Characterization of gut symbionts from wild-caught Drosophila and other Diptera: description of Utexia brackfieldae gen. nov., sp. nov., Orbus sturtevantii sp. nov., Orbus wheelerorum sp. nov, and Orbus mooreae sp. nov

Abstract

Non-culture based surveys show that the bacterial family Orbaceae is widespread in guts of insects, including wild Drosophila. Relatively few isolates have been described, and none has been described from Drosophila. We present the isolation and characterization of five strains of Orbaceae from wild-caught flies of the genera Drosophila (Diptera: Drosophilidae) and Neogriphoneura (Diptera: Lauxaniidae). Cells are generally rod-shaped, mesophilic, and measure 0.8-2.0 µm long by 0.3-0.5 µm wide. Optimal growth was observed under ambient atmosphere. Reconstruction of phylogenies from the 16S rRNA gene and from single-copy orthologs verify placement of these strains within Orbaceae. Cells exhibited similar fatty acid profiles to those of other Orbaceae. Strain lpD01^T shared 74% average nucleotide identity (ANI) with its closest relatives Ca. Schmidhempelia bombi Bimp and Zophobihabitans entericus IPMB12^T. Results from multiple genome-wide similarity comparisons indicate lpD01^T should be classified as a novel species within a novel genus. The major respiratory quinone for lpD01^T is ubiquinone Q-8. lpD02^T, lpD03, lpD04^T, and BiB^T are more closely related to Orbus hercynius CN3^T (76, 77, 76, and 77% ANI, respectively) than to other described Orbaceae. Genomic and phylogenetic analyses suggest that lpD03 and lpD04^T belong to the same species and that lpD02^T, lpD03/lpD04^T, and BiB^T are each novel species of the genus Orbus. The proposed names of these strains are Utexia brackfieldae gen. nov., sp. nov. (type strain lpD01^T =NCIMB 15517^T =ATCC TSD-399^T), Orbus sturtevantii sp. nov (type strain lpD02^T =NCIMB 15518^T =ATCC TSD-400^T), Orbus wheelerorum sp. nov. (type strain lpD04^T =NCIMB 15520^T =ATCC TSD-401^T), and Orbus mooreae sp. nov (type strain BiB^T=NCIMB 15516^T =ATCC TSD-402^T). The isolation and characterization of these strains expands the repertoire of culturable bacteria naturally associated with insects, including the model organism D. melanogaster.

Keywords: insect microbiome; Drosophila; Orbaceae; host specificity; host-associated microbiota; symbiosis.

Fig. 1.. Maximum likelihood phylogeny of the Orbales based on the 16S rRNA gene; the Enterobacterales and Pasteurellales are included as outgroups. The presently-described strains are indicated in bold with an arrow. Parentheses indicate NCBI accession numbers except for DC005 and DC022, which are from reference [3], and DM001 and DM012, which are from reference [2]. The source of isolation is indicated in brackets, and the corresponding host order is indicated according to colour. * = putatively fly-associated [6]. The scale bar represents 0.1 nucleotide substitutions per site.

Fig. 2.. Maximum likelihood phylogeny of Orbaceae based on 856 single copy orthologs and corresponding average nucleotide identity (ANIb) values. Accession numbers for assemblies are available in Table 1. Nodes with 100% bootstrap support are indicated with a black circle. The phylogeny scale bar represents 0.1 nucleotide substitutions per site. ANIb values below the suggested species cutoff of 96% are indicated in shades of red, and ANIb values at or above the suggested species cutoff are indicated in white or blue, respectively.

Fig. 3.. Scanning electron micrographs of (a), lpD01^T; (b), lpD02^T; (c), lpD03; (d), lpD04^T; and (e), BiB^T grown for 2 days at 30 °C. Scale bar=1.0 µm.