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

De novo assembly of the olive fruit fly (Bactrocera oleae) genome with linked-reads and long-read technologies minimizes gaps and provides exceptional Y chromosome assembly

Background: The olive fruit fly, Bactrocera oleae, is the most important pest in the olive fruit agribusiness industry. This is because female flies lay their eggs in the unripe fruits and upon hatching the larvae feed on the fruits thus destroying them. The lack of a high-quality genome and other genomic and transcriptomic data has hindered progress in understanding the fly’s biology and proposing alternative control methods to pesticide use. Results: Genomic DNA was sequenced from male and female Demokritos strain flies, maintained in the laboratory for over 45 years. We used short-, mate-pair-, and long-read sequencing technologies to generate a combined male-female genome assembly (GenBank accession GCA_001188975.2). Genomic DNA sequencing from male insects using 10x Genomics linked-reads technology followed by mate-pair and long-read scaffolding and gap-closing generated a highly contiguous 489 Mb genome with a scaffold N50 of 4.69 Mb and L50 of 30 scaffolds (GenBank accession GCA_001188975.4). RNA-seq data generated from 12 tissues and/or developmental stages allowed for genome annotation. Short reads from both males and females and the chromosome quotient method enabled identification of Y-chromosome scaffolds which were extensively validated by PCR. Conclusions: The high-quality genome generated represents a critical tool in olive fruit fly research. We provide an extensive RNA-seq data set, and genome annotation, critical towards gaining an insight into the biology of the olive fruit fly. In addition, elucidation of Y-chromosome sequences will advance our understanding of the Y-chromosome’s organization, function and evolution and is poised to provide avenues for sterile insect technique approaches

Cross-National Comparisons of Internalizing Problems in a Cohort of 8952 Adolescents from Five European Countries: The EU NET ADB Survey

Various factors such as family relationships, socioeconomic indicators, socio-cultural aspects, mental health problems and demographic variables, have been associated with internalizing problems in adolescence. The purpose of this study was to examine the prevalence of internalizing problems in adolescents from five European countries in comparison with risk factors. Using data from the European Network for Adolescent Behavior (EU NET ADB) survey, a cross-sectional school-based study of adolescents (from 14 to 17.9 years) was conducted. Data were retrieved from self-report questionnaires derived from Greece, Spain, the Netherlands, Iceland and Romania. Multiple logistic regression analysis overall and by country was held through estimation of odds ratios (OR) and 95% confidence intervals (95% CI). A total of 8952 adolescents participated in the study. According to the results, Greece (7.6%) and Romania (2.4%) presented with the lowest rates of internalizing problems, while Iceland presented the highest (11.1%). Internalizing problems were associated with lower school grades (adjusted OR = 1.21, 95% CI: 1.08–1.36), while the higher education level of parents was associated with lower odds of internalizing problems (adjusted OR = 0.75, 95% CI: 0.62–0.92). In country-specific analyses, factors that affected the likelihood of internalizing problems were gender, age, maternal and paternal unemployment. Variations detected in adolescents’ internalizing problems were determined by the socio-cultural context of each country. Cultural differences should be addressed thoroughly in further research, in order to better understand and intervene in aspects of internalizing problems in adolescence