giuliano gasperi

Abstract: BACKGROUND: In embryos the maternal-to-zygotic transition (MTZ) integrates post-transcriptional regulation of maternal transcripts with transcriptional activation of the zygotic genome. Although the molecular mechanisms underlying this event are being clarified in Drosophila melanogaster, little is know about the embryogenic processes in other insect species. The recent publication of expressed sequence tags (ESTs) from embryos of the global pest species Ceratitis capitata (medfly) has enabled the investigation of embryogenesis in this species and has allowed a comparison of the embryogenic processes in these two related dipteran species, C. capitata and D. melanogaster, that shared a common ancestor 80-100 mya. RESULTS: Using a novel PCR-based sexing method, which takes advantage of a putative LTR retrotransposon MITE insertion on the medfly Y chromosome, the transcriptomes of individual early male and female embryos were analysed using RT-PCR. This study is focused on two crucial aspects of the onset of embryonic development: sex determination and cellular blastoderm formation. Together with the three known medfly genes (Cctransformer, Cctransformer2 and Ccdoublesex), the expression patterns of other medfly genes that are similar to the D. melanogaster sex-determination genes (sisterlessA, groucho, deadpan, Sex-lethal, female lethal d, sans fille and intersex) and four cellular blastoderm formation genes (Rho1, spaghetti squash, slow-as-molasses and serendipity-alpha) were analyzed, allowing us to sketch a preliminary outline of the embryonic process in the medfly. Furthermore, a putative homologue of the Zelda gene has been considered, which in D. melanogaster encodes a DNA-binding factor responsible for the maternal-to-zygotic transition. CONCLUSIONS: Our novel sexing method facilitates the study of i) when the MTZ transition occurs in males and females of C. capitata, ii) when and how the maternal information of "female-development" is reprogrammed in the embryos and iii) similarities and differences in the regulation of gene expression in C. capitata and D. melanogaster. We suggest a new model for the onset of the sex determination cascade in the medfly: the maternally inherited Cctra transcripts in the female embryos are insufficient to produce enough active protein to inhibit the male mode of Cctra splicing. The slow rate of development and the inefficiency of the splicing mechanism in the pre-cellular blastoderm facilitates the male-determining factor (M) activity, which probably acts by inhibiting CcTRA protein activity.

Abstract: The medfly, Ceratitis capitata, is an invasive species in which polyandry, associated with sperm precedence, is a common behaviour in the wild. In this species, characterized by internal fertilization, we disclose how the sperm from two males are stored in the female storage organs and how they are used in terms of paternity outcome. The experiments were designed to furnish comparable and unbiased estimates of sperm numbers and progeny in twice-mated females. Results are incorporated in a model through which it is possible to relate the amount of stored sperm with the progeny of twice-mated females. The results show that polyandrous medfly females conserve equal amounts of sperm from the two males to fertilize their eggs. However, we observed a clear advantage of the second male's sperm in siring progeny, which interestingly decreases in favor of the first male as ovipositions progress. The results enable us to exclude differential sperm mortality and suggest that it is the mechanics governing the storage organs which causes the initial, but decreasing second male sperm precedence during the female reproductive life. These outcomes allow us to correlate sperm use in polyandrous females with the mating strategies and invasiveness of this fly.

Abstract: Insect transgenesis is continuously being improved to increase the efficacy of population suppression and replacement strategies directed to the control of insect species of economic and sanitary interest. An essential prerequisite for the success of both pest control applications is that the fitness of the transformant individuals is not impaired, so that, once released in the field, they can efficiently compete with or even out-compete their wild-type counterparts for matings in order to reduce the population size, or to spread desirable genes into the target population. Recent research has shown that the production of fit and competitive transformants can now be achieved and that transgenes may not necessarily confer a fitness cost. In this article we review the most recent published results of the fitness assessment of different transgenic insect lines and underline the necessity to fulfill key requirements of ecological safety. Fitness evaluation studies performed in field cages and medium/large-scale rearing will validate the present encouraging laboratory results, giving an indication of the performance of the transgenic insect genotype after release in pest control programmes.

Abstract: Phytophagous insects of the genus Bactrocera are among the most economically important invasive fruit fly pests. In 2003, an unknown Bactrocera species was found in Kenya. First identified as an 'aberrant form' of the Asian B. dorsalis complex, it was later recognized as a new species, Bactrocera invadens. Within 2 years of its discovery, the species was recorded in several African countries, becoming an important quarantine pest. As this invasive fly was discovered only recently, no data are available on its invasion pattern in Africa. This pilot study attempts to infer from genetic data the dynamic aspects of the African invasion of this pest. Using microsatellite markers, we evaluated the level of genetic diversity and the extent of common ancestry among several African populations collected across the invaded areas. A sample from the Asian Sri Lankan population was analysed to confirm the Asian origin of this pest. Genetic data cast no doubt that Sri Lanka belongs to the native range, but only a small percentage of its genotypes can be found in Africa. African populations display relatively high levels of genetic diversity associated with limited geographical structure and no genetic footprints of bottlenecks. These features are indicative of processes of rapid population growth and expansion with possible multiple introductions. In the span of relatively few years, the African invasion registered the presence of at least two uncorrelated outbreaks, both starting from the East. The results of the analyses support that invasion started in East Africa, where B. invadens was initially isolated.

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.

Abstract: BACKGROUND: The medfly, Ceratitis capitata, is a highly invasive agricultural pest that has become a model insect for the development of biological control programs. Despite research into the behavior and classical and population genetics of this organism, the quantity of sequence data available is limited. We have utilized an expressed sequence tag (EST) approach to obtain detailed information on transcriptome signatures that relate to a variety of physiological systems in the medfly; this information emphasizes on reproduction, sex determination, and chemosensory perception, since the study was based on normalized cDNA libraries from embryos and adult heads. RESULTS: A total of 21,253 high-quality ESTs were obtained from the embryo and head libraries. Clustering analyses performed separately for each library resulted in 5201 embryo and 6684 head transcripts. Considering an estimated 19% overlap in the transcriptomes of the two libraries, they represent about 9614 unique transcripts involved in a wide range of biological processes and molecular functions. Of particular interest are the sequences that share homology with Drosophila genes involved in sex determination, olfaction, and reproductive behavior. The medfly transformer2 (tra2) homolog was identified among the embryonic sequences, and its genomic organization and expression were characterized. CONCLUSION: The sequences obtained in this study represent the first major dataset of expressed genes in a tephritid species of agricultural importance. This resource provides essential information to support the investigation of numerous questions regarding the biology of the medfly and other related species and also constitutes an invaluable tool for the annotation of complete genome sequences. Our study has revealed intriguing findings regarding the transcript regulation of tra2 and other sex determination genes, as well as insights into the comparative genomics of genes implicated in chemosensory reception and reproduction.

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.

Abstract: Scaphoideus titanus Ball is a Nearctic leafhopper that was introduced for the first time in Europe probably at the beginning of the 20th century. In Europe, this species is a specialist on cultivated grapevines and is of great economic importance as the vector of Flavescence dorée (FD), a Grapevine Yellows disease caused by Candidatus Phytoplasma vitis. The Random Amplified Polymorphic DNA (RAPD) technique was employed to obtain genetic information about the diffusion and the structure of S. titanus populations. Two American and 14 European populations were analysed. A total of 188 reproducible bands, obtained from three arbitrary primers, were considered to assess the amount and the pattern of genetic variation within and among leafhopper populations. American populations showed high levels of intra-population polymorphism and dissimilarity and appeared to be the most isolated of all the tested samples. The results confirm the historical role of American samples as the sources for the more recently founded European populations. RAPD analyses revealed a weak genetic structure of European samples that could probably be explained invoking the human role in their diffusion. The non-natural spreading of S. titanus across Europe is in fact attributable to the exchange of grapevine canes and grafts carrying eggs that the insect laid under the bark to overwinter.

Abstract: The phytophagous insects of the Tephritidae family offer different case histories of successful invasions. An example is Bactrocera dorsalis sensu stricto, the oriental fruit fly which has been recognized as a key pest of Asia and the Pacific. It is known to have the potential to establish adventive populations in various tropical and subtropical areas. Despite the economic risk associated with a putative stable presence of this fly, the genetic aspects of its invasion process have remained relatively unexplored. Using microsatellite markers we have investigated the population structure and genetic variability in 14 geographical populations across the four areas of the actual species range: Far East Asia, South Asia, Southeast Asia and the Pacific Area. Results of clustering and admixture, associated with phylogenetic and migration analyses, were used to evaluate the changes in population genetic structure that this species underwent during its invasion process and establishment in the different areas. The colonization process of this fly is associated with a relatively stable population demographic structure, especially in an unfragmented habitat, rich in intensive cultivation such as in Southeast Asia. In this area, the results suggest a lively demographic history, characterized by evolutionary recent demographic expansions and no recent bottlenecks. Cases of genetic isolation attributable to geographical factors, fragmented habitats and/or fruit trade restrictions were observed in Bangladesh, Myanmar and Hawaii. Regarding the pattern of invasion, the overall genetic profile of the considered populations suggests a western orientated migration route from China to the West.

Abstract: The piggyBac transposable element is currently the vector of choice for transgenesis, enhancer trapping, gene discovery and gene function determination in both insects and mammals. However, the recent discovery of sequences with similarity to piggyBac in a wide diversity of organisms suggests that piggyBac may be horizontally transferred to distantly related species. This has raised concern on the wide-range application of piggyBac-based transformation vectors and their stability. In this paper, the presence of sequences homologous to the piggyBac transposase was investigated in 17 species belonging to six genera within the Tephritidae family, including many pest species for which transformation has already been achieved. piggyBac-like sequences, with a high degree of similarity to the original Trichoplusia ni transposase sequence were identified only in six species of the Bactrocera genus.