The Bacteriome of Bat Flies (Nycteribiidae) from the MalagasyRegion: a Community Shaped by Host Ecology, BacterialTransmission Mode, and Host-Vector Specificity

David A. Wilkinson,a,b Olivier Duron,c Colette Cordonin,a,b Yann Gomard,a,b Beza Ramasindrazana,a,b,e,f,g Patrick Mavingui,b,d

Steven M. Goodman,f,g Pablo Tortosaa,b

Centre de Recherche et de Veille sur les Maladies Émergentes dans l’Océan Indien (CRVOI), GIP CYROI, Ste Clotilde, La Réunion, Francea; Université de La Réunion, UnitéMixte de Recherche “Processus Infectieux en Milieu Insulaire Tropical (UMR PIMIT),” INSERM U 1187, CNRS 9192, IRD 249, Plateforme de Recherche CYROI, Ste Clotilde,Saint-Denis, La Réunion, Franceb; Laboratoire MIVEGEC (Maladies Infectieuses et Vecteurs: Ecologie, Génétique, Evolution et Contrôle), Centre National de la RechercheScientifique (UMR5290), Université de Montpellier, Institut pour la Recherche et le Développement (UR224), Montpellier, Francec; Université de Lyon, UMR CNRS 5557,USC INRA 1364, VetAgro Sup, Ecologie Microbienne, FR41 BioEnvironment and Health, Université Lyon 1, Villeurbanne, Franced; Institut Pasteur de Madagascar,Antananarivo, Madagascare; The Field Museum of Natural History, Chicago, Illinois, USAf; Association Vahatra, Antananarivo, Madagascarg

The Nycteribiidae are obligate blood-sucking Diptera (Hippoboscoidea) flies that parasitize bats. Depending on species, thesewingless flies exhibit either high specialism or generalism toward their hosts, which may in turn have important consequences interms of their associated microbial community structure. Bats have been hypothesized to be reservoirs of numerous infectiousagents, some of which have recently emerged in human populations. Thus, bat flies may be important in the epidemiology andtransmission of some of these bat-borne infectious diseases, acting either directly as arthropod vectors or indirectly by shapingpathogen communities among bat populations. In addition, bat flies commonly have associations with heritable bacterial endo-symbionts that inhabit insect cells and depend on maternal transmission through egg cytoplasm to ensure their transmission.Some of these heritable bacteria are likely obligate mutualists required to support bat fly development, but others are facultativesymbionts with unknown effects. Here, we present bacterial community profiles that were obtained from seven bat fly species,representing five genera, parasitizing bats from the Malagasy region. The observed bacterial diversity includes Rickettsia, Wolba-chia, and several Arsenophonus-like organisms, as well as other members of the Enterobacteriales and a widespread associationof Bartonella bacteria from bat flies of all five genera. Using the well-described host specificity of these flies and data on commu-nity structure from selected bacterial taxa with either vertical or horizontal transmission, we show that host/vector specificityand transmission mode are important drivers of bacterial community structure.

Bats are increasingly recognized as natural reservoirs of a largenumber of emerging infectious agents (1–4). It is thus implicit

that vectors of bat-borne disease will play important roles in theepidemiology and dynamics of infectious agents that can eventu-ally emerge in human populations. Further, bats are hosts to dif-ferent ectoparasites, including mites, fleas, ticks, and bat flies (5,6). Bat flies (Diptera: Hippoboscoidea) are obligate blood-suckingparasites that are classically divided into two families—the Stre-blidae and the Nycteribiidae (7). Together, the Hippoboscidae(louse or ked flies), Streblidae, and Nycteribiidae are referred to asthe Pupipara sensu stricto due to their adenotrophic viviparity,where all larval developmental stages occur within the adult fe-male’s body and the larva are nourished by milk glands until theyare ready to pupate. This particularity of the Pupipara sensu strictois thought to promote vertical parasite transmission, thus influ-encing the epidemiological role of these vectors in disease trans-mission.

To date, studies of microorganisms associated with nycteribi-ids have mainly focused on two groups of bacteria—Bartonellaspp. (8–11) and Arsenophonus-like organisms (referred to here asALOs) (12–15). These bacterial genera offer contrasting modelsystems for investigating the biotic factors driving the structures ofassociated microbial communities. Bartonella species are parasiticintracellular bacteria that infect vertebrate erythrocytes, and manyspecies are considered to be zoonotic (16) and are linked to diseasein humans (17–20), including the recently identified bat-associ-

ated Bartonella mayotimonensis (21). Nycteribiids are known tohost a wide variety of Bartonella spp. (10), and contrasting pat-terns of Bartonella-bat host associations have been describedacross Africa (8, 9, 22), South America (23–25), Europe (26), andAsia (27).

In contrast, ALOs are symbiotic organisms that are engaged incomplex interactions with a variety of arthropod species (28–30).The group making up the ALOs is a monophyletic lineage thatincludes different genera, such as Arsenophonus, Aschnera, Riesia,and other unnamed groups (ALO-1 to ALO-3) (12–15). In batflies, ALOs are primary and obligate endosymbionts that are ver-tically transmitted from infected mothers to offspring due to the

Received 29 October 2015 Accepted 23 December 2015

Accepted manuscript posted online 8 January 2016

Citation Wilkinson DA, Duron O, Cordonin C, Gomard Y, Ramasindrazana B,Mavingui P, Goodman SM, Tortosa P. 2016. The bacteriome of bat flies(Nycteribiidae) from the Malagasy region: a community shaped by host ecology,bacterial transmission mode, and host-vector specificity. Appl Environ Microbiol82:1778 –1788. doi:10.1128/AEM.03505-15.

Editor: H. Goodrich-Blair, University of Wisconsin—Madison

Address correspondence to David A. Wilkinson, dwilkin799@gmail.com.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/AEM.03505-15.

Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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presence of bacteriocytes in the milk glands of all nycteribiid spe-cies (12–15). It is thought that ALOs may play a role in the provi-sioning of B vitamins, which are deficient in vertebrate blood, theonly food source for these flies (13). The ubiquity of ALOs in batflies, in which infection is at fixation, corroborates the hypothesisof an obligate endosymbiont (12–15). Such patterns have beenfound in other exclusive blood-feeding species like bedbugs (31)and tsetse flies (32), two insect groups which rely on a single foodsource throughout their developmental cycle and harbor benefi-cial microbes that provide nutrients absent from their restricteddiets.

Nycteribiid species show various levels of bat host specificity.For example, a recent study reported a lack of host specificity andgenetic structure in Cyclopodia horsfieldi, found on several fruitbats of the genus Pteropus (Pteropodidae) in southeastern Asia(33). On islands in the western Indian Ocean, it has been observedthat members of the genera Penicillidia and Nycteribia are rela-tively promiscuous with respect to their host associations, ex-changing freely between several Miniopterus spp. (Miniopteridae)and Myotis goudoti (Vespertilionidae) (5). Interestingly, these in-sectivorous bat species are often found living in syntopy (in phys-ical contact within day roosts), which likely facilitates vector shar-ing (34). The picture is rather different for fruit bat flies belongingto the genus Eucampsipoda, which have been observed to uniquelyparasitize pteropodids of the genus Rousettus on continental Af-rica or on islands in the southwestern Indian Ocean (5) despitethese bats often sharing caves as day roost sites with other batspecies (35). Similarly, Eidolon fruit bats are infested exclusively byflies from the genus Cyclopodia, with Cyclopodia dubia and Cy-clopodia greeffi specifically parasitizing Malagasy Eidolon duprea-num and African Eidolon helvum, respectively. Thus, bat fly com-munities in Madagascar, neighboring islands in the Comorosarchipelago, and continental Africa are composed of species thateither are specialists (e.g., Eucampsipoda madagascarensis, Eu-campsipoda theodori, Eucampsipoda africana, Cyclopodia dubia,and Cyclopodia greeffi found strictly on Rousettus madagascarien-sis, Rousettus obliviosus, Rousettus aegyptiacus, Eidolon duprea-num, and Eidolon helvum, respectively) or parasitize a broaderrange of hosts, such as Penicillidia leptothrinax and Nycteribia sty-lidiopsis, which are found on several Malagasy Miniopterus spp. aswell as on Myotis goudoti. Under these interesting biological cir-cumstances, variations in the patterns of infectious agents thattransfer between mammalian and arthropod hosts are likely de-termined by a number of factors, including the specificity ofhost-vector interactions, the nature of vector-microorganism in-teractions (ranging from strict parasitism to mutualism), thetransmission mode, and the confounding factors of host ecology(Fig. 1).

Here, in order to investigate the associations between bacteriaand nycteribiids, we have obtained bacterial 16S gene pyrose-quencing data from bat flies of the genera Eucampsipoda, Penicil-lidia, Nycteribia, Cyclopodia, and Basilia (Paracyclopodia), whichwere taken from bats sampled in the western Indian Ocean region(Madagascar and the Union of the Comoros). We identify a num-ber of previously undescribed bacterial associations within theNycteribiidae, compare the phylogenetic relationships of some ofthese novel bacterial taxa, and propose a model for how host-vector-parasite interactions may shape the bacterial communitieshosted by these flies.

MATERIALS AND METHODSTaxon sampling and identification. Bats were captured using mist netsand harp traps, generally placed at cave entrances, or butterfly nets toobtain individuals from day roost sites in caves. This study was conductedin strict accordance with the terms of research permits issued by nationalauthorities (Direction du Système des Aires Protégées, Direction environ-ment et des Forêts, and Madagascar National Parks [Madagascar] andCentre National de Documentation et de Recherche Scientifique [Unionof the Comoros]), following the laws of these countries, and the associatedresearch permit numbers are listed in Acknowledgments. Upon capture,each individual cataloged bat was placed in a separate clean cloth bag untilthe collection of relevant biological data, and they were examined forectoparasites. The ectoparasites were collected with forceps and stored inseparate vials containing 70% ethanol. Morphological identification ofthe ectoparasites was carried out using published keys and descriptions

FIG 1 Host specificity between bats and bat flies (Nycteribiidae) in Madagas-car and the nearby Comoros archipelago (5).

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(36), and host bat specimens are housed in the Field Museum of NaturalHistory (FMNH) (Chicago, IL) and in the Département de Biologie Ani-male, Université d’Antananarivo (UADBA) (Antananarivo, Madagascar).A considerable portion of the nycteribiid specimens used here came froma previous study that evaluated the evolutionary origins of bat flies on theComoro Islands and Madagascar (5). New samples of Cyclopodia dubiaand Basilia (Paracyclopodia) sp. were collected on Madagascar from Eido-lon dupreanum and Scotophilus spp., respectively, as part of this study.

Nucleic acid extraction and pyrosequencing design. Nycteribiid flieswere removed from ethanol storage and dried and were then crushedusing 2- by 2-mm tungsten beads in a TissueLyser (Qiagen). Nucleic acidswere extracted using a Qiagen EZ1 robot with the DNA tissue kit, accord-ing to the manufacturer’s protocol and as previously published (5). Nu-cleic acids were pooled by species for 16S gene pyrosequencing as detailedin Table S1 in the supplemental material.

The 16S gene pyrosequencing was performed as previously described(37). Briefly, 16S V3 and V4 variable regions were amplified via PCR-specific primers targeting the upstream and downstream regions of the V3to V4 segment; the 3= end of forward (TACGGRAGGCAGCAG) and re-verse (GGACTACCAGGGTATCTAAT) bacterium-specific primers werebound at the 5= end by multiplex identifier (MID) tags, a GS FLX key, andGS FLX adapters. The quantity of each PCR product was then determinedwith PicoGreen, and all products were mixed together in equimolar con-centrations before 454 GS FLX sequencing (Genoscreen).

All reads of �250 bp in length were discarded using the Geneious Prosoftware package (38). Remaining reads were analyzed using the SILVAonline next-generation sequencing (NGS) tool (www.arb-silva.de/ngs).Raw sequence reads were aligned with a gap extension penalty of 2 and agap penalty of 5. Reads were filtered based on the following quality crite-ria: minimum length, 250 bp; minimum quality score, 30; maximum per-cent ambiguities, 1%; minimum base pair score, 30; and maximum per-cent repetitive, 2%. Remaining reads were clustered into operationaltaxonomic units (OTUs) at a threshold sequence identity of 99%. OTUswere classified by BLAST score comparison against the SILVA rRNA da-tabase version 115, with a classification similarity threshold of 93%. OTUswere used exclusively for initial taxonomic interpretations, and all quan-titative data were calculated from the numbers of original sequence reads.The statistical significance of differences in proportions of sequence datawas calculated by chi-squared comparisons with a Yates correction.

Sequence data and genetic analyses. Specific gene fragments wereamplified using primer pairs detailed in Table 1. Sequence data were an-alyzed using the Geneious Pro software package (38). All final sequencedata were deposited in GenBank, and the relevant accession numbers arelisted in Table 2. Coding sequences were aligned in-frame using the Ge-neious Pro translation align tool and the standard ClustalW cost matrix.Noncoding sequences were aligned using MUSCLE (39). Network analy-ses were performed in SplitsTree4 (40) using the neighbor-net algorithm.Maximum likelihood phylogenies were generated through RAxML v8.0.0(41) using the RAxML GUI (v1.3) with 1,000 bootstrap replicates and the

GTR�Gamma substitution model as suggested by JModelTest2 (v.2.1.6)(42). Coding sequence alignments were partitioned by base position, andsingle or multiple outgroup sequences were specified prior to analysis.The best tree outputs from RAxML were used as topology inputs in the Rsoftware package, exploiting the ape (43) and vegan (44) packages in orderto test evolutionary congruence using the ParaFit algorithm (45). Bacte-rial phylogenies used for ParaFit analysis contained only sequences ob-tained in this study and were compared against a host-nycteribiid phylog-eny generated based on cytochrome oxidase I (COI) sequence data. COIsequences were taken from reference 5, and data from Cyclopodia dubiaand Basilia (Paracyclopodia) sp. were provided by B. Ramasindrazana andS. M. Goodman (unpublished data).

Nucleotide sequence accession numbers. All PCR-generated se-quences in this study were submitted to GenBank under the accessionnumbers given in Table 2.

RESULTSBacterial community composition as revealed by 16S gene pyro-sequencing. Pyrosequencing data were obtained from seven inde-pendent samples that originated from seven nycteribiid species:Eucampsipoda madagascarensis, Eucampsipoda theodori, Penicil-lidia sp. cf. fulvida (a Penicillidia sp. that looks like Penicillidiafulvida), Penicillidia leptothrinax, Nycteribia stylidiopsis, Cyclopo-dia dubia, and Basilia (Paracyclopodia) sp. Samples containedpooled DNA that was extracted from different numbers of nyc-teribiid flies. A summary of sample composition, as well as thestatistics of pyrosequencing data acquisition, including numbersof reads obtained and quality control results, is presented in TableS1 in the supplemental material.

We then analyzed bacterial diversity at the class level. The bac-terial communities associated with all bat fly samples were domi-nated by three bacterial phyla, Alphaproteobacteria, Betaproteo-bacteria, and Gammaproteobacteria, which represented 17%, 3%,and 78% of total sequences, respectively. The vast majority ofBetaproteobacteria were observed in Eucampsipoda theodori andshowed the closest similarity to uncultured bacteria of the familyNeisseriaceae. The majority of Neisseriaceae are common com-mensal bacteria that are often associated with mammals, but Neis-seria gonorrhoeae and Neisseria meningitides are the known caus-ative agents of gonorrhea and meningococcal meningitis inhumans, respectively.

Of the sequences identified as Alphaproteobacteria, 55% camefrom Wolbachia, 26% came from Bartonella, and 17% came fromRickettsia. Nearly all Gammaproteobacteria (�99%) came fromthe order Enterobacteriales, and for all bat fly species but one (Cy-clopodia dubia), they were mostly distributed between two genera,

TABLE 1 Primers used in this study

Description Forward primer Reverse primer Reference/source

16S gene pyrosequencing TAC GGR AGG CAG CAG GGA CTA CCA GGG TAT CTA AT Genoscreen16S gene, 1,350 bp AGA GTT TGA TCM TGG CTC AG TAC GGY TAC CTT GTT ACG ACT T Unpublished data (P. Tortosa)Bartonella gltA GGG GAC CAG CTC ATG GTG G AAT GCA AAA AGA ACA GTA AAC A 23Bartonella 16S AGA GTT TGA TCM TGG CTC AG TAC GGY TAC CTT GTT ACG ACT T 15Bartonella rpoB CGC ATT GGC TTA CTT CGT ATG GTA GAC TGA TTA GAA CGC TG 62Enterobacteriales 16S GGG TTG TAA AGT ACT TTC AGT CGT CCT YTA TCT CTA AAG GMT TCG CTG GAT G 51Rickettsia gltA GGT TTT ATG TCT ACT GCT TCK TG CAT TTC TTT CCA TTG TGC CAT C 63Wolbachia wsp GTC CAA TAR STG ATG ARG AAA C CYG CAC CAA YAG YRC TRT AAA 49Wolbachia hcpA GAA ATA RCA GTT GCT GCA AA GAA AGT YRA GCA AGY TCT G 48Wolbachia fbpA GCT GCT CCR CTT GGY WTG AT CCR CCA GAR AAA AYY ACT ATT C 48Wolbachia ftsZ ATY ATG GAR CAT ATA AAR GAT AG TCR AGY AAT GGA TTR GAT AT 48

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TA

BLE

2G

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accessionn

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sequen

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a

IDb

no.

SpeciesH

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IDn

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artonellagltA

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gltA16S

wsp

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hcpA

SC1

Basilia

(Paracyclopodia)

sp.Scotophilus

marovaza

FMN

H221393

Madagascar

KT

751157K

T751105

KT

751166K

T751134

KT

751140K

T751163

SC2

Basilia

(Paracyclopodia)

sp.Scotophilus

marovaza

FMN

H221393

Madagascar

KT

751104

SC3

Basilia

(Paracyclopodia)

sp.Scotophilus

marovaza

FMN

H221393

Madagascar

KT

751103K

T751167

KT

751135

SC4

Basilia

(Paracyclopodia)

sp.Scotophilus

marovaza

FMN

H221393

Madagascar

KT

751102

1bB

asilia(P

aracyclopodia)sp.

Scotophilusrobustus

FMN

H209163

Madagascar

KT

751147K

T751101

1C

yclopodiadubia

Eidolon

dupreanumFM

NH

221295M

adagascarK

T751146

KT

751130

2C

yclopodiadubia

Eidolon

dupreanumSM

G17826/FM

NH

221296M

adagascarK

T751129

3C

yclopodiadubia

Eidolon

dupreanumSM

G17827/FM

NH

221297M

adagascarK

T751128

4C

yclopodiadubia

Eidolon

dupreanumSM

G17828/FM

NH

221298M

adagascarK

T751148

KT

751127K

T751164

KT

751132K

T751138

KT

751161

5C

yclopodiadubia

Eidolon

dupreanumSM

G17830/U

AD

BA

32975M

adagascarK

T751149

KT

751126

6C

yclopodiadubia

Eidolon

dupreanumSM

G17831/U

AD

BA

32976M

adagascarK

T751150

KT

751125K

T751165

KT

751133K

T751139

KT

751162

7C

yclopodiadubia

Eidolon

dupreanumSM

G17832/U

AD

BA

32977M

adagascarK

T751151

KT

751124

8C

yclopodiadubia

Eidolon

dupreanumSM

G17833/U

AD

BA

32978M

adagascarK

T751123

9C

yclopodiadubia

Eidolon

dupreanumSM

G17834/U

AD

BA

32979M

adagascarK

T751122

1cE

ucampsipoda

madagascarensis

Rousettus

madagascariensis

SMG

17698/FMN

H221366

Madagascar

KT

751120

3bE

ucampsipoda

madagascarensis

Rousettus

madagascariensis

SMG

17776/UA

DB

A32967

Madagascar

KT

751119

2cE

ucampsipoda

madagascarensis

Rousettus

madagascariensis

SMG

17777/UA

DB

A32968

Madagascar

KT

751158

10E

ucampsipoda

madagascarensis

Rousettus

madagascariensis

SMG

17780/UA

DB

A32971

Madagascar

KT

751118

6cE

ucampsipoda

madagascarensis

Rousettus

madagascariensis

SMG

17783/UA

DB

A32974

Madagascar

KT

751159

5cE

ucampsipoda

madagascarensis

Rousettus

madagascariensis

SMG

17781/UA

DB

A32972

Madagascar

KT

751131

14cE

ucampsipoda

madagascarensis

Rousettus

madagascariensis

SMG

17931/UA

DB

A33647

Madagascar

KT

751121

J50E

ucampsipoda

madagascarensis

Rousettus

madagascariensis

SMG

16259/FMN

H209105

Madagascar

KT

751117

J13E

ucampsipoda

theodoriR

ousettusobliviosus

SMG

16715/FMN

H220041

Un

ionofth

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omoros

KT

751116

J14E

ucampsipoda

theodoriR

ousettusobliviosus

SMG

16715/FMN

H220041

Un

ionofth

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omoros

KT

751115

J22E

ucampsipoda

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SMG

16751/FMN

H220043

Un

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omoros

KT

751156

GR

16N

ycteribiastylidiopsis

Miniopterus

gleniSM

G17841/U

AD

BA

33030M

adagascarK

T751152

KT

751108

GR

17N

ycteribiastylidiopsis

Miniopterus

gleniSM

G17841/U

AD

BA

33030M

adagascarK

T751107

GR

15N

ycteribiastylidiopsis

Miniopterus

gleniSM

G17841/U

AD

BA

33030M

adagascarK

T751109

GR

12N

ycteribiastylidiopsis

Miniopterus

gleniSM

G17846/FM

NH

221333M

adagascarK

T751110

GR

6N

ycteribiastylidiopsis

Miniopterus

griveaudiSM

G17654/U

AD

BA

32960M

adagascar

GR

8N

ycteribiastylidiopsis

Miniopterus

griveaudiSM

G17682/U

AD

BA

32962M

adagascarK

T751113

GR

9N

ycteribiastylidiopsis

Miniopterus

griveaudiSM

G17689/FM

NH

221413M

adagascarK

T751111,K

T751112

2bN

ycteribiastylidiopsis

Miniopterus

griveaudiSM

G17759/FM

NH

221343M

adagascarK

T751114

GR

19N

ycteribiastylidiopsis

Miniopterus

griveaudiSM

G17761/U

AD

BA

33004M

adagascarK

T751106

11bP

enicillidialeptothrinax

Miniopterus

griveaudiSM

G17860/FM

NH

221354M

adagascarK

T751097

11cP

enicillidialeptothrinax

Miniopterus

aelleniSM

G17922/FM

NH

221440M

adagascarK

T751143

GR

20P

enicillidialeptothrinax

Miniopterus

griveaudiSM

G17757/FM

NH

221341M

adagascarK

T751153

GR

18P

enicillidialeptothrinax

Miniopterus

griveaudiSM

G17772/U

AD

BA

33015M

adagascarK

T751093,K

T751094

KT

751171

J35P

enicillidialeptothrinax

Miniopterus

petersoniSM

G16806/FM

NH

209186M

adagascarK

T751092

J38P

enicillidialeptothrinax

Miniopterus

petersoniSM

G16806/FM

NH

209186M

adagascarK

T751160

12bP

enicillidialeptothrinax

Miniopterus

sp.SM

G17865/U

AD

BA

33032M

adagascarK

T751096

12P

enicillidialeptothrinax

Miniopterus

sp.SM

G17866/U

AD

BA

33033M

adagascarK

T751144

13P

enicillidialeptothrinax

Miniopterus

cf.ambohitrensis

SMG

17867/UA

DB

A33034

Madagascar

KT

751095

13bP

enicillidialeptothrinax

Miniopterus

sp.SM

G17866/U

AD

BA

33033M

adagascarK

T751145

KT

751169K

T751136

KT

751141

18bP

enicillidialeptothrinax

Miniopterus

sp.SM

G17884/FM

NH

221429M

adagascarK

T751170

KT

751137K

T751142

GR

21P

enicillidiasp.(cf.fulvida)

Miniopterus

griveaudiSM

G17753/FM

NH

221337M

adagascarK

T751154

KT

751098,K

T751099

9bP

enicillidiasp. (cf.fulvida)

Miniopterus

sp.SM

G17878/FM

NH

221423M

adagascarK

T751100

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.

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Arsenophonus and Aschnera, which clearly belong to the ALOgroup (12–15). The case of Cyclopodia dubia is subtly different; onthe basis of a BLAST search, most of the bacterial sequences fromthis bat fly species were found to have identity to Enterobacterialesmembers other than ALOs, such as Providencia, Erwinia, andDickeya. Interestingly, the bacteria originating from Cyclopodiadubia may actually form a new Enterobacteriales group (see be-low).

Bacterial community data are summarized in Fig. S1 in thesupplemental material.

We then further investigated bacterial taxa by sequencing ad-ditional loci. Taxa were selected because of their possible medicalimportance, such as the genera Rickettsia and Bartonella becauseof their relation to pathogenic strains causing rickettsioses andbartonellosis, respectively, or because they were common to mul-tiple species of nycteribiid fly and represented a significant pro-portion of the total number of reads obtained by pyrosequencing,such as Wolbachia and Enterobacteriales (threshold retrospectivelyset at 2.5% of the total number of reads).

Rickettsia. During pyrosequencing, 16S sequences closely re-lated to Rickettsia were obtained from Eucampsipoda madagasca-rensis and Penicillidia leptothrinax. These sequences represented5.5% and 15.3% of reads in Eucampsipoda madagascarensis andPenicillidia leptothrinax, respectively. The gltA locus of Rickettsiawas amplified from Eucampsipoda madagascarensis and Penicil-lidia leptothrinax samples. Classification using BLASTx showedthat the obtained gltA sequences showed little homology to knownspecies of Rickettsia, with the strongest match showing 90% iden-tity to gltA from Rickettsia raoultii, a bacterium identified in hardticks from China (46). In addition, a 1,350-bp fragment of the 16Sgene of Rickettsia was amplified from Eucampsipoda madagasca-rensis. Although the phylogeny of Rickettsia could only be poorlyresolved based on these data (not shown), sequence similarity was

strongest with Rickettsia gravesii, Rickettsia barbariae, and Rickett-sia tarasevichiae, bacteria previously shown to be associated withhard ticks belonging to the genera Amblyomma and Ixodes.

Wolbachia. Pyrosequencing of the 16S locus revealed thatWolbachia was present in all sampled fly species except Eucampsi-poda madagascariensis and Eucampsipoda theodori. The percent-age of reads matching Wolbachia ranged from �1% in Nycteribiastylidiopsis to nearly 40% in Basilia (Paracyclopodia) sp. PCR-based screening using the wsp system (47) was then used to testindividual nycteribiid samples for the presence of Wolbachia. Inaccordance with the pyrosequencing results, Wolbachia was iden-tified in Penicillidia leptothrinax (14/23; 61%), Penicillidia sp. (cf.fulvida) (1/1; 100%), Cyclopodia dubia (6/9; 67%), and Basilia(Paracyclopodia) sp. (5/5; 100%).

A selection of DNA fragments from the Wolbachia wsp, fbpA,ftsZ, and hcpA genes (48) was amplified from Cyclopodia dubia,Basilia (Paracyclopodia) sp., and Penicillidia leptothrinax. Se-quence data were additionally obtained from the wsp gene ofWolbachia from Penicillidia sp. (cf. fulvida), showing 100% iden-tity to those sequences obtained from Wolbachia infecting Penicil-lidia leptothrinax; however, attempts to amplify other loci wereunsuccessful for these samples. As has been observed elsewhere(49, 50), network analysis of wsp gene data against reference se-quences from the Wolbachia multilocus sequence typing(MLST) database (http://pubmlst.org/wolbachia/) suggestedhigh levels of diversification and recombination betweenWolbachia spp. from well-defined phylogenetic groups (see Fig. S2in the supplemental material), and thus, the wsp gene data wereabandoned for phylogenetic characterization of the sequencesobtained from our bat fly samples.

Network and phylogenetic analyses of concatenated sequences(fbpA, ftsZ, and hcpA) suggested that all identified bat fly Wolba-chia species belong to the F subgroup (Fig. 2). Sequence types

FIG 2 Wolbachia network analysis and group F phylogeny. The presented analyses are based on concatenation of three gene loci (fbpA::ftsZ::hcpA); reference datawere acquired from the Wolbachia MLST database (http://pubmlst.org/wolbachia/). The presented network structure was generated from aligned sequence data inSplitsTree4 using the neighbor-net algorithm. The phylogenetic tree was produced using RAxML, and bootstrap support from 1,000 replicates is indicated by dots ofdifferent sizes on each internal node. Sequences from this study are highlighted in red, orange, and blue, representing host origins from Cyclopodia dubia, Basilia(Paracyclopodia) sp., and Penicillidia leptothrinax, respectively. Asterisks indicate that the hcpA region was missing from the sequence data originating from P.leptothrinax. However, it should be noted that identical, but less well supported, topologies were generated when using only fbpA and ftsZ (data not shown).

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from different nycteribiid species were grouped by host speciesand were paraphyletic, sharing identity with Wolbachia strainsfrom multiple host types and geographical origins, which is sug-gestive of independent evolutionary histories and multiple intro-ductions of Wolbachia into bat fly populations.

Arsenophonus-like organisms. The Enterobacteriales were byfar the most predominant bacterial taxa identified in pyrose-quencing analyses, with 73% of all sequencing data identified aseither ALOs or as a new and unnamed Enterobacteriales group.Further 16S data were obtained from multiple samples of eachnycteribiid species. Network analysis was performed using refer-ence strains listed in Table S2 in the supplemental material. Nyc-teribiid endosymbionts belonging to the Enterobacteriales fell intothree distinct groups (Fig. 3). Insectivorous bat-associated Nyc-teribia and Penicillidia possessed ALOs associated with the Asch-nera subgroup, whereas fruit bat-associated Eucampsipoda andinsectivorous bat-associated Basilia (Paracyclopodia) sp. pos-sessed ALOs belonging to the Arsenophonus subgroup. Interest-ingly, network analysis suggested that the endosymbionts fromCyclopodia dubia form their own, new group within the Entero-bacteriales family, which is closely related to the known ALOmembers (Cyclopodia group in Fig. 3).

Bartonella. Bartonella sequences were detected by pyrose-quencing in all bat fly species. Eucampsipoda theodori, the only batfly in this study from the Union of the Comoros and parasitizingRousettus obliviosus, showed significantly higher proportions ofsequences from Bartonella (P � 0.0001) than those of its sisterspecies Eucampsipoda madagascarensis from Madagascar occur-ring on Rousettus madagascariensis. The percentage of reads de-rived from Bartonella ranged from �1% in Basilia (Paracyclopo-dia) sp., Penicillidia leptothrinax, and Nycteribia stylidiopsis to17.4% in Eucampsipoda theodori.

The gltA locus was amplified from these nycteribiid samples.Only 15 sequences that demonstrated clear identity to unambig-uously annotated Bartonella strains in BLAST searches and that

were monophyletic (with Bartonella reference sequences in pre-liminary phylogenetic studies) were retained for analysis. Barto-nella sequences were obtained from all bat fly species exceptEucampsipoda madagascarensis. Additional Bartonella-specificamplifications based on the 16S and rpoB loci were also at-tempted but were largely unsuccessful.

The gltA sequences obtained from the nycteribiids of Madagas-car and the Comoros archipelago were seen to cluster into fivedistinct groups (Fig. 4). Potential limitations of the gltA locus havebeen discussed elsewhere (10).

Evolutionary congruence. The global-fit method, ParaFit, wasused to test the hypothesis of evolutionary congruence betweennycteribiids and the bacteria detected in this study, by comparingphylogenetic structures to those obtained using COI data from batflies. ALOs demonstrated a significant level of congruence (P �0.001), whereas congruence was not significant for Bartonella (Ta-ble 3). As the topological structure may result from a difference inlevels of polymorphism of the chosen markers (16S for ALOs andgltA for Bartonella), we compared their nucleotide diversities (pivalue) using DnaSP v5 (http://www.ub.edu/dnasp/). This re-vealed that 16S was actually less polymorphic than gltA (pi, 0.056and 0.117 for 16S and gltA, respectively), thus allowing us to rejectthe hypothesis of an artifactual topology of ALOs resulting fromhigher resolution of the 16S locus. It was not possible to testWolbachia or Rickettsia lineages using this methodology due to thelimited number of relevant bacterial taxa identified in nycteribiidhosts.

DISCUSSION

Here, we provide characterization of the bacterial microbiota as-sociated with numerous species of Nycteribiidae bat flies occur-ring in Madagascar and the nearby Comoros archipelago and pro-vide more detailed taxonomic information for some bacterial taxaof particular interest.

Bacterial communities associated with the nycteribiid bat fly

FIG 3 Enterobacteriales network diagram generated from 16S gene sequence data. The presented network structure was generated from aligned sequence datain SplitsTree4 using the neighbor-net algorithm. The Cyclopodia group contained only sequences from this study and refers to a newly identified taxon of theEnterobacteriales, all originating from C. dubia. Sequences used to generate this representation can be found in Tables S3 and S4 in the supplemental material. Allsequences from bat flies in this study are indicated by colored circles, as identified in the key.

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FIG 4 Bartonella phylogeny based on the gltA locus, generated using 1,000 bootstrap replicates in RAxML. Dots on internal nodes represent bootstrap supportof �0.75. Sequences from this study, and similar bat- and bat fly-associated sequences from other studies, are grouped into monophyletic groups by color. Batsdrawn on leaves indicate sequences that originated from bats or bat fly specimens. The key at the bottom of the figure details bat fly and host bat origins of theassociated sequences. Abbreviations used for bat flies are as follows: Cy., Cyclopodia; Eu., Eucampsipoda; P, Penicillidia; N, Nycteribia. Abbreviations used for batsare as follows: Ei., Eidolon; Mi., Miniopterus; My., Myotis; R., Rousettus; Sc., Scotophilus. Numbers in parentheses represent the numbers of identical sequences thatwere grouped into operational taxonomic units in RAxML.

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genera Eucampsipoda, Penicillidia, Nycteribia, Cyclopodia, andBasilia (Paracyclopodia) were described by pyrosequencing of the16S locus. Variation was found in community composition indifferent species of Nycteribiidae; however, these may be due tostochastic infection effects, differences in sample pooling betweenspecies, and/or limits in sequencing repeatability. It should also benoted that taxon sampling was subject to amplification bias due tothe primers used to amplify the 16S locus (see Table S1 in thesupplemental material). Although we cannot quantify the signif-icance of these effects, our data suggest that the vast majority ofbacterial diversity in all samples can be described by the presenceof not more than two bacterial classes—the Alphaproteobacteriaand the Gammaproteobacteria. While the ALOs (Gammaproteo-bacteria) have been previously described as obligate, primary sym-bionts in bat flies (12–15), the nature of the interactions betweenother bacteria and bat flies remains unknown. The overall homo-geneity of the bacterial community structure across nycteribiidspecies, independent of bat host species, suggests that infection isnot merely opportunistic, but selected and commonly occurringtaxa likely form beneficial, positively selected interactions withtheir arthropod hosts. On the other hand, strongly significant dif-ferences between samples from Madagascar and the Comoros ar-chipelago (Betaproteobacteria in Eucampsipoda theodori) suggestthat environmental variables will also determine which interac-tions can be sustained.

Our results along with previous studies (12–15) corroboratethe widespread presence of ALOs in bat flies while identifying alikely new genus of endosymbiont of the Enterobacteriales thatinfects the fruit bat (Eidolon dupreanum)-associated nycteribiidCyclopodia dubia. While ALOs, and especially Arsenophonus, areglobally common symbionts estimated to infect ca. 5% of insectspecies (28, 51), we further confirm that bat flies harbor the high-est diversity of Enterobacteriales strains reported to date (Arseno-phonus, Aschnera, and the Cyclopodia-associated group). We alsofound congruent patterns of codivergence between bat flies andALOs, a result likely due to the association between members ofthe Nycteribiinae subfamily (which includes Penicillidia leptothri-nax, Penicillidia sp. [cf. fulvida], and Nycteribia stylidiopsis) andAschnera, which are known to have codiverged over a long evolu-tionary period (8, 26). However, the evolutionary history of theseendosymbionts in bat flies is complex; all strains found here donot cluster within a specific bat fly clade but rather exhibit distinctevolutionary origins showing that they underwent repeated hori-zontal transfer between distantly related host species. This patternis well illustrated by the presence, in some bat fly species, of Ar-senophonus strains, which are closely related to strains from verydiverse insect species, such as aphids and parasitoid wasps. Twodistinct evolutionary strategies are thus acting on ALO endosym-biosis; Aschnera is highly specialized with regard to its hosts, withancient acquisition followed by codiversification, while Arseno-

phonus is more generalist and acquired through recent horizontaltransfers. The presence of a previously unidentified group of bac-teria in Cyclopodia dubia testifies to the diverse nature of symbioticinteractions, and further work will be required to better under-stand how these findings modify our understanding of the evolu-tionary history of nycteribiid-endosymbiont interactions.

The genus Wolbachia was observed to infect Penicillidia spp.,Cyclopodia dubia, and Basilia (Paracyclopodia) sp. This commonreproductive manipulator has previously been identified in batflies (13); however, it is of note that we observe Wolbachia inmultiple nycteribiid species. Interestingly, all Wolbachia speciesthat were identified in bat flies belonged to the F supergroup, anemerging supergroup of Wolbachia that has been associated with abroad spectrum of arthropod hosts, including the orders Scorpio-nes, Blattodea, Coleoptera, Hemiptera, Isoptera, Neuroptera, Or-thoptera, Phthiraptera, Thysanoptera (52), and Diptera (the ordercontaining the Nycteribiidae), which are more commonly associ-ated with supergroups A and B (51, 53). Eucampsipoda did notharbor Wolbachia, and only a subset of bat flies infesting insectiv-orous bats tested positive for Wolbachia, showing that, unlike theALOs, Wolbachia is not an obligate endosymbiont of nycteribiidflies. Although the number of samples that could be sequenced ingreater detail was relatively limited, nycteribiid-Wolbachia se-quences appeared to be species specific but paraphyletic throughMLST analyses, suggesting multiple introductions of independentlineages to different nycteribiid species. However, a commonlineage of Wolbachia was observed in Penicillidia leptothrinax andPenicillidia sp. (cf. fulvida). Facultative association between nyc-teribiid Wolbachia suggests that their interaction over evolution-ary history is more recent than that of the ALOs despite the factthat fluorescence in situ hybridization-based physiological studiessuggest a common mechanism of vertical transmission for all nyc-teribiid bacterial endosymbionts (13). This is in keeping with ob-servations in other arthropod species, where Wolbachia strains areseen to have complex phylogenetic histories due to their dual ca-pacity to form stable coexisting mixed populations within arthro-pod communities (54, 55) and to invade entire populations bymanipulating host reproduction (56). While the potential ofWolbachia to manipulate the reproduction of bat flies is unknown,it is interesting to note that a recent study observed biased sexratios in bat fly populations (Trichobius frequens) in Puerto Rico(57).

The data generated in this study also add to the knowledge ofthe increasing diversity of Bartonella subgroups that are associatedwith bat flies and their bat hosts. It is generally thought thatBartonella species demonstrate specificity to mammalian hosts;however, the exact role of vector-host specificity in establishinghost-specific interactions is not fully understood (58). Here, thedetection of Bartonella in all tested bat fly species adds credence tothe theory that Bartonella and bat flies may form mutually bene-ficial, positively selected interactions, which may drive host spec-ificity. However, the lack of evolutionary congruence betweenBartonella and the Nycteribiidae suggests that any selective benefitis insufficient to generate stable symbiosis despite their likely abil-ity to be transferred vertically from mother to pupa. In the case ofthe insectivorous bat-infesting Nycteribiidae of Madagascar andthe Comoros, transmission through blood-feeding is likely to playa direct role in the horizontal transfer of Bartonella, as multiplenycteribiid flies (i.e., Nycteribia spp. and Penicillidia spp.) feed onthe same bat hosts (5). This was seen to be the case here as, for

TABLE 3 Evolutionary congruence between Nycteribiidae andassociated bacteriaa

Parameter Wolbachia Arsenophonus-like organisms Bartonella

ParaFit Global NAb 0.087 0.060P value NA 0.001 0.172a Congruence testing for Wolbachia was not possible due to the presence of only threeindependent taxa.b NA, not available.

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example, a single clade in our phylogenetic analysis (colored or-ange in Fig. 4) contained Bartonella variants from Nycteribia andPenicillidia as well as from five different bat species. However, thesame phylogenetic analysis also suggested exchange between Cy-clopodia dubia and Basilia (Paracyclopodia) sp. (group coloredblue in Fig. 4), taxa which are not known to interact directly orwith the same bat host species. This suggests that other direct orindirect mechanisms may promote exchange between hosts andthus drive the intraspecific diversity of Bartonella observed withinthe Nycteribiidae. This is in contrast with other members of thePupipara sensu stricto; ked flies, blood-sucking vectors of ungu-lates, are thought to form strict interactions with Bartonella, whichhave resulted in host-specific associations (10). The differencebetween these two situations is likely not related to vector hostspecificity, as members of the family Hippoboscidae are highlyadapted to their respective hosts (59). Instead, we can imagine thatthe ecology of different bat hosts may promote the diversity ofBartonella within the Nycteribiidae, where host populations thatmore frequently come into contact with other hosts undergo morefrequent horizontal exchange. This may help explain global Bar-tonella diversity as well as the previously reported regional differ-ences in host specificity between Bartonella and bats (see the in-troduction).

Overall, a relatively low diversity of bacteria was seen to beassociated with the studied bat flies from Madagascar and theComoros, with the principal infecting taxa being the Enterobacte-riales, Bartonella, and Wolbachia. Various patterns of intraspecificgenetic diversity were observed between these models, which weexplain by differences in the nature of the bacterium-nycteribiidendosymbiotic relationship, transmission mode, host-vectorspecificity, and variations in the associated bat host ecology (Fig.5). The transmission modes of these three bacterial taxa vary;while all three are thought to be transferred vertically from motherto pupa during adenotrophic viviparity, only Bartonella may betransferred in the blood of the bat hosts. Bartonella species, wherethe highest levels of intraspecific genetic diversity were observed,thus have the opportunity for direct horizontal transfer due to batfly host promiscuity and may also be transferred by bat-host in-teractions that are independent of these ectoparasites, such as withother blood-feeding vectors (e.g., Streblidae, mites, or fleas) orhabitat overlap with other hosts. The frequency of horizontaltransfer of Bartonella is thus likely to be influenced by ecologicalfactors, such as the breeding seasonality of bats as well as theirmigration patterns, geographical distribution, and colony popu-lation size and structure. In contrast, the Enterobacteriales formstrict, primary endosymbiotic relationships with their nycteribiidhosts, but evidence of horizontal transfer between hosts that infest

the same bat species may be observed, suggesting that proximitylikely drives the frequency of horizontal endosymbiont exchange.

In conclusion, while the global bacterial community structureassociated with the Nycteribiidae is likely primarily driven by theestablishment of mutually beneficial relationships between micro-organism and host, the origins and evolution of these associationsare complex. Additionally, host-associated microorganisms areknown to interact (60), and bacterial community structure in in-sects may be influenced positively by cooperation or negatively bycompetition or exclusion and in turn may affect the biology oftheir hosts (reviewed in references 61 and 12). Bartonella speciesappear to be the only bacteria with known pathogenic potentialthat form strict relationships with the tested Nycteribiidae batflies, suggesting that these arthropods may be a true reservoir ofBartonella infection. The presence of other likely pathogenic bac-teria such as Rickettsia is only anecdotal and may even remaindetrimental to the health of these arthropods. However, theseunique vectors remain fascinating from an epidemiological pointof view due to the diverse nature of the interactions that they form,especially in tropical settings.

ACKNOWLEDGMENTS

We are grateful to the Département de Biologie Animale, Universitéd’Antananarivo, the Direction du Système des Aires Protégées, DirectionGénérale de l’Environnement et des Forêts, and Madagascar NationalParks (Madagascar) and to the Centre National de Documentation et deRecherche Scientifique (Union of the Comoros) for kindly providing re-search and export permits (194/12/MEF/SG/DGF/DCB.SAP/SCB, 032/12/MEF/SG/DGF/DCB.SAP/SCBSE, 283/11/MEF/SG/DGF/DCB.SAP/SCB, 067/12/MEF/SG/DGF/DCB.SAP/SCBSE).

D. A. Wilkinson’s postdoctoral fellowship was funded by the Euro-pean Regional Development Funds ERDF-POCT, La Réunion,ParamyxOI project. B. Ramasindrazana received his postdoctoral fellow-ship from the RunEmerge project funded by the European Frame workprogram FP7 Capacities/Regpot and postdoctoral grants from “Fonds deCoopération Régionale” of the Préfecture de La Réunion and from theRalph and Marian Falk Medical Research Trust to The Field Museum ofNatural History, Chicago. This work was supported by European Re-gional Development Fund/Programme Opérationnel de CoopérationTerritoriale Réunion, Pathogènes associés à la Faune Sauvage Océan In-dien 31189.

FUNDING INFORMATIONEuropean Commission (EC) provided funding to David Arthur Wilkin-son, Colette Cordonin, Yann Gomard, Beza Ramasindrazana, and PabloTortosa under the grant RunEmerge.

REFERENCES1. Luis AD, Hayman DT, O’Shea TJ, Cryan PM, Gilbert AT, Pulliam JR,

Mills JN, Timonin ME, Willis CK, Cunningham AA, Fooks AR, Rup-

FIG 5 Factors affecting speciation patterns and monospecific microorganism diversity for different host-associated microorganisms.

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precht CE, Wood JL, Webb CT. 2013. A comparison of bats and rodentsas reservoirs of zoonotic viruses: are bats special? Proc Biol Sci 280:20122753. http://dx.doi.org/10.1098/rspb.2012.2753.

2. Lagadec E, Gomard Y, Guernier V, Dietrich M, Pascalis H, Temmam S,Ramasindrazana B, Goodman SM, Tortosa P, Dellagi K. 2012. Patho-genic Leptospira spp. in bats, Madagascar and Union of the Comoros.Emerg Infect Dis 18:1696 –1698. http://dx.doi.org/10.3201/eid1810.111898.

3. Wilkinson DA, Melade J, Dietrich M, Ramasindrazana B, SoarimalalaV, Lagadec E, le Minter G, Tortosa P, Heraud JM, de Lamballerie X,Goodman SM, Dellagi K, Pascalis H. 2014. Highly diverse morbillivirus-related paramyxoviruses in wild fauna of the southwestern Indian OceanIslands: evidence of exchange between introduced and endemic smallmammals. J Virol 88:8268–8277. http://dx.doi.org/10.1128/JVI.01211-14.

4. Lei BR, Olival KJ. 2014. Contrasting patterns in mammal-bacteria co-evolution: Bartonella and Leptospira in bats and rodents. PLoS Negl TropDis 8:e2738. http://dx.doi.org/10.1371/journal.pntd.0002738.

5. Tortosa P, Dsouli N, Gomard Y, Ramasindrazana B, Dick CW, Good-man SM. 2013. Evolutionary history of Indian Ocean nycteribiid bat fliesmirroring the ecology of their hosts. PLoS One 8(9):e75215. http://dx.doi.org/10.1371/journal.pone.0075215.

6. Dick CW, Gettinger D, Gardner SL. 2007. Bolivian ectoparasites: asurvey of bats (Mammalia chiroptera). Compar Parasitol 74:372–377.http://dx.doi.org/10.1654/4264.1.

7. Morand S, Krasnov BR, Poulin R. 2006. Micromammals and macro-parasites: from evolutionary ecology to management. Springer, NewYork, NY.

8. Bai Y, Hayman DT, McKee CD, Kosoy MY. 2015. Classification of Barto-nella strains associated with straw-colored fruit bats (Eidolon helvum) acrossAfrica using a multi-locus sequence typing platform. PLoS Negl Trop Dis9:e0003478. http://dx.doi.org/10.1371/journal.pntd.0003478.

9. Kamani J, Baneth G, Mitchell M, Mumcuoglu KY, Gutierrez R, HarrusS. 2014. Bartonella species in bats (Chiroptera) and bat flies (Nycteribii-dae) from Nigeria, West Africa. Vector Borne Zoonotic Dis 14:625– 632.http://dx.doi.org/10.1089/vbz.2013.1541.

10. Morse SF, Olival KJ, Kosoy M, Billeter S, Patterson BD, Dick CW,Dittmar K. 2012. Global distribution and genetic diversity of Bartonella inbat flies (Hippoboscoidea, Streblidae, Nycteribiidae). Infect Genet Evol12:1717–1723. http://dx.doi.org/10.1016/j.meegid.2012.06.009.

11. Billeter SA, Hayman DT, Peel AJ, Baker K, Wood JL, Cunningham A,Suu-Ire R, Dittmar K, Kosoy MY. 2012. Bartonella species in bat flies(Diptera: Nycteribiidae) from western Africa. Parasitology 139:324 –329.http://dx.doi.org/10.1017/S0031182011002113.

12. Duron O, Schneppat UE, Berthomieu A, Goodman SM, Droz B, PaupyC, Nkoghe JO, Rahola N, Tortosa P. 2014. Origin, acquisition anddiversification of heritable bacterial endosymbionts in louse flies and batflies. Mol Ecol 23:2105–2117. http://dx.doi.org/10.1111/mec.12704.

13. Hosokawa T, Nikoh N, Koga R, Sato M, Tanahashi M, Meng XY,Fukatsu T. 2012. Reductive genome evolution, host-symbiont co-speciation and uterine transmission of endosymbiotic bacteria in bat flies.ISME J 6:577–587. http://dx.doi.org/10.1038/ismej.2011.125.

14. Morse SF, Dick CW, Patterson BD, Dittmar K. 2012. Some like it hot:evolution and ecology of novel endosymbionts in bat flies of cave-roostingbats (Hippoboscoidea, Nycterophiliinae). Appl Environ Microbiol 78:8639 – 8649. http://dx.doi.org/10.1128/AEM.02455-12.

15. Morse SF, Bush SE, Patterson BD, Dick CW, Gruwell ME, DittmarK. 2013. Evolution, multiple acquisition, and localization of endosym-bionts in bat flies (Diptera: Hippoboscoidea: Streblidae and Nycteri-biidae). Appl Environ Microbiol 79:2952–2961. http://dx.doi.org/10.1128/AEM.03814-12.

16. Jiyipong T, Jittapalapong S, Morand S, Raoult D, Rolain JM. 2012.Prevalence and genetic diversity of Bartonella spp. in small mammals fromSoutheastern Asia. Appl Environ Microbiol 78:8463– 8466. http://dx.doi.org/10.1128/AEM.02008-12.

17. Margileth AM, Baehren DF. 1998. Chest-wall abscess due to cat-scratchdisease (CSD) in an adult with antibodies to Bartonella clarridgeiae: casereport and review of the thoracopulmonary manifestations of CSD. ClinInfect Dis 27:353–357. http://dx.doi.org/10.1086/514671.

18. Kordick DL, Hilyard EJ, Hadfield TL, Wilson KH, Steigerwalt AG,Brenner DJ, Breitschwerdt EB. 1997. Bartonella clarridgeiae, a newlyrecognized zoonotic pathogen causing inoculation papules, fever, andlymphadenopathy (cat scratch disease). J Clin Microbiol 35:1813–1818.

19. Kerkhoff FT, Bergmans AM, van Der Zee A, Rothova A. 1999. Dem-

onstration of Bartonella grahamii DNA in ocular fluids of a patient withneuroretinitis. J Clin Microbiol 37:4034 – 4038.

20. Bai Y, Kosoy MY, Diaz MH, Winchell J, Baggett H, Maloney SA,Boonmar S, Bhengsri S, Sawatwong P, Peruski LF. 2012. Bartonellavinsonii subsp. arupensis in humans, Thailand. Emerg Infect Dis 18:989 –991. http://dx.doi.org/10.3201/eid1806.111750.

21. Veikkolainen V, Vesterinen EJ, Lilley TM, Pulliainen AT. 2014. Bats asreservoir hosts of human bacterial pathogen, Bartonella mayotimonensis.Emerg Infect Dis 20:960 –967. http://dx.doi.org/10.3201/eid2006.130956.

22. Kosoy M, Bai Y, Lynch T, Kuzmin IV, Niezgoda M, Franka R, AgwandaB, Breiman RF, Rupprecht CE. 2010. Bartonella spp. in bats, Kenya.Emerg Infect Dis 16:1875–1881. http://dx.doi.org/10.3201/eid1612.100601.

23. Bai Y, Kosoy M, Recuenco S, Alvarez D, Moran D, Turmelle A, EllisonJ, Garcia DL, Estevez A, Lindblade K, Rupprecht C. 2011. Bartonella spp.in bats, Guatemala. Emerg Infect Dis 17:1269 –1272. http://dx.doi.org/10.3201/eid1707.101867.

24. Bai Y, Recuenco S, Gilbert AT, Osikowicz LM, Gomez J, Rupprecht C,Kosoy MY. 2012. Prevalence and diversity of Bartonella spp. in bats inPeru. Am J Trop Med Hyg 87:518 –523. http://dx.doi.org/10.4269/ajtmh.2012.12-0097.

25. Olival KJ, Dittmar K, Bai Y, Rostal MK, Lei BR, Daszak P, Kosoy M.2015. Bartonella spp. in a Puerto Rican bat community. J Wildl Dis 51:274 –278. http://dx.doi.org/10.7589/2014-04-113.

26. Concannon R, Wynn-Owen K, Simpson VR, Birtles RJ. 2005. Molecularcharacterization of haemoparasites infecting bats (Microchiroptera) inCornwall, UK. Parasitology 131:489 – 496. http://dx.doi.org/10.1017/S0031182005008097.

27. Lin JW, Hsu YM, Chomel BB, Lin LK, Pei JC, Wu SH, Chang CC. 2012.Identification of novel Bartonella spp. in bats and evidence of Asian grayshrew as a new potential reservoir of Bartonella. Vet Microbiol 156:119 –126. http://dx.doi.org/10.1016/j.vetmic.2011.09.031.

28. Novakova E, Hypsa V, Moran NA. 2009. Arsenophonus, an emergingclade of intracellular symbionts with a broad host distribution. BMC Mi-crobiol 9:143. http://dx.doi.org/10.1186/1471-2180-9-143.

29. Jousselin E, Coeur d’Acier A, Vanlerberghe-Masutti F, Duron O. 2013.Evolution and diversity of Arsenophonus endosymbionts in aphids. MolEcol 22:260 –270. http://dx.doi.org/10.1111/mec.12092.

30. Duron O, Wilkes TE, Hurst GD. 2010. Interspecific transmission of amale-killing bacterium on an ecological timescale. Ecol Lett 13:1139 –1148. http://dx.doi.org/10.1111/j.1461-0248.2010.01502.x.

31. Hosokawa T, Koga R, Kikuchi Y, Meng XY, Fukatsu T. 2010.Wolbachia as a bacteriocyte-associated nutritional mutualist. Proc NatlAcad Sci U S A 107:769 –774. http://dx.doi.org/10.1073/pnas.0911476107.

32. Akman L, Yamashita A, Watanabe H, Oshima K, Shiba T, Hattori M,Aksoy S. 2002. Genome sequence of the endocellular obligate symbiont oftsetse flies, Wigglesworthia glossinidia. Nat Genet 32:402– 407. http://dx.doi.org/10.1038/ng986.

33. Olival KJ, Dick CW, Simmons NB, Morales JC, Melnick DJ, Dittmar K,Perkins SL, Daszak P, Desalle R. 2013. Lack of population genetic struc-ture and host specificity in the bat fly, Cyclopodia horsfieldi, across speciesof Pteropus bats in Southeast Asia. Parasit Vectors 6:231. http://dx.doi.org/10.1186/1756-3305-6-231.

34. Goodman SM. 2011. Les chauves-souris de Madagascar. Association Va-hatra, Antananarivo, Madagascar.

35. Goodman SM, Raherilalao MJ. 2013. Atlas of selected land vertebrates ofMadagascar. Association Vahatra, Antananarivo, Madagascar.

36. Theodor O, Rothschild NC. 1967. An illustrated catalogue of the Roth-schild collection of Nycteribiidae (Diptera) in the British Museum (Nat-ural History); with keys and short descriptions for the identification ofsubfamilies, genera, species and subspecies. British Museum (Natural His-tory), London, United Kingdom.

37. Wilkinson DA, Dietrich M, Lebarbenchon C, Jaeger A, Le Rouzic C,Bastien M, Lagadec E, McCoy KD, Pascalis H, Le Corre M, Dellagi K,Tortosa P. 2014. Massive infection of seabird ticks with bacterial speciesrelated to Coxiella burnetii. Appl Environ Microbiol 80:3327–3333. http://dx.doi.org/10.1128/AEM.00477-14.

38. Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S,Buxton S, Cooper A, Markowitz S, Duran C, Thierer T, Ashton B,Meintjes P, Drummond A. 2012. Geneious Basic: an integrated andextendable desktop software platform for the organization and analysis ofsequence data. Bioinformatics 28:1647–1649. http://dx.doi.org/10.1093/bioinformatics/bts199.

Bacteriome Composition of Malagasy Bat Flies

March 2016 Volume 82 Number 6 aem.asm.org 1787Applied and Environmental Microbiology

on Novem

ber 16, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

39. Edgar RC. 2004. MUSCLE: multiple sequence alignment with high accu-racy and high throughput. Nucleic Acids Res 32:1792–1797. http://dx.doi.org/10.1093/nar/gkh340.

40. Huson DH, Bryant D. 2006. Application of phylogenetic networks inevolutionary studies. Mol Biol Evol 23:254 –267.

41. Stamatakis A. 2006. RAxML-VI-HPC: maximum likelihood-based phy-logenetic analyses with thousands of taxa and mixed models. Bioinformat-ics 22:2688 –2690. http://dx.doi.org/10.1093/bioinformatics/btl446.

42. Darriba D, Taboada GL, Doallo R, Posada D. 2012. jModelTest 2: moremodels, new heuristics and parallel computing. Nat Methods 9:772. http://dx.doi.org/10.1038/nmeth.2109.

43. Paradis E, Claude J, Strimmer K. 2004. APE: analyses of phylogeneticsand evolution in R language. Bioinformatics 20:289 –290. http://dx.doi.org/10.1093/bioinformatics/btg412.

44. Dixon P. 2003. VEGAN, a package of R functions for community ecology.J Vegetat Sci 14:927–930. http://dx.doi.org/10.1111/j.1654-1103.2003.tb02228.x.

45. Legendre P, Desdevises Y, Bazin E. 2002. A statistical test for host-parasite coevolution. Syst Biol 51:217–234. http://dx.doi.org/10.1080/10635150252899734.

46. Kang YJ, Diao XN, Zhao GY, Chen MH, Xiong Y, Shi M, Fu WM, GuoYJ, Pan B, Chen XP, Holmes EC, Gillespie JJ, Dumler SJ, Zhang YZ.2014. Extensive diversity of Rickettsiales bacteria in two species of ticksfrom China and the evolution of the Rickettsiales. BMC Evol Biol 14:167.http://dx.doi.org/10.1186/s12862-014-0167-2.

47. Zhou W, Rousset F, O’Neil S. 1998. Phylogeny and PCR-based classifi-cation of Wolbachia strains using wsp gene sequences. Proc Biol Sci 265:509 –515. http://dx.doi.org/10.1098/rspb.1998.0324.

48. Baldo L, Dunning Hotopp JC, Jolley KA, Bordenstein SR, Biber SA,Choudhury RR, Hayashi C, Maiden MC, Tettelin H, Werren JH. 2006.Multilocus sequence typing system for the endosymbiont Wolbachia pipi-entis. Appl Environ Microbiol 72:7098 –7110. http://dx.doi.org/10.1128/AEM.00731-06.

49. Baldo L, Lo N, Werren JH. 2005. Mosaic nature of the Wolbachia surfaceprotein. J Bacteriol 187:5406 –5418. http://dx.doi.org/10.1128/JB.187.15.5406-5418.2005.

50. Jiggins FM, von Der Schulenburg JH, Hurst GD, Majerus ME. 2001.Recombination confounds interpretations of Wolbachia evolution. ProcBiol Sci 268:1423–1427. http://dx.doi.org/10.1098/rspb.2001.1656.

51. Duron O, Bouchon D, Boutin S, Bellamy L, Zhou L, Engelstadter J,Hurst GD. 2008. The diversity of reproductive parasites among arthro-pods: Wolbachia do not walk alone. BMC Biol 6:27. http://dx.doi.org/10.1186/1741-7007-6-27.

52. Ros VI, Fleming VM, Feil EJ, Breeuwer JA. 2009. How diverse is thegenus Wolbachia? Multiple-gene sequencing reveals a putatively newWolbachia supergroup recovered from spider mites (Acari: Tetranychi-dae). Appl Environ Microbiol 75:1036 –1043.

53. Werren JH, Windsor DM. 2000. Wolbachia infection frequencies in in-sects: evidence of a global equilibrium? Proc Biol Sci 267:1277–1285. http://dx.doi.org/10.1098/rspb.2000.1139.

54. Atyame CM, Labbe P, Rousset F, Beji M, Makoundou P, Duron O,Dumas E, Pasteur N, Bouattour A, Fort P, Weill M. 2015. Stablecoexistence of incompatible Wolbachia along a narrow contact zone inmosquito field populations. Mol Ecol 24:508 –521. http://dx.doi.org/10.1111/mec.13035.

55. Raychoudhury R, Baldo L, Oliveira DC, Werren JH. 2009. Modes ofacquisition of Wolbachia: horizontal transfer, hybrid introgression, andcodivergence in the Nasonia species complex. Evolution 63:165–183. http://dx.doi.org/10.1111/j.1558-5646.2008.00533.x.

56. Werren JH, Baldo L, Clark ME. 2008. Wolbachia: master manipulators ofinvertebrate biology. Nat Rev Microbiol 6:741–751. http://dx.doi.org/10.1038/nrmicro1969.

57. Dittmar K, Morse S, Gruwell M, Mayberry J, DiBlasi E. 2011. Spatialand temporal complexities of reproductive behavior and sex ratios: a casefrom parasitic insects. PLoS One 6:e19438. http://dx.doi.org/10.1371/journal.pone.0019438.

58. Vayssier-Taussat M, Le Rhun D, Bonnet S, Cotte V. 2009. Insights inBartonella host specificity. Ann N Y Acad Sci 1166:127–132. http://dx.doi.org/10.1111/j.1749-6632.2009.04531.x.

59. Pospischil R. 2015. Hippoboscidae, louse flies/keds, p 1– 4. In Encyclope-dia of parasitology. Springer, Berlin, Germany.

60. Telfer S, Lambin X, Birtles R, Beldomenico P, Burthe S, Paterson S,Begon M. 2010. Species interactions in a parasite community drive infec-tion risk in a wildlife population. Science 330:243–246. http://dx.doi.org/10.1126/science.1190333.

61. Minard G, Tran FH, Dubost A, Tran-Van V, Mavingui P, Moro CV.2014. Pyrosequencing 16S rRNA genes of bacteria associated with wildtiger mosquito Aedes albopictus: a pilot study. Front Cell Infect Microbiol4:59.

62. Renesto P, Gouvernet J, Drancourt M, Roux V, Raoult D. 2001. Use ofrpoB gene analysis for detection and identification of Bartonella species. JClin Microbiol 39:430 – 437. http://dx.doi.org/10.1128/JCM.39.2.430-437.2001.

63. Davis MJ, Ying Z, Brunner BR, Pantoja A, Ferwerda FH. 1998. Rick-ettsial relative associated with papaya bunchy top disease. Curr Microbiol36:80 – 84. http://dx.doi.org/10.1007/s002849900283.

Wilkinson et al.

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