Draft Genome Sequence of the Bactrocera oleae Symbiont “Candidatus Erwinia dacicola”

“Candidatus Erwinia dacicola” is a Gammaproteobacterium that forms a symbiotic association with the agricultural pest Bactrocera oleae. Here, we present a 2.1-Mb draft hybrid genome assembly for “Ca. Erwinia dacicola” generated from single-cell and metagenomic data.

T he association between Bactrocera oleae (Tephritidae) and a bacterial symbiont was first discovered in 1909 (1). Several studies have since identified this organism as "Candidatus Erwinia dacicola" (Enterobacteriaceae) (2)(3)(4). "Ca. Erwinia dacicola" plays a role in nutrient provisioning, particularly during juvenile development in unripe olives, where it is essential for larval survival (5)(6)(7)(8). However, due to a lack of genomic resources and the inability to culture "Ca. Erwinia dacicola," the metabolic basis of its association with B. oleae remains elusive. We present a draft of the "Ca. Erwinia dacicola" genome sequence that will inform future investigations into the functional and evolutionary foundations of the symbiosis.
Multiple single-cell (eight) and metagenomic (two) libraries were used to generate a draft hybrid genome assembly of "Ca. Erwinia dacicola." Single-cell libraries were prepared from the guts of adult female flies collected in Heraklion, Greece, stained with CellTracker deep red, and sorted on a Sony SH800. Genomic DNA was amplified using the REPLI-g kit (Qiagen) and was validated as "Ca. Erwinia dacicola" by amplification of the 16S rRNA gene, followed by digestion with the restriction enzyme PstI (9). Shotgun libraries were then prepared with the NEBNext Ultra DNA kit (New England Biosciences) and sequenced on an Illumina MiSeq sequencer at the Centre for Genomic Research, University of Liverpool, United Kingdom. Metagenomic shotgun and mate-pair (2-to 6-kb) libraries were prepared from gastric ceca dissected from third-instar larvae isolated in Israel from unripe olives and from a mixture of ripe and unripe olives, respectively. DNA for the shotgun library was extracted using an adapted cetyltrimethylammonium bromide (CTAB) method (10) with additional bead beating and lysozyme digestion, and the library was prepared with the Ovation rapid DR multiplex system (NuGen). The mate-pair library was prepared with the gel-free NexteraMate protocol from DNA extracted with the Chemagic DNA bacteria kit (Chemagen). Both libraries were prepared and sequenced on an Illumina MiSeq sequencer by LGC Genomics GmbH (Berlin, Germany).
Reads were assembled with SPAdes version 3.7.1 (11) in singlecell mode. Using Blobology (12), contigs identified as belonging to other organisms based on coverage and GϩC content were excluded, and the 12,519,932 reads that mapped back to putative "Ca. Erwinia dacicola" contigs were extracted and reassembled with SPAdes. The result was a 2.1-Mb assembly comprising 333 scaffolds (Ͼ500 bp) at~1,000ϫ coverage, with an N 50 of 9,998 bp. The assembly was assessed as in reference 13 and was found to be 92% complete in comparison to free-living bacteria and 100% complete in comparison to the endosymbiotic bacteria of aphids and tsetse flies, Buchnera aphidicola and Wigglesworthia glossinidia, respectively. Its GϩC content (53.5%) is similar to that observed in other members of the Erwinia genus (14) and higher than that of other vertically transmitted endosymbiotic bacteria (15). The genome contains 2,407 protein-coding genes and 28 RNA-coding genes, based on annotation with PROKKA version 1.5.2 (16).
Accession number(s). This whole-genome shotgun project has been deposited at DDBJ/ENA/GenBank under the accession no. MAZB00000000. The version described in this paper is version MAZB01000000 and BioProject no. PRJNA326914.

ACKNOWLEDGMENTS
We thank Anastasia Kampouraki for technical support and sample collection. The single-cell isolation and sequencing were conducted in the Single Cell Lab at the Centre for Genomic Research, University of Liverpool.
The funders had no role in the study design, data analysis, decision to publish, or preparation of the manuscript.