The bat flies P. jenynsii were collected from the host eastern bent-winged bats M. fuliginosus caught at Kawabe County, Hyogo Prefecture, Japan, on 14 May and 16 September 2022 in field bat monitoring conducted by Nobutaka Urano and Shunji Fujita (permission number 環近地野許第 2107301 号, the Ministry of the Environment, Japan). The collected bat flies were placed in plastic tubes with wet tissue paper, kept in a cooling box, brought to laboratory, and used for experiments.

Each adult female P. jenynsii was fixed to a dissection stage assembled with a silicon rubber disk and a plastic Petri dish by pinning at the center of its flat thorax. Legs of the insect were removed using ophthalmological microscissors in a phosphate buffered saline (PBS: 0.8% NaCl, 0.02% KCl, 0.115% Na₂HPO₄, 0.02% KH₂PO₄, pH 7.4), and their dorsal abdomen was cut open along the midline to expose the internal organs of the insect. Fat bodies, milk glands, and digestive tract were all carefully removed not to break the stomach full of blood meal, by which whitish bacteriomes packed in the paired projections on the fifth ventral plate were exposed and carefully collected using fine forceps. In total, 60 bacteriomes from 30 insects were collected and subjected to DNA extraction using a DNA tissue mini kit (Qiagen).

The DNA sample extracted from the bacteriomes was subjected to the library preparation for long-read sequencing by using Ligation sequencing kit SQK-LSK109 (Oxford Nanopore Technologies). The library prepared was analyzed on a MinION sequencer implemented with the flow cell R9.4.1 (Oxford Nanopore Technologies). The DNA sample was also subjected to short-read sequencing. The short-read library was prepared using NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolab), and 150 bp paired-end reads were obtained by NovaSeq 6000 (Illumina). Because excessive short-read data were obtained, a 10% subset was extracted from the original read library. Furthermore, the subset was quality-trimmed on CLC genomics workbench version 23 (Qiagen). A short- and long-read hybrid assembling was conducted by using Unicycler version 0.5.0 (Wick et al., 2017). Assemble errors in the resultant draft genome sequence were searched and manually corrected with the assistance of CLC genomics workbench. The circularity of the assembled genome was confirmed by mapping the long reads to the rotated contigs and checking the resultant read maps. Annotation of the corrected genome was performed using DFAST version 1.2.18 (Tanizawa et al., 2018). The BUSCO scores were calculated with the enterobacterales_odb10 database using BUSCO version 5.5.0 (Manni et al., 2021). The genome map was drawn using DNAplotter in Artemis version 18.0.0 (Carver et al., 2009).

Deduced protein sequences inferred from the Aschnera genome and 31 bacterial genomes retrieved from DNA databases (GenBank, accessed in July, 2023) were prepared from the annotated genome sequences using CLC genomic workbench. BCGtree pipeline (Ankenbrand and Keller, 2016) was used to assemble and concatenate the multiple alignments of 107 bacterial core protein genes from 32 bacterial genomes with the following parameter: –min-proteomes = 2. The resultant concatenated alignment and corresponding partition information were used to infer the maximum likelihood tree with 1,000 bootstrap replicates using IQ-TREE version 2.2.2.7 (Minh et al., 2020) with the following parameter: -m MFP+MERGE. The phylogenetic tree was visualized using TreeViewer version 2.1.0 (Bianchini and Sánchez-Baracaldo, 2023).

The metabolic capability of Aschnera was predicted based on the genome sequence using GhostKOALA web service (https://www.kegg.jp/ghostkoala/) (Kanehisa et al., 2016). The metabolic models of the endosymbiont of human louse, Riesia pediculicola strain USDA (T number of the KEGG database: T01218), the endosymbiont of louse fly Arsenophonus lipoptenae strain CB (T04248), the endosymbiont of tsetse fly Wigglesworthia glossinidia (T00101), the endosymbiont of bedbug Wolbachia sp. strain wCle (T03628), the endosymbiont of aphid Buchnera aphidicola strain APS (T00036), the gut symbiont of plataspid stinkbug Ishikawaella capsulata (T04149), and Escherichia coli strain K-12 MG1655 (T00007) were acquired from the KEGG database (https://www.kegg.jp/) (Kanehisa et al., 2022). The metabolic pathways were compared essentially based on KEGG modules with references to the EcoCyc database (Keseler et al., 2021).

The abdomen of each adult female P. jenynsii was cut to make a slit at the midline using ophthalmological microscissors in PBS. Then, the insects were fixed in PBS containing 4% paraformaldehyde at 4°C overnight. After several washes with PBS, the specimens were subjected to wFISH as previously described (Koga et al., 2009). The specimens were hybridized with Al555-EUB338 (5'-Alexa Fluor 555- GCT GCC TCC CGT AGG AGT-3') targeting bacterial 16S ribosomal RNA. After overnight hybridization at room temperature, the specimens were washed with PBT (PBS containing 0.1% Tween-20) containing 165 nM Alexa Fluor 488 phalloidin and 2 μg/mL 4',6-diamidino-2-phenylindole (DAPI) for 30 min at room temperature and subsequently washed with PBT three times. The hybridized specimens were dissected and pictured in PBS under a fluorescence dissection microscope M165 controlled by LAS version 4.13.0 (Leica). The bacteriomes on the abdominal cuticle were mounted in PBS-50% glycerol and observed with a laser scanning microscope LSM700 controlled with Zen 2011 version 7.0 (Zeiss). Digital images were manually manipulated using Affinity Photo version 2.1.1 (Affinity).

The bat fly P. jenynsii (Figure 1A) is a blood-sucking ectoparasite of the eastern bent-winged bat M. fuliginosus (Figure 1B). On the ventral side of the abdomen of a female insect, the fifth ventral plate bears paired projections on both sides (Figure 1C), each of which contains the bacteriome consisting of the bacteriocytes harboring the symbiotic bacteria (Hosokawa et al., 2012). When the abdomen of a female insect was dissected from the dorsal side (Figure 1D) and deprived of the internal organs (alimentary tract, gonad, fat body, etc.), the bacteriome was seen as a whitish cell mass inside each of the projections (Figure 1E). FISH targeting bacterial 16S rRNA detected the symbiont signals within the bacteriomes in the abdominal projections (Figures 1F, G). The bacteriomes were carefully isolated (Figure 1H), wherein the symbiotic bacteria within numerous bacteriocytes were visualized using FISH (Figure 1I).

The isolated bacteriomes collected from 30 adult female P. jenynsii were subjected to DNA extraction, library construction, long- and short-read DNA sequencing, and genome assembly, whereby the complete Aschnera genome was determined as a 748,020 bp circular chromosome and a 18,747 bp circular plasmid. The latter was identified as the plasmid because of the presence of the replication initiation protein A (repA) gene in this contig. The genome size approximately agreed with a previous estimation of the genome size of Aschnera as 0.76 Mbp by pulsed field gel electrophoresis (Hosokawa et al., 2012). The 0.75 Mbp chromosome, consisting of 23.1% G + C, encoded 603 putative protein-coding sequences (CDSs), of which 3 were predicted as pseudogenes, 33 transfer RNAs, and 1 copy of 16S/23S/5S ribosomal RNA operon (Figure 2; Table 1; Supplementary Table S1). The 18 kbp plasmid, consisting of 22.3% G + C, encoded only 10 genes, whose biological relevance was elusive (Figure 2; Table 1; Supplementary Table S2). The Aschnera genome, 0.77 Mbp in size, is around 1/5–1/7 of the E. coli genomes of 4–6 Mbp (Lukjancenko et al., 2010). The BUSCO score of A. chinzeii was 81.1%. This score was higher than those of R. pediculicola USDA (66.8%) and R. sp. GBBU (61.8%). These results confirmed that Aschnera has experienced reductive genome evolution in the process of intimate co-evolution with nycteribiid bat flies (Hosokawa et al., 2012).

Molecular phylogenetic analysis based on 109 bacterial core protein sequences revealed that Aschnera is placed in the γ-proteobacterial clade consisting of Arsenophonus and allied bacterial lineages that embraces many insect-associated endosymbiotic bacteria (Nováková et al., 2009) (Figure 3). Among them, Aschnera was closely related to the endosymbionts of primate lice Riesia spp. (Kirkness et al., 2010; Boyd et al., 2014) and other endosymbionts of blood-sucking insects, such as Lightella of rodent lice (Ríhová et al., 2022), Puchtella of monkey lice (Boyd et al., 2017), and Wigglesworthia of tsetse flies (Akman et al., 2002; Rio et al., 2012). Arsenophonus endosymbionts of louse flies (Nováková et al., 2016) were placed outside of them in the phylogenetic tree. These phylogenetic patterns strongly suggested that although tsetse flies (Glossinidae), louse flies (Hippoboscidae), and bat flies (Nycteribiidae) belong to the same well-defined superfamily Hippoboscoidea (Petersen et al., 2007), their current endosymbiotic bacterial associates are likely of independent evolutionary origins presumably via symbiont acquisitions and replacements, as previously suggested (Nováková et al., 2009; Hosokawa et al., 2012; Duron et al., 2014; Ríhová et al., 2023).

In accordance with the drastic genome reduction, the Aschnera genome has lost many important metabolic pathways (Figure 4). For example, synthetic pathway genes for purines and pyrimidines are mostly missing. Strikingly, synthetic pathway genes for essential amino acids are completely lost from the Aschnera genome, which was also observed in the primary endosymbionts of other blood-sucking insects, including Riesia of human louse, Arsenophonus of louse fly, Wigglesworthia of tsetse fly, and Wolbachia of bedbug (Figure 4). These patterns may be relevant to the feeding habit of the host bat fly P. jenynsii as an obligatory blood-sucking ectoparasite of bats (Funakoshi, 1977; Lehane, 2005), considering that the blood meal is very rich in proteins and nucleic acids.

On the other hand, the blood meal is deficient in some nutrients, such as B vitamins. Therefore, obligatory blood-feeding insects, such as tsetse flies, lice, and bedbugs, rely on their specific bacterial symbiont for supply of B vitamins (Akman et al., 2002; Kirkness et al., 2010; Nikoh et al., 2014). Consequently, such symbiont genomes retain the synthetic pathway genes for some, if not all, vitamins and cofactors (Rio et al., 2016; Husnik, 2018; Duron and Gottlieb, 2020; Fukatsu et al., 2023). The Aschnera genome retained complete or near-complete synthetic pathway genes for biotin (vitamin B7), tetrahydrofolate (vitamin B9), riboflavin (vitamin B2), and pyridoxal 5'-phosphate (vitamin B6) (Figures 5A–E). The Aschnera genome also encoded synthetic pathway genes for such cofactors as coenzyme A (from pantothenate) and nicotinamide adenine dinucleotide (NAD) (from nicotinate) (Figures 5A, F, G). On the other hand, the Aschnera genome was incapable of synthesizing thiamine (vitamin B1) and pantothenate (vitamin B5) (Figures 5A, F, H). Based on these results, it seems plausible that Aschnera provides the host insect with biotin, pyridoxal, riboflavin, and tetrahydrofolate to compensate for nutritional deficiency of the blood meal. It is notable that not only Aschnera of the bat fly but also Riesia of human lice, Arsenophonus of louse flies, and Wigglesworthia of tsetse flies exhibit similar retention patterns of the synthesis pathway genes for B vitamins (Figure 5A), which may be either due to convergent evolution in the blood-sucking host insects or reflecting the genomic architecture of γ -proteobacterial Arsenophonus-allied bacteria. In this context, it seems meaningful that the α-proteobacterial mutualistic Wolbachia endosymbiont of bedbug retains synthesis pathway genes for biotin and riboflavin only (Figure 5A).