USING SURFACE WASHING TO REMOVE THE ENVIRONMENTAL COMPONENT FROM FLEA MICROBIOME ANALYSIS.

Microbial metabarcoding is a common method to study the biology of blood-feeding arthropods and identify patterns of potential pathogen transmission. Before DNA extraction, specimens are often surface washed to remove environmental contaminants. While surface washing is common, its effects on microbial diversity remain unclear. We characterized the microbiome of the flea species Ceratophyllus idius, an avian ectoparasite, and a potential vector of pathogens, using high-throughput 16S rRNA sequencing. Half of the nests from which fleas were collected were subjected to an environmental manipulation in which nesting materials were periodically replaced. In a crossed study design we surface washed half of the flea samples from each environmental condition to produce 4 experimental conditions. Environmental manipulations resulted in significant differences in the diversity and structure of the flea microbiome, but these differences were unapparent when specimens were surface washed. Furthermore, differential abundance testing of the experimental groups revealed that surface washing predominantly affected the abundance of bacterial groups that are characterized as environmental contaminants. These findings suggest that environmental changes primarily affect the surface microbiome of arthropods and that surface washing is a useful tool to reduce the footprint of the external microbiome on analysis.

Cascading effects of habitat loss on ectoparasite-associated bacterial microbiomes.

Suitable habitat fragment size, isolation, and distance from a source are important variables influencing community composition of plants and animals, but the role of these environmental factors in determining composition and variation of host-associated microbial communities is poorly known. In parasite-associated microbial communities, it is hypothesized that evolution and ecology of an arthropod parasite will influence its microbiome more than broader environmental factors, but this hypothesis has not been extensively tested. To examine the influence of the broader environment on the parasite microbiome, we applied high-throughput sequencing of the V4 region of 16S rRNA to characterize the microbiome of 222 obligate ectoparasitic bat flies (Streblidae and Nycteribiidae) collected from 155 bats (representing six species) from ten habitat fragments in the Atlantic Forest of Brazil. Parasite species identity is the strongest driver of microbiome composition. To a lesser extent, reduction in habitat fragment area, but not isolation, is associated with an increase in connectance and betweenness centrality of bacterial association networks driven by changes in the diversity of the parasite community. Controlling for the parasite community, bacterial network topology covaries with habitat patch area and exhibits parasite-species specific responses to environmental change. Taken together, habitat loss may have cascading consequences for communities of interacting macro- and microorgansims.

Visual system characterization of the obligate bat ectoparasite Trichobius frequens (Diptera: Streblidae).

As an obligate ectoparasite of bats, the bat fly Trichobius frequens (Diptera: Streblidae) inhabits the same subterranean environment as their nocturnal bat hosts. In this study, we characterize the macromorphology, optical architecture, rhabdom anatomy, photoreceptor absorbance, and opsin expression of the significantly reduced visual system in T. frequens resulting from evolution in the dark. The eyes develop over a 21-22 day pupal developmental period, with pigmentation appearing on pupal day 11. After eclosion as an adult, T. frequens eyes consist of on average 8 facets, each overlying a fused rhabdom consisting of anywhere from 11 to 18 estimated retinula cells. The dimensions of the facets and fused rhabdoms are similar to those measured in other nocturnal insects. T. frequens eyes are functional as shown by expression of a Rh1 opsin forming a visual pigment with a peak sensitivity to 487 nm, similar to other dipteran Rh1 opsins. Future studies will evaluate how individuals with such reduced capabilities for spatial vision as well as sensitivity still capture enough visual information to use flight to maneuver through dark habitats.

Expanding our view of Bartonella and its hosts: Bartonella in nest ectoparasites and their migratory avian hosts.

BACKGROUND: Bartonella is a genus of Gram-negative facultative intracellular Alphaproteobacteria of public health importance. Although they are known to mainly infect mammalian hosts with some blood-feeding arthropods having been confirmed as vectors, there is some evidence of Bartonella association with non-mammalian hosts including birds. METHODS: Here we used high-throughput sequencing of 16S rRNA and Sanger sequencing of the citrate synthase (gltA) genes to test for the presence of Bartonellaceae in the blood of three migratory cavity nesting bird species, purple martins (Progne subis), tree swallows (Tachycineta bicolor) and eastern bluebirds (Sialia sialis) and their most prevalent and abundant nest ectoparasites, Dermanyssus prognephilus (mite), Ceratophyllus idius (flea) and Protocalliphora sialia (bird blow fly larva). We constructed maximum likelihood phylogenetic trees to verify the placement of the resulting sequences in the Bartonellaceae. RESULTS: We found evidence of Bartonella in all three bird species and all three arthropod species tested. We report multiple instances of identical Bartonella sequences in both birds and parasites, leading to the likely hypothesis that these ectoparasites are potential vectors of Bartonella. Our phylogenetic analysis suggests that 'avian Bartonella' may form its own sub-clade within the genus Bartonella. CONCLUSIONS: To the best of our knowledge, we provide the first confirmation of overlapping Bartonella strains among bird hosts and various species of nest-associated ectoparasites from the same system, suggesting a possible Bartonella host-vector relationship between these arthropods and a non-mammalian host. Our study adds to the growing appreciation of the Bartonellaceae as a phylogenetically diverse group with a wide range of hosts.

A Fly on the Cave Wall: Parasite Genetics Reveal Fine-scale Dispersal Patterns of Bats.

Dispersal influences the evolution and adaptation of organisms, but it can be difficult to detect. Host-specific parasites provide information about the dispersal of their hosts and may be valuable for examining host dispersal that does not result in gene flow or that has low signals of gene flow. We examined the population connectivity of the buffy flower bat, Erophylla sezekorni (Chiroptera: Phyllostomidae), and its associated obligate ectoparasite, Trichobius frequens (Diptera: Streblidae), across a narrow oceanic channel in The Bahamas that has previously been implicated as a barrier to dispersal in bats. Due to the horizontal transmission of T. frequens, we were able to test the hypothesis that bats are dispersing across this channel, but this dispersal does not result in gene flow, occurs rarely, or started occurring recently. We developed novel microsatellite markers for the family Streblidae in combination with previously developed markers for bats to genotype individuals from 4 islands in The Bahamas. We provide evidence for a single population of the host, E. sezekorni, but 2 populations of its bat flies, potentially indicating a recent reduction of gene flow in E. sezekorni, rare dispersal, or infrequent transportation of bat flies with their hosts. Despite high population differentiation in bat flies indicated by microsatellites, mitochondrial DNA shows no polymorphism, suggesting that bacterial reproductive parasites may be contributing to mitochondrial DNA sweeps. Parasites, including bat flies, provide independent information about their hosts and can be used to test hypotheses of host dispersal that may be difficult to assess using host genetics alone.

Osteochondromatosis (multiple cartilaginous exostoses) in an immature killer whale Orcinus orca.

An immature killer whale Orcinus orca found dead on the southeastern Brazilian coast had multiple bone proliferations: on the skull, vertebrae, hemal arches, and ribs. The bony formations were characterized as multiple osteochondromas, as defined by osteochondromatosis. The diagnosis was based on macroscopic and radiographic observations. These benign osseocartilaginous tumors affect young individuals and grow until skeletal maturity is achieved. Case reports of this condition, besides humans, include other mammals, with most reports for pets and domestic mammals such as cattle, and a report in a fossil canid (Hesperocyon) from the Oligocene. The etiology, diagnosis, developmental characteristics, and occurrence of osteochondromas are distinct among different species. This report describes the first case of multiple osteochondromas in a wild cetacean.

Elucidating transmission dynamics and host-parasite-vector relationships for rodent-borne Bartonella spp. in Madagascar.

Bartonella spp. are erythrocytic bacteria transmitted via arthropod vectors, which infect a broad range of vertebrate hosts, including humans. We investigated transmission dynamics and host-parasite-vector relationships for potentially zoonotic Bartonella spp. in invasive Rattus rattus hosts and associated arthropod ectoparasites in Madagascar. We identified five distinct species of Bartonella (B. elizabethae 1, B. elizabethae 2, B. phoceensis 1, B. rattimassiliensis 1, and B. tribocorum 1) infecting R. rattus rodents and their ectoparasites. We fit standard epidemiological models to species-specific age-prevalence data for the four Bartonella spp. with sufficient data, thus quantifying age-structured force of infection. Known zoonotic agents, B. elizabethae 1 and 2, were best described by models exhibiting high forces of infection in early age class individuals and allowing for recovery from infection, while B. phoceensis 1 and B. rattimassiliensis 1 were best fit by models of lifelong infection without recovery and substantially lower forces of infection. Nested sequences of B. elizabethae 1 and 2 were recovered from rodent hosts and their Synopsyllus fonquerniei and Xenopsylla cheopsis fleas, with a particularly high prevalence in the outdoor-dwelling, highland-endemic S. fonquerniei. These findings expand on force of infection analyses to elucidate the ecological niche of the zoonotic Bartonella elizabethae complex in Madagascar, hinting at a potential vector role for S. fonquerniei. Our analyses underscore the uniqueness of such ecologies for Bartonella species, which pose a variable range of potential zoonotic threats.

Host-Parasite Associations in Small Mammal Communities in Semiarid Savanna Ecosystems of East Africa.

Despite the established importance of rodents as reservoirs of vector-borne zoonoses in East Africa, there is relatively limited information regarding the infestation parameters and host associations of ectoparasites that vector many such pathogens among small mammals in this region. Between 2009 and 2013, small mammals were live-trapped in the semiarid savanna of Kenya. A subset of these individual hosts, including 20 distinct host taxa, was examined for ectoparasites, which were identified to species. Species of fleas, ticks, mites, and sucking lice were recorded. Based on these data, we calculated host-specific infestation parameters, documented host preferences among ectoparasites, conducted a rarefaction analysis and extrapolation to determine if ectoparasites were adequately sampled, and assessed nestedness for fleas to understand how pathogens might spread in this system. We found that the flea community structure was significantly nested. Understanding the ectoparasite network structure may have significant human relevance, as at least seven of the ectoparasite species collected are known vectors of pathogens of medical importance in the region, including Yersinia pestis, Rickettsia spp., and Theileria parva, the causative agents of plague, spotted fevers and other rickettsial illnesses in humans, and theileriosis, respectively.

On the probability of dinosaur fleas.

Recently, a set of publications described flea fossils from Jurassic and Early Cretaceous geological strata in northeastern China, which were suggested to have parasitized feathered dinosaurs, pterosaurs, and early birds or mammals. In support of these fossils being fleas, a recent publication in BMC Evolutionary Biology described the extended abdomen of a female fossil specimen as due to blood feeding.We here comment on these findings, and conclude that the current interpretation of the evolutionary trajectory and ecology of these putative dinosaur fleas is based on appeal to probability, rather than evidence. Hence, their taxonomic positioning as fleas, or stem fleas, as well as their ecological classification as ectoparasites and blood feeders is not supported by currently available data.

Fleas (Siphonaptera) are Cretaceous, and evolved with Theria.

Fleas (order Siphonaptera) are highly-specialized, diverse blood-feeding ectoparasites of mammals and birds with an enigmatic evolutionary history and obscure origin. We here present a molecular phylogenetic study based on a comprehensive taxon sampling of 259 flea taxa, representing 16 of the 18 extant families of this order. A Bayesian phylogenetic tree with strong nodal support was recovered, consisting of seven sequentially derived lineages with Macropsyllidae as the earliest divergence, followed by Stephanocircidae. Divergence times of flea lineages were estimated based on fossil records and host specific associations to bats (Chiroptera), suggesting that the common ancestor of extant Siphonaptera diversified during the Cretaceous. However, most of the intraordinal divergence into extant lineages took place after the K-Pg boundary. Ancestral states of host association and biogeographical distribution were reconstructed, suggesting with high likelihood that fleas originated in the southern continents (Gondwana) and migrated from South America to their extant distributions in a relatively short time frame. Theria (placental mammals and marsupials) represent the most likely ancestral host group of extant Siphonaptera, with marsupials occupying a more important role than previously assumed. Major extant flea families evolved in connection to post K-Pg diversification of Placentalia. The association of fleas with monotremes and birds is likely due to later secondary host association. These results suggest caution in casually interpreting recently discovered Mesozoic fossil "dinosaur fleas" of Northeast Asia as part of what we currently consider Siphonaptera.

Bartonella spp. in fruit bats and blood-feeding Ectoparasites in Madagascar.

We captured, ectoparasite-combed, and blood-sampled cave-roosting Madagascan fruit bats (Eidolon dupreanum) and tree-roosting Madagascan flying foxes (Pteropus rufus) in four single-species roosts within a sympatric geographic foraging range for these species in central Madagascar. We describe infection with novel Bartonella spp. in sampled Eidolon dupreanum and associated bat flies (Cyclopodia dubia), which nest close to or within major known Bartonella lineages; simultaneously, we report the absence of Bartonella spp. in Thaumapsylla sp. fleas collected from these same bats. This represents the first documented finding of Bartonella infection in these species of bat and bat fly, as well as a new geographic record for Thaumapsylla sp. We further relate the absence of both Bartonella spp. and ectoparasites in sympatrically sampled Pteropus rufus, thus suggestive of a potential role for bat flies in Bartonella spp. transmission. These findings shed light on transmission ecology of bat-borne Bartonella spp., recently demonstrated as a potentially zoonotic pathogen.

Drivers of Intensity and Prevalence of Flea Parasitism on Small Mammals in East African Savanna Ecosystems.

The relative importance of environmental factors and host factors in explaining variation in prevalence and intensity of flea parasitism in small mammal communities is poorly established. We examined these relationships in an East African savanna landscape, considering multiple host levels: across individuals within a local population, across populations within species, and across species within a landscape. We sampled fleas from 2,672 small mammals of 27 species. This included a total of 8,283 fleas, with 5 genera and 12 species identified. Across individual hosts within a site, both rodent body mass and season affected total intensity of flea infestation, although the explanatory power of these factors was generally modest (<10%). Across host populations in the landscape, we found consistently positive effects of host density and negative effects of vegetation cover on the intensity of flea infestation. Other factors explored (host diversity, annual rainfall, anthropogenic disturbance, and soil properties) tended to have lower and less consistent explanatory power. Across host species in the landscape, we found that host body mass was strongly positively correlated with both prevalence and intensity of flea parasitism, while average robustness of a host species to disturbance was not correlated with flea parasitism. Cumulatively, these results provide insight into the intricate roles of both host and environmental factors in explaining complex patterns of flea parasitism across landscape mosaics.

Bartonella spp. in a Puerto Rican bat community.

We captured and sampled 68 bats of six species from a shared roosting site in Puerto Rico in April 2012. Bats were screened for Bartonella spp. by culture and confirmed by PCR and sequencing for the gltA gene. Bartonella cultures were obtained from blood specimens of 9/51 (18%) individuals from three species (Artibeus jamaicensis, Brachyphylla cavernarum, and Monophyllus redmani). Phylogenetic analysis of the gltA sequences showed that M. redmani was infected with multiple, diverse Bartonella strains, and A. jamaicensis was infected with a strain related to a strain from a congeneric host. Ectoparasite load could possibly explain observed differences in Bartonella diversity and prevalence between bat species in this community, and we suggest future research to substantiate these preliminary findings.

Horizontal transfers and gene losses in the phospholipid pathway of bartonella reveal clues about early ecological niches.

Bartonellae are mammalian pathogens vectored by blood-feeding arthropods. Although of increasing medical importance, little is known about their ecological past, and host associations are underexplored. Previous studies suggest an influence of horizontal gene transfers in ecological niche colonization by acquisition of host pathogenicity genes. We here expand these analyses to metabolic pathways of 28 Bartonella genomes, and experimentally explore the distribution of bartonellae in 21 species of blood-feeding arthropods. Across genomes, repeated gene losses and horizontal gains in the phospholipid pathway were found. The evolutionary timing of these patterns suggests functional consequences likely leading to an early intracellular lifestyle for stem bartonellae. Comparative phylogenomic analyses discover three independent lineage-specific reacquisitions of a core metabolic gene-NAD(P)H-dependent glycerol-3-phosphate dehydrogenase (gpsA)-from Gammaproteobacteria and Epsilonproteobacteria. Transferred genes are significantly closely related to invertebrate Arsenophonus-, and Serratia-like endosymbionts, and mammalian Helicobacter-like pathogens, supporting a cellular association with arthropods and mammals at the base of extant Bartonella spp. Our studies suggest that the horizontal reacquisitions had a key impact on bartonellae lineage specific ecological and functional evolution.

HGTector: an automated method facilitating genome-wide discovery of putative horizontal gene transfers.

BACKGROUND: First pass methods based on BLAST match are commonly used as an initial step to separate the different phylogenetic histories of genes in microbial genomes, and target putative horizontal gene transfer (HGT) events. This will continue to be necessary given the rapid growth of genomic data and the technical difficulties in conducting large-scale explicit phylogenetic analyses. However, these methods often produce misleading results due to their inability to resolve indirect phylogenetic links and their vulnerability to stochastic events. RESULTS: A new computational method of rapid, exhaustive and genome-wide detection of HGT was developed, featuring the systematic analysis of BLAST hit distribution patterns in the context of a priori defined hierarchical evolutionary categories. Genes that fall beyond a series of statistically determined thresholds are identified as not adhering to the typical vertical history of the organisms in question, but instead having a putative horizontal origin. Tests on simulated genomic data suggest that this approach effectively targets atypically distributed genes that are highly likely to be HGT-derived, and exhibits robust performance compared to conventional BLAST-based approaches. This method was further tested on real genomic datasets, including Rickettsia genomes, and was compared to previous studies. Results show consistency with currently employed categories of HGT prediction methods. In-depth analysis of both simulated and real genomic data suggests that the method is notably insensitive to stochastic events such as gene loss, rate variation and database error, which are common challenges to the current methodology. An automated pipeline was created to implement this approach and was made publicly available at: https://github.com/DittmarLab/HGTector. The program is versatile, easily deployed, has a low requirement for computational resources. CONCLUSIONS: HGTector is an effective tool for initial or standalone large-scale discovery of candidate HGT-derived genes.

New distribution and host records for Hectopsylla pulex Haller (Siphonaptera, Tungidae) with notes on biology and morphology.

Hectopsylla pulex Haller is documented for the first time from Cochise County, Arizona on the Lesser Long-nosed bat, Leptonycteris yerbabuenae Martinez and Villa (Phyllostomidae). This represents the first record of this flea on this Chiropteran Family. The most favorable site of attachment of H. pulex appears to be the head, particularly the ears and tragus. The potential interference of echolocation caused by flea attachment at or near the tragus is discussed in addition to host preferences and specialized morphological features. It is demonstrated that expansion of abdominal segments during egg development is a function of mechanical design and not neosomy such as occurs in Tunga monositus Barnes and Radovsky, Neotunga euloidea Smit and some vermipsyllid fleas.

Declines in large wildlife increase landscape-level prevalence of rodent-borne disease in Africa.

Populations of large wildlife are declining on local and global scales. The impacts of this pulse of size-selective defaunation include cascading changes to smaller animals, particularly rodents, and alteration of many ecosystem processes and services, potentially involving changes to prevalence and transmission of zoonotic disease. Understanding linkages between biodiversity loss and zoonotic disease is important for both public health and nature conservation programs, and has been a source of much recent scientific debate. In the case of rodent-borne zoonoses, there is strong conceptual support, but limited empirical evidence, for the hypothesis that defaunation, the loss of large wildlife, increases zoonotic disease risk by directly or indirectly releasing controls on rodent density. We tested this hypothesis by experimentally excluding large wildlife from a savanna ecosystem in East Africa, and examining changes in prevalence and abundance of Bartonella spp. infection in rodents and their flea vectors. We found no effect of wildlife removal on per capita prevalence of Bartonella infection in either rodents or fleas. However, because rodent and, consequently, flea abundance doubled following experimental defaunation, the density of infected hosts and infected fleas was roughly twofold higher in sites where large wildlife was absent. Thus, defaunation represents an elevated risk in Bartonella transmission to humans (bartonellosis). Our results (i) provide experimental evidence of large wildlife defaunation increasing landscape-level disease prevalence, (ii) highlight the importance of susceptible host regulation pathways and host/vector density responses in biodiversity-disease relationships, and (iii) suggest that rodent-borne disease responses to large wildlife loss may represent an important context where this relationship is largely negative.

Lack of population genetic structure and host specificity in the bat fly, Cyclopodia horsfieldi, across species of Pteropus bats in Southeast Asia.

BACKGROUND: Population-level studies of parasites have the potential to elucidate patterns of host movement and cross-species interactions that are not evident from host genealogy alone. Bat flies are obligate and generally host-specific blood-feeding parasites of bats. Old-World flies in the family Nycteribiidae are entirely wingless and depend on their hosts for long-distance dispersal; their population genetics has been unstudied to date. METHODS: We collected a total of 125 bat flies from three Pteropus species (Pteropus vampyrus, P. hypomelanus, and P. lylei) from eight localities in Malaysia, Cambodia, and Vietnam. We identified specimens morphologically and then sequenced three mitochondrial DNA gene fragments (CoI, CoII, cytB; 1744 basepairs total) from a subset of 45 bat flies. We measured genetic diversity, molecular variance, and population genetic subdivision (FST), and used phylogenetic and haplotype network analyses to quantify parasite genetic structure across host species and localities. RESULTS: All flies were identified as Cyclopodia horsfieldi with the exception of two individuals of Eucampsipoda sundaica. Low levels of population genetic structure were detected between populations of Cyclopodia horsfieldi from across a wide geographic range (~1000 km), and tests for isolation by distance were rejected. AMOVA results support a lack of geographic and host-specific population structure, with molecular variance primarily partitioned within populations. Pairwise FST values from flies collected from island populations of Pteropus hypomelanus in East and West Peninsular Malaysia supported predictions based on previous studies of host genetic structure. CONCLUSIONS: The lack of population genetic structure and morphological variation observed in Cyclopodia horsfieldi is most likely due to frequent contact between flying fox species and subsequent high levels of parasite gene flow. Specifically, we suggest that Pteropus vampyrus may facilitate movement of bat flies between the three Pteropus species in the region. We demonstrate the utility of parasite genetics as an additional layer of information to measure host movement and interspecific host contact. These approaches may have wide implications for understanding zoonotic, epizootic, and enzootic disease dynamics. Bat flies may play a role as vectors of disease in bats, and their competence as vectors of bacterial and/or viral pathogens is in need of further investigation.

Evolution, multiple acquisition, and localization of endosymbionts in bat flies (Diptera: Hippoboscoidea: Streblidae and Nycteribiidae).

Bat flies are a diverse clade of obligate ectoparasites on bats. Like most blood-feeding insects, they harbor endosymbiotic prokaryotes, but the origins and nature of these symbioses are still poorly understood. To expand the knowledge of bacterial associates in bat flies, the diversity and evolution of the dominant endosymbionts in six of eight nominal subfamilies of bat flies (Streblidae and Nycteribiidae) were studied. Furthermore, the localization of endosymbionts and their transmission across developmental stages within the family Streblidae were explored. The results show diverse microbial associates in bat flies, with at least four ancestral invasions of distantly related microbial lineages throughout bat fly evolution. Phylogenetic relationships support the presence of at least two novel symbiont lineages (here clades B and D), and extend the geographic and taxonomic range of a previously documented lineage ("Candidatus Aschnera chinzeii"; here clade A). Although these lineages show reciprocally monophyletic clusters with several bat fly host clades, their phylogenetic relationships generally do not reflect current bat fly taxonomy or phylogeny. However, within some endosymbiont clades, congruent patterns of symbiont-host divergence are apparent. Other sequences identified in this study fall into the widely distributed, highly invasive, insect-associated Arsenophonus lineage and may be the result of symbiont replacements and/or transient infections (here clade C). Vertical transmission of endosymbionts of clades B and D is supported by fluorescent signal (fluorescent in situ hybridization [FISH]) and microbial DNA detection across developmental stages. The fluorescent bacterial signal is consistently localized within structures resembling bacteriomes, although their anatomical position differs by host fly clade. In summary, the results suggest an obligate host-endosymbiont relationship for three of the four known symbiont clades associated with bat flies (clades A, B, and D).