The genome of the stable fly, Stomoxys calcitrans, reveals potential mechanisms underlying reproduction, host interactions, and novel targets for pest control.

BACKGROUND: The stable fly, Stomoxys calcitrans, is a major blood-feeding pest of livestock that has near worldwide distribution, causing an annual cost of over $2 billion for control and product loss in the USA alone. Control of these flies has been limited to increased sanitary management practices and insecticide application for suppressing larval stages. Few genetic and molecular resources are available to help in developing novel methods for controlling stable flies. RESULTS: This study examines stable fly biology by utilizing a combination of high-quality genome sequencing and RNA-Seq analyses targeting multiple developmental stages and tissues. In conjunction, 1600 genes were manually curated to characterize genetic features related to stable fly reproduction, vector host interactions, host-microbe dynamics, and putative targets for control. Most notable was characterization of genes associated with reproduction and identification of expanded gene families with functional associations to vision, chemosensation, immunity, and metabolic detoxification pathways. CONCLUSIONS: The combined sequencing, assembly, and curation of the male stable fly genome followed by RNA-Seq and downstream analyses provide insights necessary to understand the biology of this important pest. These resources and new data will provide the groundwork for expanding the tools available to control stable fly infestations. The close relationship of Stomoxys to other blood-feeding (horn flies and Glossina) and non-blood-feeding flies (house flies, medflies, Drosophila) will facilitate understanding of the evolutionary processes associated with development of blood feeding among the Cyclorrhapha.

Colony-level behavioural variation correlates with differences in expression of the foraging gene in red imported fire ants.

Among social insects, colony-level variation is likely to be widespread and has significant ecological consequences. Very few studies, however, have documented how genetic factors relate to behaviour at the colony level. Differences in expression of the foraging gene have been associated with differences in foraging and activity of a wide variety of organisms. We quantified expression of the red imported fire ant foraging gene (sifor) in workers from 21 colonies collected across the natural range of Texas fire ant populations, but maintained under standardized, environmentally controlled conditions. Colonies varied significantly in their behaviour. The most active colonies had up to 10 times more active foragers than the least active colony and more than 16 times as many workers outside the nest. Expression differences among colonies correlated with this colony-level behavioural variation. Colonies with higher sifor expression in foragers had, on average, significantly higher foraging activity, exploratory activity and recruitment to nectar than colonies with lower expression. Expression of sifor was also strongly correlated with worker task (foraging vs. working in the interior of the nest). These results provide insight into the genetic and physiological processes underlying collective differences in social behaviour. Quantifying variation in expression of the foraging gene may provide an important tool for understanding and predicting the ecological consequences of colony-level behavioural variation.

Effect of Quorum Sensing by Staphylococcus epidermidis on the Attraction Response of Female Adult Yellow Fever Mosquitoes, Aedes aegypti aegypti (Linnaeus) (Diptera: Culicidae), to a Blood-Feeding Source.

Aedes aegypti, the principal vector of yellow fever and dengue fever, is responsible for more than 30,000 deaths annually. Compounds such as carbon dioxide, amino acids, fatty acids and other volatile organic compounds (VOCs) have been widely studied for their role in attracting Ae. aegypti to hosts. Many VOCs from humans are produced by associated skin microbiota. Staphyloccocus epidermidis, although not the most abundant bacteria according to surveys of relative 16S ribosomal RNA abundance, commonly occurs on human skin. Bacteria demonstrate population level decision-making through quorum sensing. Many quorum sensing molecules, such as indole, volatilize and become part of the host odor plum. To date, no one has directly demonstrated the link between quorum sensing (i.e., decision-making) by bacteria associated with a host as a factor regulating arthropod vector attraction. This study examined this specific question with regards to S. epidermidis and Ae. aegypti. Pairwise tests were conducted to examine the response of female Ae. aegypti to combinations of tryptic soy broth (TSB) and S. epidermidis wildtype and agr- strains. The agr gene expresses an accessory gene regulator for quorum sensing; therefore, removing this gene inhibits quorum sensing of the bacteria. Differential attractiveness of mosquitoes to the wildtype and agr- strains was observed. Both wildtype and the agr- strain of S. epidermidis with TSB were marginally more attractive to Ae. aegypti than the TSB alone. Most interestingly, the blood-feeder treated with wildtype S. epidermidis/TSB attracted 74% of Ae. aegypti compared to the agr- strain of S. epidermidis/TSB (P ≤ 0.0001). This study is the first to suggest a role for interkingdom communication between host symbiotic bacteria and mosquitoes. This may have implications for mosquito decision-making with regards to host detection, location and acceptance. We speculate that mosquitoes "eavesdrop" on the chemical discussions occurring between host-associated microbes to determine suitability for blood feeding. We believe these data suggest that manipulating quorum sensing by bacteria could serve as a novel approach for reducing mosquito attraction to hosts, or possibly enhancing the trapping of adults at favored oviposition sites.

A cell-based screening platform identifies novel mosquitocidal toxins.

Pesticides currently in widespread use often lack species specificity and also become less effective as resistance emerges. Consequently, there is a pressing need to develop novel agents that are narrowly targeted and safe to humans. A cell-based screening platform was designed to discover compounds that are lethal to mosquito (Anopheles and Aedes) cells but show little or no activity against other insect (Drosophila) or human cell lines. Mosquito-specific, aqueous-stable cytotoxins were recovered at rare frequencies. Three of these were profiled for structure-activity relationships and also assessed in whole-animal toxicity assays. In at least one test case, species-specific cytotoxicity seen in culture effectively translated to the whole-animal level, with potent toxicity against Anopheles yet none against Drosophila. Therefore, this initiative has the potential to advance novel mosquitocidal agents and, in a broader sense, could establish a versatile platform for developing customized pesticides that selectively target other disease vectors as well.

Chimeric piggyBac transposases for genomic targeting in human cells.

Integrating vectors such as viruses and transposons insert transgenes semi-randomly and can potentially disrupt or deregulate genes. For these techniques to be of therapeutic value, a method for controlling the precise location of insertion is required. The piggyBac (PB) transposase is an efficient gene transfer vector active in a variety of cell types and proven to be amenable to modification. Here we present the design and validation of chimeric PB proteins fused to the Gal4 DNA binding domain with the ability to target transgenes to pre-determined sites. Upstream activating sequence (UAS) Gal4 recognition sites harbored on recipient plasmids were preferentially targeted by the chimeric Gal4-PB transposase in human cells. To analyze the ability of these PB fusion proteins to target chromosomal locations, UAS sites were randomly integrated throughout the genome using the Sleeping Beauty transposon. Both N- and C-terminal Gal4-PB fusion proteins but not native PB were capable of targeting transposition nearby these introduced sites. A genome-wide integration analysis revealed the ability of our fusion constructs to bias 24% of integrations near endogenous Gal4 recognition sequences. This work provides a powerful approach to enhance the properties of the PB system for applications such as genetic engineering and gene therapy.

Helper-independent piggyBac plasmids for gene delivery approaches: strategies for avoiding potential genotoxic effects.

Efficient integration of functional genes is an essential prerequisite for successful gene delivery such as cell transfection, animal transgenesis, and gene therapy. Gene delivery strategies based on viral vectors are currently the most efficient. However, limited cargo capacity, host immune response, and the risk of insertional mutagenesis are limiting factors and of concern. Recently, several groups have used transposon-based approaches to deliver genes to a variety of cells. The piggyBac (pB) transposase in particular has been shown to be well suited for cell transfection and gene therapy approaches because of its flexibility for molecular modification, large cargo capacity, and high transposition activity. However, safety considerations regarding transposase gene insertions into host genomes have rarely been addressed. Here we report our results on engineering helper-independent pB plasmids. The single-plasmid gene delivery system carries both the piggyBac transposase (pBt) expression cassette as well as the transposon cargo flanked by terminal repeat element sequences. Improvements to the helper-independent structure were achieved by developing new plasmids in which the pBt gene is rendered inactive after excision of the transposon from the plasmid. As a consequence, potentially negative effects that may develop by the persistence of an active pBt gene posttransposition are eliminated. The results presented herein demonstrate that our helper-independent plasmids represent an important step in the development of safe and efficient gene delivery methods that should prove valuable in gene therapy and transgenic approaches.

Genetic transformation of midgut bacteria from the red imported fire ant (Solenopsis invicta).

In our previous study we isolated 10 bacterial species from fourth-instar larval midguts of the red imported fire ant, Solenopsis invicta. Here we report the genetic transformation and reintroduction of three species (Kluyvera cryocrescens, Serratia marcescens, and isolate 38) into the fire ant host. All three species were transformed with the plasmid vector, pZeoDsRed. High expression levels of DsRed were observed and the plasmid is maintained in these bacteria at 37 degrees C in the absence of antibiotic selection for at least 9 days of subculturing. The transformed bacteria were successfully reintroduced into fire ant larvae and survived in the fire ant gut for at least 7 days. Upon pupal emergence, 7 days after reintroduction, transformed bacteria can still be isolated, however, most were passed out in the meconium. We further demonstrated that the engineered bacteria could be spread within the colony by feeding this meconium to naive larvae with the aid of worker fire ants.

Steps toward targeted insertional mutagenesis with class II transposable elements.

Insertional mutagenesis can be achieved by a variety of approaches, including both random and targeted methods. In contrast to chemical mutagenesis, insertional mutagens provide a molecular tag, thereby allowing rapid identification of the mutated genomic region. Integration into defined genomic locations has great utility for both gene insertion and mutagenesis. Our laboratories have explored targeted integration through the use of transposases coupled to defined DNA-binding domains. This technology holds great promise for targeted insertional mutagenesis by biasing integration events to regions recognized by the chosen DNA-binding domain. Herein, we provide a brief background on targeted transposon integration and detailed protocols for testing chimeric transposases in both mammalian cell culture and insect embryos.

Active integration: new strategies for transgenesis.

This paper presents novel methods for producing transgenic animals, with a further emphasis on how these techniques may someday be applied in gene therapy. There are several passive methods for transgenesis, such as pronuclear microinjection (PNI) and Intracytoplasmic Sperm Injection-Mediated Transgenesis (ICSI-Tr), which rely on the repair mechanisms of the host for transgene (tg) insertion. ICSI-Tr has been shown to be an effective means of creating transgenic animals with a transfection efficiency of approximately 45% of animals born. Furthermore, because this involves the injection of the transgene into the cytoplasm of oocytes during fertilization, limited mosaicism has traditionally occurred using this technique. Current active transgenesis techniques involve the use of viruses, such as disarmed retroviruses which can insert genes into the host genome. However, these methods are limited by the size of the sequence that can be inserted, high embryo mortality, and randomness of insertion. A novel active method has been developed which combines ICSI-Tr with recombinases or transposases to increase transfection efficiency. This technique has been termed "Active Transgenesis" to imply that the tg is inserted into the host genome by enzymes supplied into the oocyte during tg introduction. DNA based methods alleviate many of the costs and time associated with purifying enzyme. Further studies have shown that RNA can be used for the transposase source. Using RNA may prevent problems with continued transposase activity that can occur if a DNA transposase is integrated into the host genome. At present piggyBac is the most effective transposon for stable integration in mammalian systems and as further studies are done to elucidate modifications which improve piggyBac's specificity and efficacy, efficiency in creating transgenic animals should improve further. Subsequently, these methods may someday be used for gene therapy in humans.

PiggyBac transposon-mediated gene transfer in human cells.

Transposons are mobile genetic elements that can be used to integrate transgenes into host cell genomes. The piggyBac transposon system has been used for transgenesis of insects and for germline mutagenesis in mice. We compared transposition activity of piggyBac with Sleeping Beauty (SB), a widely used transposon system for preclinical gene therapy studies. An engineered piggyBac transposon with minimal length 5' and 3' terminal repeats exhibited greater transposition activity in transfected cultured human cells than a well-characterized hyperactive SB system. PiggyBac excision was very precise as evidenced by the typical absence of "footprint" mutations at the site of transposon excision. We mapped 575 piggyBac integration sites in human cells to determine site selectivity of genomic integration. PiggyBac demonstrated non-random integration site selectivity that differed from that previously reported for SB, including a higher preference for integrations in regions surrounding transcriptional start sites and within long terminal repeat elements. Importantly, overproduction inhibition was not observed with piggyBac, a major limitation of the SB system. This permitted the generation of combination "helper-independent" piggyBac transposase-transposon vectors that exhibited a 2-fold increase of transposition activity in human cells as compared with cells transfected with separate transposon and transposase plasmids. We conclude that piggyBac is a transposon system with certain properties, including high efficiency and lack of overproduction inhibition that are advantageous in preclinical development of transposon-based gene therapy.

piggyBac is a flexible and highly active transposon as compared to sleeping beauty, Tol2, and Mos1 in mammalian cells.

A nonviral vector for highly efficient site-specific integration would be desirable for many applications in transgenesis, including gene therapy. In this study we directly compared the genomic integration efficiencies of piggyBac, hyperactive Sleeping Beauty (SB11), Tol2, and Mos1 in four mammalian cell lines. piggyBac demonstrated significantly higher transposition activity in all cell lines whereas Mos1 had no activity. Furthermore, piggyBac transposase coupled to the GAL4 DNA-binding domain retains transposition activity whereas similarly manipulated gene products of Tol2 and SB11 were inactive. The high transposition activity of piggyBac and the flexibility for molecular modification of its transposase suggest the possibility of using it routinely for mammalian transgenesis.

Mutant Mos1 mariner transposons are hyperactive in Aedes aegypti.

The development of genetic strategies to control the spread of mosquito-borne diseases through the use of class II transposons has been hampered by suboptimal rates of transformation and the absence of post-integration mobility for all transposons evaluated to date. Two Mos1 mariner transposase mutants were produced by the site-directed mutagenesis of amino acids, E137 and E264, to K and R, respectively. The effects of these mutations on the transpositional activities of Mos1-derived transposon constructs were evaluated by interplasmid transposition assays in Escherichia coli and Aedes aegypti. The transpositional activities of two Mos1 transposons, one with imperfect wild type inverted terminal repeats (ITRs) and another that contained two perfectly matched 3' ITRs, were increased when the mutant transposases were supplied in trans in E. coli. The use of the perfect repeat transposon with wild type transposase did not result in an increase in transposition frequency in Ae. aegypti. However, an improvement in the integrity of the transposition process did occur, as evidenced by a lower rate of recombination events in which the transgene was transferred. An increase in the transpositional activity of the perfect repeat transposon was observed in the mosquito in the presence of either mutant transposase, and in the case of the E264R transposase, the observed increase in transposition frequency was also accompanied by a further improvement in the integrity of transposition. We discuss the possible contributions of these mutant residues to the transposition of the perfect repeat Mos1 transposon, the implications of these results with respect to the molecular evolution of Mos1, and the potential uses of the perfect repeat transposon and mutant transposases for the improvement of Mos1 mediated germ line transformation of Ae. aegypti.

Isolation, characterization, and molecular identification of bacteria from the red imported fire ant (Solenopsis invicta) midgut.

Bacteria were isolated and cultured from the red imported fire ant (Solenopsis invicta) midgut. The small-subunit ribosomal RNA gene, (16s rRNA gene, approximately 1500 bp) was amplified from bacterial genomic DNA using the polymerase chain reaction and consensus sequence primers. Restriction fragment length polymorphism analysis revealed 10 unique profiles, indicating that at least 10 different bacteria are present in red imported fire ant midguts. The 16s rRNA gene sequence was determined for these isolates and queried against the NCBI genetic database. The results identified all isolates to at least the genus level. Antibiotic resistance profiles and biochemical activities were also determined for these species. This work provides the basis for a wider characterization of bacterial distributions in fire ant colonies and provides strains suitable for genetic manipulation to develop novel methods of fire ant control.

Site-directed genome modification: derivatives of DNA-modifying enzymes as targeting tools.

The modification of mammalian genomes is an important goal in gene therapy and animal transgenesis. To generate stable genetic and biochemical changes, the therapeutic genes or transgenes need to be incorporated into the host genome. Ideally, the integration of the foreign gene should occur at sites that ensure their continual expression in the absence of any unwanted side effects on cellular metabolism. In this article, we discuss the opportunities provided by natural DNA-modifying enzymes, such as transposases, recombinases and integrases, to mediate the stable integration of foreign genes into host genomes. In addition, we discuss the approaches that have been taken to improve the efficiency and to modify the site-specificity of these enzymes.

Site-directed genome modification: nucleic acid and protein modules for targeted integration and gene correction.

A variety of technological advances in recent years have made permanent genetic manipulation of an organism a technical possibility. As the details of natural biological processes for genome modification are elucidated, the enzymes catalyzing these events (transposases, recombinases, integrases and DNA repair enzymes) are being harnessed or modified for the purpose of intentional gene modification. Targeted integration and gene repair can be mediated by the DNA-targeting specificity inherent to a particular enzyme, or rely on user-designed specificities. Integration sites can be defined by using DNA base-pairing or protein-DNA interaction as a means of targeting. This review will describe recent progress in the development of 'user-targetable' systems, particularly highlighting the application of custom DNA-binding proteins or nucleic acid homology to confer specificity.

Cloning and characterization of cDNAs encoding putative CTCFs in the mosquitoes, Aedes aegypti and Anopheles gambiae.

BACKGROUND: One of the many ascribed functions of CCCTC-binding factor (CTCF) in vertebrates is insulation of genes via enhancer-blocking. Insulation allows genes to be shielded from "cross-talk" with neighboring regulatory elements. As such, endogenous insulator sequences would be valuable elements to enable stable transgene expression. Recently, CTCF joined Su(Hw), Zw5, BEAF32 and GAGA factor as a protein associated with insulator activity in the fruitfly, Drosophila melanogaster. To date, no known insulators have been described in mosquitoes. RESULTS: We have identified and characterized putative CTCF homologs in the medically-important mosquitoes, Aedes aegypti and Anopheles gambiae. These genes encode polypeptides with eleven C2H2 zinc fingers that show significant similarity to those of vertebrate CTCFs, despite at least 500 million years of divergence. The mosquito CTCFs are constitutively expressed and are upregulated in early embryos and in the ovaries of blood-fed females. We have uncovered significant bioinformatics evidence that CTCF is widespread, at least among Drosophila species. Finally, we show that the An. gambiae CTCF binds two known insulator sequences. CONCLUSION: Mosquito CTCFs are likely orthologous to the widely-characterized vertebrate CTCFs and potentially also serve an insulating function. As such, CTCF may provide a powerful tool for improving transgene expression in these mosquitoes through the identification of endogenous binding sites.

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.

Detection of differences in oligonucleotide-influenced aggregation of colloidal gold nanoparticles using absorption spectroscopy.

A rapid, simple, and reproducible assay is described that can be used to detect differences in the ability of oligonucleotides to influence the aggregation of colloidal gold nanoparticles. The aggregation reaction of the gold colloid was monitored through UV-visible absorption spectroscopy. Single isolated colloidal gold particles have a surface plasmon resonance manifested as a single absorbance peak at approximately 520 nm, and aggregated gold complexes develop new red-shifted peaks/shoulders depending on the nature and extent of the aggregated complex. A simple ratiometric study of the area under the single and aggregated plasmon resonance peaks thus gives information about the extent of the aggregation. It is postulated that differences in dynamic flexibility of the oligonucleotides affect their influence on the aggregation state of the gold nanoparticles. The results of this study provide new clues toward unraveling the causes behind the preferential affinity of the Hermes transposable element for certain insertion sites compared to other sequences that also contain recognizable target sites. The technique is robust and thus can potentially be used to study similar questions for numerous transposable elements and target sequences.

Promoter and piggyBac activities within embryos of the potato tuber moth, Phthorimaea operculella, Zeller (Lepidoptera: Gelechiidae).

Potato production in tropical and subtropical countries suffers from damage caused by the potato tuber moth (PTM), Phthorimaea operculella. The aim of this research was the development of the components required for a germline transformation system for the PTM. We tested three components that are critical to genetic transformation systems for insects: promoter activity, marker gene expression, and transposable element function. We compared the transcriptional activities of five different promoters, hsp70, hsp82, actin5C, polyubiquitin and immediate early 1 gene (ie1), within PTM embryos. The ie1 promoter, flanked by the hr5 enhancer element, showed a very high level of transcriptional activity compared to the other promoters. The fluorescence activity of EGFP was also determined and transient expression of EGFP was detected in 57% of injected embryos. The transpositional activity of the piggyBac transposable element was tested in an interplasmid transposition assay. The piggyBac element was shown to be mobile within the embryonic soma of the PTM with a transposition frequency of 4.2 x 10(-5) transpositions/donor plasmid. Incorporating a transactivator plasmid expressing the immediate early protein (IE1) from the Bombyx mori nuclear polyhedrosis virus enhanced the efficiency of piggyBac mobility.