Posted on Authorea 2 Nov 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.160425924.49795319/v2 — This a preprint and has not been peer reviewed. Data may be preliminary. [email protected] 92161 CA Jolla, La Drive Gilman 9500 Diego San California of University Pediatrics of Department Lutz L. Holly Author *Corresponding 4 3 2 1 symbionts microbial and HL parasites, Lutz eukaryotic bats, Afrotropical between parasites. microbial associations that and Tripartite hypothesis hosts eukaryotic the among in support parasitism results differences of Our striking mediators bats. as indirect the miniopterid well community between as among as bacterial links serve parasites identify lineages, may between malarial vertebrate also symbionts bat correlations of We the presence major significant bats. that and four non-ectoparasitized identify hypothesis microbiome and We across the oral ectoparasitized prevalence between test system. ectoparasite characteristics to network dipteran ectoparasitic wild microbial model hematophagous and a skin a bats, skin in as Afrotropical the parasitism flies use of to bat we composition study, linked by vectored this is have Hypotheses In parasites microbiome and (malarial) animals, vertebrates. host parasites. domestic haemosporidian wild malarial and and in humans and flies, tested among microbiota influence bat been tested gut also never been knowledge animal may rarely have our between bacteria mediated parasitism to interactions be eukaryotic Host-associated in to and symbionts observed hypothesized parasitism. bacterial been been as influence relating have of serve has specifically indirectly VOCs to fur as and thereby known meals. directly, and may specialized, are blood parasitism skin which (VOCs) diverse, of The compounds bacteria, phylogenetically sources organic host-associated are compatible environment. volatile by to which external parasites, parasites of dipteran the leading many hematophagous against attractants, Among ectoparasites, important barrier of hosts. defensive variety their first to wide the adapted a and host organ can mammalian mammals largest the is Skin Abstract 2020 2, November 2 1 Lutz Holly symbionts microbial and eukaryotic parasites, bats, Afrotropical between associations Tripartite etr etcyUniversity Kentucky Diego Western San California of University eateto ilg,WsenKnuk nvriy oln re,Knuk,USA Kentucky, Green, Bowling University, USA Kentucky Illinois, Western Chicago, Biology, History, of USA Natural Department California, of Jolla, Museum La Field Diego, Center, San Research California Integrative USA of California, University Jolla, Oceanography, La of Diego, Institution San Scripps California of University Pediatrics, of Department 1,2,3 ibr JA Gilbert , 1 akGilbert Jack , 1,2 ikCW Dick , 1 n alDick Carl and , ,3,4 2 1 Posted on Authorea 2 Nov 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.160425924.49795319/v2 — This a preprint and has not been peer reviewed. Data may be preliminary. a isaentiinlydpneto hi ot n anancnattruhu oto hi lives, their of most throughout contact maintain and in hosts frequently their less on observed (23). dependent are locations nutritionally but geographic are host-specific different flies typically across flies. Bat also bat species ectoparasitic are single to flies host-specific a relative typically bat within bats primary are by many the widely are flies vectored vary also Bat parasites can and 20) prevalence Malarial Haemosporida). (19, and relevance (Apicomplexa: human 22) parasites of par- (21, malarial pathogens blood-feeding several bat-specific light obligate transmit in are for to which concern hosts making known increasing Hippoboscoidea), are of (16-18), bats, (Arthropoda: and area viruses of flies Inter- an of Bat is asites relevant. animals number more pandemics. other the zoonotic a and recent all bats for of transmission among reservoirs ( pathogen pathogens as and of health parasite transmission bats human intraspecific regulating Afrotropical for factors of of importance understanding role (8- of our the helminths pathogens and supported microbial have haemosporidia, surveys with insects, they dipteran associated and only as also (second testing, such , are mammals hypothesis parasites Bats of for eukaryotic sym- framework orders of microbial 11). phylogenetic diversity speciose between comparative great most interactions diverse the a a the among harbor providing examining are Rodentia), Bats for the system hosts. to ideal and parasites, an eukaryotic are bionts, Chiroptera) have interactions (Mammalia: such vertebrates. Bats parasites, wild eukaryotic in and studied com- symbionts, well organic microbial been volatile hosts, not produce animal between that of interactions vectors microbes facilitate of of arthropod skin-associated that models to of ways rodent attractive abundance in Con- in pound with microbes observed correlates (4). host-associated been positively populations of human has sitism abundance on as relative and pressures transmission, the ( selective human parasite mosquito influence pathogens imposing of may influence potentially vector-borne studies parasites can and of example, versely, microbiome etc.), patterns For malaria, skin transmission dengue, human (3). affecting fever, the evolution thereby that host (1, preference, found host-parasite origins shape mediating feeding have ancient for ultimately interactions have responsible could mosquito be hosts anthropophilic that may bacteria, and symbionts ways viruses, bacterial parasites in that including eukaryotic interactions suggests symbionts between evidence interdependent mounting associations and biologically Many 2), myriad eukarya. hosting and of archaea, capable are Animals Afrotropics INTRODUCTION Haemosporidia, malaria, 1. parasites. hypothesis Hippoboscoidea, and the Chiroptera, hosts eukaryotic support bats, among results microbiome, parasitism of Our Keywords: mediators bats. indirect as miniopterid oral network serve among may the microbial symbionts parasites between skin microbial links malarial in that ec- identify of differences system. also dipteran presence striking We wild and and as a bats. well microbiome skin in non-ectoparasitized as the and parasitism lineages, ectoparasitized to of bat between linked composition characteristics major is community four microbiome across bacterial host prevalence Afrotropical between as vertebrate toparasite use flies the correlations we bat that significant study, by hypothesis this identify vectored the We In Hypotheses parasites test (malarial) vertebrates. to haemosporidian wild parasites. model and domestic in a malarial flies, and tested bat humans been and ectoparasitic among never hematophagous microbiota knowledge tested bats, our been gut which to rarely animal have bacteria, have and parasitism between host-associated animals, eukaryotic interactions directly, by and parasitism symbionts mediated in influence compatible bacterial be observed also relating to may to been parasites bacteria hypothesized has di- Host-associated leading been phylogenetically as attractants, parasitism. have are influence important VOCs indirectly which as thereby of meals. may serve volatile many to blood parasites, The ectoparasites, dipteran known of hematophagous of environment. are sources Among variety external (VOCs) hosts. wide the their compounds a against to organic adapted host barrier specifically defensive can and first mammals specialized, the verse, of and fur organ and mammalian skin largest the is Skin ABSTRACT Pasteurella AScrnvrss lvrss ais edaadNphvrss(21),adserological and (12-15)), viruses Nipah and Hendra rabies, filaviruses, coronaviruses, SARS , Plasmodium 2 57.Dsieteptnileouinr significance evolutionary potential the Despite (5-7). Plasmodium rnmsini hc para- which in transmission e.g. ..Bartonella e.g. N,yellow WNV, Posted on Authorea 2 Nov 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.160425924.49795319/v2 — This a preprint and has not been peer reviewed. Data may be preliminary. irr nomto spoie nTbeS.Cd o eunepoesn n nlsscnb iwdat viewed be can analyses and processing sequence for Code S2. Table sequence in additional reads, 11815 data PRJEB32520; provided https://github.com/hollylutz/BatMP. (ID) 1000 no. number sequence identifier is reads, accession rRNA study under 5000 information 16S (catalog the (EBI) of library All under Institute depth (https://qiita.ucsd.edu) Bioinformatics estimates. kit read European platform curve QIITA the even rarefaction following and the an protocol on isolation Deblur via to based available respectively, using rarefied publicly Project library, DNA were identified are per libraries pipeline were Microbiome reads gut Ecology soil (ASVs) 1000 Microbial and and variants into Earth oral, Insights well sequence Skin, Quantitative the standard (34). Amplicon using (QIIME2) de- 96 filtering (2019). the quality previously al. and as PowerSoil demultiplexing et following standard performed Lutz were MoBio USA) in sequencing scribed and CA, the amplification PCR Carlsbad, using (http://www.earthmicrobiome.org/). MoBio, extracted Natural 12955-4; of generation was Museum data Field and DNA the preparation, library at extraction, accessioned DNA Museum are Microbial Field vouchers 2.2 the S1;S2). parasite (Table with screened USA) and accordance by and IL, followed in Host (Chicago, CA) confirmation, conducted IACUC. In History sequencing Valencua, was (2015). History Sanger sampling (Qiagen, and al. Natural All DNeasy taxonomy. PCR et of triplicate Qiagen parasite Lutz using confirm using in parasites to blood malarial described BLASTn of whole as presence Honolulu. from determined the Museum, extracted were for Bishop was identity P. Bernice taxonomic DNA the and brief, and presence Chicago reference History, parasite to Natural and Malarial of identified alpha-literature, the Museum were Collected in Field flies descriptions was the microscope. to Bat of parasites stereozoom keys, ethyl identification. taxonomic collections MZ16 dipteran relevant in taxonomic Leica to of fumigated for a compared absence were using were ethanol or specimens magnification 95% bats under Presence into examination sampling, ectoparasites. by collected for blood morphologically were stored examined and blood parasites was then bats sample skin, 2-3 and and blood and euthanized noted, euthanasia, minutes each extraction, Following from 15 and of acid lN2. remainder for blood root) nucleic The in acetate for of to analyses. placed cards microscopic ˜2-4mL apex FTA cryovials for collected (from Whatman prepared in We were on collected individual analysis. placed were per was microbiome tongues (Figure films Blood chest oral Whole puncture. the for ethanol. cardiac of ethanol 95% via region 95% sterile interclavicular in in the ear, from stored (Integrastored the punches one from and biopsy one and combined membrane, disposable back, were tail sterile the from 3mm of one using membrane, region wing points 1) a interscapular multiple from using the from biopsies 2016, fur from three and included October specimens one skin voucher These to of as body. August sampling collected the post-mortem from bats extensive on from Uganda for taken allowing and were inventories, Kenya Samples biodiversity in for hand-netting. sites and field mist-netting of sixteen combination at conducted was Sampling sampling parasite and Host 2.1 host-specific the MATERIALS obligate, test two & we by METHODS data, parasitism to these 2. linked Using is bats. gut) of and involving oral, lineages parasites. (skin, widespread processes eukaryotic microbiome four characterize bat metabolic to in the 33) haemosporidia via that (32, and microbes hypothesis including bat-associated flies Afrotropical ways, bat 31). of by studies of (30, parasitism broad-scale skin number previous the on a build proximal on we other in or Here, The cavity, chemical in produced oral observed host-associated gut, involve 25). be been the may has can (21, in (29)), as cues role bacteria flies but Chemical a tse-tse protein unknown, How (26-28), also play blood mosquitoes is cues. (24). to but hosts (e.g. periods thought unknown, “preferred” parasites reproductive are is locate hematophagous brief parasite parasites time for which evolutionary and only by over host hosts mechanism bats their between leaving with immunocompatibility and host-specificity and membranes maintain skin parasites on these and fur in living 3 TM Miltex ® .Bos ape rmec individual each from samples Biopsy ). Posted on Authorea 2 Nov 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.160425924.49795319/v2 — This a preprint and has not been peer reviewed. Data may be preliminary. iesau fhsssgicnl upromdnl oes(iipeia suoQ pseudo ectopara- (Miniopteridae included models that null models outperformed Q found significantly pseudo Songbird hosts using of microbiome status skin site the of analysis regression Multinomial taxa. UniFrac weighted rare not of but unweighted ( variance using metrics microbiota oral UniFrac and ( unweighted gut metric the and among weighted observed parasitized only ( between both were observed Differences levels for were betadiversity bats non- species bacterial or non-parasitized or skin-associated ectoparasitized and in family and differences alphadiversity host significant microbial but the gut subtle or at oral, bats skin, between ectoparasitized observed and were ectoparasitism. molecular differences and No by microbiota bat haemosporidia the for Cytochrome between negative a Associations were to 3.2 similarity bats sequence other S1). 98% All (Table exhibited Uganda. analyses and microscopic in Nycteria identified genus previously the lineage to belonged which parasites, species the of individuals miniopterid profiling genus b 51% microbiota haemosporidian of for prevalence the ASVs, 53% reads) average (mean to (3,361 an 1000 47-65% samples with to from oral taxa, ranged rarefied host 202 prevalence ASVs, all reads), (5,771 from 5000 recovered samples to were (SD gut rarefied ectoparasites 230 ASVs, Hippoboscoid and 1). (29,270 (Table reads), samples 1000 skin resulted to libraries 237 rRNA Mini- rarefied of 16S (Hipposideridae, of retention families filtering the quality chiropteran and in four Rarefaction from Pteropodidae). species and Rhinolophidae, eight opteridae, representing detection. individuals 283 and sampled sampling We parasite malarial and ectoparasite, Microbiome, and 3.1 degree) and centrality stability betweenness RESULTS Network by 3. (44). con- (ordered igraph produced and nodes ( and results dataset connectivity network Network (43) respective CAVnet natural removing status. packages each observing ectoparasite sequentially R within and by the family individuals assessed using host three was filtered summarized by least were were grouped at datasets SPIEC-EASI libraries network in with microbial All appeared skin (42). and that of (SPIEC-EASI) sisted parasitized ASVs Covarian- Inference between only 0.50. Inverse Sparse Association contain structure of package prior Ecological to and R differential the for stability using a networks Estimation in and microbial ce differ skin 1e-5, reconstructed communities of we microbial rate bats, non-parasitized learning skin a (41). whether 5, Qurro examine program of To epochs count the 100,000 using feature for visualized minimum run were 3, were differentials of regressions Ranked estimated measures size multinomial to absolute on batch for Songbird referred relies a need groupings. the (hereafter with (40), surpasses between thereby Songbird coefficients differentiation and program regression groupings feature sample the multinomial of between non-parasitized features using ranking of and implemented ratios by approach, log parasitized level centered This between family differentials). ASVs host ranked microbial the as at associated permutations. grouped skin 1000 bats among with were (36), differences betadiversity vegan2.4-2 evaluated in package We differences (R fa- for function within tests adonis2 PERMANOVA bats the test. non-parasitized Kruskal-Wallis using measures and the performed alphadiversity ectoparasitized using mean between in assessed metrics) Differences were index (39). milies ggplot2 Shannon and and packa- (38), and richness dplyr (35) (observed R (37), language phyloseq programming (36), the vegan2.4-2 using ges performed were analyses betadiversity and Alphadiversity analyses Statistical 2.3 iegspeiul dnie nKna n gnamnotrdbt 1) eosre nytonon- two only observed We (11). bats miniopterid Uganda and Kenyan in identified previously lineages ± 3) aailprsts a etitdams nieyt h aiyMnotrde ihnwhich within Miniopteridae, family the to entirely almost restricted was parasitism Malarial 13%). p 2 < .8 hnlpia suoQ pseudo Rhinolophidae 0.28, = .0,PRAOA Tbe2,sgetn htdffrne eedie ytecompositional the by driven were differences that suggesting 2), (Table PERMANOVA) 0.002, Polychromophilus i.e. ievleo h rp daec arx sndsaeremoved. are nodes as matrix) adjacency graph the of eigenvalue hnlpu lvssacrotis clivosus Rhinolophus 2 ± .7 trpddeped Q pseudo Pteropodidae 0.17, = 1) l aailprstsosre nmnotrd belonged miniopterids in observed parasites malarial All 11%). n hrd9-0%sqec iiaiyt Cytochrome to similarity sequence 99-100% shared and , 4 p > .5 rsa-als Fgr ) However, 2). (Figure Kruskal-Wallis) 0.05, Rioohde ob oiiefrmalarial for positive be to (Rhinolophidae) 2 .6,alwn st identify to us allowing 0.26), = p < 2 .0,PERMANOVA). 0.005, .2 Hipposideridae 0.12, = b Posted on Authorea 2 Nov 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.160425924.49795319/v2 — This a preprint and has not been peer reviewed. Data may be preliminary. bevdi ua-oqioitrcin,i hc niiul ihlwrbceildvriyo h skin been the have on patterns diversity Similar bacterial four bats. lower all fruit with by pteropodid individuals but shared which all were that for in clusters statistics significant interactions, fewer network were exhibiting human-mosquito and these bats in study in non-parasitized observed the with differences in clusters, The families microbial diversity. bat skin microbial of greater reduction stability a contained with and associated size significantly was the parasites hippoboscoid in of presence humans that showed to analyses Network attraction needed. certainly mosquito VOCs, is anopheline similar validation with producing Further of be (45). space associated may head genera octasulfur) the these associating characterize dimethyltetrasulfide, in in consistently or identified methylsulfide, ASVs quantify them compounds bacterial not find sulfur-containing the did that as to we hypothesize such interesting host-seeking Although we study, therefore families. other this is bat in to divergent it VOCs across attractive and hippoboscoid are (46), blood-feeding disease) that with Chagas and VOCs of 45) produce (26, vector to mosquitoes primary anopheline experimentally including Actinomy- shown arthropods included been hematophagus hippoboscoid ectoparasitism have with with genera genera associations correlating these the bacterial positively in of convergence Bacteria ASVs possible hosts. cetales ectoparasitism suggesting with these studied, associated among positively families were ectoparasitism bat that ASVs four genus as all well the as in to families, mediated bat belonging multiple part ASVs across in microbiota of ectoparasites be number the may a between parasitism found links that We examine hypothesis bats to the among support first bacteria. parasites and the host-associated bats malarial are by wild by results we in infection These microbiome, parasitism and eukaryotic bat Miniopteridae. of microbiota and the family oral skin and the the the ectoparasitism microbiota. to between between on belonging oral links association bacteria these and significant transient non- to a gut of apparently of addition skin found the and In microbiome the disconnected ectoparasites. in on skin bacteria relatively harboring taxa the of bats to rare clusters and compared rich between flies taxonomically bats, association and bat ectoparasitized stable, limited ectoparasitic consistent, identified and obligate analyses families, between Network bat associations Afrotropical identify we four study this In 5). (Figure Pasteurellaceae family DISCUSSION the 4. belon- to bats with belonged positive parasites associated malaria malarial ASVs in of increase bacterial absence species proportional of greatest the number the to a ( exhibiting ged identified bats ASVs unweighted Two non-malarial analyses mi- between parasitism. and gut regression differences malarial malarial and Multinomial significant between oral, identified 4). microbiota skin, parasitism but (Table oral the NOVA) status malarial the of infection exhibited alphadiversity of malarial Miniopteridae betadiversity in on family UniFrac differences based the no bats to observed of We belonging crobiota analysis. species statistical only for sampled, adequate taxa bat the Of parasitism haemosporidian and microbiome Host 3.3 4). (Figure exception) ( the degree being para- (pteropodids node ( in median size ( microbiome cluster and connectivity skin in network the test) decrease of sum significant stability rank a and revealing Wilcoxon topology bats, the Pseudomonadales, non-parasitized in versus Burkholderiales, differences sitized orders striking remaining revealed and the analyses Firmicutes) 3). with Network (Table Actinomycetales, (phylum and Proteobacteria) order Bacillales (phylum the parasitized Sphingomonadales order or to between and presence belonged the the Rhizobiales, abundant families with to host differentially associated belonging four consistently were Actinomyce- all be six to in order that found ectoparasites bacterial ASVs ASVs of The fourteen absence of of 3). eight number (Figure Indeed, bats. ectoparasitism greatest non-parasitized with the associated exhibited potentially tales ASVs of number a ate agglomerans Pantoea p < 0.05, Corynebacterium t et o aaiie asfo he ftefu a aiisexamined families bat four the of three from bats parasitized for test) n h genus the and , Dermacoccus p Mycoplasma 5 < Acinetobacter 0.05, , Janibacter t et,a ela infiatrdcinin reduction significant a as well as test), htwr soitdwt h rsneof presence the with associated were that oyeatru minutissimum Corynebacterium Ssms togyascae with associated strongly most ASVs . hdisprolixus Rhodnius and , Kocuria p < .5 Mann-Whitney- 0.05, p < oebcei in bacteria Some . isn us(the bugs kissing .0,PERMA- 0.002, ( e.g. di- Posted on Authorea 2 Nov 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.160425924.49795319/v2 — This a preprint and has not been peer reviewed. Data may be preliminary. niiul ilpoiemc-eddisgtit h ffc flclmgaino knmcoilcommunity microbial skin on migration that local of suggested analyses of Longitudinal been prevalence. effect (57). has ectoparasite the pathogens thereby and into it and and insight parasites composition and much-needed of bacteria, caves, avoidance provide between skin the composition will have with migration of individuals bacterial bats associated localized composition of be and exhibit the may studies behavior conditions species driving behavioral this many and local been in Ecological that previously role Thus, parasitized. observed has important (56). become also microbiome an time individuals skin playing which over bat likely influencing share level the potentially are to colony and roosts found determining the (55), bat been in material have at of role plant species shift greater bat and of to a multiple soil composition known play documented America, with community variables the North genera microbial environmental Given in bacterial in local environment. Indeed, many signal that bats. the in phylogenetic is from associations explanation host have acquired host-bacteria may possible of been bats one absence have parasitized (33), apparent may with skin and or associated locality themselves Bacteria bacteria. of flies but these level, effect bat of family the host origin in the the at ascertain originated well bats as to non-parasitized betadiversity unable and microbial were ample parasitized in as we providing between differences population significant hosts, bacteria specific host identified abundant chiropteran the host-species microbiota differentially utilizing their some skin as effectively even the of (54) Moreover, of fur individuals fly. analysis host and bat Our intraspecific habitat. and skin between bat transfer the Bat between readily on (53). microbes flies microbiome living bat of fly lives exchange bat their the hippoboscoid for of the from opportunity of majority result constituent the common could spend a can indeed composition are flies study, ectoparasites – microbial our which hosts in in by ectoparasitism to our mechanism differences with study, parasites this a observed from in provide Alternatively, transfer skin may hosts. microbial of differences distinguish composition these or microbial that the locate hypothesis in the differences support of causality results ascertain robust cannot more we human reveal and Although 52). rodent may controlled 51, with species 48, experiments fur- malaria-positive (5-7, numerous relationship in infections of this documented malaria sampling explore been have to greater as unable incorporate associations, were with microbial experienced we that groups associated analyses, studies bat statistical is other Future for bacterium no ther. adequate As this investigation. parasitism why in-depth malarial more and of requires How bats rates (50). malarial of pathogens microbiome viral oral and the fungal, bacterial, from against ‘Immunopotentiator midgut, effective of dipteran the production ( of the parasites constituent with malarial common A of (49). transmission mosquitoes the culicine controlling bacterial at a aimed found periments Interestingly, we malaria. abundances, species microbiota with the differential infected of to among UniFrac investigation rare belonging unweighted relatively further are by ASV Upon differences supported observed microbiota. were to oral contributing parasitism ASVs the malarial that suggesting and analysis, microbiome metric diversity oral the between VOCs the byAssociations of consuming (suggested characterization approaches. former populations, be and metabolomics the wild other of sampling may in those and correlations including insects mask these spectrometry consider that underlying such rely mechanisms own should with the their insects experiments understand of with the associated future better VOCs To which associated producing negatively (27)). on be al. positively are may VOCs et Verhulst or bacteria that producing former, bat- 48), Bacteria be the of may the 27, hosts. products selection in their (7, host play identify interactions dipteran to at human-mosquito blood-feeding of of be and rates (47). studies may VOCs increased dipterans by mechanisms of of suggested history Similar production evolutionary humanAs 28). their shared decrease the via 27, or given increase mosquitoes (7, particularly can attracting system, (27). mosquitoes composition ectoparasite community diversity in blood-seeking microbial higher role skin to with known in attractiveness variation individuals a that than play shown have mosquitoes bacteria studies blood-seeking skin to humans, attractive In more significantly are ate agglomerans Pantoea .agglomerans P. .agglomerans P. Mycoplasma ob otsrnl soitdwt iipei bats miniopterid with associated strongly most be to 6 a entetre fnmru aarngnssex- paratransgenesis numerous of target the been has atra hc eecmol associated commonly were which bacteria, ’(PP1,abodsetu antibiotic broad-spectrum a (IP-PA1), 1’ Plasmodium .agglomerans P. p. naohln and anopheline in spp.) nvivo in a enassociated been has hog mass through Posted on Authorea 2 Nov 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.160425924.49795319/v2 — This a preprint and has not been peer reviewed. Data may be preliminary. .. od ..(03 coyo ontcifciu iessi as urn nweg n uuedirec- future and knowledge current Webb, bats: A., in Gilbert, diseases A.J., infectious Peel, zoonotic T.J., of O’Shea, Ecoogy tions. G.F., (2013) McCracken, P.M., J.L. Cryan, Wood, R.A., C.T., economy. Bowen, meets DT, ecology Hayman where Guinea: 13. in disease virus Dis Ebola Trop of Negl Outbreak PLoS (2014) L. Schwarz, DG, Bausch 12. rodents. and primates in parasites malaria HL Lutz 11. taxa. Plasmodium J Schaer 10. (2006) D. Eo- B. the Patterson, Management from CW, evolution Dick fly 9. Bat (2015) K.). D. U. Bruce Cambridge, Patterson, Nycteribiidae). Press, and Phylogenetics W., Meets Streblidae Carl Ecology (Hippoboscoidea, Dick, Evolutionary present of F., the preferences Solo to host Morse, cene in Variation K, (2017) volatiles. Plasmodium- Dittmar NO bacterial of Verhulst 8. skin & Mechanisms by WR, (2017) mediated Mukabana is JG DEB, mosquitoes JG Boer malaria W, de Takken AO, & Busula W, 7. Takken mating Vectors. T, and Mosquito Bousema odor of NO, influence Attraction Verhulst Enhanced genes AO, complex histocompatibility Busula major 6. do How (1998) WK teach Potts can preferences? & genetics human D what Penn and 5. genome human the affected malaria. has about malaria us How (2005) DP Kwiatkowski 4. A S U Sci Acad Natl M McFall-Ngai 3. pathogenic human the of tions origins divergent DB and Larremore ancient 2. cruzi. The T. (1999) WC and Gibson brucei Trypanosoma & trypanosomes, HA, Noyes JR, Stevens 1. guided JAG manuscript. analyses. the of data REFERENCES writing and to hippoboscoid laboratory of contributed identification work, authors taxonomic provided field All CWD parasites. support. performed laboratory and provided and study, analyses statistical designed and conceived HLL Science of National 1611948). Kweyu CONTRIBUTIONS the number by AUTHOR (award Phausia For supported Biology was Centers Kenya, support. in HLL the Fellowship assistance. of of logistical Research logistical Tiller Museums their Postdoctoral his Rebecca for Foundation Makerere and National Prevention Towner, for the and Jonathan the of Control Kenya Amman, Akoth Disease Brian of Cissy of for Drs. and Bartonjo and Museums Sebuliba, Museum, Solomon Mike National Babyesiza, Zoological University Sadic thank the Kityo, research we Robert conduct of Dr. University, field, to Musila Karatina permission the for Simon Authority in and Wildlife assistance Uganda parks, the national and Service in Wildlife Kenya the thank We ACKNOWLEDGMENTS 6(1). onssadPbi Health Public and Zoonoses tal. et , tal. et , dacsi Immunology in Advances Srne,Kt umih,Japan). Bunmeisha, Kato (Springer, tal. et , tal. et , rceig fteNtoa cdm fSciences of Academy National the of Proceedings m .Hm Genet. Hum. J. Am. 21)Hg iest fWs fia a aai aaie n ih ikwt rodent with link tight a and parasites malaria bat African West of diversity High (2013) 21)Dvresmln fEs fia amsoiin eel hrpea rgnof origin chiropteran reveals haemosporidians African East of sampling Diverse (2016) 8(7):e3056. 110(9):3229-3236. 21)Aeprst rgn fhmnmlravrlnegenes. virulence malaria human of origins parasite Ape (2015) 21)Aiasi atra ol,anwiprtv o h iesciences. life the for imperative new a world, bacterial a in Animals (2013) 60:2-21. 69:411-436. irmmasadMcoaaie:Fo vltoayEooyto Ecology Evolutionary From Macroparasites: and Micromammals 77:171-190. rnsi Parasitology in Trends dS oadBK .T .Ltlwo CmrdeUniversity (Cambridge Littlewood J. T. D. BRK, Morand S. ed , o hlgntEvol Phylogenet Mol Parasitology 7 e e Entomol Vet Med 118:107-116. 99:7-15. 33(12):961-973. 100:17415-17419. aaieDvriyadDiversification: and Diversity Parasite 31(3):320-326. aueCommunica- Nature Proc Posted on Authorea 2 Nov 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.160425924.49795319/v2 — This a preprint and has not been peer reviewed. Data may be preliminary. ol .Lt W ayR rvs rgr upry akA ibr,SannJ akt, ei P. Kevin Hackett,, J. JMM, Shannon McCracken G. Gilbert, Kevin A. Miller,, Jack Matthew Humphrey, Braile, Thomas Gregory Withrow, Graves, Kurtis,mJack M. R. Sarah Mazel,, Gary Florent HRS, Taylor, KW, White W. micro- Michael Lutz the Amato, L. and Katherine Fr´ed´eric behavior Holly Metcalf, JGS, Animal Jessica Song (2012) Delsuc, Jin JB Se Xavier 32. & M, Medina DW, Inouye biome. NM, Gerardo VO, morsitans. Ezenwa flyGlossina 31. tsetse the of (2013) antenna MJ the Klowden in 30. Sciences coding of Odor Academy (2019) National JR Carlson the & of Proceedings JS, Chahda N, Soni 29. NO Verhulst 28. interactions mosquitoes. of NO ecology Verhulst Chemical (2010) 27. RC mosquitoes. Smallegange and & microbiota G, skin Schraa 179-194. human M, pp Dicke between Tokyo), bats. W, (Springer, Takken of R NO, Verhulst ectoparasites Poulin 26. & Obligate B, (2006) Krasnov Knowledge BD S, Current Patterson Morand Microparasites: eds & Their CW and Dick Flies 25. Bat Flies (2019) Bat O of Glaizot Distribution. & Parasitism and and P, (2019) Infracommunities, Christe Prevalence, G Szentiv´anyi T, Lower 24. Graciolli Environments: & Urban CME, in Carvalho Bats EACD, 131-155. Specificity. on pp Anjos Streblidae) 5, JM, and specifi- Vol Torres (Nycteribiidae Heidelberg), host GL, Berlin Nycteribiidae): (Springer-Verlag, Urbieta and H 23. Streblidae Melhorn (Diptera: & S flies Klimpel bat dispersal-prone eds Parasitic vectors. Monographs, in (2014) as specificity K potential host Dittmar and high city & Explaining CW odds: Dick all 22. Against (2007) BD Patterson Transmission, parasites. & Diversity, Parasitizing CW (2020) Flies Dick Uganda. TL Bat District, 21. Goldberg Nycteribiid Bundibugyo & in and Novel Bats KA, Ledantevirus) Fruit A Bishop-Lilly (Rhabdoviridae: Soft-Furred Angolan JH, Ledanteviruses Virus: Kanyawara Kuhn of Cophylogeny AC, (2017) Paskey and Pteropodid CA AJ, Unknown Chapman Bennett Previously & 20. Infesting JH, Flies Kuhn Bat Uganda. Nycteribiid R, in Discovered Kityo Bats Newly AJ, Infecting Bennett Rhabdovirus Uganda. TL, in Goldberg Locations Multiple 19. at Bats directions. Fruit future aegyptiacus Rousettus and in knowledge current BR bats: Amman 18. in Filoviruses (2014) DT Hayman 6(4):1759-1788. & KJ Olival 17. Pathog Virus. JS Nipah Towner and 16. Hendra (2008) M. Czubet HM, Biology. Weingartl RC, B Sawatsky 15. Parasitology SA Billeter 14. 5(7):e1000536. Science CitrAaei rs,Nrok UK). Norfolk, Press, Academic (Caister , nentoa ora o Parasitology for Journal International caChiropterologica Acta 139(3):324-329. LSOne PLoS tal. et , tal. et , 338(6104):198-199. tal. et , tal. et , tal. et , cetfi Reports Scientific rnir nVtrnr Science Veterinary in Frontiers 20)Ioaino eeial ies abr iue rmEyta ri bats. fruit Egyptian from viruses Marburg diverse genetically of Isolation (2009) 6(12):e28991. 21)Broel pce nbtfle Dpea ytrbia)fo etr Africa. western from Nycteribiidae) (Diptera: flies bat in species Bartonella (2012) 21)ARcnl icvrdPtoei aayoiu,Ssg iu,i Present is Virus, Sosuga Paramyxovirus, Pathogenic Discovered Recently A (2015) 20)Clue knmcoit trcsmlramosquitoes. malaria attracts microbiota skin Cultured (2009) hsooia ytm nInsects in Systems Physiological 21)Cmoiino ua knmcoit ffcsatatvns omalaria to attractiveness affects microbiota skin human of Composition (2011) as(hrpea sVctso iessadParasities, and Diseases of Vecotrs as (Chiroptera) Bats 20(2):511. 7(1). 37:871-876. ESMcoilEcol Microbiol FEMS 116(28):14300-14308. 6. 8 Aaei rs,Lno)TidEiinEd. Edition Third London) Press, (Academic Microorganisms irmmasadMacroparasites and Micromammals id Dis Wildl J 74(1):1-9. nmlVrss Molecular Viruses: Animal 8(5):750. 51(3):774-779. aaiooyResearch Parasitology aa J Malar 8:302. Viruses PLoS , Posted on Authorea 2 Nov 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.160425924.49795319/v2 — This a preprint and has not been peer reviewed. Data may be preliminary. interactions. JG Boer de 52. A S U Sci Acad Natl CM Moraes (2016) De 51. J Milanowski & M, Medicine Golec Environmental MK, Lemieszek B, ans Mackiewicz Malaria. against J, Tool Dutkiewicz Paratransgenic 50. a as Application Their for N maculipennis Djadid Dinparast 49. A S U Sci Acad A Robinson 48. skin. BM facial Wiegmann human 47. from isolated bacteria by vitro in M Tabares 46. Bacteria. Skin Human Research. NO Network Verhulst Complex 45. for Package Software Igraph The Systems (2006) Networks). Complex T. of Nepusz, Visualization G, and Csardi Analysis 44. Creation CAVNet: (2017) C Cardona 43. Biol Comput PLoS ZD Kurtz 42. Bioinformatics and MW Fedarko 41. frames. JT Morton 40. and (2016) H analysis Wickham interactive 39. M reproducible & for L, package 0.7.6. Henry version R Fran¸coispackage R, H, an Wickham phyloseq: 38. (2013) data. S Ecology census Community Holmes microbiome vegan: B., of & (2018) R. graphics H. O’Hara, PJ Wagner, R., McMurdie E., P. Minchin, Szoecs, 37. D., H., McGlinn, H. P., M. Legendre, Stevens, 2.5-1. R., P., Package), Kindt, Solymos, M., L., Friendly, G. Statistical G., Simpson, for F. Foundation JB, (R Oksanen computing 36. statistical for environment and Austria). language Vienna, A Computing, R: (2019) RC Team 35. 2. QIIME E Bolyen 34. Comparative microbiome. (2020) HL RK bats. and McKenzie Lutz birds J. between 33. Valerie convergence & reveal microbiomes Blanton,, gut M. vertebrate Jessica of Williams, analyses Allison Ezenwa, O. Vanessa < /i > aueCommunications Nature ytrosbceimo vladgo.Pr V eeca effects. Beneficial IV. Part good. and evil of bacterium mysterious a : aueBiotechnology Nature c Rep Sci tal. et , tal. et , mSystems tal. et , tal. et , tal. et , tal. et , tal. et , 115(18):E4209-E4218. tal. et , tal. et , 1695. tal. et , tal. et , 11(5):e1004226. 21)Rpouil,itrcie clbeadetnil irboedt cec using science data microbiome extensible and scalable interactive, Reproducible, (2019) 2(2). 21)Bhvoa epne fRonu rlxst oaieognccmonsreleased compounds organic volatile to prolixus Rhodnius of responses Behavioral (2018) 7(1):9283. 111(30):11079-11084. 21)Eooyadhs dniyoteg vltoayhsoyi hpn h bat the shaping in history evolutionary outweigh identity host and Ecology (2019) 21)Oor fPamdu acprmifce atcpnsiflec mosquito-host influence participants falciparum-infected Plasmodium of Odours (2017) 21)Sas n opstoal outifrneo irba clgclnetworks. ecological microbial of inference robust compositionally and Sparse (2015) LSONE PLoS 21)Pamdu-soitdcagsi ua dratatmosquitoes. attract odor human in changes Plasmodium-associated (2018) 4(6):e00511-00519. 21)DffrnilAtato fMlraMsute oVltl lnsPoue by Produced Blends Volatile to Mosquitoes Malaria of Attraction Differential (2010) 22)Vsaiig’mcfauernig n o-aisuigQurro. using log-ratios and rankings feature ’omic Visualizing (2020) glt:EeatGahc o aaAnalysis Data for Graphics Elegant ggplot2: 21)Etbihn irba opsto esrmn tnad ihreference with standards measurement composition microbial Establishing (2019) tal. et , 23(2):206-222. 21)Eioi aitosi h yte flife. of tree fly the in radiations Episodic (2011) 21)Mlraidcdcagsi otoosehnemsut attraction. mosquito enhance odors host in changes Malaria-induced (2014) 21)Ietfiaino h igtMcoit fA.sehniadAn. and stephensi An. of Microbiota Midgut the of Identification (2011) 37(8):852-857. 10(1). 5(12):e15829. LSOne PLoS le 21)dlr rma fDt aiuain,R Manipulation), Data of Grammar A dplyr: (2018) K uller ¨ 8(4):e61217. LSNgetdToia Diseases Tropical Neglected PLOS 9 Srne-elg e ok NY). York, New (Springer-Verlag, PNAS LSONE PLoS naso giutrland Agricultural of Annals mBio 108(14):5690-5695. < i > 12(4):e0006423. 11(1):e02901-02919. ate agglomer- Pantoea 6(12):e28484. A Genomics NAR InterJournal, rcNatl Proc Proc Posted on Authorea 2 Nov 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.160425924.49795319/v2 — This a preprint and has not been peer reviewed. Data may be preliminary. otdfile Hosted associations-between-afrotropical-bats-eukaryotic-parasites-and-microbial-symbionts Table2.pdf htt- file Hosted at viewed ac- be associations-between-afrotropical-bats-eukaryotic-parasites-and-microbial-symbionts under can of (EBI) Museum Table1.pdf analyses Field Institute and the (https://qiita.ucsd.edu) Bioinformatics file at processing platform Hosted European accessioned USA). QIITA sequence are the IL, vouchers the (Chicago, for and History parasite via Natural Code 11815 and available Host (ID) PRJEB32520. publicly ps://github.com/hollylutz/BatMP. identifier number are study data cession the sequence rRNA under 16S All STATEMENT AVAILABILITY DATA green)). (dark the family ( Pasteurellaceae are parasites Highlighted the malarial bats. in of miniopterid malarial presence of with acter oral associated with features associated highest-ranked features two differential Ranked indicates clusters. 5. network (* Figure individual indicates density by bats (* colored size non-parasitized distribution topology degree and cluster network B) ectoparasitized Reingold A) test), among sum including microbiome rank family, Wilcoxon skin host the by of grouped characteristics (orange), Network families 4. bat clarity. four Figure for the of enlarged three been in have observed features genus features those Highlighted the highlighted (red), inset. to families gray belonging bat in features full four shown and family; all features) host in by associated observed grouped negatively those bats (including include ectoparasitized features of ranked skin with of associated range ectoparasite features and differential family Ranked haemosporidian by 3. grouped malarial Figure bats by of microbiota infected gut cells and oral, blood skin, status. red the bat among Alphadiversity C) 2. indicating @MEMIllustration. fly, Figure Mayfield circles bat Erin orange hippoboscoid Madison with region B) by sampling host, Illustration bat. interscapular indicates parasites. Bat of circle dashed A) side collected; sampling. were dorsal samples and on fur and associations skin which host-parasite from locations of Diagram 1. Figure Review. A Bats: LEGENDS of FIGURE Fidelity Roost (1995) SE Lewis 57. time. O Kolodny 56. 52: ronment. Panama. in CV Streblidae) Avena (Diptera: 55. aranea Megistopoda batfly the of mediate Host-relations diversity 1–20. community (1980) local WL and Overal microbiomes. identity 54. bacterial species (Parasite on al. fragmentation et habitat Brown, of Alexis effects TT, Speer A. Kelly 53. a clEvol Ecol Nat nvriyo assSineBulletin Science Kansas of University p oag),adtems bnatfaue soitdwt bec fmlra aaiim(ASVs parasitism malarial of absence with associated features abundant most the and (orange)), sp. rn Microbiol Front tal. et , tal. et , vial at available at available 3(1):116-124. 21)Dcntutn h a knMcoim:Iflecso h otadteEnvi- the and Host the of Influences Microbiome: Skin Bat the Deconstructing (2016) 21)Codntdcag ttecln ee nfutbtfrmcoimsthrough microbiomes fur bat fruit in level colony the at change Coordinated (2019) 7:1753. https://authorea.com/users/372035/articles/490187-tripartite- https://authorea.com/users/372035/articles/490187-tripartite- Mycoplasma 52:1-20. htwr losae ytreo orbtfmle.Br of Bars families. bat four of three by shared also were that 10 ora fMammalogy of Journal Authorea p -value . < 0.05, p -value .agglomerans P. t- 76(2):481-496. et,adC Fruchterman- C) and test), < .5 Mann-Whitney- 0.05, (red), Acinetob- Posted on Authorea 2 Nov 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.160425924.49795319/v2 — This a preprint and has not been peer reviewed. Data may be preliminary. ndra ieo a.B ipbsodbtfl,C a e lo el netdb aailhaemosporidian malarial by infected cells blood red bat C) indicating fly, @MEMIllustration. Mayfield bat circles Erin orange hippoboscoid Madison with B) region by sampling host, Illustration interscapular bat. Bat indicates parasites. of circle A) dashed side collected; sampling. dorsal were samples and on fur and associations skin which host-parasite from of locations Diagram 1: Figure associations-between-afrotropical-bats-eukaryotic-parasites-and-microbial-symbionts Table4.pdf file Hosted associations-between-afrotropical-bats-eukaryotic-parasites-and-microbial-symbionts Table3.pdf vial at available at available figures/Figure1/Figure1-eps-converted-to.pdf https://authorea.com/users/372035/articles/490187-tripartite- https://authorea.com/users/372035/articles/490187-tripartite- 11 Posted on Authorea 2 Nov 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.160425924.49795319/v2 — This a preprint and has not been peer reviewed. Data may be preliminary. iue2: Figure lhdvriyaogtesi,oa,adgtmcoit fbt rue yfml n ectopar- and family by grouped bats of microbiota gut and status. oral, asite skin, the among Alphadiversity

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Hipposideridae

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Pteropodidae Parasite status Parasite Parasitized Non-parasitized Posted on Authorea 2 Nov 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.160425924.49795319/v2 — This a preprint and has not been peer reviewed. Data may be preliminary. etrsicuetoeosre nalfu a aiis(e) hs bevdi he ftefu a families bat clarity. four Highlighted the for of enlarged inset. three been in have gray observed features in those highlighted family; genus (red), shown of the families host Bars features) to bat by belonging four associated grouped features all and negatively bats in (orange), ectoparasitized (including observed of those features include skin ranked features with of associated range features differential full Ranked 3: Figure figures/Figure3/Figure3-eps-converted-to.pdf Mycoplasma 13 htwr losae ytreo orbtfamilies. bat four of three by shared also were that Posted on Authorea 2 Nov 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.160425924.49795319/v2 — This a preprint and has not been peer reviewed. Data may be preliminary. rue yhs aiy nldn )cutrsz est *indicates clusters network (* individual indicates density by bats (* colored size non-parasitized distribution topology degree and cluster network B) ectoparasitized Reingold A) test), among sum including microbiome rank family, skin Wilcoxon host the by of characteristics grouped Network 4: Figure A B C

Non-parasitized Parasitized Frequency Density 0.0 0.5 1.0 1.5 0.00 0.05 0.10 0.15 0.20 1 0.0 Miniopteridae Cluster size (ln) 5 2.5 Degree ** ** 10 * 5.0 525 15 20 7.5 30 0.0 0.1 0.2 0.3 0.4 0.5 0.00 0.05 0.10 0.15 0.20 1 0.0 Hipposideridae Cluster size (ln) 5 2.5 Degree * 10 5.0 15 7.5 20 25 14 0.0 0.1 0.2 0.3 0.4 0.5 0.00 0.05 0.10 0.15 0.20 0 0.0 Rhinolophidae Cluster size (ln) 10 2.5 Degree * 20 5.0 30 p 7.5 40 -value 50 0.00 0.05 0.10 0.15 0.20 0.0 0.1 0.2 0.3 0.4 0.5 0 Pteropodidae 0.0 Cluster size (ln) < 20 Degree 2.5 40 0.05, p 5.0 60 -value 80 7.5 Parasitized Non-parasitized 100 t- et,adC Fruchterman- C) and test), < .5 Mann-Whitney- 0.05, Posted on Authorea 2 Nov 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.160425924.49795319/v2 — This a preprint and has not been peer reviewed. Data may be preliminary. ntePserlaeefml dr green)). (dark ( the family parasites Pasteurellaceae are Highlighted malarial the in of bats. miniopterid presence malarial with of bacter associated oral with features associated highest-ranked features two differential Ranked 5: Figure p oag),adtems bnatfaue soitdwt bec fmlra aaiim(ASVs parasitism malarial of absence with associated features abundant most the and (orange)), sp. figures/Figure5/Figure5-eps-converted-to.pdf 15 .agglomerans P. (red), Acineto-