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

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

Primer sequences and parameters of the RT-PCR and qPCR assay.

<p>Primer sequences and parameters of the RT-PCR and qPCR assay.</p

Analysis of BoR300 transcription.

<p>Total RNA from male (lane 1) and female (lane 2) adult <i>B. oleae</i> flies were extracted and reverse-transcribed using random oligonucleotides. Satellite transcripts were amplified by PCR using BoR300-F and BoR300-R primers. M represents the molecular marker (1000 bp/1 kb BLUE DNA Ladder, GeneON). The epic175F and epic175R primers were also used, to check the presence of any DNA contamination on both male and female cDNAs (lanes 4 & 5 respectively). The amplification results were compared to those obtained using the genomic DNA as template (lane 7) at which the product size was 550 bp.</p

qPCR analysis data of the relative estimation of BoR300 repeats using a reference standard curve.

a<p>Initial template concentration of the <i>B</i>. <i>oleae</i> genomic DNA (pg) used at the qPCR reactions.</p>b<p>Mean copy number of BoR300 repeats which was estimated for the initial template concentration of the genomic DNA (a) based on the standard curve.</p>c<p><i>B. oleae</i> haploid genome size: 0.352 pg.</p>d<p>Standard Error (SE) for the triplicate measurements (n = 3).</p

Species distribution of BoR300 repeats.

<p>HaeIII-digested genomic DNA of representative species of the Diptera family and the genus Bactrocera was analyzed by Southern hybridization using as probe the biotinylated monomer (298 bp) of the repeat. (<b>A</b>) Southern blot analysis of digested genomic DNA from the following dipteran species: <i>B. oleae</i> (lane 1), <i>C. capitata</i> (lane 2), <i>D. melanogaster</i> (lane 3) and <i>An. gambiae</i> (lane 4). <b>B</b>) Southern blot analysis of digested genomic DNA from the following Bactrocera species: <i>B. oleae</i> (lane 1), <i>B. correcta</i> (lane 3), <i>B. cucurbitae</i> (lane 4), and <i>B. dorsalis</i> (lane 5). In Lane 2 is the cloned monomer of the repeat. M represents the molecular marker (SM0331, Fermentas).</p

FISH with the BoR300 probe on mitotic and polytene nuclei of <i>Bactrocera oleae.</i>

<p>Chromosomes were counterstained with DAPI (blue). Female (a) and male (b) metaphase showing strong hybridization signals (red) on the centromeres of chromosomes 4 and 5. Polytene complement (c) showing strong hybridization signals (red) on the centromeric heterochromatic blocks (C) of chromosomes III and IV (arrows). Bar  = 3 µm (a, b), 20 µm (c).</p

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

Standard curves for the reference single copy gene (<i>ace</i>) and the repeats of BoR300.

<p>The construction of the curves was based on serial 10-fold dilutions of the genomic DNA template used (10 pg, 100 pg, 1 ng). For each amplicon, qPCR determined Ct values were plotted against the logarithm of their initial concentration (1, 2 and 3 values respectively).</p

CENP-B like motif.

<p>Comparison of the CENP-B like motif (237–252 bp) found in BoR300 with the degenerate motif considered to bind the CENP-B protein.</p