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Page 6 of 16 Stuehler et al. Microbiome Res Rep. 2025;4:19 https://dx.doi.org/10.20517/mrr.2024.84

Figure 1. Unrooted Maximum Likelihood tree for Wolbachia MLST concatenated sequences supporting placement of wLfag-FL in
Supergroup B. MLST: Multilocus sequence typing; wLfag-FL: Wolbachia endosymbiont of Leuronota fagarae.

Transcriptome analysis
Of the 1,359 transcripts mapped to reference Wolbachia genomes by HISAT2, only six sequences did not
match Wolbachia through BLASTn to NCBI nt; however, these 6 did match Wolbachia sequences through
BLASTx to NCBI nr with the greatest E-value reported as 4e-08. To assess transcriptome completeness of
the assembled Wolbachia cDNA sequences, Benchmark Universal Single Copy Ortholog (BUSCO) v5.2.2
[26]
was used . In total, 1,332 non-redundant transcripts were compared against the rickettsiales_odb10
database, using transcriptome mode. BUSCO calculated only 37 Complete BUSCOs (10.7%), 14 Fragmented
BUSCOs (4.1%), and 294 Missing BUSCOs (85.2%). Seqkit v0.16.1 was used to calculate a GC content of
[35]
34.8% and an N50 value of 348 nt for Trinity assembled Wolbachia transcripts. When aligning the wLfag-FL
transcripts to the annotated sequences of wDcit dawsonii and the wSpic, we identified 634 and 560
alignments to unique transcripts, respectively, and revealed, on average, a 45% annotation rate of our wLfag-
FL Trinity transcripts. Roughly 2.5 times more Wolbachia sequences were recovered from the female L.
fagarae dataset compared to orthologs recovered in the male dataset. To visualize sequence comparisons
between wLfag-FL and other closely related Wolbachia strains, we used AliTv and the Blast Ring Image
[36]
[37]
Generator (BRIG) . An AliTv alignment mapping figure was generated with concatenated wLfag-FL
transcripts, post CAP3 assembly, and Wolbachia reference genomes Spodoptera picta Spic B and Diaphorina
citri dawsonii [Figure 3]. To generate the BRIG image [Figure 4], Supergroup B Wolbachia genomes from
the MLST phylogeny were reused.