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Bats harbor diverse intracellular Bartonella bacteria, but there is limited understanding of the factors that influence transmission over time. Investigation of Bartonella dynamics in bats could reveal general factors that control transmission of multiple bat-borne pathogens, including viruses. We used molecular methods to detect Bartonella DNA in paired bat (Pteropus medius) blood and bat flies in the family Nycteribiidae collected from a roost in Faridpur, Bangladesh between September 2020 and January 2021. We detected high prevalence of Bartonella DNA in bat blood (35/55, 64%) and bat flies (59/60, 98%), with sequences grouping into three phylogenetic clades. Prevalence in bat blood increased over the study period (33% to 90%), reflecting an influx of juvenile bats in the population and an increase in the prevalence of bat flies. Discordance between infection status and the clade/genotype of detected Bartonella was also observed in pairs of bats and their flies, providing evidence that bat flies take blood meals from multiple bat hosts. This evidence of bat fly transfer between hosts and the changes in Bartonella prevalence during a period of increasing nycteribiid density support the role of bat flies as vectors of bartonellae. The study provides novel information on comparative prevalence and genetic diversity of Bartonella in pteropodid bats and their ectoparasites, as well as demographic factors that affect Bartonella transmission and potentially other bat-borne pathogens.

Bartonella species are emerging human pathogens. Bats are known to carry diverse Bartonella species, some of which are capable of infecting humans. However, as the second largest mammalian group by a number of species, the role of bats as the reservoirs of Bartonella species is not fully explored, in term of their species diversity and worldwide distribution. China, especially Northern China, harbors a number of endemic insectivorous bat species; however, to our knowledge, there are not yet studies about Bartonella in bats in China. The aim of the study was to investigate the prevalence and genetic diversity of Bartonella species in bats in Northern China. Bartonella species were detected by PCR amplification of gltA gene in 25.2% (27/107) bats in Mengyin County, Shandong Province of China, including 1/3 Rhinolophus ferrumequinum, 2/10 Rhinolophus pusillus, 9/16 Myotis fimbriatus, 1/5 Myotis ricketti, 14/58 Myotis pequinius. Phylogenetic analysis showed that Bartonella species detected in bats in this study clustered into ten groups, and some might be novel Bartonella species. An association between Bartonella species and bat species was demonstrated and co-infection with different Bartonella species in a single bat was also observed. Our findings expanded our knowledge on the genetic diversity of Bartonella in bats, and shed light on the ecology of bat-borne Bartonella species.

Bat flies (Hippoboscoidea: Nycteribiidae and Streblidae) are obligate hematophagous ectoparasites of bats. We collected streblid bat flies from the New World (México) and the Old World (Uganda), and used metagenomics to identify their viruses. In México, we found méjal virus (Rhabdoviridae; Vesiculovirus), Amate virus (Reoviridae: Orbivirus), and two unclassified viruses of invertebrates. Méjal virus is related to emerging zoonotic encephalitis viruses and to the agriculturally important vesicular stomatitis viruses (VSV). Amate virus and its sister taxon from a bat are most closely related to mosquito- and tick-borne orbiviruses, suggesting a previously unrecognized orbivirus transmission cycle involving bats and bat flies. In Uganda, we found mamucuso virus (Peribunyaviridae: Orthobunyavirus) and two unclassified viruses (a rhabdovirus and an invertebrate virus). Mamucuso virus is related to encephalitic viruses of mammals and to viruses from nycteribiid bat flies and louse flies, suggesting a previously unrecognized orthobunyavirus transmission cycle involving hippoboscoid insects. Bat fly virus transmission may be neither strictly vector-borne nor strictly vertical, with opportunistic feeding by bat flies occasionally leading to zoonotic transmission. Many “bat-associated” viruses, which are ecologically and epidemiologically associated with bats but rarely or never found in bats themselves, may actually be viruses of bat flies or other bat ectoparasites.

In recent years, bat-associated pathogens, such as 2019 novel coronavirus, have been ravaging the world, and ectoparasites of bats have received increasing attention. Penicillidia jenynsii is a member of the family Nycteribiidae which is a group of specialized ectoparasites of bats. In this study, the complete mitochondrial genome of P. jenynsii was sequenced for the first time and a comprehensive phylogenetic analysis of the superfamily Hippoboscoidea was conducted. The complete mitochondrial genome of P. jenynsii is 16 165 base pairs (bp) in size, including 13 protein-coding genes (PCGs), 22 transfer RNA genes, 2 ribosomal RNA genes and 1 control region. The phylogenetic analysis based on 13 PCGs of the superfamily Hippoboscoidea known from the NCBI supported the monophyly of the family Nycteribiidae, and the family Nycteribiidae was a sister group with the family Streblidae. This study not only provided molecular data for the identification of P. jenynsii, but also provided a reference for the phylogenetic analysis of the superfamily Hippoboscoidea.

The family Streblidae is a significant grouping of dipteran insects within the superfamily Hippoboscoidea, which parasitizes the body surface of bats. With the global spread of bat-related pathogens in recent years, Streblidae has gained increasing attention due to its potential for pathogen transmission. A sample of Brachytarsina amboinensis was sequenced on the B. amboinensis were obtained, compared with available Streblidae mitogenomes, and the phylogeny of Hippoboscoidea was reconstructed. The results indicate that the mitochondrial genome of B. amboinensis exhibits a relatively high degree of conservation, with an identical gene count, arrangement, and orientation as the ancestral insect's genome. Base composition analysis revealed a strong bias towards A and T in the base composition. Selection pressure analysis indicated strong purifying selection acting on cox1 . Pairwise genetic distance analysis showed that cox1 evolved at a relatively slow rate. Regarding phylogenetic relationships, the constructed phylogenetic trees using Bayesian inference and Maximum Likelihood methods supported the monophyly of the Hippoboscoidea, Glossinidae, Hippoboscidae, and Nycteribiidae clades, with high nodal support values. Our research confirmed the paraphyly of the families Streblidae. In the familial relations between Nycteribiidae and Streblidae, New World Streblidae share a closer kinship with Nycteribiidae. This contrasts with prior findings which indicated that Old World Streblidae share a closer kinship with Nycteribiidae. This study not only enhances the molecular database for bat flies but also provides a valuable reference for the identification and phylogenetic analysis of Streblidae.

The family Streblidae comprises a monophyletic group of Hippoboscoidea, hematophagous dipterans that parasitize bats. Bartonella spp. and Rickettsia spp. have been reported in bats sampled in Europe, Africa, Asia, North, Central and South America. However, there are few reports on the Bartonella and Rickettsia bacteria infecting Hippoboscoidea flies and mites. While Spinturnicidae mites are ectoparasites found only in bats, those belonging to the family Macronyssidae comprise mites that also parasitize other mammal species. This study investigates the occurrence and assesses the phylogenetic positioning of Bartonella spp. and Rickettsia spp. found in Streblidae flies and Spinturnicidae and Macronyssidae mites collected from bats captured in Brazil. From May 2011 to April 2012 and September 2013 to December 2014, 400 Streblidae flies, 100 Macronyssidaes, and 100 Spinturnicidae mites were collected from bats captured in two sites in northeastern Nova Iguaçu, Rio de Janeiro, southeastern Brazil. Forty (19.8%) out of 202 Streblidae flies were positive for Bartonella spp. in qPCR assays based on the nuoG gene. Among the flies positive for the bacterium, six (18%) were Paratrichobius longicrus, seven (29%) Strebla guajiro, two (40%) Aspidoptera phyllostomatis, five (11%) Aspidoptera falcata, one (10%) Trichobius anducei, one (25%) Megistopoda aranea, and 18 (32%) Trichobius joblingi, and collected from bats of the following species: Artibeus lituratus, Carollia perspicillata, Artibeus planirostris, Sturnira lilium, and Artibeus obscurus. Six sequences were obtained for Bartonella (nuoG [n = 2], gltA [n = 2], rpoB [n = 1], ribC = 1]). The phylogenetic analysis based on gltA (750pb) gene showed that the Bartonella sequences clustered with Bartonella genotypes detected in bats and ectoparasites previously sampled in Latin America, including Brazil. Only one sample (0.49%) of the species Trichobius joblingi collected from a specimen of Carollia perspicillata was positive for Rickettsia sp. in cPCR based on the gltA gene (401bp). This sequence was clustered with a 'Candidatus Rickettsia andaenae\" genotype detected in an Amblyomma parvum tick collected from a rodent in the southern region of Brazilian Pantanal. The sampled Macronyssidae and Spinturnicidae mites were negative for Bartonella spp. and Rickettsia spp. This study demonstrated the first occurrence of Bartonella spp. and Rickettsia spp. DNA in Streblidae flies collected from bats in Brazil.

Flies belonging to the families Streblidae and Nycteribiidae are highly specialized arthropods that feed on the blood of bats. Their morphology varies and has adapted throughout their coevolutionary history with hosts. Bat flies are often associated with specific bat species and can establish distinct infracommunities. Interaction networks have been used to better understand these associations, revealing interaction modules between bats and their parasites. The Magdalena River basin is the largest in Colombia, encompassing a wide variety of climatic and ecological conditions, with up to 98 bat species reported. We conducted field trips to capture bats and bat flies in different locations along the basin and reviewed literature records and biological collections to gather additional data on interactions between bats and bat flies in this region. We found a high diversity of bats and bat flies in the Magdalena River basin, revealing a medium specialization and modularity in these interactions. We identified bat fly infracommunities and negative associations between certain bat fly species, suggesting competition for resources within hosts. The specialization is similar to that reported in degraded and fragmented habitats where the availability of shelters decreases, favoring the overcrowding of bats, forming multi-species colonies. In conclusion, our study provides important information on the interactions between bats and bat flies in the Magdalena River basin, expanding knowledge about the diversity and structure of these communities in inter-Andean landscapes.

Increasing anthropization is detrimental to the natural environment and the quality of life, affecting populations, communities, and the relationships between organisms. One of the most unique relationships in the animal world is parasitism, which often involves tightly specialized interactions between pairs of species. Bat flies, for example, are obligate ectoparasites represented by two highly adapted dipteran families that usually parasite a single bat species or genus. Recent studies have shown that bat flies could carry pathogens such as bacteria and viruses, transmitting them among bat individuals in a colony. Because host roost characteristics can influence bat-fly parasitism, we aimed to assess whether the ecological networks between parasites and their host bats are influenced by the degree of habitat anthropization. Our hypothesis was that bat-fly interaction networks would be less specialized and more nested in highly anthropized sites. We collected bat fly individuals from bats captured at 21 sampling sites located in the Federal District of Brazil and quantified the amount of natural and anthropized area within a 3-km buffer from the sampling site. Areas consisting of agriculture, construction, mining, roads, or any man-made structure were considered anthropized. Sites presented different degrees of anthropization, with areas ranging from 100% anthropized to areas retaining full natural cover. We built bat-bat fly networks for each of the sites and excluded those with sampling completeness values smaller than 0.7. We calculated key weighted structural metrics for each network, such as nestedness, specialization, and modularity. The effect of the reduction in natural cover on structural metrics was assessed through GLMMs, controlling for network size and ectoparasite diversity. Nestedness increased with the amount of anthropization, while specialization and modularity did not change and were overall high in all networks. This result suggests that anthropization may influence the assembly of bat-bat fly networks, leading to the emergence of a hierarchical assembly of interactions as parasites become less specialized and interact with a wider variety of hosts. Less specialized relationships could influence parasite fitness or even increase the likelihood of transmitting pathogens between populations of different bat species.

We sequenced the complete mitochondrial genomes of two bat fly species within the Nycteribiidae (Diptera: Hippoboscoidea) - Dipseliopoda setosa (Cyclopodiinae) and Basilia ansifera (Nycteribiinae). Both mitogenomes were complete and contained 13 protein-coding genes, 22 tRNAs, and two rRNAs. Relative to the inferred ancestral gene order of dipteran mitochondrial genomes, no rearrangements were identified in either species. There were large differences in size between the two genomes, with D. setosa having a larger genome (19,164 bp) than B. ansifera (16,964 bp); both species had larger genomes than two previously published Streblidae bat fly species (e.g., Paradyschiria parvula and Paratrichobius longicrus). The increased genome sizes were due to expansions in the control region and the non-coding region downstream of the light-strand origin of replication. Additional differences between the two mitogenomes included a significantly longer cox3 gene in B. ansifera and a longer nad1 gene in D. setosa. Interestingly, both genomes also had the lowest GC content (D. setosa - 15.9%; B. ansifera - 17.0%) of any available Hippoboscoidea mitochondrial genome (18.8-23.9%). These mitogenomes represent the first sequences from species within the bat fly family Nycteribiidae. The sequence data here will provide a foundation for continued studies of genome evolution more generally within obligate blood-feeding ectoparasites, and specifically for the bat flies as vectors of significant 'bat-associated' viruses and microorganisms.

Symbiotic interactions between insects and bacteria are ubiquitous and form a continuum from loose facultative symbiosis to greatly intimate and stable obligate symbiosis. In blood-sucking insects living exclusively on vertebrate blood, obligate endosymbionts are essential for hosts and hypothesized to supplement B-vitamins and cofactors missing from their blood diet. The role and distribution of facultative endosymbionts and their evolutionary significance as seeds of obligate symbioses are much less understood. Here, using phylogenetic approaches, we focus on the Hippoboscidae phylogeny as well as the stability and dynamics of obligate symbioses within this bloodsucking group. In particular, we demonstrate a new potentially obligate lineage of Sodalis co-evolving with the Olfersini subclade of Hippoboscidae. We also show several likely facultative Sodalis lineages closely related to Sodalis praecaptivus (HS strain) and suggest repeated acquisition of novel symbionts from the environment. Similar to Sodalis , Arsenophonus endosymbionts also form both obligate endosymbiotic lineages co-evolving with their hosts (Ornithomyini and Ornithoica groups) as well as possibly facultative infections incongruent with the Hippoboscidae phylogeny. Finally, we reveal substantial diversity of Wolbachia strains detected in Hippoboscidae samples falling into three supergroups: A, B, and the most common F. Altogether, our results prove the associations between Hippoboscoidea and their symbiotic bacteria to undergo surprisingly dynamic, yet selective, evolutionary processes strongly shaped by repeated endosymbiont replacements. Interestingly, obligate symbionts only originate from two endosymbiont genera, Arsenophonus and Sodalis , suggesting that the host is either highly selective about its future obligate symbionts or that these two lineages are the most competitive when establishing symbioses in louse flies.