac gene-transfer system from the cabbage looper moth, Trichoplusia ni. Using a self-regulated transposase helper and a white marked vector, a transformation frequency of 1-3% per fertile G0 was obtained, similar to that previously achieved in the medfly. Use of an hsp70-regulated helper increased this frequency more than eight-fold. Transformation with a vector marked with white and green fluorescent protein (GFP) under polyubiquitin-nuclear localizing sequence regulation yielded seventy G1 transformants which all expressed GFP, but only twenty-seven of these expressed eye pigmentation that would have allowed their selection based on white+ expression. PiggyBac transformation in two distantly related dipteran species and efficient expression of the gfp marker supports the potential use of this system in other dipterans, and perhaps insects in general.

The lepidopteran transposon vector, piggyBac, mediates germ-line transformation in the Mediterranean fruit fly.

The piggyBac (IFP2) short inverted terminal repeat transposable element from the cabbage looper Trichoplusia ni was tested for gene transfer vector function as part of a bipartite vector-helper system in the Mediterranean fruit fly Ceratitis capitata. A piggyBac vector marked with the medfly white gene was tested with a normally regulated piggyBac transposase helper at two different concentrations in a white eye host strain. Both experiments yielded transformants at an approximate frequency of 3-5%, with a total of six lines isolated having pigmented eyes with various levels of coloration. G1 transformant siblings from each line shared at least one common integration, with several sublines having an additional second integration. For the first transformant line isolated, two integrations were determined to be stable for 15 generations. For five of the lines, a piggyBac-mediated transposition was verified by sequencing the insertion site junctions isolated by inverse PCR that identified a characteristic piggyBac TTAA target site duplication. The efficient and stable transformation of the medfly with a lepidopteran vector represents transposon function over a relatively large evolutionary distance and suggests that the piggyBac system will be functional in a broad range of insects.

P element excision in Drosophila is stimulated by gamma-irradiation in transient embryonic assays.

The influence of gamma-irradiation on P element excision and excision-site repair mechanisms was directly tested by embryonic somatic excision assays. Preblastoderm P[ry+, delta 2-3](99B) embryos, having a stable source of somatically active P transposase, were irradiated previous to injection with P excision indicator plasmids. Frequencies of precise or nearly precise P excision increased with gamma-ray doses ranging from 0.5 to 3.5 Gy. Higher doses resulted in frequencies close to that in unirradiated embryos, though considerable embryonic lethality was also evident at these doses. A direct positive interaction between gamma-irradiation and P element activity is concluded.

A Drosophila melanogaster hobo-white(+) vector mediates low frequency gene transfer in D. virilis with full interspecific white(+) complementation.

Transformation of a Drosophila virilis white mutant host strain was attempted using a hobo vector containing the D. melanogaster mini-white(+) cassette (H[w(+), hawN]) and an unmodified or heat shock regulated hobo transposase helper. Two transformant lines were recovered with the unmodified helper (HFL1), one containing only the white(+) marked vector, and a sibling line containing the vector as well as an HFL1 helper integration. An approximate total transformation frequency of 1% is deduced. A high frequency of wing and eye morphology mutants were also observed, suggesting that hobo may have mobilized a related element in D. virilis. The data reaffirms a relatively low transformation vector activity for the hobo transposon in D. virilis; however, nearly full interspecific expression white(+) marker supports its possible function in other species as well.

A new hobo, Ac, Tam3 transposable element, hopper, from Bactrocera dorsalis is distantly related to hobo and Ac.

A new transposable element from the hobo, Ac, Tam3 transposon family was isolated as a genomic clone from the oriental fruit fly, Bactrocera dorsalis. It is approximately 3.1 kb in length with 19-bp inverted terminal repeat sequences having a single mismatch. Though sharing several amino acid sequence identities with other hAT elements, it is distantly related to both hobo and Ac. Among hAT elements thus far described in insects, it is apparently the most distantly related to hobo.

Developmental regulation of yolk protein gene expression in Anastrepha suspensa.

A partial cDNA clone for the 48,000 dalton yolk polypeptide gene from Anastrepha suspensa was isolated from a cDNA expression library using a yolk polypeptide antibody probe and hybridization to the Drosophila melanogaster yolk protein 1 gene. The sequenced DNA has greatest homology to the yolk protein genes from Ceratitis capitata, D. melanogaster, and Calliphora erythrocephala and, similar to these genes, shares amino acid sequence domains with those from lipases. RNA hybridization studies indicated that the yolk protein gene expression is completely female-specific and limited to the ovaries, without apparent regulation by 20-hydroxyecdysone or juvenile hormone. This is in contrast to an earlier study which suggested, based on immunological probes, that a very low level of yolk protein synthesis occurred in fat body and was not sex-specific.