Isolation and characterization of microsatellite markers from the olive fly, Bactrocera oleae, and their cross-species amplification in the Tephritidae family

<p>Abstract</p> <p>Background</p> <p>The Tephritidae family of insects includes the most important agricultural pests of fruits and vegetables, belonging mainly to four genera (<it>Bactrocera, Ceratitis, Anastrepha </it>and <it>Rhagoletis</it>). The olive fruit fly, <it>Bactrocera oleae</it>, is the major pest of the olive fruit. Currently, its control is based on chemical insecticides. Environmentally friendlier methods have been attempted in the past (Sterile Insect Technique), albeit with limited success. This was mainly attributed to the lack of knowledge on the insect's behaviour, ecology and genetic structure of natural populations. The development of molecular markers could facilitate the access in the genome and contribute to the solution of the aforementioned problems. We chose to focus on microsatellite markers due to their abundance in the genome, high degree of polymorphism and easiness of isolation.</p> <p>Results</p> <p>Fifty-eight microsatellite-containing clones were isolated from the olive fly, <it>Bactrocera oleae</it>, bearing a total of sixty-two discrete microsatellite motifs. Forty-two primer pairs were designed on the unique sequences flanking the microsatellite motif and thirty-one of them amplified a PCR product of the expected size. The level of polymorphism was evaluated against wild and laboratory flies and the majority of the markers (93.5%) proved highly polymorphic. Thirteen of them presented a unique position on the olive fly polytene chromosomes by <it>in situ </it>hybridization, which can serve as anchors to correlate future genetic and cytological maps of the species, as well as entry points to the genome. Cross-species amplification of these markers to eleven Tephritidae species and sequencing of thirty-one of the amplified products revealed a varying degree of conservation that declines outside the <it>Bactrocera </it>genus.</p> <p>Conclusion</p> <p>Microsatellite markers are very powerful tools for genetic and population analyses, particularly in species deprived of any other means of genetic analysis. The presented set of microsatellite markers possesses all features that would render them useful in such analyses. This could also prove helpful for species where SIT is a desired outcome, since the development of effective SIT can be aided by detailed knowledge at the genetic and molecular level. Furthermore, their presented efficacy in several other species of the Tephritidae family not only makes them useful for their analysis but also provides tools for phylogenetic comparisons among them.</p

Sex and the single embryo: early deveopment in the Mediterranean fruit fly, Ceratitis capitata

<p>Abstract</p> <p>Background</p> <p>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 <it>Drosophila melanogaster</it>, 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 <it>Ceratitis capitata </it>(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, <it>C. capitata </it>and <it>D. melanogaster</it>, that shared a common ancestor 80-100 mya.</p> <p>Results</p> <p>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 (<it>Cctransformer</it>, <it>Cctransformer2 </it>and <it>Ccdoublesex</it>), the expression patterns of other medfly genes that are similar to the <it>D. melanogaster </it>sex-determination genes (<it>sisterlessA, groucho, deadpan, Sex-lethal, female lethal d, sans fille </it>and <it>intersex</it>) and four cellular blastoderm formation genes (<it>Rho1, spaghetti squash, slow-as-molasses </it>and <it>serendipity-α</it>) were analyzed, allowing us to sketch a preliminary outline of the embryonic process in the medfly. Furthermore, a putative homologue of the <it>Zelda </it>gene has been considered, which in <it>D. melanogaster </it>encodes a DNA-binding factor responsible for the maternal-to-zygotic transition.</p> <p>Conclusions</p> <p>Our novel sexing method facilitates the study of i) when the MTZ transition occurs in males and females of <it>C. capitata</it>, 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 <it>C. capitata </it>and <it>D. melanogaster</it>. We suggest a new model for the onset of the sex determination cascade in the medfly: the maternally inherited <it>Cctra </it>transcripts in the female embryos are insufficient to produce enough active protein to inhibit the male mode of <it>Cctra </it>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.</p

Conditional embryonic lethality to improve the sterile insect technique in Ceratitis capitata (Diptera: Tephritidae)

<p>Abstract</p> <p>Background</p> <p>The sterile insect technique (SIT) is an environment-friendly method used in area-wide pest management of the Mediterranean fruit fly <it>Ceratitis capitata </it>(Wiedemann; Diptera: Tephritidae). Ionizing radiation used to generate reproductive sterility in the mass-reared populations before release leads to reduction of competitiveness.</p> <p>Results</p> <p>Here, we present a first alternative reproductive sterility system for medfly based on transgenic embryonic lethality. This system is dependent on newly isolated medfly promoter/enhancer elements of cellularization-specifically-expressed genes. These elements act differently in expression strength and their ability to drive lethal effector gene activation. Moreover, position effects strongly influence the efficiency of the system. Out of 60 combinations of driver and effector construct integrations, several lines resulted in larval and pupal lethality with one line showing complete embryonic lethality. This line was highly competitive to wildtype medfly in laboratory and field cage tests.</p> <p>Conclusion</p> <p>The high competitiveness of the transgenic lines and the achieved 100% embryonic lethality causing reproductive sterility without the need of irradiation can improve the efficacy of operational medfly SIT programs.</p

The whole genome sequence of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann), reveals insights into the biology and adaptive evolution of a highly invasive pest species

The Mediterranean fruit fly (medfly), Ceratitis capitata, is a major destructive insect pest due to its broad host range, which includes hundreds of fruits and vegetables. It exhibits a unique ability to invade and adapt to ecological niches throughout tropical and subtropical regions of the world, though medfly infestations have been prevented and controlled by the sterile insect technique (SIT) as part of integrated pest management programs (IPMs). The genetic analysis and manipulation of medfly has been subject to intensive study in an effort to improve SIT efficacy and other aspects of IPM control

Cytogenetic and symbiont analysis of five members of the B. dorsalis complex (Diptera, Tephritidae): no evidence of chromosomal or symbiont-based speciation events

The Bactrocera dorsalis species complex, currently comprising about 90 entities has received much attention. During the last decades, considerable effort has been devoted to delimiting the species of the complex. This information is of great importance for agriculture and world trade, since the complex harbours several pest species of major economic importance and other species that could evolve into global threats. Speciation in Diptera is usually accompanied by chromosomal rearrangements, particularly inversions that are assumed to reduce/eliminate gene flow. Other candidates currently receiving much attention regarding their possible involvement in speciation are reproductive symbionts, such as Wolbachia, Spiroplasma, Arsenophonus, Rickettsia and Cardinium. Such symbionts tend to spread quickly through natural populations and can cause a variety of phenotypes that promote pre-mating and/or post-mating isolation and, in addition, can affect the biology, physiology, ecology and evolution of their insect hosts in various ways. Considering all these aspects, we present: (a) a summary of the recently gained knowledge on the cytogenetics of five members of the B. dorsalis complex, namely B. dorsaliss.s., B. invadens, B. philippinensis, B. papayae and B. carambolae, supplemented by additional data from a B. dorsaliss.s. colony from China, as well as by a cytogenetic comparison between the dorsalis complex and the genetically close species, B. tryoni, and, (b) a reproductive symbiont screening of 18 different colonized populations of these five taxa. Our analysis did not reveal any chromosomal rearrangements that could differentiate among them. Moreover, screening for reproductive symbionts was negative for all colonies derived from different geographic origins and/or hosts. There are many different factors that can lead to speciation, and our data do not support chromosomal and/or symbiotic-based speciation phenomena in the taxa under study

Cryopreservation of Embryos of the Mediterranean Fruit Fly Ceratitis capitata Vienna 8 Genetic Sexing Strain.

The Mediterranean fruit fly, Ceratitis capitata, is one of the most serious pests of fruit crops world-wide. During the last decades, area-wide pest management (AW-IPM) approaches with a sterile insect technique (SIT) component have been used to control populations of this pest in an effective and environment-friendly manner. The development of genetic sexing strains (GSS), such as the Vienna 8 strain, has been played a major role in increasing the efficacy and reducing the cost of SIT programs. However, mass rearing, extensive inbreeding, possible bottleneck phenomena and hitch-hiking effects might pose major risks for deterioration and loss of important genetic characteristics of domesticated insect. In the present study, we present a modified procedure to cryopreserve the embryos of the medfly Vienna 8 GSS based on vitrification and used this strain as insect model to assess the impact of the cryopreservation process on the genetic structure of the cryopreserved insects. Forty-eight hours old embryos, incubated at 24°C, were found to be the most suitable developmental stage for cryopreservation treatment for high production of acceptable hatch rate (38%). Our data suggest the absence of any negative impact of the cryopreservation process on egg hatch rate, pupation rates, adult emergence rates and stability of the temperature sensitive lethal (tsl) character on two established cryopreserved lines (flies emerged from cryopreserved embryos), named V8-118 and V8-228. Taken together, our study provides an optimized procedure to cryopreserve the medfly Vienna 8 GSS and documents the absence of any negative impact on the genetic structure and quality of the strain. Benefits and sceneries for utilization of this technology to support operational SIT projects are discussed in this paper

<i>Achilles</i>, a New Family of Transcriptionally Active Retrotransposons from the Olive Fruit Fly, with Y Chromosome Preferential Distribution

<div><p>Sex chromosomes have many unusual features relative to autosomes. The in depth exploration of their structure will improve our understanding of their origin and divergence (degeneration) as well as the evolution of genetic sex determination pathways which, most often are attributed to them. In Tephritids, the structure of Y chromosome, where the male-determining factor M is localized, is largely unexplored and limited data concerning its sequence content and evolution are available. In order to get insight into the structure and organization of the Y chromosome of the major olive insect pest, the olive fly <i>Bactrocera oleae</i>, we characterized sequences from a Pulse Field Gel Electrophoresis (PFGE)-isolated Y chromosome. Here, we report the discovery of the first olive fly LTR retrotransposon with increased presence on the Y chromosome. The element belongs to the <i>BEL-Pao</i> superfamily, however, its sequence comparison with the other members of the superfamily suggests that it constitutes a new family that we termed <i>Achilles</i>. Its ~7.5 kb sequence consists of the 5’LTR, the 5’non-coding sequence and the open reading frame (ORF), which encodes the polyprotein Gag-Pol. <i>In situ</i> hybridization to the <i>B</i>. <i>oleae</i> polytene chromosomes showed that <i>Achilles</i> is distributed in discrete bands dispersed on all five autosomes, in all centromeric regions and in the granular heterochromatic network corresponding to the mitotic sex chromosomes. The between sexes comparison revealed a variation in <i>Achilles</i> copy number, with male flies possessing 5–10 copies more than female (CI range: 18–38 and 12–33 copies respectively per genome). The examination of its transcriptional activity demonstrated the presence of at least one intact active copy in the genome, showing a differential level of expression between sexes as well as during embryonic development. The higher expression was detected in male germline tissues (testes). Moreover, the presence of <i>Achilles</i>-like elements in different species of the Tephritidae family suggests an ancient origin of this element.</p></div

Incipient speciation revealed in Anastrepha fraterculus (Diptera; Tephritidae) by studies on mating compatibility, sex pheromones, hybridization, and cytology

It has long been proposed that the nominal species Anastrepha fraterculus is a species complex and earlier studies showed high levels of pre-zygotic isolation between two laboratory strains from Argentina and Peru. Further experiments were carried out on the same populations and on their reciprocal hybrids, including pre- and post-zygotic isolation studies, pheromone analysis, and mitotic and polytene chromosome analysis. A high level of pre-zygotic isolation had been maintained between the parental strains despite 3 years of laboratory rearing under identical conditions. The level of pre-zygotic isolation was reduced in matings with hybrids. There were also differences in other components of mating behaviour. There were quantitative and qualitative differences in the sex pheromone of the two strains with the hybrids producing a mixture. The pre-zygotic isolation barriers were complemented by high levels of post-zygotic inviability and sex ratio distortion, most likely not due to Wolbachia, although there was evidence of some cytoplasmic factor involved in sex ratio distortion. Analysis of polytene chromosomes revealed a high level of asynapsis in the hybrids, together with karyotypic differences between the parental strains. The combined results of the present study indicate that these two strains belong to different biological entities within the proposed A. fraterculus complex.Fil: Cáceres, Carlos. International Atomic Energy Agency; AustriaFil: Segura, Diego Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires; ArgentinaFil: Vera, María Teresa. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Gobierno de Tucumán. Ministerio de Desarrollo Productivo. Estación Experimental Agroindustrial Obispo Colombres; ArgentinaFil: Wornoayporn, Viwat. International Atomic Energy Agency; AustriaFil: Cladera, Jorge Luis. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires; ArgentinaFil: Teal, Peter. US Department of Agriculture Agricultural Research Service; Estados UnidosFil: Sapountzis, Panagiotis. University of Ioannina; GreciaFil: Bourtzis, Kostas. University of Ioannina; GreciaFil: Zacharopoulou, Antigone. International Atomic Energy Agency; Austria. University of Patras; GreciaFil: Robinson, Alan S.. International Atomic Energy Agency; Austri

Cytogenetic analysis of the South American fruit fly Anastrepha fraterculus (diptera:tephritidae) species complex : construction of detailed photographic polytene chromosome maps of the Argentinian af. sp.1 member

Genetic and cytogenetic studies constitute a significant basis for understanding the biology of insect pests and the design and the construction of genetic tools for biological control strategies. Anastrepha fraterculus is an important pest of the Tephritidae family. It is distributed from southern Texas through eastern Mexico, Central America and South America causing significant crop damage and economic losses. Currently it is considered as a species complex; until now seven members have been described based on multidisciplinary approaches. Here we report the cytogenetic analysis of an Argentinian population characterized as Af. sp.1 member of the Anastrepha fraterculus species complex. The mitotic karyotype and the first detailed photographic maps of the salivary gland polytene chromosomes are presented. The mitotic metaphase complement consists of six (6) pairs of chromosomes, including one pair of heteromorphic sex chromosomes, with the male being the heterogametic sex. The analysis of the salivary gland polytene complement shows a total number of five long chromosomes that correspond to the five autosomes of the mitotic karyotype and a heterochromatic network corresponding to the sex chromosomes. Comparison of the polytene chromosome maps between this species and Anastrepha ludens shows significant similarity. The polytene maps presented here are suitable for cytogenetic studies that could shed light on the species limits within this species complex and support the development of genetic tools for sterile insect technique (SIT) applications.Inst. de Genética "Ewald A. Favret"- IGEAFFil: Gariou-Papalexiou, Aggeliki. University of Patras. Biology Department; GreciaFil: Giardini, Maria Cecilia. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Genética "Ewald A. Favret"; ArgentinaFil: Augustinos, Antonios A. University of Patras. Biology Department; Grecia. Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture. Insect Pest Control; AustriaFil: Drosopoulou, Elena. Aristotle University of Thessaloniki. School of Biology, Faculty of Sciences. Department of Genetics, Development and Molecular Biology; GreciaFil: Lanzavecchia, Silvia Beatriz. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Genética "Ewald A. Favret"; ArgentinaFil: Cladera, Jorge Luis. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Genética "Ewald A. Favret"; ArgentinaFil: Caceres, Carlos. Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture. Insect Pest Control; AustriaBourtzis, Kostas. Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture. Insect Pest Control; AustriaFil: Mavragani-Tsipidou, Penelope. Aristotle University of Thessaloniki. School of Biology, Faculty of Sciences. Department of Genetics, Development and Molecular Biology; GreciaFil: Zacharopoulou, Antigone. University of Patras. Biology Department; Greci