Wolbachia pipientis is a widespread obligate intracellular bacterium and an effective biocontrol agent being deployed in the field against mosquito-borne viral diseases. Investigating the transcriptomic dynamics of Wolbachia within host cells is highly desired but is fundamentally challenged by the overwhelming abundance of eukaryotic host RNA and bacterial ribosomal RNA. Here, we demonstrate that Cappable-seq specifically captures the active, primary transcriptome of Wolbachia while drastically reducing host background. We applied this methodology to monitor Wolbachia gene expression changes over a single host cell passage cycle, tracking the transition from early infection to a high-titer maintenance phase. Differential gene expression analysis identified the upregulation of rpoH transcription factor, stress-response chaperones and WO prophage elements. This was coupled with a continuous downregulation of key host-manipulation effectors, including Ankyrin-repeat domain-containing proteins and secretion systems, alongside the broad suppression of metabolic transporters over the time-course. In addition, we discovered that Cappable-seq concurrently co-enriches the transcripts and genomes from various single-stranded RNA viruses that are known to be present in these mosquito cells. In summary, Cappable-seq technology provides a robust framework to investigate complex, tripartite host-microbe-virus interactions, demonstrating that Wolbachia actively senses and adapts to the dynamic constraints of its intracellular environment by regulating its transcriptome.

Bacterial endosymbionts are highly prevalent among invertebrate animals, in which they can confer fitness benefits such as pathogen defence and/or act as reproductive manipulators, inducing phenotypes including cytoplasmic incompatibility (CI). For the alpha-proteobacterium Wolbachia, its wide distribution among macroparasites and disease-transmitting arthropods coupled with mutualistic roles, reduction of vector competence and CI has found recent applications in the control of several vector-borne tropical diseases. However, in common with other bacterial endosymbionts, which often lose regulatory elements during genomic erosion, the degree to which Wolbachia can respond to environmental or pharmacological stressors is poorly understood. Here, we apply Cappable-Seq methodology to achieve unprecedented depth and resolution of transcriptional start sites (TSS) in two Wolbachia strains (wMelPop-CLA and wAlbB) that have been used to transinfect mosquitoes for arbovirus control. We exposed Wolbachia in mosquito cell lines to temperature stress (both strains) or antibiotics (wAlbB only) and observed that all classes of TSS (including antisense) exhibited differential regulation, some of which were associated with mobile elements and may control ncRNA expression. Of the three antibiotics used as pharmacological stressors (doxycycline, rifampicin and moxifloxacin), doxycycline had the greatest impact on differential expression from primary TSS. Cappable-Seq also resolved the organization of the bicistronic cifA/cifB operon that is responsible for inducing CI in Wolbachia hosts. The use of Cappable-Seq in this study enabled the resolution of the primary transcriptome of an obligate intracellular bacterium in unparalleled detail. Moreover, this methodology shows great promise for revealing regulation of symbiont functions in whole invertebrates.

BACKGROUND:Trombiculid mites are globally distributed, highly diverse arachnids that largely lack molecular resources such as whole mitogenomes for the elucidation of taxonomic relationships. Trombiculid larvae (chiggers) parasitise vertebrates and can transmit bacteria (Orientia spp.) responsible for scrub typhus, a zoonotic febrile illness. Orientia tsutsugamushi causes most cases of scrub typhus and is endemic to the Asia-Pacific Region, where it is transmitted by Leptotrombidium spp. chiggers. However, in Dubai, Candidatus Orientia chuto was isolated from a case of scrub typhus and is also known to circulate among rodents in Saudi Arabia and Kenya, although its vectors remain poorly defined. In addition to Orientia, chiggers are often infected with other potential pathogens or arthropod-specific endosymbionts, but their significance for trombiculid biology and public health is unclear.RESULTS:Ten chigger species were collected from rodents in southwestern Saudi Arabia. Chiggers were pooled according to species and screened for Orientia DNA by PCR. Two species (Microtrombicula muhaylensis and Pentidionis agamae) produced positive results for the htrA gene, although Ca. Orientia chuto DNA was confirmed by Sanger sequencing only in P. agamae. Metagenomic sequencing of three pools of P. agamae provided evidence for two other bacterial associates: a spirochaete and a Wolbachia symbiont. Phylogenetic analysis of 16S rRNA and multi-locus sequence typing genes placed the spirochaete in a clade of micromammal-associated Borrelia spp. that are widely-distributed globally with no known vector. For the Wolbachia symbiont, a genome assembly was obtained that allowed phylogenetic localisation in a novel, divergent clade. Cytochrome c oxidase I (COI) barcodes for Saudi Arabian chiggers enabled comparisons with global chigger diversity, revealing several cases of discordance with classical taxonomy. Complete mitogenome assemblies were obtained for the three P. agamae pools and almost 50 SNPs were identified, despite a common geographic origin.CONCLUSIONS:P. agamae was identified as a potential vector of Ca. Orientia chuto on the Arabian Peninsula. The detection of an unusual Borrelia sp. and a divergent Wolbachia symbiont in P. agamae indicated links with chigger microbiomes in other parts of the world, while COI barcoding and mitogenomic analyses greatly extended our understanding of inter- and intraspecific relationships in trombiculid mites.

Fosmids and cosmids are vectors frequently used in functional metagenomic studies. With a large insert capacity (around 30 kb) they can encode dozens of cloned genes or in some cases, entire biochemical pathways. Fosmids with cloned inserts can be transferred to heterologous hosts and propagated to enable screening for new enzymes and metabolites. After screening, fosmids from clones with an activity of interest must be de novo sequenced, a critical step toward the identification of the gene(s) of interest. In this work, we present a new approach for rapid and high-throughput fosmid sequencing directly from Escherichia coli colonies without liquid culturing or fosmid purification. Our sample preparation involves fosmid amplification with phi29 polymerase and then direct nanopore sequencing using the Oxford Nanopore Technologies system. We also present a bioinformatics pipeline termed "phiXXer" that facilitates both de novo read assembly and vector trimming to generate a linear sequence of the fosmid insert. Finally, we demonstrate the accurate sequencing of 96 fosmids in a single run and validate the method using two fosmid libraries that contain cloned large insert (similar to 30-40 kb) genomic or metagenomic DNA. IMPORTANCE Large-insert clone (fosmids or cosmids) sequencing is challenging and arguably the most limiting step of functional metagenomic screening workflows. Our study establishes a new method for high-throughput nanopore sequencing of fosmid clones directly from lysed Escherichia coli cells. It also describes a companion bioinformatic pipeline that enables de novo assembly of fosmid DNA insert sequences. The devised method widens the potential of functional metagenomic screening by providing a simple, high-throughput approach to fosmid clone sequencing that dramatically speeds the pace of discovery.

Supplementary Data from MLL-Rearranged B Lymphoblastic Leukemias Selectively Express the Immunoregulatory Carbohydrate-Binding Protein Galectin-1

While fleas are often perceived simply as a biting nuisance and cause of allergic dermatitis, they represent important disease vectors worldwide, especially for bacterial zoonoses such as plague (transmitted by rodent fleas) and some of the rickettsioses and bartonelloses. The cosmopolitan cat ( Ctenocephalides felis ) and dog ( C. canis ) fleas, as well as C. orientis (restricted to tropical and subtropical Asia), breed in human dwellings and are vectors of cat-scratch fever (caused by Bartonella spp.) and Rickettsia spp. of the so-called "transitional group". The latter includes R. felis (agent of flea-borne spotted fever) and R. asembonensis , an emerging pathogen. The relatively depauperate flea microbiome can also contain arthropod-specific endosymbionts, including a diverse range of Wolbachia strains. Here, we present circularized genome assemblies for two C. orientis -associated pathogens ( Bartonella clarridgeiae and R. asembonensis ) from Malaysia, a novel Wolbachia strain ( w Cori), and the C. orientis mitochondrion; all obtained by direct metagenomic sequencing of flea tissues. Moreover, we isolated two Wolbachia strains from Malaysian C. felis into tick cell culture and recovered circularized genome assemblies for both, one of which ( w CfeF) is newly sequenced. We demonstrate that the three Wolbachia strains are representatives of different major clades ("supergroups"), two of which appear to be flea-specific. These Wolbachia genomes exhibit unique combinations of features associated with reproductive parasitism or mutualism, including prophage WO, cytoplasmic incompatibility factors, and the biotin operon of obligate intracellular microbes. The first circularized assembly for R. asembonensis includes a plasmid with a markedly different structure and gene content compared to the published plasmid; moreover, this novel plasmid was also detected in cat flea metagenomes from the US. Analysis of loci under positive selection in the transitional group revealed genes involved in host-pathogen interactions that may facilitate host switching. Finally, the first B. clarridgeiae genome from Asia exhibited largescale genome stability compared to isolates from other continents, except for SNPs in regions predicted to mediate interactions with the vertebrate host. These findings highlight the paucity of data on the genomic diversity of Ctenocephalides -associated bacteria and raise questions regarding how interactions between members of the flea microbiome might influence vector competence.

Mpox is a neglected zoonotic disease endemic in West and Central Africa. The Mpox outbreak with more than 90,000 cases worldwide since 2022 generated great concern about future outbreaks and highlighted the need for a simple and rapid diagnostic test. The Mpox virus, MPV, is a member of the Orthopoxvirus (OPV) genus that also contains other pathogenic viruses including variola virus, vaccinia virus, camelpox virus, and cowpox virus. Phylogenomic analysis of 200 OPV genomes identified 10 distinct phylogroups with the New World OPVs placed on a very long branch distant from the Old World OPVs. Isolates derived from infected humans were found to be distributed across multiple phylogroups interspersed with isolates from animal sources, indicating the zoonotic potential of these viruses. In this study, we developed a simple and sensitive colorimetric LAMP assay for generic detection of Old World OPVs. We also developed an MPV-specific probe that differentiates MPV from other OPVs in the N1R LAMP assay. In addition, we described an extraction-free protocol for use directly with swab eluates in LAMP assays, thereby eliminating the time and resources needed to extract DNA from the sample. Our direct LAMP assays are well-suited for low-resource settings and provide a valuable tool for rapid and scalable diagnosis and surveillance of OPVs and MPV.

Background Mansonellosis is an undermapped insect-transmitted disease caused by filarial nematodes that are estimated to infect hundreds of millions of people. Despite their prevalence, there are many outstanding questions regarding the general biology and health impacts of the responsible parasites. Historical reports suggest that the Colombian Amazon is endemic for mansonellosis and may serve as an ideal location to pursue these questions. Methods We deployed molecular and classical approaches to survey Mansonella prevalence among adults belonging to indigenous communities along the Amazon River and its tributaries near Leticia, Colombia. Results Loop-mediated isothermal amplification (LAMP) assays on whole-blood samples detected a much higher prevalence of Mansonella ozzardi infection (approximately 40%) compared to blood smear microscopy or LAMP performed using plasma, likely reflecting greater sensitivity and the ability to detect low microfilaremias and occult infections. Mansonella infection rates increased with age and were higher among men. Genomic analysis confirmed the presence of M. ozzardi that clusters closely with strains sequenced in neighboring countries. We successfully cryopreserved M. ozzardi microfilariae, advancing the prospects of rearing infective larvae in controlled settings. Conclusion These data suggest an underestimation of true mansonellosis prevalence, and we expect that these methods will help facilitate the study of mansonellosis in endemic and laboratory settings.

The filarial parasites Mansonella ozzardi and Mansonella perstans , causative agents of mansonellosis, infect hundreds of millions of people worldwide, yet remain among the most understudied of the human filarial pathogens. M. ozzardi is highly prevalent in Latin American countries and Caribbean Islands, while M. perstans is predominantly found in sub-Saharan Africa as well as in a few areas in South America. In addition to the differences in their geographical distribution, the two parasites are transmitted by different insect vectors, as well as exhibit differences in their responses to commonly used anthelminthic drugs. The lack of genome information has hindered investigations into the biology and evolution of Mansonella parasites and understanding the molecular basis of the clinical differences between species. In the current study, high quality genomes of two independent clinical isolates of M. perstans from Cameroon and two M. ozzardi isolates one from Brazil and one from Venezuela are reported. The genomes are approximately 76 Mb in size, encode about 10,000 genes each, and are largely complete based on BUSCO scores of about 90%, similar to other completed filarial genomes. These sequences represent the first genomes from Mansonella parasites and enabled a comparative genomic analysis of the similarities and differences between Mansonella and other filarial parasites. Horizontal DNA transfers (HDT) from mitochondria (nuMTs) as well as transfers from genomes of endosymbiotic Wolbachia bacteria (nuWTs) to the host nuclear genome were identified and analyzed. Sequence comparisons and phylogenetic analysis of known targets of anti-filarial drugs diethylcarbamazine (DEC), ivermectin and mebendazole revealed that all known target genes were present in both species, except for the DEC target encoded by gon-2 gene, which is fragmented in genome assemblies from both M. ozzardi isolates. These new reference genome sequences will provide a valuable resource for further studies on biology, symbiosis, evolution and drug discovery.

NPM1 is among the most frequently mutated genes in acute myeloid leukemia (AML). Mutations in the NPM1 gene result in the increased export of NPM1 to the cytoplasm (NPM1c) and are associated with multiple transforming events including the aberrant upregulation of MEIS1 that maintains stem cell and cell cycle-associated pathways in NPM1c AML. However, another consequence of the NPM1c mutation is the inadequate levels of NPM1 wild-type in the nucleus and nucleolus, caused by the loss of one wild-type allele in addition to enforced NPM1 nuclear export. The contribution of NPM1 haploinsufficiency independently of the NPM1 mutation to AML development and its relationship with MEIS1 function is poorly understood. Using mouse models, our study shows that NPM1 haploinsufficiency paired with MEIS1 overexpression is sufficient to induce a fully penetrant AML in mice that transcriptionally resembles human NPM1c AML. NPM1 haploinsufficiency alters MEIS1-binding occupancies such that it binds the promoter of the oncogene structural maintenance of chromosome protein 4 (SMC4) in NPM1 haploinsufficient AML cells but not in NPM1 wild-type-harboring Hoxa9/Meis1-transformed cells. SMC4 is higher expressed in haploinsufficient and NPM1c+ AML cells, which are more vulnerable to the disruption of the MEIS1-SMC4 axis compared with AML cells with nonmutated NPM1. Taken together, our study underlines that NPM1 haploinsufficiency on its own is a key factor of myeloid leukemogenesis and characterizes the MEIS1-SMC4 axis as a potential therapeutic target in this AML subtype.

AbstractThe filarial parasitesMansonella ozzardiandMansonella perstans, causative agents of mansonellosis, infect hundreds of millions of people worldwide, yet remain among the most understudied of the human filarial pathogens.M. ozzardiis highly prevalent in Latin American countries and Caribbean Islands, whileM. perstansis predominantly found in sub-Saharan Africa as well as in a few areas in South America. In addition to the differences in their geographical distribution, the two parasites are transmitted by different insect vectors, as well as exhibit differences in their responses to commonly used anthelminthic drugs. The lack of genome information has hindered investigations into the biology and evolution ofMansonellaparasites and understanding the molecular basis of the clinical differences between species. In the current study, high quality genomes of two independent clinical isolates ofM. perstansfrom Cameroon and twoM. ozzardiisolates one from Brazil and one from Venezuela are reported. The genomes are approximately 76 Mb in size, encode about 10,000 genes each, and are largely complete based on BUSCO scores of about 90%, similar to other completed filarial genomes. These sequences represent the first genomes fromMansonellaparasites and enabled a comparative genomic analysis of the similarities and differences betweenMansonellaand other filarial parasites. Horizontal DNA transfers (HDT) from mitochondria (nuMTs) as well as transfers from genomes of endosymbioticWolbachiabacteria (nuWTs) to the host nuclear genome were identified and analyzed. Sequence comparisons and phylogenetic analysis of known targets of anti-filarial drugs diethylcarbamazine (DEC), ivermectin and mebendazole revealed that all known target genes were present in both species, except for the DEC target encoded bygon-2gene, which is fragmented in genome assemblies from bothM. ozzardiisolates. These new reference genome sequences will provide a valuable resource for further studies on biology, symbiosis, evolution and drug discovery.

Supplementary Figure 1. SURVEYOR assay assessing indel mutations of sgRNAs that target the N-HINGE-LOOP and the MBM-LBM locus. Supplementary Figure 2. Summary of negative selection experiments with sgRNAs targeting exons encoding specific MLL1 and MLL2 protein domains in a second independent OCI-AML3-Cas9 clone (OCI-AML3-Cas9-C8). Supplementary Figure 3. Dose responsive curves from cell viability assays of murine Npm1^CA/+ FIt3^ITD/+ after 7 days of MI-2-2 or MI-503 treatment and compared to MII-Af9 leukemia cells treated for 7 days with MI-503. Supplementary Figure 4. Apoptosis in OCI-AML3 cells treated for 7 days with MI-503 or DMSO as assessed by Annexin V / SYTOX blue staining. Supplementary Figure 5. Cytospins from cultured murine Npm1^CA/+ FIt3^ITD/+ comparing cells treated for 7 days with MI-503 versus DMSO. Supplementary Figure 6. Kaplan-Meier survival estimates from mice transplanted with secondary Npm1^CA/+ FIt3^ITD/+ leukemia cells (n=5 mice/group) and treated in vivo for ten consecutive days with either MI-503 or vehicle control. Supplementary Figure 7. Kaplan-Meier estimates of mice transplanted with secondary Npm1^CA/+ FIt3^ITD/+ leukemia cells (n=10 mice/group) that had been pretreated with either 10 days of EPS4777 [10 microM] or vehicle prior to transplantation. Supplementary Figure 8. Schematic of RNA sequencing detecting an MLL-AF6 fusion transcript in the DNMT3A mutated AML cell line OCI-AML2. Supplementary Figure 9. RNA sequencing of the OCI-AML2 cells reveals a HOX gene expression pattern as it is typically found in MLL-rearranged leukemias with predominant HOXA and MEIS1 expression but lack of HOXB cluster expression. Supplementary Figure 10. Isobolograms showing drug synergism for (A) MI-2-2 and EPZ4777 in the OCI-AML3 cells and (B) MI-503 and EPZ4777 in the murine Npm1^CA/+ FIt3^ITD/+ cells. Supplementary Figure 11. ChIP-PCR assessing RNA-Polymerase II enrichment after vehicle, single drug (MI-503 or EPZ4777) or combination treatment at the MEIS1 locus. Supplementary Figure 12. Cytologic analysis of human OCI-AML3 cells treated with DMSO, MI-2-2 (12 microM), EPZ4777 (10 microM), or combination therapy (MI-2-2 + EPZ4777). Supplementary Figure 13. Number of morphologically differentiated cells of two primary AML samples (ID1 and ID3) that had been treated in a human co-culture assay for 10 days with DMSO, EPZ4777, MI-503, or EPZ4777 and MI-503 (combo). Supplementary Figure 14. Engraftment of Npm1^CA/+ Rosa^SB/+ leukemia cells in the peripheral blood of tertiary recipient animals 22 days after transplantations. Supplementary Table 1. MLL1 single guide RNAs (sgRNA). Supplementary Table 2. MLL2 single guide RNAs. Supplementary Table 3. Synergy analysis of OCI-AML3 cells (day 7). Supplementary Table 4. Synergy analysis of Npm1^CA/+ FIt3^ITD/+ leukemias (day 11). Supplementary Table 5. Primary AML samples.

Mpox is a neglected zoonotic disease endemic in West and Central Africa. The 2022 Mpox outbreak with more than 18,000 cases worldwide generated great concern about future outbreaks and highlighted the need for a simple and rapid diagnostic test. The Mpox virus, MPXV, is a member of the Orthopoxvirus genus that also contains other pathogenic viruses including variola virus, vaccinia virus, camelpox virus, and cowpox virus. Phylogenomic analysis of all available Orthopoxvirus genomes identified 10 distinct phylogroups, with isolates from infected humans distributed across various phylogroups interspersed with isolates from animal sources, indicating the zoonotic potential of these viruses. In this study, we developed a simple and sensitive colorimetric pan-Orthopoxvirus LAMP assay for broader Orthopoxvirus detection. We also developed an MPXV-specific probe that differentiates MPXV from other Orthopoxviruses on the N1R gene which differs by only a few nucleotides. In addition, we described an extraction-free protocol for use directly with swab eluates in LAMP assays, thereby eliminating the time and resources needed to extract DNA from the sample. Our direct LAMP assays are well-suited for low-resource settings and provide a valuable tool for rapid and scalable diagnosis and surveillance of Orthopoxviruses and MPXV.

Abstract The intracellular endosymbiotic proteobacteria Wolbachia have evolved across the phyla nematoda and arthropoda. In Wolbachia phylogeny, supergroup F is the only clade known so far with members from both arthropod and filarial nematode hosts and therefore can provide unique insights into their evolution and biology. In this study, four new supergroup F Wolbachia genomes have been assembled using a metagenomic assembly and binning approach, wMoz and wMpe from the human filarial parasites Mansonella ozzardi and M. perstans, and wOcae and wMoviF from the blue mason bee Osmia caerulescens and the sheep ked Melophagus ovinus, respectively. A comprehensive phylogenomic analysis revealed two distinct lineages of filarial Wolbachia in supergroup F, indicating multiple horizontal transfer events between arthropod and nematode hosts. The analysis also reveals that the evolution of Wolbachia-filaria symbioses is accompanied by a convergent pseudogenization and loss of the bacterioferritin gene, a phenomenon found to be shared by all filarial Wolbachia, even those outside supergroup F. These observations indicate that differences in heme metabolism might be a key feature distinguishing filarial and arthropod Wolbachia. The new genomes provide a valuable resource for further studies on symbiosis, evolution, and the discovery of new antibiotics to treat mansonellosis.

RNA-binding proteins (RBPs) form a large and diverse class of factors many members of which are overexpressed in hematological malignancies. RBPs participate in various processes of mRNA metabolism and prevent harmful DNA:RNA hybrids or R-loops. Here we report that PIWIL4, a germ stem cell-associated RBP belonging to the RNase H-like superfamily, is overexpressed in acute myeloid leukemia patients and is essential for leukemic stem cell function and AML growth, but dispensable for healthy human hematopoietic stem cells. In AML cells, PIWIL4 binds to a small number of known piwi-interacting RNA. It instead largely interacts with mRNA annotated to protein-coding genic regions and enhancers that are enriched for genes associated with cancer and human myeloid progenitor gene signatures. PIWIL4 depletion in AML cells downregulates human myeloid progenitor signature and LSC-associated genes and upregulates DNA damage signalling. We demonstrate that PIWIL4 is an R-loop resolving enzyme that prevents R-loop accumulation on a subset of AML and LSC-associated genes, and maintains their expression. It also prevents DNA damage, replication stress, and activation of the ATR pathway in AML cells. PIWIL4 depletion potentiates sensitivity to pharmacological inhibition of the ATR pathway and creates a pharmacologically actionable dependency in AML cells.

While fleas are often perceived simply as a biting nuisance and a cause of allergic dermatitis, they represent important disease vectors worldwide, especially for bacterial zoonoses such as plague (transmitted by rodent fleas) and some of the rickettsioses and bartonelloses. The cosmopolitan cat ( Ctenocephalides felis ) and dog ( Ctenocephalides canis ) fleas, as well as Ctenocephalides orientis (restricted to tropical and subtropical Asia), breed in human dwellings and are vectors of cat-scratch fever (caused by Bartonella spp.) and Rickettsia spp., including Rickettsia felis (agent of flea-borne spotted fever) and Rickettsia asembonensis , a suspected pathogen. These Rickettsia spp. are members of a phylogenetic clade known as the ‘transitional group’, which includes both human pathogens and arthropod-specific endosymbionts. The relatively depauperate flea microbiome can also contain other endosymbionts, including a diverse range of Wolbachia strains. Here, we present circularized genome assemblies for two C. orientis -derived pathogens ( Bartonella clarridgeiae and R. asembonensis ) from Malaysia, a novel Wolbachia strain ( w Cori), and the C. orientis mitochondrion; all were obtained by direct metagenomic sequencing of flea tissues. Moreover, we isolated two Wolbachia strains from Malaysian C. felis into tick cell culture and recovered circularized genome assemblies for both, one of which ( w CfeF) is newly sequenced. We demonstrate that the three Wolbachia strains are representatives of different major clades (‘supergroups’), two of which appear to be flea-specific. These Wolbachia genomes exhibit unique combinations of features associated with reproductive parasitism or mutualism, including prophage WO, cytoplasmic incompatibility factors and the biotin operon of obligate intracellular microbes. The first circularized assembly for R. asembonensis includes a plasmid with a markedly different structure and gene content compared to the published plasmid; moreover, this novel plasmid was also detected in cat flea metagenomes from the USA. Analysis of loci under positive selection in the transitional group revealed genes involved in host–pathogen interactions that may facilitate host switching. Finally, the first B. clarridgeiae genome from Asia exhibited large-scale genome stability compared to isolates from other continents, except for SNPs in regions predicted to mediate interactions with the vertebrate host. These findings highlight the paucity of data on the genomic diversity of Ctenocephalides -associated bacteria and raise questions regarding how interactions between members of the flea microbiome might influence vector competence.