A transposon-based genetic marker for conspecific identity within the Bactrocera dorsalis species complex.

Here we describe a molecular approach to assess conspecific identity that relies on the comparison of an evolved mutated transposable element sequence and its genomic insertion site in individuals from closely related species. This was explored with the IFP2 piggyBac transposon, originally discovered in Trichoplusia ni as a 2472 bp functional element, that was subsequently found as mutated elements in seven species within the Bactrocera dorsalis species complex. In a B. dorsalis [Hendel] strain collected in Kahuku, Hawaii, a degenerate 2420 bp piggyBac sequence (pBacBd-Kah) having ~ 94.5% sequence identity to IFP2 was isolated, and it was reasoned that common species, or strains within species, should share the same evolved element and its precise genomic insertion site. To test this assumption, PCR using primers to pBacBd-Kah and adjacent genomic sequences was used to isolate and compare homologous sequences in strains of four sibling species within the complex. Three of these taxa, B. papayae, B. philippinensis, and B. invadens, were previously synonymized with B. dorsalis, and found to share nearly identical pBacBd-Kah homologous elements (> 99% nucleotide identity) within the identical insertion site consistent with conspecific species. The fourth species tested, B. carambolae, considered to be a closely related yet independent species sympatric with B. dorsalis, also shared the pBacBd-Kah sequence and insertion site in one strain from Suriname, while another divergent pBacBd-Kah derivative, closer in identity to IFP2, was found in individuals from French Guiana, Bangladesh and Malaysia. This data, along with the absence of pBacBd-Kah in distantly related Bactrocera, indicates that mutated descendants of piggyBac, as well as other invasive mobile elements, could be reliable genomic markers for common species identity.

The hAT family hopper transposon exists as highly similar yet discontinuous elements in the Bactrocera tephritid fly genus.

The hAT family transposable element, hopper, was originally discovered as a defective 3120-bp full-length element in a wild-type strain of the oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae), and subsequently a functional 3131-bp element, hopperBdwe, was isolated from a white eye mutant strain. The latter study showed that closely related elements exist in melonfly, Zeugodacus cucurbitae (Coquillett) (Diptera: Tephritidae), a closely related subgenus, suggesting that hopper could have a widespread presence in the Bactrocera genus. To further understand the distribution of hopper within and beyond the B. dorsalis species complex, primer pairs from hopperBdwe and its adjacent genomic insertion site were used to survey the presence and relatedness of hopper in five species within the complex and four species beyond the complex. Based on sequence identity of a 1.94 kb internal nucleotide sequence, the closest relationships were with mutated elements from B. dorsalis s.s. and species synonymized with B. dorsalis including B. papayae, B. philippinensis and B. invadens, ranging in identity between 88.4% and 99.5%. Notably, Bactrocera carambolae (Drew & Hancock) (Diptera: Tephritidae), which is most closely related to B. dorsalis beyond the synonymized species, shared hopper identities of 97.3%-99.5%. Beyond the B. dorsalis complex, Z. cucurbitae, Bactrocera tryoni (Froggatt) (Diptera: Tephritidae) and Bactrocera zonata (Saunders) (Diptera: Tephritidae) shared identities of 83.1%-97.1%, while hopper was absent from the Bactrocera oleae (Gmelin) (Diptera: Tephritidae) strain tested. While the functional autonomous hopperBdwe element was not detected in these species, another closely related hopper element isolated from a B. dorsalis genetic sexing strain has an uninterrupted transposase open reading frame. The discontinuous presence of hopper in the Bactrocera genus has implications for its use for genomic manipulation and understanding the phylogenetic relationship of these species.

Sequence and expression analysis of the spermatogenesis-specific gene cognates, wampa and Prosα6T, in Drosophila suzukii.

The sterile insect technique (SIT) is a highly effective biologically-based method for the population suppression of highly invasive insect pests of medical and agricultural importance. The efficacy of SIT could be significantly enhanced, however, by improved methods of male sterilization that avoid the fitness costs of irradiation. An alternative sterilization method is possible by gene-editing that targets genes essential for sperm maturation and motility, rendering them nonfunctional, similar to the CRISPR-Cas9 targeting of ß2-tubulin in the genetic model system, Drosophila melanogaster. However, since genetic strategies for sterility are susceptible to breakdown or resistance in mass-reared populations, alternative targets for sterility are important for redundancy or strain replacement. Here we have identified and characterized the sequence and transcriptional expression of two genes in a Florida strain of Drosophila suzukii, that are cognates of the D. melanogaster spermatocyte-specific genes wampa and Prosalpha6T. Wampa encodes a coiled-coil dynein subunit required for axonemal assembly, and the proteasome subunit gene, Prosalpha6T, is required for spermatid individualization and nuclear maturation. The reading frames of these genes differed from their NCBI database entries derived from a D. suzukii California strain by 44 and 8 nucleotide substitutions/polymorphisms, respectively, though all substitutions were synonymous resulting in identical peptide sequences. Expression of both genes is predominant in the male testis, and they share similar transcriptional profiles in adult males with ß2-tubulin. Their amino acid sequences are highly conserved in dipteran species, including pest species subject to SIT control, supporting their potential use in targeted male sterilization strategies.

Mitochondrial superoxide dismutase overexpression and low oxygen conditioning hormesis improve the performance of irradiated sterile males.

The Sterile Insect Technique (SIT) is a successful autocidal control method that uses ionizing radiation to sterilize insects. However, irradiation in normal atmospheric conditions can be damaging for males, because irradiation generates substantial biological oxidative stress that, combined with domestication and mass-rearing conditions, may reduce sterile male sexual competitiveness and quality. In this study, biological oxidative stress and antioxidant capacity were experimentally manipulated in Anastrepha suspensa using a combination of low-oxygen conditions and transgenic overexpression of mitochondrial superoxide dismutase (SOD2) to evaluate their role in the sexual behavior and quality of irradiated males. Our results showed that SOD2 overexpression enhances irradiated insect quality and improves male competitiveness in leks. However, the improvements in mating performance were modest, as normoxia-irradiated SOD2 males exhibited only a 22% improvement in mating success compared to normoxia-irradiated wild type males. Additionally, SOD2 overexpression did not synergistically improve the mating success of males irradiated in either hypoxia or severe hypoxia. Short-term hypoxic and severe-hypoxic conditioning hormesis, per se, increased antioxidant capacity and enhanced sexual competitiveness of irradiated males relative to non-irradiated males in leks. Our study provides valuable new information that antioxidant enzymes, particularly SOD2, have potential to improve the quality and lekking performance of sterile males used in SIT programs.

Toxicological risk assessment of some commonly used insecticides on Cotesia flavipes, a larval parasitoid of the spotted stem borer Chilo partellus.

Cotesia flavipes Cameron is an important larval parasitoid exploited for the control of the spotted stem borer, Chilo partellus (Swinhoe). Several studies have evaluated the toxic effects of insecticides on C. partellus, however, little is known about non-target effects of insecticides on this parasitoid, when used to control C. partellus. This laboratory study evaluated the lethal and sublethal effects of twelve insecticides on C. flavipes. Residual toxicity tests showed that organophosphates (chlorpyrifos, triazophos and profenofos) exhibited highest contact toxicity to C. flavipes adults with a LC50 range from 0.63 to 1.05 mg a.i/l, while neonicotinoids (nitenpyram, acetamiprid and imidacloprid) were less toxic to C. flavipes with a LC50 range from 1.27 to 139.48 mg a.i/l. Sugar-insecticide feeding bioassays showed that organophosphates, pyrethroids (cypermethrin, bifenthrin and lambda-cyhalothrin) and carbamates (thiodicarb, carbaryl and methomyl) were highly toxic to C. flavipes adults and caused 100% mortality at 48 h of exposure, while imidacloprid caused 66% mortality at 48 h of exposure. Risk quotient analysis showed that among all tested insecticides, imidacloprid and acetamiprid were less toxic to C. flavipes adults with a risk quotient value of 0.88 and 1.6, respectively. Furthermore, exposure of immature C. flavipes through their host bodies significantly decreased the parasitism rate at their F1 and F2 generations. Risk quotient analysis of insecticides indicated that imidacloprid and acetamiprid were the least toxic to C. flavipes. This study provides important information that will be used in incorporating the most suitable insecticides in integrated pest management programs with reduced negative impacts on non-target beneficial arthropods.

The hAT-family transposable element, hopper, from Bactrocera dorsalis is a functional vector for insect germline transformation.

BACKGROUND: The hopper hAT-family transposable element isolated from the Oriental fruit fly, Bactrocera dorsalis, is distantly related to both the Drosophila hobo element and the Activator element from maize. The original 3120 bp hopperBd-Kah element isolated from the Kahuku wild-type strain was highly degenerate and appeared to have a mutated transposase and terminal sequences, while a second 3131 bp element, hopperBd-we, isolated from a white eye mutant strain had an intact transposase reading frame and terminal sequences consistent with function. RESULTS: The hopperBd-we element was tested for function by its ability to mediate germline transformation in two dipteran species other than B. dorsalis. This was achieved by creating a binary vector/helper transformation system by linking the hopperBd-we transposase reading frame to a D. melanogaster hsp70 promoter for a heat-inducible transposase helper plasmid, and creating vectors marked with the D. melanogaster mini-white+ or polyubiquitin-regulated DsRed fluorescent protein markers. CONCLUSIONS: Both vectors were successfully used to transform D. melanogaster, and the DsRed vector was also used to transform the Caribbean fruit fly, Anastrepha suspensa, indicating a wide range of hopper function in dipteran species and, potentially, non-dipteran species. This vector provides a new tool for insect genetic modification for both functional genomic analysis and the control of insect populations.

Genetic breakdown of a Tet-off conditional lethality system for insect population control.

Genetically modified conditional lethal strains have been created to improve the control of insect pest populations damaging to human health and agriculture. However, understanding the potential for the genetic breakdown of lethality systems by rare spontaneous mutations, or selection for inherent suppressors, is critical since field release studies are in progress. This knowledge gap was addressed in a Drosophila tetracycline-suppressible embryonic lethality system by analyzing the frequency and structure of primary-site spontaneous mutations and second-site suppressors resulting in heritable survivors from 1.2 million zygotes. Here we report that F1 survivors due to primary-site deletions and indels occur at a 5.8 × 10-6 frequency, while survival due to second-site maternal-effect suppressors occur at a ~10-5 frequency. Survivors due to inherent lethal effector suppressors could result in a resistant field population, and we suggest that this risk may be mitigated by the use of dual redundant, albeit functionally unrelated, lethality systems.

Transcriptome Analysis of the Oriental Fruit Fly Bactrocera dorsalis Early Embryos.

: The oriental fruit fly, Bactrocera dorsalis (Hendel), is one of the most devastating and highly invasive agricultural pests world-wide, resulting in severe economic loss. Thus, it is of great interest to understand the transcriptional changes that occur during the activation of its zygotic genome at the early stages of embryonic development, especially the expression of genes involved in sex determination and the cellularization processes. In this study, we applied Illumina sequencing to identify B. dorsalis sex determination genes and early zygotic genes by analyzing transcripts from three early embryonic stages at 0-1, 2-4, and 5-8 h post-oviposition, which include the initiation of sex determination and cellularization. These tests generated 13,489 unigenes with an average length of 2185 bp. In total, 1683, 3201 and 3134 unigenes had significant changes in expression levels at times after oviposition including at 2-4 h versus 0-1 h, 5-8 h versus 0-1 h, and 5-8 h versus 2-4 h, respectively. Clusters of gene orthology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) annotations were performed throughout embryonic development to better understand the functions of differentially expressed unigenes. We observed that the RNA binding and spliceosome pathways were highly enriched and overrepresented during the early stage of embryogenesis. Additionally, transcripts for 21 sex-determination and three cellularization genes were identified, and expression pattern analysis revealed that the majority of these genes were highly expressed during embryogenesis. This study is the first assembly performed for B. dorsalis based on Illumina next-generation sequencing technology during embryogenesis. Our data should contribute significantly to the fundamental understanding of sex determination and early embryogenesis in tephritid fruit flies, and provide gene promoter and effector gene candidates for transgenic pest-management strategies for these economically important species.

miRNA-1-3p is an early embryonic male sex-determining factor in the Oriental fruit fly Bactrocera dorsalis.

Regulation of male sexual differentiation by a Y chromosome-linked male determining factor (M-factor) is one of a diverse array of sex determination mechanisms found in insects. By deep sequencing of small RNAs from Bactrocera dorsalis early embryos, we identified an autosomal-derived microRNA, miR-1-3p, that has predicted target sites in the transformer gene (Bdtra) required for female sex determination. We further demonstrate by both in vitro and in vivo tests that miR-1-3p suppresses Bdtra expression. Injection of a miR-1-3p mimic in early embryos results in 87-92% phenotypic males, whereas knockdown of miR-1-3p by an inhibitor results in 67-77% phenotypic females. Finally, CRISPR/Cas9-mediated knockout of miR-1-3p results in the expression of female-specific splice variants of Bdtra and doublesex (Bddsx), and induced sex reversal of XY individuals into phenotypic females. These results indicate that miR-1-3p is required for male sex determination in early embryogenesis in B. dorsalis as an intermediate male determiner.

Gene content evolution in the arthropods.

BACKGROUND: Arthropods comprise the largest and most diverse phylum on Earth and play vital roles in nearly every ecosystem. Their diversity stems in part from variations on a conserved body plan, resulting from and recorded in adaptive changes in the genome. Dissection of the genomic record of sequence change enables broad questions regarding genome evolution to be addressed, even across hyper-diverse taxa within arthropods. RESULTS: Using 76 whole genome sequences representing 21 orders spanning more than 500 million years of arthropod evolution, we document changes in gene and protein domain content and provide temporal and phylogenetic context for interpreting these innovations. We identify many novel gene families that arose early in the evolution of arthropods and during the diversification of insects into modern orders. We reveal unexpected variation in patterns of DNA methylation across arthropods and examples of gene family and protein domain evolution coincident with the appearance of notable phenotypic and physiological adaptations such as flight, metamorphosis, sociality, and chemoperception. CONCLUSIONS: These analyses demonstrate how large-scale comparative genomics can provide broad new insights into the genotype to phenotype map and generate testable hypotheses about the evolution of animal diversity.

Overexpression of an antioxidant enzyme improves male mating performance after stress in a lek-mating fruit fly.

In many species, courtship displays are reliable signals of male quality, and current hypotheses suggest that these displays allow females to choose males with high cellular function. Environmental stressors generate excess reactive oxygen species (ROS) that impair cellular function, and thus antioxidant pathways that remove ROS are probably critical for preserving complex sexual behaviours. Here, we test the hypothesis that enhanced antioxidant activity in mitochondria preserves mating performance following oxidative stress. Using a transgenic approach, we directly manipulated mitochondrial antioxidant activity in the Caribbean fruit fly, Anastrepha suspensa, a lek-mating species with elaborate sexual displays and intense sexual selection that is also a model for sterile insect technique programmes. We generated seven transgenic lines that overexpress mitochondrial superoxide dismutase (MnSOD). Radiation is a severe oxidative stressor used to induce sterility for sterile insect programmes. After radiation treatment, two lines with intermediate MnSOD overexpression showed enhanced mating performance relative to wild-type males. These improvements in mating corresponded with reduced oxidative damage to lipids, demonstrating that MnSOD overexpression protects flies from oxidative stress at the cellular level. For lines with improved mating performance, overexpression also preserved locomotor activity, as indicated by a laboratory climbing assay. Our results show a clear link between oxidative stress, antioxidant capacity and male performance. Our work has implications for fundamentally understanding the role of antioxidants in sexual selection, and shows promise for using transgenic approaches to enhance the field performance of insects released for area-wide pest management strategies and improving performance of biological control agents in general.

CRISPR/Cas9-mediated gene editing in an exogenous transgene and an endogenous sex determination gene in the Caribbean fruit fly, Anastrepha suspensa.

CRISPR/Cas9-mediated gene-editing, using injected Cas9 protein, was achieved in the Caribbean fruit fly, Anastrepha suspensa, by initially targeting an exogenous transgene, polyubiquitin-regulated EGFP (PUb-EGFP), for heritable non-homologous end-joining (NHEJ) knock-outs using an individual sgRNA. Multiple deletion mutations, ranging from two to five nts proximal to the target site, were identified phenotypically by the loss of green fluorescence in transgenic flies that were also marked with PUb-DsRed. This represented a relatively high efficiency rate of 29% for germ-line mutations. Similar conditions were then used to target an endogenous sex-determination gene, As-transformer-2 (Astra-2), using two sgRNAs that targeted independent exon sequences 671 bp apart. Somatic mutations were identified phenotypically in G0 adult flies at a frequency of 81% based upon intersexual genital morphology, expected to occur only in XX females since Astra-2 knock-outs by dsRNA do not have a phenotypic effect in XY males. Consistent with this expectation, twelve types of short indels, ranging from -15 nts to +5 nts, were identified proximal to the 5' sgRNA-1 target site in intersexual adults. However, the 3' sgRNA-2 target was only associated with a single 774 bp deletion extending from the sgRNA-1 target site to 100 bp downstream of the sgRNA-2 target. This is encouraging for the eventual use of dual target sites for homology-directed repair (HDR) insertions, but suggests that the sgRNA-2 target site tested may not be optimal for Astra-2 HDR modification.

Recommendations for Laboratory Containment and Management of Gene Drive Systems in Arthropods.

Versatile molecular tools for creating driving transgenes and other invasive genetic factors present regulatory, ethical, and environmental challenges that should be addressed to ensure their safe use. In this article, we discuss driving transgenes and invasive genetic factors that can potentially spread after their introduction into a small proportion of individuals in a population. The potential of invasive genetic factors to increase their number in natural populations presents challenges that require additional safety measures not provided by previous recommendations regarding accidental release of arthropods. In addition to providing physical containment, invasive genetic factors require greater attention to strain management, including their distribution and identity confirmation. In this study, we focus on insects containing such factors with recommendations for investigators who are creating them, institutional biosafety committees charged with ensuring safety, funding agencies providing support, those managing insectaries handling these materials who are responsible for containment, and other persons who will be receiving insects-transgenic or not-from these facilities. We give specific examples of efforts to modify mosquitoes for mosquito-borne disease control, but similar considerations are relevant to other arthropods that are important to human health, the environment, and agriculture.

Temperature-dependent sex-reversal by a transformer-2 gene-edited mutation in the spotted wing drosophila, Drosophila suzukii.

Female to male sex reversal was achieved in an emerging agricultural insect pest, Drosophila suzukii, by creating a temperature-sensitive point mutation in the sex-determination gene, transformer-2 (tra-2), using CRISPR/Cas9 (clustered regularly interspaced palindromic repeats/CRISPR-associated) homology-directed repair gene-editing. Ds-tra-2 ts2 mutants developed as normal fertile XX and XY adults at permissive temperatures below 20 °C, but at higher restrictive temperatures (26 to 29 °C) chromosomal XX females developed as sterile intersexuals with a predominant male phenotype, while XY males developed with normal morphology, but were sterile. The temperature-dependent function of the Ds-TRA-2ts2 protein was also evident by the up- and down-regulation of female-specific Ds-Yolk protein 1 (Ds-Yp1) gene expression by temperature shifts during adulthood. This study confirmed the temperature-dependent function of a gene-edited mutation and provides a new method for the more general creation of conditional mutations for functional genomic analysis in insects, and other organisms. Furthermore, it provides a temperature-dependent system for creating sterile male populations useful for enhancing the efficacy of biologically-based programs, such as the sterile insect technique (SIT), to control D. suzukii and other insect pest species of agricultural and medical importance.

Cre/lox-Recombinase-Mediated Cassette Exchange for Reversible Site-Specific Genomic Targeting of the Disease Vector, Aedes aegypti.

Site-specific genome modification (SSM) is an important tool for mosquito functional genomics and comparative gene expression studies, which contribute to a better understanding of mosquito biology and are thus a key to finding new strategies to eliminate vector-borne diseases. Moreover, it allows for the creation of advanced transgenic strains for vector control programs. SSM circumvents the drawbacks of transposon-mediated transgenesis, where random transgene integration into the host genome results in insertional mutagenesis and variable position effects. We applied the Cre/lox recombinase-mediated cassette exchange (RMCE) system to Aedes aegypti, the vector of dengue, chikungunya, and Zika viruses. In this context we created four target site lines for RMCE and evaluated their fitness costs. Cre-RMCE is functional in a two-step mechanism and with good efficiency in Ae. aegypti. The advantages of Cre-RMCE over existing site-specific modification systems for Ae. aegypti, phiC31-RMCE and CRISPR, originate in the preservation of the recombination sites, which 1) allows successive modifications and rapid expansion or adaptation of existing systems by repeated targeting of the same site; and 2) provides reversibility, thus allowing the excision of undesired sequences. Thereby, Cre-RMCE complements existing genomic modification tools, adding flexibility and versatility to vector genome targeting.

Enhancing the stability and ecological safety of mass-reared transgenic strains for field release by redundant conditional lethality systems.

The genetic manipulation of agriculturally important insects now allows the development of genetic sexing and male sterility systems for more highly efficient biologically-based population control programs, most notably the Sterile Insect Technique (SIT), for both plant and animal insect pests. Tetracycline-suppressible (Tet-off) conditional lethal systems may function together so that transgenic strains will be viable and fertile on a tetracycline-containing diet, but female-lethal and male sterile in tetracycline-free conditions. This would allow their most efficacious use in a unified system for sterile male-only production for SIT. A critical consideration for the field release of such transgenic insect strains, however, is a determination of the frequency and genetic basis of lethality revertant survival. This will provide knowledge essential to evaluating the genetic stability of the lethality system, its environmental safety, and provide the basis for modifications ensuring optimal efficacy. For Tet-off lethal survival determinations, development of large-scale screening protocols should also allow the testing of these modifications, and test the ability of other conditional lethal systems to fully suppress propagation of rare Tet-off survivors. If a dominant temperature sensitive (DTS) pupal lethality system proves efficient for secondary lethality in Drosophila, it may provide the safeguard needed to support the release of sexing/sterility strains, and potentially, the release of unisex lethality strains as a form of genetic male sterility. Should the DTS Prosß2(1) mutation prove effective for redundant lethality, its high level of structural and functional conservation should allow host-specific cognates to be created for a wide range of insect species.

The role of the transformer gene in sex determination and reproduction in the tephritid fruit fly, Bactrocera dorsalis (Hendel).

Transformer (tra) is a switch gene in the somatic sex-determination hierarchy that regulates sexual dimorphism based on RNA splicing in many insects. In tephritids, a Y-linked male determining gene (M) controls sex in the sex-determination pathway. Here, homologues of Drosophila tra and transformer-2 (tra-2) genes were isolated and characterized in Bactrocera dorsalis (Hendel), one of the most destructive agricultural insect pests in many Asian countries. Two male-specific and one female-specific isoforms of B. dorsalis transformer (Bdtra) were identified. The presence of multiple TRA/TRA-2 binding sites in Bdtra suggests that the TRA/TRA-2 proteins are splicing regulators promoting and maintaining, epigenetically, female sex determination by a tra positive feedback loop in XX individuals during development. The expression patterns of female-specific Bdtra transcripts during early embryogenesis shows that a peak appears at 15 h after egg laying. Using dsRNA to knock-down Bdtra expression in the embryo and adult stages, we showed that sexual formation is determined early in the embryo stage and that parental RNAi does not lead to the production of all male progeny as in Tribolium castaneum. RNAi results from adult abdominal dsRNA injections show that Bdtra has a positive influence on female yolk protein gene (Bdyp1) expression and fecundity.

Pro-apoptotic gene regulation and its activation by gamma-irradiation in the Caribbean fruit fly, Anastrepha suspensa.

Transcriptional activation of pro-apoptotic genes in response to cytotoxic stimuli is a conserved feature of the cell death pathway in metazoans. However, understanding the extent of this conservation in insects has been limited by the lack of known pro-apoptotic genes in non-drosophilids. Recently, we described the pro-apoptotic genes, Asrpr and Ashid, from the tephritid, Anastrepha suspensa, that now allow us to explore the conservation of pro-apoptotic gene regulation between a tephritid and drosophilids. In this study, we determined the developmental profiles of Asrpr and Ashid transcripts during embryogenesis and in embryos exposed to γ-irradiation. Transcript levels of both genes determined by qRT-PCR were low throughout embryogenesis, with strong Ashid expression occurring during early to mid-embryogenesis and Asrpr expression peaking in late embryogenesis. This correlated to acridine orange stained apoptotic cells first appearing at 17 h and increasing over time. However, when irradiated at 16 h post-oviposition embryos exhibited significant levels of apoptosis consistent with strong induction of Asrpr and Ashid transcript levels by γ-irradiation in young embryos <24 h post-oviposition. Furthermore, embryos irradiated <24 h post-oviposition failed to hatch, those irradiated between 24 and 32 h had increased hatching rates, but between 48 and 72 h irradiation had no effect on egg hatching. This indicates a transition of embryos from an irradiation-sensitive to irradiation-resistance stage between 24 and 48 h. Throughout post-embryonic development, the two pro-apoptotic genes share similar patterns of up-regulated gene expression, which correlate to ecdysone-induced developmental events, especially during metamorphosis. Together these results provide the first direct evidence for a conserved molecular mechanism of the programmed cell death pathway in insects.

Male-specific Y-linked transgene markers to enhance biologically-based control of the Mexican fruit fly, Anastrepha ludens (Diptera: Tephritidae).

BACKGROUND: Reliable marking systems are critical to the prospective field release of transgenic insect strains. This is to unambiguously distinguish released insects from wild insects in the field that are collected in field traps, and tissue-specific markers, such as those that are sperm-specific, have particular uses such as identifying wild females that have mated with released males. For tephritid fruit flies such as the Mexican fruit fly, Anastrepha ludens, polyubiquitin-regulated fluorescent protein body markers allow transgenic fly identification, and fluorescent protein genes regulated by the spermatocyte-specific ß2-tubulin promoter effectively mark sperm. For sterile male release programs, both marking systems can be made male-specific by linkage to the Y chromosome. RESULTS: An A. ludens wild type strain was genetically transformed with a piggyBac vector, pBXL{PUbnlsEGFP, Asß2tub-DsRed.T3}, having the polyubiquitin-regulated EGFP body marker, and the ß2-tubulin-regulated DsRed.T3 sperm-specific marker. Autosomal insertion lines effectively expressed both markers, but a single Y-linked insertion (YEGFP strain) expressed only PUbnlsEGFP. This insertion was remobilized by transposase helper injection, which resulted in three new autosomal insertion lines that expressed both markers. This indicated that the original Y-linked Asß2tub-DsRed.T3 marker was functional, but specifically suppressed on the Y chromosome. The PUbnlsEGFP marker remained effective however, and the YEGFP strain was used to create a sexing strain by translocating the wild type allele of the black pupae (bp+) gene onto the Y, which was then introduced into the bp- mutant strain. This allows the mechanical separation of mutant female black pupae from male brown pupae, that can be identified as adults by EGFP fluorescence. CONCLUSIONS: A Y-linked insertion of the pBXL{PUbnlsEGFP, Asß2tub-DsRed.T3} transformation vector in A. ludens resulted in male-specific expression of the EGFP fluorescent protein marker, and was integrated into a black pupae translocation sexing strain (T(YEGFP/bp+), allowing the identification of male adults when used in sterile male release programs for population control. A unique observation was that expression of the Asß2tub-DsRed.T3 sperm-specific marker, which was functional in autosomal insertions, was specifically suppressed in the Y-linked insertion. This may relate to the Y chromosomal regulation of male-specific germ-line genes in Drosophila.

Fitness cost implications of PhiC31-mediated site-specific integrations in target-site strains of the Mexican fruit fly, Anastrepha ludens (Diptera: Tephritidae).

Site-specific recombination technologies are powerful new tools for the manipulation of genomic DNA in insects that can improve transgenesis strategies such as targeting transgene insertions, allowing transgene cassette exchange and DNA mobilization for transgene stabilization. However, understanding the fitness cost implications of these manipulations for transgenic strain applications is critical. In this study independent piggyBac-mediated attP target-sites marked with DsRed were created in several genomic positions in the Mexican fruit fly, Anastrepha ludens. Two of these strains, one having an autosomal (attP_F7) and the other a Y-linked (attP_2-M6y) integration, exhibited fitness parameters (dynamic demography and sexual competitiveness) similar to wild type flies. These strains were thus selected for targeted insertion using, for the first time in mexfly, the phiC31-integrase recombination system to insert an additional EGFP-marked transgene to determine its effect on host strain fitness. Fitness tests showed that the integration event in the int_2-M6y recombinant strain had no significant effect, while the int_F7 recombinant strain exhibited significantly lower fitness relative to the original attP_F7 target-site host strain. These results indicate that while targeted transgene integrations can be achieved without an additional fitness cost, at some genomic positions insertion of additional DNA into a previously integrated transgene can have a significant negative effect. Thus, for targeted transgene insertions fitness costs must be evaluated both previous to and subsequent to new site-specific insertions in the target-site strain.