Papers by Kostas Mathiopoulos
ABSTRACTFull-length transcript sequencing remains a main goal of RNA sequencing. However, even th... more ABSTRACTFull-length transcript sequencing remains a main goal of RNA sequencing. However, even the application of long-read sequencing technologies such as Oxford Nanopore Technologies still fail to yield full-length transcript sequencing for a significant portion of sequenced reads. Since these technologies can sequence reads that are far longer than the longest known processed transcripts, the lack of efficiency to obtain full-length transcripts from good quality RNAs stems from library preparation inefficiency rather than the presence of degraded RNA molecules. It has previously been shown that addition of inverted terminal repeats in cDNA during reverse transcription followed by single-primer PCR creates a PCR suppression effect that prevents amplification of short molecules thus enriching the library for longer transcripts. We adapted this method for Nanopore cDNA library preparation and show that not only is PCR efficiency increased but gene body coverage is dramatically impro...
Long non-coding RNA (lncRNA) research has emerged as an independent scientific field in recent ye... more Long non-coding RNA (lncRNA) research has emerged as an independent scientific field in recent years. Despite their association with critical cellular and metabolic processes in plenty of organisms, lncRNAs are still a largely unexplored area in mosquito research. We propose that they could serve as exceptional tools for pest management due to unique features they possess. These include low inter-species sequence conservation and high tissue specificity. In the present study, we investigated the role of ovary-specific lncRNAs in the reproductive ability of the Asian tiger mosquito, Aedes albopictus. Through the analysis of transcriptomic data, we identified several lncRNAs that were differentially expressed upon blood feeding; we called these genes Norma (NOn-coding RNA in Mosquito ovAries). We observed that silencing some of these Normas resulted in significant impact on mosquito fecundity and fertility. We further focused on Norma3 whose silencing resulted in 43% oviposition reduc...
a,b<p>Intercept and slope of the linear regression lines (<a href="http://www.ploso... more a,b<p>Intercept and slope of the linear regression lines (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079393#pone-0079393-g006" target="_blank">Figure 6</a>).</p>c<p>The efficiency of the reaction was estimated by the equation: E = 10<sup>−1/slope</sup>-1.</p>d<p>ΔCt value is obtained by comparing the y-axis intercepts derived from the two lines-of-best-fit of the target sequences BoR300 and <i>ace</i>.</p>e<p>According to the mean efficiency of the reactions (94.05%) the amplification factor F was 1.881.</p
Additional file 1. Supplementary materials.
Supplementary Tables S1–S26. Table S1 C. capitata genome and RNA-seq source material and sequenci... more Supplementary Tables S1–S26. Table S1 C. capitata genome and RNA-seq source material and sequencing runs. Table S2 Pluralibacter gergoviae genome metrics. Table S3 P. gergoviae genes associated with general functional categories. Table S4 BUSCO genome assembly comparisons between C. capitata, D. melanogaster, and Bactrocera species. Table S5a Orthology tables - Copy numbers. Table S5b Orthology tables - Orthologous groups. Table S5c Orthology tables - Counts by species. Table S6 Chromosomal positions for mapped scaffolds. Table S7 C. capitata transposable element sequences. Table S8 C. capitata microRNA sequences. Table S9 microRNA/siRNA/piRNA machinery in C capitata. Table S10 C. capitata odorant-binding protein (OBP) genes. Table S11 C. capitata odorant receptor (OR) genes. Table S12 C. capitata gustatory receptor (GR) gene assignments. Table S13 C. capitata ionotrophic receptor (IR) gene assignments. Table S14 C. capitata aquaporin genes. Table S15 Immunity-related gene compariso...
Supplementary material A. C. capitata genome sequencing approaches, B. Automated annotations, C. ... more Supplementary material A. C. capitata genome sequencing approaches, B. Automated annotations, C. Detection of bacterial sequence contamination and D. Screening the C. capitata genome sequence for potential horizontal gene transfer events. (DOCX 601 kb)
Supplementary material: C. capitata chemoreceptor genes. (DOCX 194 kb)
Supplementary figures S1–S9. Figure S1 Odorant-binding protein (OBP) genes phylogenetic tree. Fig... more Supplementary figures S1–S9. Figure S1 Odorant-binding protein (OBP) genes phylogenetic tree. Figure S2 Ionotrophic receptor (IR) genes phylogenetic tree. Figure S3 Opsin genes phylogenetic tree. Figure S4 CYP gene clusters A and B. Figure S5 Glutathione S-transferase genes phylogenetic tree. Figure S6 cysLGIC genes superfamily phylogenetic tree. Figure S7 Cuticle protein gene clusters. Figure S8 CPLCA cuticle protein genes phylogenetic tree. Figure S9 Seminal fluid protein functional classes. (PDF 9425 kb)
Scientific Reports, 2021
The olive fruit fly, Bactrocera oleae, is the most important pest for the olive fruit but lacks a... more The olive fruit fly, Bactrocera oleae, is the most important pest for the olive fruit but lacks adequate transcriptomic characterization that could aid in molecular control approaches. We apply nanopore long-read RNA-seq with internal RNA standards allowing absolute transcript quantification to analyze transcription dynamics during early embryo development for the first time in this organism. Sequencing on the MinION platform generated over 31 million reads. Over 50% of the expressed genes had at least one read covering its entire length validating our full-length approach. We generated a de novo transcriptome assembly and identified 1768 new genes and a total of 79,810 isoforms; a fourfold increase in transcriptome diversity compared to the current NCBI predicted transcriptome. Absolute transcript quantification per embryo allowed an insight into the dramatic re-organization of maternal transcripts. We further identified Zelda as a possible regulator of early zygotic genome activat...
Archives of Insect Biochemistry and Physiology, 2019
Agricultural pest control using genetic-based methods provides a species-specific and environment... more Agricultural pest control using genetic-based methods provides a species-specific and environmentally harmless way for population suppression of fruit flies. One way to improve the efficiency of such methods is through self-limiting, female-eliminating approaches that can alter an insect populations' sex ratio toward males. In this microreview, we summarize recent advances in synthetic sex ratio distorters based on X-chromosome shredding that can induce male-biased progeny. We outline the basic principles to guide the efficient design of an X-shredding system in an XY heterogametic fruit fly species of interest using CRISPR/Cas gene editing, newly developed computational tools, and insect genetic engineering. We also discuss technical aspects and challenges associated with the efficient transferability of this technology in fruit fly pest populations, toward the potential use of this new class of genetic control approaches for pest management purposes.
Entomologia Experimentalis et Applicata, 2017
In insects, rapidly evolving primary sex-determining signals are transduced by a conserved regula... more In insects, rapidly evolving primary sex-determining signals are transduced by a conserved regulatory module producing sex-specific proteins that direct sexual differentiation1-4. In the agricultural pest Ceratitis capitata (medfly), a Y-linked Maleness factor (M) is thought to repress the autoregulatory splicing of transformer (Cctra), which is required in XX individuals to establish and maintain female sex determination5,6. Despite the sequencing of the medfly genome, its M factor has remained elusive7. Here, we report the identification of a Y-linked gene, Maleness-on the-Y (MoY), and show that it encodes a small and novel protein which is both necessary and sufficient for medfly male sex determination. CRISPR-induced MoY mutants can develop into fertile XY females and expression of MoY in XX individuals can produce fertile males. indicating that, irrespective of its karyotype, sexual fate of the germline follows that of the soma. In contrast to the molecular diversity of the Y-l...
Insect Biochemistry and Molecular Biology, 2008
Scientific reports, Apr 3, 2017
Real-time quantitative-PCR has been a priceless tool for gene expression analyses. The reaction, ... more Real-time quantitative-PCR has been a priceless tool for gene expression analyses. The reaction, however, needs proper normalization with the use of housekeeping genes (HKGs), whose expression remains stable throughout the experimental conditions. Often, the combination of several genes is required for accurate normalization. Most importantly, there are no universal HKGs which can be used since their expression varies among different organisms, tissues or experimental conditions. In the present study, nine common HKGs (RPL19, tbp, ubx, GAPDH, α-TUB, β-TUB, 14-3-3zeta, RPE and actin3) are evaluated in thirteen different body parts, developmental stages and reproductive and olfactory tissues of two insects of agricultural importance, the medfly and the olive fly. Three software programs based on different algorithms were used (geNorm, NormFinder and BestKeeper) and gave different ranking of HKG stabilities. This confirms once again that the stability of common HKGs should not be taken...
Entomologia Experimentalis et Applicata
Bactrocera oleae (Rossi) (Diptera: Tephritidae) is the main pest of olive trees (Olea europaea L.... more Bactrocera oleae (Rossi) (Diptera: Tephritidae) is the main pest of olive trees (Olea europaea L.), causing major damages in olive crops. Improvement of mass rearing is a prerequisite for the successful development of large‐scale sterile insect technique (SIT) applications. This can be achieved through the enrichment of artificial diets with gut bacteria isolates. We assessed the efficiency of three gut bacteria previously isolated from Ceratitis capitata (Wiedemann), and four isolated from B. oleae, as larval diet additives in both live and inactivated/dead forms. Our results showed that dead Enterobacter sp. AA26 increased pupal weight, whereas both live and dead cells increased pupal and adult production and reduced immature developmental time, indicating that its bacterial cells serve as a direct nutrient source. Live Providencia sp. AA31 improved pupal and adult production, enhanced male survival under stress conditions, and delayed immature development. Dead Providencia sp. AA31, however, did not affect production rates, indicating that live bacteria can colonize the insect gut and biosynthesize nutrients essential for larval development. Live and dead Bacillus sp. 139 increased pupal weight, accelerated immature development, and increased adult survival under stress. Moreover, live Bacillus sp. 139 improved adult production, indicating that Bacillus cells are a direct source of nutrients. Dead Serratia sp. 49 increased pupal and adult production and decreased male survival under stress conditions whereas live cells decreased insect production, indicating that the live strain is entomopathogenic, but its dead cells can be utilized as nutrient source. Klebsiella oxytoca, Enterobacter sp. 23, and Providencia sp. 22 decreased pupal and subsequent adult production and were harmful for B. oleae. Our findings indicate that dead Enterobacter sp. AA26 is the most promising bacterial isolate for the improvement of B. oleae mass rearing in support of future SIT or related population suppression programs.
BMC Genetics
Background The olive fruit fly (Bactrocera oleae) is the most destructive pest of the olive culti... more Background The olive fruit fly (Bactrocera oleae) is the most destructive pest of the olive cultivation worldwide causing significant production losses and olive fruit impoverishment, as its larvae feed exclusively on the olive fruit. Reproductive and sexual behavior, as well as host-plant recognition of the fly, are highly dependent on its chemosensory system. Therefore, exploring the role of genes that play a critical role in olfaction, could reveal potential molecular targets that determine species-specific features on chemical communication and could be used to impair sexual behavior. Results In this study we identified the gene that encodes the conserved olfactory co-receptor Orco (Odorant receptor co-receptor), which interacts with all divergent insect odorant receptors, and investigated how disruption of its expression affects chemoreception. We initially searched the expression profile of Bo-Orco in both sexes during sexual maturation, as well as pre- and post-mating communi...
BMC Genomics
Background The olive fruit fly, Bactrocera oleae, is the most important pest in the olive fruit a... more 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 GC...
A great deal of behavioral and molecular interactions between male and female insects takes place... more A great deal of behavioral and molecular interactions between male and female insects takes place during insect reproduction. Here, we comprehensively analyze the reproductive system of the olive fruit fly. Specifically, transcriptomic and genomic analysis of the reproductive tissues from virgin and once mated insects were performed. Genes encoding proteins implicated in immune response, mucins, antigen 5 proteins, proteases inhibitors and proteins with putative secretory activity were identified. Comparison of the transcriptomes between virgin and mated insects resulted in the identification of genes that are up- or down-regulated after mating. In testes 106 genes were up-regulated and 344 genes were down-regulated, whereas in male accessory glands with ejaculatory bulb 1,607 genes were up-regulated and 384 genes were down-regulated in mated male insects. Respectively, in mated females 1,705 genes were up-regulated and 120 genes were down-regulated in mated insects. To get a deeper...
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Papers by Kostas Mathiopoulos