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ABSTRACT: The gene nanos (nos) is a maternal-effect gene that plays an important role in posterior patterning and germ cell development in early stage embryos. nos is known from several diverse insect species, but has so far not been described for any Tephritid fruit fly. Here, we report the molecular cloning and expression pattern of the nos orthologous gene, Ccnos, in the Mediterranean fruit fly Ceratitis capitata, which is a destructive pest of high agricultural importance. CcNOS contains 398 amino acids and has a C-terminal region with two conserved CCHC zinc-binding motifs known to be essential for NOS function. Transcripts of Ccnos were confirmed by in situ hybridization to be maternally-derived and localized to the posterior pole of early stage embryos. Regulatory regions of nos have been employed in genetic engineering in some dipterans such as Drosophila and mosquitoes. Given the similarity in spatial and temporal expression between Ccnos and nos orthologs from other dipterans, its regulatory regions will be valuable to generate additional genetic tools that can be applied for engineering purposes to improve the fight against this devastating pest.
Gene Expression Patterns 04/2013; · 2.02 Impact Factor
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Christian Schmitt,
Peter Atkinson,
Erica Sodergren,
Qingsong Zhu,
Jennifer Hume,
Shalini N Jhangiani,
Mimi N Chandrabose,
Jim Beidler,
Michael Schoppmeier,
Selina Vattahil, [......],
Jeremy A Lynch,
Peer Bork,
Rodrigo Nunes da Fonseca,
Manuel Aranda,
Nico Posnien,
Riyue Bao,
Rolf Reuter,
Anke Beermann,
Nicola Berns,
Johannes B Schinko
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ABSTRACT: Fruit fly pest species have been successfully controlled and managed via the Sterile Insect Technique (SIT), a control strategy that uses infertile matings of sterile males to wild females to reduce pest populations. Biological efficiency in the field is higher if only sterile males are released in SIT programs and production costs are also reduced. Sexing strains developed in the Mediterranean fruit fly Ceratitis capitata (medfly) through classical genetics are immensely beneficial to medfly SIT programs but exhibit reduced fertility and fitness. Moreover, transfer of such classical genetic systems to other tephritid species is difficult. Transgenic approaches can overcome this limitation of classical genetic sexing strains (GSSs), but had resulted so far in transgenic sexing strains (TSSs) with dominant lethality at late larval and pupal stages. Here we present a transgene-based female-specific lethality system for early embryonic sexing in medfly. The system utilizes the sex-specifically spliced transformer intron to restrict ectopic mRNA translation of the pro-apoptotic gene hid(Ala5) to females only. The expression of this lethal effector gene is driven by a tetracycline-repressible transactivator gene tTA that is under the control of promoters/enhancers of early acting cellularization genes. Despite observed position effects on the sex-specific splicing, we could effectively establish this early acting transgenic sexing system in the medfly C. capitata. After satisfactory performance in large-scale tests, TSSs based on this system will offer cost-effective sexing once introduced into SIT programs. Moreover, this approach is straight forward to be developed also for other insect pest and vector species.
Insect biochemistry and molecular biology 11/2012; · 3.25 Impact Factor
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ABSTRACT: Evolution often results in morphologically similar solutions in different organisms, a phenomenon known as convergence. However, there is little knowledge of the processes that lead to convergence at the genetic level. The genes of the Hox cluster control morphology in animals. They may also be central to the convergence of morphological traits, but whether morphological similarities also require similar changes in Hox gene function is disputed. In arthropods, body subdivision into a region with locomotory appendages ("thorax") and a region with reduced appendages ("abdomen") has evolved convergently in several groups, e.g., spiders and insects. In insects, legs develop in the expression domain of the Hox gene Antennapedia (Antp), whereas the Hox genes Ultrabithorax (Ubx) and abdominal-A mediate leg repression in the abdomen. Here, we show that, unlike Antp in insects, the Antp gene in the spider Achaearanea tepidariorum represses legs in the first segment of the abdomen (opisthosoma), and that Antp and Ubx are redundant in the following segment. The down-regulation of Antp in A. tepidariorum leads to a striking 10-legged phenotype. We present evidence from ectopic expression of the spider Antp gene in Drosophila embryos and imaginal tissue that this unique function of Antp is not due to changes in the Antp protein, but likely due to divergent evolution of cofactors, Hox collaborators or target genes in spiders and flies. Our results illustrate an interesting example of convergent evolution of abdominal leg repression in arthropods by altering the role of distinct Hox genes at different levels of their action.
Proceedings of the National Academy of Sciences 03/2012; 109(13):4921-6. · 9.68 Impact Factor
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ABSTRACT: In Drosophila, trunk metamerization is established by a cascade of segmentation gene activities: the gap genes, the pair rule genes, and the segment polarity genes. In the anterior head, metamerization requires also gap-like genes and segment polarity genes. However, because the pair rule genes are not active in this part of the embryo, the question on which gene activities are fulfilling the role of the second order regulator genes still remains to be solved. Here we provide first molecular evidence that the Helix-Loop-Helix-COE transcription factor Collier fulfills this role by directly activating the expression of the segment polarity gene hedgehog in the posterior part of the intercalary segment. Collier thereby occupies a newly identified binding site within an intercalary-specific cis-regulatory element. Moreover, we identified a direct physical association between Collier and the basic-leucine-zipper transcription factor Cap'n'collar B, which seems to restrict the activating input of Collier to the posterior part of the intercalary segment and to lead to the attenuation of hedgehog expression in the intercalary lobes at later stages.
Developmental Biology 12/2011; 360(2):403-14. · 4.07 Impact Factor
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ABSTRACT: Anterior head segmentation is governed by different regulatory mechanisms than those that control trunk segmentation in Drosophila. For segment polarity genes, both initial mode of activation as well as cross-regulatory interactions among them differ from the typical genetic circuitry in the trunk and are unique for each of the procephalic segments. In order to better understand the segment-specific gene network responsible for the procephalic expression of the earliest active segment polarity genes wingless and hedgehog, we started to identify and analyze cis-regulatory DNA elements of these genes. For hedgehog, we could identify a cis-regulatory element, ic-CRE, that mediates expression specifically in the posterior part of the intercalary segment and requires promoter-specific interaction for its function. The intercalary stripe is the last part of the metameric hedgehog expression pattern that appears during embryonic development, which probably reflects the late and distinct establishment of this segment. The identification of a cis-regulatory element that is specific for one head segment supports the mutant-based observation that the expression of segment polarity genes is governed by a unique gene network in each of the procephalic segments. This provides further indication that the anterior-most head segments represent primary segments, which are set up independently, in contrast to the secondary segments of the trunk, which resemble true repetitive units.
Archiv für Entwickelungsmechanik der Organismen 03/2011; 221(1):1-16. · 1.77 Impact Factor
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ABSTRACT: Transposon-based vectors currently provide the most suitable gene transfer systems for insect germ-line transformation and are used for molecular improvement of the Sterile Insect Technique. However, the long time stability of genome-integrated transposon constructs depends on the absence of transposase activity that could remobilize the transposon-embedded transgenes. To achieve transgene stability transposon vectors are usually non-autonomous, lacking a functional transposase gene, and chosen so that endogenous or related transposon activities are not present in the host. Nevertheless, the non-autonomous transposon-embedded transgenes could become unstable by the unintended presence of a mobilizing transposase that may have been undetected or subsequently entered the host species by horizontal gene transfer. Since the field release of transgenic insects will present environmental concerns relating to large populations and high mobility, it will be important to ensure that transgene constructs are stably integrated for maintaining strain integrity and eliminating the possibility for unintentional transfer into the genome of another organism. Here we review efficient methods to delete or rearrange terminal repeat sequences of transposons necessary for their mobility, subsequent to their initial genomic integration. These procedures should prevent transposase-mediated remobilization of the transgenes, ensuring their genomic stability.
Genetica 01/2011; 139(1):71-8. · 2.15 Impact Factor
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ABSTRACT: Non-traditional model systems need new tools that will enable them to enter the field of functional genetics. These tools should enable the exploration of gene function, via knock-downs of endogenous genes, as well as over-expression and ectopic expression of transgenes.
We constructed a new vector called Pogostick that can be used to over-express or down-regulate genes in organisms amenable to germ line transformation by the piggyBac transposable element. Pogostick can be found at www.addgene.org, a non-profit plasmid repository. The vector currently uses the heat-shock promoter Hsp70 from Drosophila to drive transgene expression and, as such, will have immediate applicability to organisms that can correctly interpret this promotor sequence. We detail how to clone candidate genes into this vector and test its functionality in Drosophila by targeting a gene coding for the fluorescent protein DsRed. By cloning a single DsRed copy into the vector, and generating transgenic lines, we show that DsRed mRNA and protein levels are elevated following heat-shock. When cloning a second copy of DsRed in reverse orientation into a flanking site, and transforming flies constitutively expressing DsRed in the eyes, we show that endogenous mRNA and protein levels drop following heat-shock. We then test the over-expression vector, containing the complete cDNA of Ultrabithorax (Ubx) gene, in an emerging model system, Bicyclus anynana. We produce a transgenic line and show that levels of Ubx mRNA expression rise significantly following a heat-shock. Finally, we show how to obtain genomic sequence adjacent to the Pogostick insertion site and to estimate transgene copy number in genomes of transformed individuals.
This new vector will allow emerging model systems to enter the field of functional genetics with few hurdles.
PLoS ONE 01/2011; 6(4):e18659. · 4.09 Impact Factor
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ABSTRACT: The establishment of broadly applicable insect transgenesis systems will enable the analyses of gene function in diverse insect
species. This will greatly increase our understanding of diverse aspects of biology so far not functionally addressable. Moreover,
insect transgenesis will provide novel strategies for insect pest management and the means to impair transmission of pathogens
by human disease vectors. Especially the Sterile Insect Technique (SIT) might be improved by the use of transgenic approaches.
The SIT represents an effective and ecologically safe method for area-wide pest control that reduces the pest population by
mass release of sterilized organisms, leading to infertile matings and in consequence to a decline of the pest population.
Although the SIT is already successfully applied for some species, each of its steps – mass-rearing, sex-separation for male-only
releases, sterilization, and marking for monitoring – can be improved biotechnologically to optimize the efficiency and to
reduce the costs of ongoing programs or to transfer this effective technique to a wider range of species. However, this powerful
transgenic technology must be applied with great care to avoid harm to our environment.
KeywordsTransposable elements-Pest management-GMO safety
12/2010: pages 169-194;
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ABSTRACT: The insect intercalary segment represents a small and appendage-less head segment that is homologous to the second antennal segment of Crustacea and the pedipalpal segment in Chelicerata, which are generally referred to as "tritocerebral segment." In Drosophila, the gene collier (col) has an important role for the formation of the intercalary segment. Here we show that in the beetle Tribolium castaneum col is required for the activation of the segment polarity genes hedgehog (hh), engrailed (en) and wingless (wg) in the intercalary segment, and is a regulatory target of the intercalary segment specific Hox gene labial (lab). Loss of Tc col function leads to increased cell death in the intercalary segment. In the milkweed bug Oncopeltus fasciatus, the loss of col function has a more severe effect in lacking the intercalary segment and also affecting the adjacent mandibular and antennal segments. By contrast, col is not expressed early in the second antennal segment in the crustacean Parhyale hawaiensis or in the pedipalpal segment of the spider Achaearanea tepidariorum. This suggests that the early expression of col in a stripe and its role in tritocerebral segment development is insect-specific and might correlate with the appendage-less morphology of the intercalary segment.
Developmental Biology 05/2010; 344(1):363-76. · 4.07 Impact Factor
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ABSTRACT: The Sp-family of transcription factors are evolutionarily conserved zinc finger proteins present in many animal species. The orthology of the Sp genes in different animals is unclear and their evolutionary history is therefore controversially discussed. This is especially the case for the Sp gene buttonhead (btd) which plays a key role in head development in Drosophila melanogaster, and has been proposed to have originated by a recent gene duplication. The purpose of the presented study was to trace orthologs of btd in other insects and reconstruct the evolutionary history of the Sp genes within the metazoa.
We isolated Sp genes from representatives of a holometabolous insect (Tribolium castaneum), a hemimetabolous insect (Oncopeltus fasciatus), primitively wingless hexapods (Folsomia candida and Thermobia domestica), and an amphipod crustacean (Parhyale hawaienis). We supplemented this data set with data from fully sequenced animal genomes. We performed phylogenetic sequence analysis with the result that all Sp factors fall into three monophyletic clades. These clades are also supported by protein domain structure, gene expression, and chromosomal location. We show that clear orthologs of the D. melanogaster btd gene are present even in the basal insects, and that the Sp5-related genes in the genome sequence of several deuterostomes and the basal metazoans Trichoplax adhaerens and Nematostella vectensis are also orthologs of btd.
All available data provide strong evidence for an ancestral cluster of three Sp-family genes as well as synteny of this Sp cluster and the Hox cluster. The ancestral Sp gene cluster already contained a Sp5/btd ortholog, which strongly suggests that btd is not the result of a recent gene duplication, but directly traces back to an ancestral gene already present in the metazoan ancestor.
BMC Evolutionary Biology 03/2010; 10:88. · 3.52 Impact Factor
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ABSTRACT: The red flour beetle Tribolium castaneum has developed into an insect model system second only to Drosophila. Moreover, as a coleopteran it represents the most species-rich metazoan taxon which also includes many pest species. The genetic toolbox for Tribolium research has expanded in the past years but spatio-temporally controlled misexpression of genes has not been possible so far.
Here we report the establishment of the GAL4/UAS binary expression system in Tribolium castaneum. Both GAL4 Delta and GAL4VP16 driven by the endogenous heat shock inducible promoter of the Tribolium hsp68 gene are efficient in activating reporter gene expression under the control of the Upstream Activating Sequence (UAS). UAS driven ubiquitous tGFP fluorescence was observed in embryos within four hours after activation while in-situ hybridization against tGFP revealed expression already after two hours. The response is quick in relation to the duration of embryonic development in Tribolium - 72 hours with segmentation being completed after 24 hours - which makes the study of early embryonic processes possible using this system. By comparing the efficiency of constructs based on Tribolium, Drosophila, and artificial core promoters, respectively, we find that the use of endogenous core promoters is essential for high-level expression of transgenic constructs.
With the established GAL4/UAS binary expression system, ectopic misexpression approaches are now feasible in Tribolium. Our results support the contention that high-level transgene expression usually requires endogenous regulatory sequences, including endogenous core promoters in Tribolium and probably also other model systems.
BMC Developmental Biology 01/2010; 10:53. · 2.79 Impact Factor
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ABSTRACT: Insect transgenesis is mainly based on the random genomic integration of DNA fragments embedded into non-autonomous transposable elements. Once a random insertion into a specific location of the genome has been identified as particularly useful with respect to transgene expression, the ability to make the insertion homozygous, and lack of fitness costs, it may be advantageous to use that location for further modification. Here we describe an efficient method for the modification of previously inserted transgenes by the use of the site-specific integration system from phage phiC31 in a tephritid pest species, the Mediterranean fruit fly Ceratitis capitata. First, suitable transgenic strains with randomly integrated attP landing sites within transposon-based vectors were identified by molecular and functional characterization. Second, donor plasmids containing an attB site, with additional markers, and transposon ends were integrated into attP sites by phiC31 integrase-mediated recombination. Third, transposase-encoding 'jumpstarter' strains were created and mated to transgenic strains resulting in the postintegrational excision of transposon ends, which left stably integrated transgene insertions that could not be remobilized. This three-step integration and stabilization system will allow the combination of several transgene-encoded advantageous traits at evaluated genomic positions to generate optimized strains for pest control that minimize environmental concerns.
Proceedings of the National Academy of Sciences 10/2009; 106(43):18171-6. · 9.68 Impact Factor
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ABSTRACT: INTRODUCTION Tribolium castaneum is a small, low-maintenance beetle that has emerged as a sophisticated model system for studying the evolution of development and that complements (in some cases, even rivals) Drosophila for functional genetic analysis of basic biological questions. Although Tribolium and Drosophila are both holometabolous insects, they differ fundamentally in larval and adult morphology. Even generally conserved developmental features, such as body segmentation, are achieved by quite different means. Thus, comparison of developmental mechanisms between these two insects can address many interesting questions concerning the evolution of morphology and other characters. Genetic tools available for Tribolium include genetic maps for visible and molecular markers, chromosomal rearrangements that enable lethal mutations to be balanced in true-breeding stocks, transposon-based transformation systems, a completed and annotated genome sequence, and systemic RNA interference (RNAi), which makes it possible to knock down any given gene and even particular splice variants in the offspring or in any tissue of the injected animal. Inactivating gene functions at various developmental stages provides new opportunities to investigate post-embryonic development, as well as larval and adult physiology, including hormonal control, host-parasite interactions, and pesticide resistance.
Cold Spring Harbor Protocols 08/2009; 2009(8):pdb.emo126. · 4.63 Impact Factor
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ABSTRACT: The head gap genes orthodenticle (otd), empty spiracles (ems) and buttonhead (btd) are required for metamerization and segment specification in Drosophila. We asked whether the function of their orthologs is conserved in the red flour beetle Tribolium castaneum which in contrast to Drosophila develops its larval head in a way typical for insects. We find that depending on dsRNA injection time, two functions of Tc-orthodenticle1 (Tc-otd1) can be identified. The early regionalization function affects all segments formed during the blastoderm stage while the later head patterning function is similar to Drosophila. In contrast, both expression and function of Tc-empty spiracles (Tc-ems) are restricted to the posterior part of the ocular and the anterior part of the antennal segment and Tc-buttonhead (Tc-btd) is not required for head cuticle formation at all. We conclude that the gap gene like roles of ems and btd are not conserved while at least the head patterning function of otd appears to be similar in fly and beetle. Hence, the ancestral mode of insect head segmentation remains to be discovered. With this work, we establish Tribolium as a model system for arthropod head development that does not suffer from the Drosophila specific problems like head involution and strongly reduced head structures.
Developmental Biology 06/2008; 317(2):600-13. · 4.07 Impact Factor
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Stephen Richards,
Richard A Gibbs,
George M Weinstock,
Susan J Brown,
Robin Denell,
Richard W Beeman,
Richard Gibbs,
Gregor Bucher,
Markus Friedrich,
Cornelis J P Grimmelikhuijzen, [......],
Joachim Schachtner,
Peter Verleyen,
Florian Raible,
Kimberly K O Walden,
Sergio Angeli,
Sylvain Forêt,
Stefan Schuetz,
Ryszard Maleszka,
Sherry C Miller,
Daniela Grossmann
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ABSTRACT: Tribolium castaneum is a member of the most species-rich eukaryotic order, a powerful model organism for the study of generalized insect development, and an important pest of stored agricultural products. We describe its genome sequence here. This omnivorous beetle has evolved the ability to interact with a diverse chemical environment, as shown by large expansions in odorant and gustatory receptors, as well as P450 and other detoxification enzymes. Development in Tribolium is more representative of other insects than is Drosophila, a fact reflected in gene content and function. For example, Tribolium has retained more ancestral genes involved in cell-cell communication than Drosophila, some being expressed in the growth zone crucial for axial elongation in short-germ development. Systemic RNA interference in T. castaneum functions differently from that in Caenorhabditis elegans, but nevertheless offers similar power for the elucidation of gene function and identification of targets for selective insect control.
Nature 05/2008; 452(7190):949-55. · 36.28 Impact Factor
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Stephen Richards,
Richard A. Gibbs,
George M. Weinstock,
Susan J. Brown,
Robin Denell,
Richard W. Beeman,
Richard Gibbs,
Gregor Bucher,
Markus Friedrich,
Cornelis J. P. Grimmelikhuijzen, [......],
Joachim Schachtner,
Peter Verleyen,
Florian Raible,
Kimberly K. O. Walden,
Sergio Angeli,
Sylvain For|[ecirc]|t,
Stefan Schuetz,
Ryszard Maleszka,
Sherry C. Miller,
Daniela Grossmann
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ABSTRACT: Tribolium castaneum is a member of the most species-rich eukaryotic order, a powerful model organism for the study of generalized insect development, and an important pest of stored agricultural products. We describe its genome sequence here. This omnivorous beetle has evolved the ability to interact with a diverse chemical environment, as shown by large expansions in odorant and gustatory receptors, as well as P450 and other detoxification enzymes. Development in Tribolium is more representative of other insects than is Drosophila, a fact reflected in gene content and function. For example, Tribolium has retained more ancestral genes involved in cell–cell communication than Drosophila, some being expressed in the growth zone crucial for axial elongation in short-germ development. Systemic RNA interference in T. castaneum functions differently from that in Caenorhabditis elegans, but nevertheless offers similar power for the elucidation of gene function and identification of targets for selective insect control.
Nature 03/2008; 452(7190):949-955. · 36.28 Impact Factor
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ABSTRACT: The Sterile Insect Technique (SIT) involving area-wide release of mass-reared and sterilized pest insects has proven successful to reduce, control and eradicate economically important pest species, such as the Mediterranean fruit fly (medfly). For the efficient application, effective monitoring to assess the number and mating success of the released medflies is essential. Here, we report sperm-specific marking systems based on the spermatogenesis-specific Ceratitis capitata beta2-tubulin (Ccbeta2t) promoter. Fluorescent sperm can be isolated from testes or spermathecae. The marking does not cause general disadvantages in preliminary laboratory competitiveness assays. Therefore, transgenic sperm marking could serve as a major improvement for monitoring medfly SIT programs. The use of such harmless transgenic markers will serve as an ideal initial condition to transfer insect transgenesis technology from the laboratory to field applications. Moreover, effective and easily recognizable sperm marking will make novel studies possible on medfly reproductive biology which will help to further improve SIT programs.
New Biotechnology 02/2008; 25(1):76-84. · 2.76 Impact Factor
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ABSTRACT: Binary expression systems are of key interest to functional gene analysis by over- or misexpression. The application of such systems in diverse organisms would allow the study of many biological problems not addressable in model organisms. Here we report a set of constructs and an effective kinetic approach to quantitatively compare a series of diverse binary expression systems based on GAL4/UAS, LexA/(LL)(4) and tetracycline-controlled tTA/TRE. By the use of these constructs, we could show that in Drosophila melanogaster the yeast-derived GAL4/UAS systems are more effective in activating responder gene expression than the bacterial-derived LexA/(LL)(4) and tTA/TRE systems. The constructs are embedded in broad-range piggyBac-based transposon vectors and the transactivators are driven by the widely applicable 3xP3 promoter. These constructs should therefore be transferable to evaluate the functionality of binary expression systems in non-model insect species.
Insect Biochemistry and Molecular Biology 04/2007; 37(3):246-54. · 3.25 Impact Factor
