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.

Knocking down the sex peptide receptor by dsRNA feeding results in reduced oviposition rate in olive fruit flies.

Insect pests can cause crop damage in yield or quality, resulting in profit losses for farmers. The primary approach to control them is still the use of chemical pesticides resulting in significant hazards to the environment and human health. Biological control and the sterile insect technique are alternative strategies to improve agriculture protection. However, both strategies have significant limitations. A newly introduced approach that could be both effective and species-specific is the RNA interference mechanism. One key point for the success of this strategy is the delivery method of double-strand RNA (dsRNA) to the insects. A method of dsRNA delivery to insects with potential use in the field is the oral delivery, feeding the insects engineered microorganisms that produce dsRNA. Here, we present the first protocol for dsRNA feeding using modified bacteria, in the olive fruit fly, the most important insect pest of cultivated olives. We chose to target the sex peptide receptor gene. The sex peptide receptor interacts with the sex peptide, a peptide that is responsible for the postmating behavior in the model organism, Drosophila melanogaster. Feeding the female olive fruit fly with bacteria that produced dsRNA for the sex peptide receptor gene resulted in the development of female insects with significantly lower oviposition rates. Administration of dsRNA producing bacteria in insect diet against target genes that lead to genetic sexing or female-specific lethality could be added in the armory of control methods.

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.

Effect of macronutrients and of anaerobic digestate on the heterotrophic cultivation of Chlorella vulgaris grown with glycerol.

The aim of this work was to investigate the kinetics of the heterotrophic growth of Chlorella vulgaris as a means of producing bio-oil for biodiesel production. Glycerol was used as the sole organic carbon substrate. Anaerobic digestate from a local plant was used to examine its effect on the kinetics and the protein and lipid content of the biomass. The effect of the initial carbon and nitrogen concentrations on the carbon uptake rate was studied independently. In the one set of five experiments, the organic carbon in the form of glycerol varied from 0.27 to 5.36 g L-1, while the concentration of atomic nitrogen was held constant and equal to 45.4 mg L-1. The Co/No ratio varied from 6 to 118.1. In the second set, also of five experiments, the organic carbon was held constant and equal to 3.3 g L-1 and atomic nitrogen varied from 22.7 to 450 mg L-1. The Co/No ratio varied from 7.3 to 145.4. In the third set of experiments, anaerobic digestate was added in increasing amounts into the culture media from 4 to 16%. It was found that the carbon uptake rate as well as the lipid and protein content depended on the Co/No ratio. Increasing ratios of Co/No led to higher carbon uptake rates, higher lipid content, and lower protein content. The initial nitrogen concentration was also found to affect the growth rate of C. vulgaris. The addition of anaerobic digestate did not affect appreciably the protein and lipid content of the biomass, while the addition of anaerobic digestate up to 16% in the culture medium increased the carbon uptake rate by about 24%.

A species-specific lncRNA modulates the reproductive ability of the asian tiger mosquito.

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 reduction, in smaller ovaries and 53% hatching reduction of the laid eggs, compared to anti-GFP controls. Moreover, a significant downregulation of 2 mucins withing a neighboring (∼100 Kb) mucin cluster was observed in smaller anti-Norma3 ovaries, indicating a potential mechanism of in-cis regulation between Norma3 and the mucins. Our work constitutes the first experimental proof-of-evidence connecting lncRNAs with mosquito reproduction and opens a novel path for pest management.

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.