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.

Anosmic flies: what Orco silencing does to olive fruit flies.

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 communication by relative quantitative real time PCR (qRT-PCR) analysis suggesting that Bo-Orco was abundantly expressed in sexually mature adults. We further investigated the functional role of Bo-Orco in mating and oviposition behavior via transient gene silencing that was performed through in vivo dsRNA hemolymph injections in sexually mature flies 7 days after eclosion. Orco-knockdown phenotypes in both sexes showed reduced copulation rates in mating competitiveness tests, possibly through impaired olfactory-mediated detection of sex pheromone. In addition, oviposition was significantly inhibited in dsRNA-Orco injected females in a post-mating behavior test. CONCLUSIONS: Our results demonstrate that Orco plays a crucial role in the reproductive behavior of the olive fruit fly, since pre- and post-mating processes were affected. This is the first report in the olive fruit fly that links the chemosensory pathway with the mating behavior and the reproductive potential at a molecular basis, rendering this gene a potential target for the improvement of the olive fruit fly population control techniques.

Developing CRISPR-based sex-ratio distorters for the genetic control of fruit fly pests: A how to manual.

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.

Targeted somatic mutagenesis through CRISPR/Cas9 ribonucleoprotein complexes in the olive fruit fly, Bactrocera oleae.

The olive fruit fly, Bactrocera oleae (Diptera: Tephritidae), is the most destructive insect pest of olive cultivation, causing significant economic and production losses. Here, we present the establishment of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 methodology for gene disruption in this species. We performed targeted mutagenesis of the autosomal gene white (Bo-we), by injecting into early embryos in vitro preassembled and solubilized Cas9 ribonucleoprotein complexes loaded with two gene-specific single-guide RNAs. Gene disruption of Bo-we led to somatic mosaicism of the adult eye color. Large eye patches or even an entire eye lost the iridescent reddish color, indicating the successful biallelic mutagenesis in somatic cells. Cas9 induced either indels in each of the two simultaneously targeted Bo-we sites or a large deletion of the intervening region. This study demonstrates the first efficient implementation of the CRISPR/Cas9 technology in the olive fly, providing new opportunities towards the development of novel genetic tools for its control.

Olive fly transcriptomics analysis implicates energy metabolism genes in spinosad resistance.

BACKGROUND: The olive fly, Bactrocera oleae, is the most devastating pest of cultivated olives. Its control has been traditionally based on insecticides, mainly organophosphates and pyrethroids. In recent years, the naturalyte spinosad is used against the olive fly. As with other insecticides, spinosad is subject to selection pressures that have led to resistance development. Mutations in the α6 subunit of the nicotinic acetylcholine receptor (nAChR) have been implicated in spinosad resistance in several species (e.g., Drosophila melanogaster) but excluded in others (e.g., Musca domestica). Yet, additional mechanisms involving enhanced metabolism of detoxification enzymes (such as P450 monooxygenases or mixed function oxidases) have also been reported. In order to clarify the spinosad resistance mechanisms in the olive fly, we searched for mutations in the α6-subunit of the nAChR and for up-regulated genes in the entire transcriptome of spinosad resistant olive flies. RESULTS: The olive fly α6-subunit of the nAChR was cloned from the laboratory sensitive strain and a spinosad selected resistant line. The differences reflected silent nucleotide substitutions or conserved amino acid changes. Additionally, whole transcriptome analysis was performed in the two strains in order to reveal any underlying resistance mechanisms. Comparison of over 13,000 genes showed that in spinosad resistant flies nine genes were significantly over-expressed, whereas ~40 were under-expressed. Further functional analyses of the nine over-expressed and eleven under-expressed loci were performed. Four of these loci (Yolk protein 2, ATP Synthase FO subunit 6, Low affinity cationic amino acid transporter 2 and Serine protease 6) showed consistently higher expression both in the spinosad resistant strain and in wild flies from a resistant California population. On the other side, two storage protein genes (HexL1 and Lsp1) and two heat-shock protein genes (Hsp70 and Hsp23) were unfailingly under-expressed in resistant flies. CONCLUSION: The observed nucleotide differences in the nAChR-α6 subunit between the sensitive and spinosad resistant olive fly strains did not advocate for the involvement of receptor mutations in spinosad resistance. Instead, the transcriptome comparison between the two strains indicated that several immune system loci as well as elevated energy requirements of the resistant flies might be necessary to lever the detoxification process.

The molecular biology of the olive fly comes of age.

BACKGROUND: Olive cultivation blends with the history of the Mediterranean countries since ancient times. Even today, activities around the olive tree constitute major engagements of several people in the countryside of both sides of the Mediterranean basin. The olive fly is, beyond doubt, the most destructive pest of cultivated olives. The female fly leaves its eggs in the olive fruit. Upon emergence, the larvae feed on the olive sap, thus destroying the fruit. If untreated, practically all olives get infected. The use of chemical insecticides constitutes the principal olive fly control approach. The Sterile Insect Technique (SIT), an environmentally friendly alternative control method, had been tried in pilot field applications in the 1970's, albeit with no practical success. This was mainly attributed to the low, non-antagonistic quality of the mixed-sex released insects. Many years of experience from successful SIT applications in related species, primarily the Mediterranean fruit fly, Ceratitis capitata, demonstrated that efficient SIT protocols require the availability of fundamental genetic and molecular information. RESULTS: Among the primary systems whose understanding can contribute towards novel SIT approaches (or its recently developed alternative RIDL: Release of Insects carrying a Dominant Lethal) is the reproductive, since the ability to manipulate the reproductive system would directly affect the insect's fertility. In addition, the analysis of early embryonic promoters and apoptotic genes would provide tools that confer dominant early-embryonic lethality during mass-rearing. Here we report the identification of several genes involved in these systems through whole transcriptome analysis of female accessory glands (FAGs) and spermathecae, as well as male testes. Indeed, analysis of differentially expressed genes in these tissues revealed higher metabolic activity in testes than in FAGs/spermathecae. Furthermore, at least five olfactory-related genes were shown to be differentially expressed in the female and male reproductive systems analyzed. Finally, the expression profile of the embryonic serendipity-α locus and the pre-apoptotic head involution defective gene were analyzed during embryonic developmental stages. CONCLUSIONS: Several years of molecular studies on the olive fly can now be combined with new information from whole transcriptome analyses and lead to a deep understanding of the biology of this notorious insect pest. This is a prerequisite for the development of novel embryonic lethality female sexing strains for successful SIT efforts which, combined with improved mass-reared conditions, give new hope for efficient SIT applications for the olive fly.

Decoding the Reproductive System of the Olive Fruit Fly, Bactrocera oleae.

In most diploid organisms, mating is a prerequisite for reproduction and, thus, critical to the maintenance of their population and the perpetuation of the species. Besides the importance of understanding the fundamentals of reproduction, targeting the reproductive success of a pest insect is also a promising method for its control, as a possible manipulation of the reproductive system could affect its destructive activity. Here, we used an integrated approach for the elucidation of the reproductive system and mating procedures of the olive fruit fly, Bactrocera oleae. Initially, we performed a RNAseq analysis in reproductive tissues of virgin and mated insects. A comparison of the transcriptomes resulted in the identification of genes that are differentially expressed after mating. Functional annotation of the genes showed an alteration in the metabolic, catalytic, and cellular processes after mating. Moreover, a functional analysis through RNAi silencing of two differentially expressed genes, yellow-g and troponin C, resulted in a significantly reduced oviposition rate. This study provided a foundation for future investigations into the olive fruit fly's reproductive biology to the development of new exploitable tools for its control.

Nanopore long-read RNA-seq and absolute quantification delineate transcription dynamics in early embryo development of an insect pest.

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 activation in B. oleae and provide further insights into the maternal-to-zygotic transition. These data show the utility of long-read RNA in improving characterization of non-model organisms that lack a fully annotated genome, provide potential targets for sterile insect technic approaches, and provide the first insight into the transcriptome landscape of the developing olive fruit fly embryo.

Maleness-on-the-Y (MoY) orchestrates male sex determination in major agricultural fruit fly pests.

In insects, rapidly evolving primary sex-determining signals are transduced by a conserved regulatory module controlling sexual differentiation. In the agricultural pest Ceratitis capitata (Mediterranean fruit fly, or Medfly), we identified a Y-linked gene, Maleness-on-the-Y (MoY), encoding a small protein that is necessary and sufficient for male development. Silencing or disruption of MoY in XY embryos causes feminization, whereas overexpression of MoY in XX embryos induces masculinization. Crosses between transformed XY females and XX males give rise to males and females, indicating that a Y chromosome can be transmitted by XY females. MoY is Y-linked and functionally conserved in other species of the Tephritidae family, highlighting its potential to serve as a tool for developing more effective control strategies against these major agricultural insect pests.

Housekeeping in Tephritid insects: the best gene choice for expression analyses in the medfly and the olive fly.

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, ß-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 for granted and demonstrates the caution that is needed in the choice of the appropriate HKGs. Finally, by estimating the average of a standard score of the stability values resulted by the three programs we were able to provide a useful consensus key for the choice of the best HKG combination in various tissues of the two insects.

Achilles, a New Family of Transcriptionally Active Retrotransposons from the Olive Fruit Fly, with Y Chromosome Preferential Distribution.

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 Bactrocera oleae, 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 BEL-Pao superfamily, however, its sequence comparison with the other members of the superfamily suggests that it constitutes a new family that we termed Achilles. 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. In situ hybridization to the B. oleae polytene chromosomes showed that Achilles 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 Achilles 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 Achilles-like elements in different species of the Tephritidae family suggests an ancient origin of this element.

Molecular characterization and chromosomal distribution of a species-specific transcribed centromeric satellite repeat from the olive fruit fly, Bactrocera oleae.

Satellite repetitive sequences that accumulate in the heterochromatin consist a large fraction of a genome and due to their properties are suggested to be implicated in centromere function. Current knowledge of heterochromatic regions of Bactrocera oleae genome, the major pest of the olive tree, is practically nonexistent. In our effort to explore the repetitive DNA portion of B. oleae genome, a novel satellite sequence designated BoR300 was isolated and cloned. The present study describes the genomic organization, abundance and chromosomal distribution of BoR300 which is organized in tandem, forming arrays of 298 bp-long monomers. Sequence analysis showed an AT content of 60.4%, a CENP-B like-motif and a high curvature value based on predictive models. Comparative analysis among randomly selected monomers demonstrated a high degree of sequence homogeneity (88%-97%) of BoR300 repeats, which are present at approximately 3,000 copies per haploid genome accounting for about 0.28% of the total genomic DNA, based on two independent qPCR approaches. In addition, expression of the repeat was also confirmed through RT-PCR, by which BoR300 transcripts were detected in both sexes. Fluorescence in situ hybridization (FISH) of BoR300 on mitotic metaphases and polytene chromosomes revealed signals to the centromeres of two out of the six chromosomes which indicated a chromosome-specific centromeric localization. Moreover, BoR300 is not conserved in the closely related Bactrocera species tested and it is also absent in other dipterans, but it's rather restricted to the B. oleae genome. This feature of species-specificity attributed to BoR300 satellite makes it a good candidate as an identification probe of the insect among its relatives at early development stages.

Spinosad resistance development in wild olive fruit fly Bactrocera oleae (Diptera: Tephritidae) populations in California.

BACKGROUND: Among target pests of the insecticide spinosad is the olive fruit fly, Bactrocera oleae (Rossi) (Diptera: Tephritidae). In Cyprus, spinosad has been sporadically used since its registration in 2002, whereas in Greece its use has been very limited since its registration in 2004, particularly in biological olive cultivars in Crete. By contrast, in California it has been the only insecticide used against the olive fruit fly since its registration in 2004. This study aimed at examining the resistance status of the olive fruit fly to spinosad. RESULTS: Populations from California, Greece and Cyprus, plus a laboratory population, were tested. Bioassays were performed by oral or topical application of different concentrations of the insecticide. Cypriot populations demonstrated no resistance as compared with that of the laboratory population. Among the Greek populations, only one from Crete demonstrated a fourfold increase in resistance, whereas five populations from California demonstrated a 9-13-fold increase. CONCLUSION: The observed resistance increase was associated with spinosad applications in the respective areas. These values are relatively low and do not yet pose a serious control problem in the field. However, the observed variation documents that spinosad tolerance has increased in areas where the insecticide has been more extensively used.