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Stuehler et al. Microbiome Res Rep. 2025;4:19 https://dx.doi.org/10.20517/mrr.2024.84 Page 7 of 16

Figure 2. Unrooted Maximum Likelihood tree for Wolbachia wsp sequences highlighting the placement of wLfag-FL in Supergroup B with
a distinct evolutionary lineage. wLfag-FL: Wolbachia endosymbiont of Leuronota fagarae.

Functional annotation
As a result of the KEGG ontology analysis, 465 transcripts were assigned a K number, of which 279 were
unique of the 828 total sequences examined. The map pathway function available in the KO Database was
used to group the unique K number assignments into their associated pathways. The KEGG Orthology
Metabolic pathway (ko01100) contained the highest number of assignments with 108 sequences. The bar
graph [Figure 5] depicts an abundance of 13 KEGG Orthology pathways containing at least 10 sequence
assignments. Supplementary Table 1 contains a complete list of K numbers from all unique wLfag-FL
sequences assigned to 146 pathways, as well as their assigned sequence names. Analysis of ANK domain-
containing proteins performed on cDNA sequences of wLfag-FL revealed five sequences containing ankyrin
domains. This was expected though, as BUSCO analysis revealed only 10.7% of the endosymbiont’s
transcriptome was recovered from the four datasets, and that other closely related Wolbachia strains of
Supergroup B contain around 50-60 ANK domain proteins. Our identification of 5 ANK domain-
containing proteins is consistent with our BUSCO score and average number of ANK domain proteins in
Supergroup B strains which predicts that wLfag-FL encodes roughly 50 ANK domain proteins.

The reproductive advantages provided by Wolbachia-host symbiosis are critical aspects of the symbiotic
relationship; however, Wolbachia provides another method to increase host fitness by supporting
nutritional provisioning. A 5’ portion of the Wolbachia bacterioferritin CDS was present in wLfag-FL,
having the closest similarity to bacterioferritin characterized in the Wolbachia endosymbiont of
Homalodisca vitripennis (MBR9983968.1). To verify the identity of the partial bacterioferritin transcript, we
used ExPASy translate to identify the correct protein sequence and aligned previously characterized
bacterioferritin protein sequences, discovered by methods other than bioinformatic analysis, with MUSCLE.
We found the highly conserved residues within the ferroxidase center to align between all 8 sequences