This article isThis article by protected copyright. All rights reserved. 10.1111/mec.14219doi: differences to lead between the of this version cite thisarticle and asVersion Record.Please copyediting, been throughthe typese This article acceptedhas been for publication andundergone fullpeer review buthasnot † 7 6 5 4 3 2 1 G. Mitchell Jurkevitch Ben Michael the Host Article :Original Article type : (Orcid ID 0000 DR. SONIA KLEINDORFER : (Orcid ID 0000 Y RACHAEL DUDANIEC DR. Joint first authorship first Joint Island, Cruz Santa Foundation, Darwin Charles Department of Chemistry and Physics, Arcadia University, Glenside, Philadelphia, 19038, USA 19038, Philadelphia, University, Glenside, Physics, Arcadia and Chemistry of Department Australia Adelaide, 5001, University, Flinders Sciences, School ofBiological and Microbiology of Department Australia 2109, University,Sydney, Sciences, Biological Macquarie of Department University Hebrew Environment,The and Food Agriculture, of Faculty Entomology, of Department Facultad De Ciencias Agrícolas, Universidad Central Del Ecuador, Quito, Ecuador Del Ecuador, Central Universidad Agrícolas, Ciencias Facultad De AcceptedRehovot Jerusalem, of University Hebrew The Rehovot Jerusalem, of Article Galápagos Islands Galápagos - specific associations affect the microbiome of of themicrobiome affect associations specific 3 , 4

Renee - , Sonia Kleindorfer , Sonia Yosef J. 1† , Doron S. Y. Zaada Y. S. , Doron

Smith

,

76100, Israel 76100,

4 , Charlotte E. Causton E. , Charlotte

4 *, *,

Plant Pathology, Faculty of Agriculture, Food and Environment, and Food Agriculture, Facultyof Plant Pathology, Boaz

tting, pagination and proofreadingtting, process,may which 1† Yuval - , Rachael Y. Dudaniec , RachaelY. 0001 - 0002 , 1 - Galápa

5130 - 76100, Israel 76100, 1854 5 , M. Piedad Lincango , M.Piedad - Philornis downsi Philornis 3122) - gos 6808)

Islands, Ecuador Islands,

2 , Zohar , an introduced parasite to to parasite introduced , an 5,6

Pasternak ,

Shanan

3 S.

,

Edouard Edouard Tobe 4,7 , J ames

This article isThis article by protected copyright. All rights reserved. to according across islan microbiomes similar finches) harbored (ground the host same on feeding microbiome, aslarvae larval finches, ( warblerfinches insectivorous from strictly larvae retrieved between differed significantly structure which that show host finch species four we nestlings developing tissue of and andconsume finches blood Darwin’s of Larvae infestnests birds. other land and finches Darwin’s fly parasitic invasive microbiome an of the investigate Herewe hosts. impact affected in and prevalence can therefo pathogens of microbiome host and pathogenicity host differential for implications have parasiticorganisms of microbiome the bacteriaforming of and diversity composition The Abstract: interactions. Keywords: +61 Phone: Sonia author* Corresponding Accepted olivacea Certhidea Article Philornis downsi Philornis characterize the bacterial microbiota associ thebacterial characterize Kleindorfer significantly differ significantly

Geospiza

larval and adult microbiomes are dominated by are dominated microbiomes larval and adult ds

- microbiome,

dietary composition or or composition dietary 8 . Our results suggest that the microbiome of microbiome of the that results suggest Our -

8201 5232 8201

: and School of Biological Sciences, Flinders University, University, Sciences, Flinders Biological of School ) and ) and , recently introduced to the Galápagos Islands, where it poses extinction extinction itposes where Islands, Galápagos the to introduced , recently

, inhabiting two islandsand two , inhabiting Camarhynchus

those those

Philornis downsi Philornis between life stages between life ;

Email: , and , and parasitizing hosts with hostswith parasitizing have [email protected] nutritional needs nutritional re have consequences that are relevant for managing disease managing for are relevant that consequences re have

spp. respectively). Finally, we found no spatial effects on spatialon no effects wefound Finally, respectively). spp.

severe mortality impacts mortality severe , Galápagos

in the in ated with with ated

representing two ecologically distinct groups. We groups. We distinct ecologically representingtwo ir

, and is significantly affected by host affected by is significantly , and distribution broader dietary preferences (ground andtree (ground preferences dietary broader

Islands, Darwin’s finches, host finches, Darwin’s Islands, - parasite co parasite P. downsi P. P. downsi P.

the phyla

s. . Using

adults and larvae sourced fromlarvae sourced and adults Additionally, bacterial community bacterial community Additionally, - evolutionary interactions. The The interactions. evolutionary

changes during during changes Proteobacteria and Firmicutes, Firmicutes, and Proteobacteria Adelaide, South Australia 5001 South Australia Adelaide,

16s

r RNA sequencing data data RNA sequencing its - parasite development, - related threat to

the the .

This article isThis article by protected copyright. All rights reserved. ( the means a provide andmay pathogenicity, of patterns understanding into agateway offers (e. intern species, and host their of from that externalinfluences environmental their of much derive parasites, which ( differ throughout of organisms changes diversity microbial (e.g., hosts affected impact in and disease prevalence managing and 2015 host and pathogenicity parasitic organisms bacteria of composition within varying and A diverse ( parasiticorganisms inthe of case particularly complexity, additional micro between the acclima thermal pathogens, resistance against reproduction, in nutrition, hosts their bacteria assist Symbiotic ( distinguished canbe relationships andevolutionary taxonomic boundaries Clemente ( trajectories evolutionary and characteristics ecological their with of organisms diversity microbial the link feasible to makingit increasingly is Metagenomics Introduction of to development the contribute stage thelarval factors during Weiss 2015 Douglas Newton

Accepted Weiss g., Article progress of co of progress ). Therefore, & tion tion et al.

Aksoy 2011 Aksoy et al. and host range expansion (e.g. expansion hostrange and et al. et

) and environmental ) and 201

2012

evolutionary relationships in novel novel long in or relationships evolutionary 2013 understanding understanding biomes of parasitic organisms with parasiticorganisms of biomes ally, from the physiological or chemical defenses that hosts evolve against them against evolve hosts that chemicaldefenses or fromally, physiological the 0 ). ). Microbiomes).

; Sandionigi ; Sandionigi - ). Characterizing parasite co . Unraveling the ecological significance ecological significance the Unraveling

microbial diversity of pathogens of pathogens diversity microbial novel, effective control strategies. novel, effective control - evolutionary interactions ( interactions evolutionary et al. conditions conditions can now can

the microbiome of parasites across host species th host across parasites microbiome of the 2015 Engel & Moran 2013 Engel

). be characterized at sufficient resolution such thatsuch resolution be characterized atsufficient (

Moran 2015

their

ent life stage ent own ; Zolnik ; Zolnik - Zilber standing host standing Wang Wang

host’s ecology and evolution evolution and adds ecology host’s ;

Douglas 2015 isimportant step an -

Rosenberg Rosenberg et al. et al. Newton Newton that affect multiple host species multiple host affect that of of s and according to according s and bacteria for bacteria

2012; 2012; 2016 can lead to to can lead - parasite interactions interactions parasite Ley et al. ). This is also true for for alsotrue ). Thisis & ). However, the link ). link the However, Sandionigi Sandionigi et al.

Rosenberg 2008 Rosenberg

2010

this parasite this

differential host differential host 2008 for predicting predicting for ). The of measuring measuring of erefore ). dietary dietary et al.

will ;

This article isThis article by protected copyright. All rights reserved. their parasitic,nest to fruit asopposed decaying on 1997 ( in nests finch Darwin’s in wasfirstobserved but to the introduced nest in the (reviewed birds within nestling fly, parasitic impactsintroduced an from withenormous mortality confronted isbeing finches, Darwin’s (Francesconi direconse canhave and widespread wildlife. and humans diseasein managing for outcomes organisms pathogenic ( ticks fitnessin lower and (e.g. (e.g. microbiomes ( to ontogeny according Lymethe indisease changes documented have 2011 Jurkevitch ( pests agricultural diseaseand vectorsof tool againstboth novel an emerging 2011; & Aksoy as transmission, pathogen for vectors insectdisease within the microbiome of on the role focusing 2013 in (reviewed groups protistan or distinctfungal with symbioses specific document and 2013 species important economically

Accepted Articlemalaria Philornis downsi downsi Philornis , ). ), termites(

via antibiotic treatment) has also been proposed as a method to decrease disease susceptibility susceptibility todecrease method disease as a alsobeenproposed has treatment) antibiotic via Insights into Insights The major The The parasitic infestation of live vertebrates with dipterous fly larvae, termed larvae, fly dipterous with vertebrates of live infestation parasitic The

and trypanosome trypanosome and &

; Weiss Lupi Warnecke Warnecke Zindel Galápagos ity of insect microbiome research has focused on emerging modelemerging systems on focused microbiome has research insect of ity ,

Varroa Varroa

pa

2012 (Diptera: Muscidae), which which (Diptera: Muscidae),

therefore offers evolutionary and ecological insights, as well as applied aswellapplied insights, andecological evolutionary offers therefore thogen et al. Zolnik Zolnik et al. et al. Clayton Clayton ). On the ). the On mites honeybees of et al.

2013 parasites Islands circa 1960s ( 1960s circa Islands

ecology via metagenomic methods are methodsare metagenomic via ecology

2011 et al.

including honeybees ( honeybees including

2007 ) .

et al.

). quences for the health of humans, livestock and wild animals wild and humans,livestock of health the for quences Galápagos

Further to this, m Further this, to 2016

Microbiome studies of arthropod ectoparasites are arthropod of Microbiome studies in mosquito in ), and

2015; 2015; ), and report ectoparasite and ), Kleindorfer Kleindorfer fruit fruit

, - Gall Islands in Ecuador, the world Ecuador, in Islands causing tick ( tick causing Sandionigi Sandionigi flies ( Causton e develops as larva by feeding by aslarva develops - s dwelling larvae, which require frequent blood blood frequent require larvae, which dwelling et al. , and anipulation of the anipulation K e.g. e.g. & Fessl

w

Tset 2016 Dudaniec 2016 Dudaniec o et al. et al. ng & Moran 2016) &Moran ng Behar et al. Ixodes scapularis Ixodes et al. s ). Characterizing

e flies 2006

2015 et al.

- 2001 induced changes in host host changesin induced , respectively ; increasing ). Microbiome manipulation manipulation Microbiome ). Kleindorfer Kleindorfer

200 microbiome of pest insects is pest microbiome of ). Adult ). ). The fly was accidentally ). flywas The accidentally 9 ; -

renown group of birds, of birds, group renown Hoffman Hoffman Staubach , ants (

) microbiome ) microbiome on blood andtissue of on blood , with much work much , with work

the microbiome in microbiome in the P. downsi P.

& ( reviewed in Weiss in reviewed

Kautz Sulloway 2016 Sulloway myiasis, is Newton Newton et al. et al. et al. et al. few, and and few,

flies feed et al. al. et

2011 or or 2013 et al. such such ; ), ),

This article isThis article by protected copyright. All rights reserved. et al. Knutie 2014; species invasive against measure used et al. battle speciesconservation critical invasive declines recent cause of Galapag of survival to the threat size naris enlarged haveseverely generally that fledge fewhosts the deformation; massive cause and beak the nestling ( ( 2017 & Kleindorfer and ( (1) the endemics: island endangered critically are finchspecies Darwin’s P. downsi w devastating, been has populations host The on impact in parasitismlarval (reviewed to due nestlings of at55% this is documented period over mortality direct theof 2000, number Since me Fessl et al. 2010 etal. Fessl heliobates Camarhynchus Camarhynchus Kleindorfer Kleindorfer

Accepted Article the in ( before nest pupating nestlings fromdeveloping als declined 52% between 2004 to 2013 2004to2013 between 52% declined

2017 2017 . To date, control control date, . To The The

larvae per nest ( per nest larvae ) ). ,

Galápagos and et al. & pauper

; trapping adult flies ( adult flies trapping Dudaniec 2016 Dudaniec Cunninghame Cunninghame

) 2014; 2014; ( ; (2) ; Kleindorfer Kleindorfer

) Islands are a protected area and a World Heritage Site, HeritageSite, aWorld areaand aprotected are Islands

Philornis downsi Philornis has the highest the has in in options O’Connor etal.2014 O’Connor O’Connor et al. etal. 2010 O’Connor ), P. downsi P. Galápagos whose population barely persists with persists barely population whose P. downsi P.

& ). os land birds ( land os et al include

Sulloway 2016 Sulloway

. 2017 . Causton Causton larvae per endemic finch nest has increased by 46% in nests, and innests,and 46% by increased has larvae nest endemicfinch per

passerines

P. downsi treating nests with permethrin permethrin with nests treating

flies is a major threat to the persis to threat is amajor ( O’Connor O’Connor ). ( Causton Causton

The first instar larvae of larvae instar first The et al. must be highly specific highly mustbe Causton etal. 2006 Causton ) a , ; biological control using natural natural enemies using biological control ,

Dvorak et2012 al. Dvorak ). making this novel host making

intensity of any finch species on Floreana Island Island Floreana on finch species ofany intensity

2013; Kleindorfer et al. 2016), while Kleindorfer2016), etal. 2013; High by caused mortality etal. et al. ith severe declines in avian hosts with many withmany avianhosts in declines ith severe

2010 2013 Fessl.

a/b ;

Kleindorfer ) andis ; Kleindorfer ; Kleindorfer e circa circa ; t al.

Peters to to P. downsi insecticide tence of the tence ofthe

avoid threatening avoid - 100 birds 100 2006 m parasite association a association parasite thought to be the principal principal bethe thought to edium finch tree edium & & P. downsi P. ;

and and Kleindorfer 2017). Kleindorfer 2017). Lahuatte

Dudanie feed on the inside theinside of feed on et al.

( o any control any control Causton Causton mangrove mangrove n Isabela Island Island n Isabela

2014

is

c 2016 et al. there is there

( the biggest biggest the Bulgarella Bulgarella endemic a/b

et al.

2016 ; finch ; Pe

Fessl

Two ters ).

This article isThis article by protected copyright. All rights reserved. co of mechanisms may which islands, communiti bacterial disparate we aim identify to above, the nestsof samples from envi or ecological inherent to dietary ( terrestrial finches finches, tree Darwin’s ( arborealfinches host groups; distinct ecologically to two relation in communities non and of microbiome stud been genus associated the with specificcommunity the (e.g. Enterobacteriaceae, of communities harbor to flies areknown a the parasitemicrobiome of requirescharacterization control (e.g defen host to resistance decrease parasite’s to the order microbiome fly’s in the of via be manipulation could Technique. ( program breeding fly trapping more develop effective researchto ongoing Accepted Article Causton . Wang Wang ses or environmental stressors, as similarly proposed for insect vectors of transmissible diseases diseases transmissible of insectvectors proposedfor similarly as stressors, ses environmental or preferences Here, we implement high throughput throughput high implement Here, we - parasitic adult developmental stages of the fly, (2) divergence in in divergence (2) fly, the of stages developmental parasitic adult ied. et al.

et al. A novel approach that may be an instrumental and targeted method to control to control method targeted and beaninstrumental may that approach A novel

2013 P. downsi P.

2012; 2012; - Small evolutionary change evolutionary

and physiology and indicate indicate ;

Lahuatte is underway is underway s Camar mall ground finches across two islands (Santa Cruz, Floreana). Cruz, (Santa islands finches acrosstwo mall ground Clayton

ground finch; ground and differential pathogenicity to hosts or parasites and thus, thus, or parasites and hosts to pathogenicity differential ronmental differences. We test this by comparing the microbiota in microbiota in the comparing by testthis We ronmental differences. h determine ynchus et al. et al. to to understand .

Finally, (3) if the (3)if Finally,

2016 2015

spp.

Geospiza fuliginosa Geospiza or : (1) changes in bacterial community across parasitic larval across parasiticlarval bacterial community in changes (1) :

) to ) to targets for microbiome manipulation microbiome targets for

) and onespeciand . However, moving towards this potential avenue of of avenue potential this towards moving . However, 16s evaluate the possibility of using Sterile usingInsect of evaluate possibility the the fly’s the rRNA

Philornis techniques techniques P. downsi downsi P. sequencing es of warbler fi warbler es of

reproductive biology in greater detail in greaterdetail reproductive biology ), which differ in in whichdiffer ), es across ecologically divergent hosts divergent or ecologically across es , or any other avian parasitic fly, has never hasnever parasitic avian fly, anyother , or cross hosts and locations. While While locations. muscid and hosts cross

(Cha microbiota differs across islands due microbiota acrossislands differs Su Su

to characterize the gut characterize gut to the et al. et al. nch P. downsi P.

2016)

2010; 2010; Certhidea olivaceae Certhidea their .

.

foraging behavior foraging At the two Gupta Gupta

bacterial potential potential In achieving achieving In species of of species

same time et al. P. downsi

2011 ) and ) and ,

), ,

a

This article isThis article by protected copyright. All rights reserved. ( al. 2016 papaya using 2014 February in trapped = 5) were females (n posterio colorofthe and presence the based on thelaboratory backin verified fieldand assessedinthe was each of larva class The age ethanol. 95% in stored immediately tweezers and with extracted aseptically were inspections nest detected during zip inaplastic sealed collected and nest was endoscope) camera boroscope non were inspected (e.g. protocols Cruz Floreana: during Philornis d season nesting the during particularly diet generalist broad hasa but also onthe ground, forages that primarily Kleindorfer 2015) parvulus Camarhynchus and insects both feed or on ( insectivorous areeitherstrictly and habitats arboreal ofthe andFloreana SantaCruz of Philornis sampling Species and Methods Accepted0°44′3 Article :

0°37′ 34″ S, 90°23′ 10″ W 90°23′ 10″ 34″ S, 0°37′ February and February 4.1″S, 90°18′10.4″W, elevation 90°18′10.4″W, 4.1″S,

for methods) positioned at several locations around El Barranco on the island of Santa Cruz Cruz islandofSanta onthe ElBarranco around locations at several positioned methods) for downsi

1°17’48.4”S, 90°27’07.0”W 1°17’48.4”S, ownsi Kleindorfer Kleindorfer

larvae (n = 20) were collected from nests of four finch species inhabiting the islands the inhabiting finch species four nests of from collected = 20)were (n larvae 2 . The fourth species ( species . Thefourth nd

- and 3 and invasiv March March

, C rd a et al.

ely using a wireless endoscope snake camera snake endoscope wireless a ely using marhynchus pauper marhynchus instar larvae were collected from were collected instar larvae in in vegetative resources vegetative resources 2006 . Once nestling activity had ceased (nestlings died or fledged), the orfledged), (nestlings died ceased had activity Oncenestling .

, 2014a

elevation Galápagos

( Santa Cruz Santa

, elevation ~350 m.a.s.l. ~350 , elevation ( : s ). Finch nesting was monitored every two days; nest contents days;nest two every monitored was nesting ). Finch Kleindorfer

mall ground finch, SGF, SGF, finch, mall ground 15 – :

41 m.a.s.l. 41 4 -

archipelago (Table 1). Three Three 1). (Table archipelago 00 lock bag for dismantling dismantling for bag lock – , respectively

r spiracles(e.g 0°37′ 34″ S, 90°23′ W 90°23′ 10″ 34″ S, 0°37′ 700 700 m. ( s et al. mall and mall ) , and g a.s.l. reen warbl reen

2006

preserved ) - m ) baited Mc baited ; Chaves ; Chaves following well following

nests )

( edium tree edium

. Tebbich Tebbich and February 2014 and 2015 and2015 February 2014 and Geospiza fuliginosa Geospiza Kleindorfer Kleindorfer er in the highlands of twoislands of thehighlands in - finch, WF, finch, in thelaboratory in

etal. in 95% ethanol until until 95%ethanol in P et al. hail traps (see (see traps hail

(

including insects, insects, including host host e.g. 2.4GHz WiFi video WiFi 2.4GHz e.g. finches, STF and MTF, MTF, STF and finches, , elevation ~550 m.a.s.l.; , elevation

- et al. 2016) established field field established

2004; Peters 2004; species inhabit inhabit species Certhidea Certhidea olivacea

2016 .

) is a seed feeder feeder isaseed )

). . L Lahuatte et et Lahuatte

Adult Adult arvae arvae &

( Santa Santa

) This article isThis article by protected copyright. All rights reserved. hood. flow alaminar in sterileconditions under wereperformed procedures extraction andDNA Dissections in females adult from preparations intracellular, possible to test for order In instructions. themanufacturer’s to according DNA to extract processed and individual ofeach out dissected saline sterile times in USA, one Germany). the using insects from extracted Total DNAwas sequencing and DNA extraction T locations annual rainfall Comparable islands Santa Cruz and Floreana unpublished m (408 Island Santa Cruz of the highlands mm in 70 data) and unpublished m (340 Island Floreana of highlands inthe was57mm rainfall March 2014, m (200 Island Cruz ofSanta inthehighlands mm and 6 m (340 Island Floreana of thehighlands mmin was13 March rainfall (respectively). trap nest or unique fromeach oradult, larva either onespecimen, we used analysis, For processed.

Accepted Articleable 1.

.

minute), surface sterilized in 1% sodium hypochlorite hypochlorite sodium 1% sterilized in surface minute), Further details regarding sample source, host species and collection dates are provided in provided dates are collection species and host source, sample detailsregarding Further

Prio

data). Within the same year, highland highland year, the Within same data).

Rainfall showed comparable patterns within years and across locations. locations. and across within years patterns comparable showed Rainfall r toDNAextraction

solution suggests suggests . The intestinal tract, including the mid the including tract, intestinal . The –

irrespective irrespective , cluded the ovaries in addition to the gut tissue of each individual. individual. each of tissue to thegut in addition theovaries cluded

insects were rinsed in a mild deterg a in rinsed were insects that similar nutritional resources were available to hosts across across hosts to available were resources nutritional similar that of whether whether of

Qiagen blood and tissue and blood Qiagen rainfall during March was comparable between between comparable was March during rainfall maternally transmitted maternally . a . s . it was a dry year (2006) or wet year (2014). (2014). (2006) orwet year dry year a it was l . ) (CDF Meteorolo ) (CDF

(1 minute), and finally washed three washed andfinally minute), (1

. and hindgut and a . s ent solution (1% Alconox, (1% Alconox, ent solution .

l or MoBio or . ) ( . . a a O’Connor et al. 2010 et al. O’Connor . . s gical Database). In gical Database). s

symbionts, DNA DNA symbionts, . . l l . . ) ) (Kleindorfer (Kleindorfer )

( , Causton

was subsequently subsequently was soil

kit In 2006, 2006, In s ,

(Hilden, c )

This article isThis article by protected copyright. All rights reserved. the location genus, and / finch group sample. each in abundance Bray USA) using each sample. diversity in Holmes taxonom genus and the similarity on97% based (OTU) units taxonomic operational into clustered were deleted. Sequences and mitochondria, chloroplast, (Edgar method UCHIME ofthe implementation Huse algorithm of the based on sequenc errors, sequencing reduce Tofurther overhangs. no with overlapped (Quast database alignment reference SILVA to the aligned perreads sampl 2013). et al. de and trimming control, ( protocol MiSeq the to according standard (Schloss v1.31 MOTHUR in and analyzed wereprocessed sequences Raw rRNA gene. by Standard USA). (Illumina, platform MiSeq Illumina the USA)using (Chicago, ofIllinois University the of Services Facility The Bacterial 16S

Accepted Article the 515F etal.(2015) with Green M b acterial acterial ulti

Statistical analyses and diversity indices were performed PC were in performed indices anddiversity analyses Statistical (2014) y. Genus abundance data were not rarefied, following the recommendation recommendation the following rarefied, not data were abundance Genus y. - R esponse esponse

After trimming and quality filtering, the resulting datasets included on average 37134 average37134 on included datasets the resulting filtering, quality and After trimming

diversity of each sample was analyzed by high analyzedby was sample diversity each of rRNA - . Rarefaction curves indicated that sequencing covered over 99% of the microbial ofthemicrobial over99% covered sequencing that indicated curves . Rarefaction level taxonomic affiliation of each OTU was identified according to current SILVA SILVA to current according identified OTU was ofeach level taxonomicaffiliation - e (min: 10272, max: 62013, table 1) spanning table1) spanning max:62013, 10272, e (min: Curtis distances. All data were relativized according to sample total, i.e. the sum of i.e.thesum total, to sample were according data relativized All distances. Curtis

gene gene P ermutation ermutation -

diversity analyses diversity T noising were performed as previously outlined ( outlined previously performed as noising were argeted A priori

‘unknown’ ‘unknown’ et al. et P -

rocedure rocedure - island related differences among groups) were performed with performed were amonggroups) relateddifferences island

A comparisons aimed to test for specific hypotheses ( specific hypotheses for test aimedto comparisons - 806R primer pair targeting the V4 region of the bacterial 16s bacterial the of theV4region targeting pair primer 806R mplicon mplicon

(2010). Finally, chimeric reads were removed with MOTHUR’s MOTHUR’s with wereremoved chimeric reads Finally, (2010). s http:// equences equences

( MRPP; Mielke 1984). Ordinations were performed with with were performed Ordinations Mielke 1984). MRPP; S equencing (TAS) approach was employ was (TAS) approach equencing et al. et al. www.mothur.org/wiki/MiSeq_SOP 2011) and all 2011) and (i.e. unclassified at the kingdom level) the kingdom at unclassified (i.e. etal. - throughput sequencing at the DNA theDNA at sequencing throughput 250 bp in length. All sequences were All sequences bpinlength. 250

2013) and filtered so that they all they that filteredso and 2013)

non - - ORD v6.08 (MjM Software, (MjM Software, v6.08 ORD bacterial Schloss et 2011; al. Schloss es were pre es

reads ). Quality ). Quality et al. et al. of McMurdie McMurdie of ed as described described as ed , including i.e. - clustered clustered

host host 2009) 2009) ,

were Kozich Kozich -

& This article isThis article by protected copyright. All rights reserved. ofthemicrobiome minor part arelatively for accounting Proteobacteria with ofallreads), 4.16% primarily The P. downsi re eachsample, comprising taxa the among distributed unevenly by Accordingly representatives. few by a dominated commonly and were sections). following distributed were respectively,and eachsample, readsin all ± 5.90%of and24.24 74.02±5.96% for accounted average On representatives. other dominant microbiota andadult larval level taxonomic at some unidentified 4 between distributed all sampl across affiliation to taxonomic according sequences nests of community bacterial The gut of diversity and Composition Results arereported. errors standard corresponding niche, the text ( linkages flexible beta with were performed analyses Cluster measure. asimilarity as N on

Accepted indices Simpson and Shannon Article - microbial communities communities microbial metric

of four four of

and do not differ in their in notdiffer do and

- 0.25

of

a differently differently

dult dult Multi Firmicutes (mostly Lactobacillaceae and Enterococcaceae: 57.07 Enterococcaceae: and Lactobacillaceae (mostly Firmicutes ). Darwin

Data from from Data fly - Dimensional Scaling Scaling Dimensional

microbiome The The ’s

finch species finch in phyla, phyla, microbiomes microbiomes larva 2 nd s associated associated were and 3 and 14 the e

and habitat or or habitat five adult female flies female five adult class ,

P. downsi P. and evenness measures indicated that reads were reads indicated that measures evenness and comprised mainly mainly comprised rd

were

adult instar larvae instar . Groups with less than 100 reads were reads excluded. than 100 less Groups with . es of i of (NMDS; Mather 1976)at Mather (NMDS; with adult f with adult , 24

ndividual s examined examined , nutrition.

and

orders, 27 families and 12 groups of bacteria bacteria of 12groups familiesand 27 orders, microbiome microbiome

( ±

in larvae retrieved from from retrieved larvae in s.e.)

were

emale adults and larvae and adults

via

Throughout the text Throughout sequences affiliated to these two phyla two phyla these to affiliated sequences of

and 20 individual larvae parasitizing parasitizing larvae and 20individual

Proteobacteria and Firmicutes, with few with Firmicutes, and Proteobacteria gr Illumina Miseq sequencing. Clustering Clustering sequencing. Miseq Illumina

P. downsi ouped ouped es identified a total of 64 taxa 64 taxa of total a es identified

500 iterations, using Sorensen index index Sorensen using iterations, 500 as both as gardless of their source (T source their of gardless

flies

included 22 22 included ,

alpha diversity alpha

(n = 5) (n = different share thesame share ,

means and their their and means -

82.64%, mean: 71.31 ± 71.31± mean: 82.64%, were were -

host

48 bacterial taxa, bacterial 48

comprised comprised generally generally β = β s ,

Generally, Generally, as measured measured as (see (see

ecological ecological throughout throughout that that discrete able 1). able were were

This article isThis article by protected copyright. All rights reserved. of microbiome Escherichia dataset, data Car Rickettsia, Wolbachia, adults: (2 phylum occurred randomly and12.90% 14.12 present for accounted bacteria These Enterobacteriaceae). (Gammaproteobacteria: main genera,the by several low foundin usually and females detected in (s with associated larvae commonly (Enterococcaceae), (51.48 all females in high numbers occurring in value:18.03) stress 2a, adult defined 2). mic larval from significantly and differed group aseparate as together clustered microbiomes these Accordingly, Firmicutes. by dominated ( taxa common sharedseveral larvae adults and 17.33 Enterobacteriaceae: (mainly

Accepted Article Non ,

Indicator Species Analysis Species Analysis Indicator - Among the Firmicutes, the Firmicutes, Among , metric multidimensional scaling multidimensional metric

Lactobacillus but were inconsistently distributed among females among distributed were inconsistently but - Shigella - derived microbiomes as a distinct cluster sepa cluster distinct a as microbiomes derived larvae ( larvae

13.88 and 19.44% of all reads;F all 19.44%of and 13.88 and at > at , Unclassified OTU 4, OTU 4, , Unclassified

was uniquely associated with adult flies (IV = 100, P = 0.0002 = 100,P (IV adult flies with associated uniquely was IV > 84.9, P < 0.049 > 84.9,P< IV 1 .

10% of the gut ofthe 10%

female dinium, members members Lactobacillus

revealed at at and 33.74% 33.74% -

42.86%, mea 42.86%,

of which were whichwere of Siroplasma populations were populations

robiomes (MR robiomes ordination ordination Enterococcus that , see following section , seefollowing ( F

bacteria (Lactobacillaceae) were most prevalent, prevalent, most were (Lactobacillaceae) bacteria igure 1). Other Pr Other 1). igure , numbers (F numbers

among the five most prevalent prevalent most five the among - F

74.53% of all reads). allreads). of 74.53% igure igure n: 27.15 ± 4.39% of all of ±4.39% n: 27.15 , igure 1). igure were not detected. not were of the dataset confirmed these differences and differences these confirmed the dataset of Kluyvera

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igure 1). Proteobacteria were represented wererepresented Proteobacteria 1). igure

designated adult microbiotas were distinctly microbiotas adult The The Kluyvera , rated from larval microbiomes (F microbiomes larval from rated

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This article isThis article by protected copyright. All rights reserved. all reads). 11.36%of 1larva: Comamonadaceae; and allreads), 19.17%of larva: 1 Pseudomonadaceae; ofallrea 28.48% 1larva: Micrococcaceae; allreads), 12.23%of 1larva: Brucellaceae; (Alphaproteobacteria: high numbers detected in contrib were (Staphylococcaceae) 0.1 comprising (F numbers and ofallreads) ± 3.22% 5.71 mean: 46.92%, Acinetobacter others of components major (F examined larvae 92.36%, 1) to belonged inmost larvae revealed that classification m theof larval a for accounted usually and bacteria abundant most second the were Enterococcaceae Firmicutes lownumbers. in detected wereusually Proteobacteria contributed bacteria These most were bacteria Moraxellaceae also insome larvae and Enterobacteriaceae, larvae females, toadult Contrary P. downsi .

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This article isThis article by protected copyright. All rights reserved. respectively). groups, other and WF allreads, of ±5.82% 13.31 ± 0.45and 1.94 (Enterobacteriaceae, 0.85 (Enterobacteriaceae, microbiotas n =4)harbored (WF, finches warbler insectivorous from retrieved Thus, larvae microbiomes. from nest treefinch and inground developing host according to clusters distinct into microbiomes separated A=0.028, d retriev treefinches 0.001; pre dietary withbroader hosts feeding on counterparts that of from different significantly microbiota, distinct a harbored (WF) finch warbler insectivorous strictly parasitizingthe larvae that indicated comparisons Pairwise composition. tests:A= (MRPP weresampled larvae microbiome microbiomes inlarval differences to contribute the that We hypothesized host and Spatial

Acceptedietary resources Article

larvae e Host Host host to according data Grouping d from ground and tree finches ( treefinches and fromground d P

= 0.10)

sampled that were were that composition composition differences differences

retrieved from ground finches ( finches fromground retrieved - specific patterns in patterns specific )

. were similar in composition and statistically indistinguishable (MRPP tests: tests: (MRPP indistinguishable andstatistically composition in similar were

– Corresponding with these comparisons, NMDS ordinations of the dataset theof dataset ordinations NMDS comparisons, these with Corresponding

STF and MTF, A=0.137, A=0.137, andMTF, STF from warbler finch nests (F nests finch warbler from distinguished by by distinguished were associated associated were remained consistent within the host, regardless of the island on which which islandon the of regardless the host, within consistent remained

± 0.66 and 31.47 ± 7.12% of all reads), and unclassified OTU 4 bacteria 4bacteria OTU andunclassified allreads), of ±7.12% 31.47 ± 0.66and s patial isolation of collection sites collection of patial isolation m icrobiome composition. Island composition. icrobiome

the - 0.02, P= 0.70). 0.02, a which feed on a combination of acombination on feed which

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and vegetative vegetative and

for larval larval for P wi

= ll This article isThis article by protected copyright. All rights reserved. compo species bacterialin a significantshift with be transient, Accepted tree nests. or finch ground sampled from insecti strictly the of from nests larvae sourced species. Specifically, host the parasitized to according of gutmicrobiomes foundthat study Our Discussion host species of effect the with compared Accordingly, (0.94±0.05). samples among difference larger much a showed WF_2014/2015, vs. STF_2014 microbiome simi comparing available dataset the However, respectively). and 0.70±0.2, (0.61±0.12 differences relatively small exhibited WF_2015, vs. WF_2014 and vs.STF_2014 i.e.STF_2006 years, indifferent hosts the same among similarities comparing datasets available Thetwo years. Bray the we compared microbiome onlarval year sampling of effect possible the to estimate In order ArticleBray marginally was associ 'OTU 4', Enterobacteriaceae 98 (IV: finches withwarbler associated uniquely nests tree finch and ground retrieved from larvae patterns. these confirmed and (Comamonadaceae) families: genera ofother Instead - - Curtis distances for for distances Curtis Curtis distances Curtis vorous vorous ,

the Enterobacterial population was dominated by dominated by was population the Enterobacterial

in our dataset ourdataset in w

non arbler finch harbored significantly different microbiomes compared to counterparts to microbiomes compared different significantly harbored arbler finch - significant (IV: 87.3 87.3 (IV: significant

among host species and years and species host among

P. downsi P. Pseudomonas Acinetobacter Acinetobacter Kluyvera sampling year had a smaller effect on the microbiome of themicrobiome effecton smaller hada year sampling larities in larvae feeding on different hosts at the same year, i.e. at sameyear,i.e. the hosts different feeding larvae on larities in

ated mainly with larvae retrieved from ground and tree finches, tree finches, and ground retrieved from larvae with ated mainly microbiota retrieved from different host species in different different in species host different from retrieved microbiota sp.

was assigned as asp as assigned was (Pseudomonadaceae, F (Pseudomonadaceae, (Moraxellaceae), (Moraxellaceae), P Additionally, . P. downsi P.

= 0.06).

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the gut microbiome of microbiome of gut the Ochrobactrum

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= 0.0016), whereas whereas 0.0016), = sition between the parasitic larval and and larval parasitic between the sition

igure 1). igure larval microbiomes retrieved from retrieved microbiomes larval sp . or completely or

Indicator Species Analysis SpeciesAnalysis Indicator (Brucellaceae), P. downsi P. Serratia Serratia P. downsi P.

replaced by by replaced

was found to was found Delftia sp.

was was

larvae larvae This article isThis article by protected copyright. All rights reserved. environmental present bacteria to exposed on bouts the feeding nocturnal their resting sitesto diurnal thetheir from around nest, time muchtheir roving of spend and parasites asfollows. mechanisms one the weoutline to of be attributed instead species. host of microbiomes irrespective larval i larvae,agreatersimilarity and and alladults to bacteriacommon specific of detection in result transmitted vertically o bacteria otherwise common the of replacement microbiome ( carried adistinct species ecologically microbiome affects larval the upon which host finch Darwin’s that the results indicate Our Host due possibly development, Additiona tooffspring. fly adult bacteriafrom of maternaltransmission islimited there (b) therefore and related influences, host or environmental hostspecies, indicating finch divergent ecologically among differentiated fin (ground same host the larvaein parasitizing notmicrobiome Islands did affect Floreana composition Cruzand Santa non r

Accepted results bacteria,our siteswith oviposition Article – - parasitic adult developmental stages. Finally, we show that the large spatial barrier between between the spatialbarrier that large weshow Finally, stages. developmental adult parasitic species effects species

Firstly, nesting material may affect the larval microbiome. Larval microbiome.Larval larval affectthe may material nesting Firstly, harbored similar gut misimilar gut harbored (e.g. (e.g. - diverged multi diverged Serratia

ches). Together, our results our results ches). Together, en masse sp. )

in larvae feeding on inlarvae feeding to the shift in diet composition or or needs nutritional in shift composition diet the to - host, island system island host, , suggesting uniquely evolving host evolving uniquely , suggesting P

host (O’Connor (O’Connor host crobial communities ( communities crobial by adult flies to offspring, which offspring, adultflies to by lly, (c) the gut microbiome composition of of microbiome the composition (c) gut lly,

≤ 0.005, Figure 2b). Figure ≤ 0.005, Kluyvera

in the nest, which may inherently vary according to to vary according inherently may nest,which the in

support the notion that the larval microbiome is not microbiome larval that the isnot notion the support

w et al. . While larvae retrieved from ground and treefinch from ground retrieved larvae . While

We propose that the source of these differences may may these theofdifferences that source We propose spp. and the Enterobacteriacae "OTU 4" by by other "OTU 4" Enterobacteriacaethe and spp. arbler finches. arbler finches. suggest that, (a) that, suggest

2010b; O’Connor O’Connor 2010b; P This difference was mainly attributed to attributed mainly was difference This

= 0.10), those that had fed on those on fed had that = 0.10), P.

downsi Although adults may inoculate eggs eggs inoculate adults may Although

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et al. larvae feed P. downsi downsi P. P. downsiP.

larvalmicrobiome is 2014). As 2014). would be predicted to be would of larvae and adult and larvae of

significantly are

w changes during during changes

arbler finches arbler finches such, they are they such, external external P. downsi P.

n - s .

This article isThis article by protected copyright. All rights reserved. (O’Connor with associated ( unsanitary nests aretypically inhabit that may parasitic nests. Although unsanitary in particularly behavior, feeding coprophagic 2006; Kleindorfer & Dudaniec ( parasitism internal towards genus this of inthe progression position evolutionary occupiesintermediate an and hosts, avian some of nests neotropical in as scavengers develop who diet to according vary these withbacteria, and abound feces Bird matter. hostfecal isvia of which one pathways, putative several maythrough establish link on it. This feeding parasites the the of microbiome and diet microbiome of gut the our observations. of source resemblance bears no on plumage found previously the community of bacterial composition the as grows load bacterial this that evidence species be may bacteria, which communitiesenvironmental of associatedwith istypically birds of plumageThe microbiome. 2007 (Kleindorfer nesting anewevery for build nest and a newnest) theymay usurp nests(though reuse not s within werefound microbiota larval the in significant differences no because thisnotion support species Accepted Article finchesdo Darwin’s importantly, area,and thestudy throughout randomly were sampled pecies that - Thirdly Secondly, specific nest sites and lead to differences in the gut microbiome. However, our findings do not do findings our microbiome. However, gut the in differences to lead and sites nest specific et al. , larval nutrition may determine its microbiome. Diet is awell microbiome. Diet may determine , larval nutrition its some

2010b)

; Kleindorfer & Dudaniec 2009 ; &Dudaniec Kleindorfer differences in the feather microbiota of host species may affect the larval larval affectthe of may host species microbiota thefeather differences in

host to the larval g larval the to ( animals (Ley (Ley animals Glünder 2002 . Accordingly

species

- Couri specific to some to specific that remove fecal matter and other organic debris from nests debris from organic other matter and fecal remove that ut community found in our study, and cannot be pointed toasthe cannot pointed be and foundinour study, community ut et al. et al. ). The parasitic The ). et a , the opportunities for for , theopportunities l. the the

2008) 2007). As such larvae may retain some of their ancestral their some of retain may As larvae 2007). such nesting period advances (Kilgas advances period nesting Löwenberg degree ( degree ,

). and there may be may andthere

P. downsi P. Bisson Bisson - Neto 2008 developing larvae to encounter feces encounter in larvaeto developing

evolved from evolved et al. et

a link between the host nestling’s nestling’s host between the a link

2007 ) ,

in thein

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P. attempt

2007 in in species

dow

) nsi

is This article isThis article by protected copyright. All rights reserved. Accepted insectivorous the of infesting nests larvae consis are ourresults further research, to open being therelationship of mechanism the exact Despite period. larval remaining the throughout residents thus dietmay present Bacteria food. to regurgitated nares the feeding in larvae of exposure facilitating easy slit connect choanal the median birds, theof Firstinstar well. bacteriaas associated their differ in probably andwhich diet, natural oftheir typical items withfood nestlings their provide Adult finches consequ specific and larvae ishost feeding co their and Article afflicted nestlings from taken on samples focusing studies, samples. ourFurther of only inone found flies ( myiasis causing a number of with associated been bacteriumis such sameOne the individual. of wounds infected nest( the of bottom the to on small feed nestlings usually source Larvae bacteria. of abundant inflict feeding sterile, larval considered is hosts of healthy blood theof Although host. avian andwounds theblood larvaeisthrough colonize for bacteria to pathway dietary second A determined. be to microbiome remains larval the reflectedin nest( the feces in this and behavior remain exhibit mechanism. this by affected finches nestsof the tent with a scenario involving host dietary effects, as we found that the gut microbiota of microbiota the gut that aswefound effects, dietary host involving tent with ascenario host (Fessl (Fessl host host that wesuggest Finally, - occurring parasites, will determine if wound microbiome composition encountered by the the by encountered microbiome composition ifwound determine parasites, will occurring

be transferred onto be transferred et al. may be

2006) O’Connor O’Connor

less

Nevertheless ,

may , and therefore larval microbiome microbiome larval therefore , and

first be dir et al. diet determines the gut bacterial community in larval in larval community the gut bacterial dietdetermines s - s wounds that can rapidly become infected and provideand an become can that infected s rapidly wounds

instar larvae and consequently perpetuate as stable gut gut asstable perpetuate and larvae consequently instar the nasal cavity with the buccal cavity and the pharynx, pharynx, the and cavity the buccal with cavity the nasal ectly exposed to these food these to ectly exposed , other hosts, such as the such hosts, , other

2010b) w ently reflected in the larval microbiome. microbiome. larval the reflectedin ently arbler finch was strikingly different from the microbiota the microbiota from different strikingly was arbler finch

and Lincan

P. downsi P. therefore therefore Toth Toth g o , unpubl et al.

Ignatzschineria larvae may repeatedly feed on the exist the on repeatedly feed may may be less be may Galápagos

2006; Gupta Gupta 2006; that that ished data ished - , associated bacteria. Indeed, in Indeed,in bacteria. associated

which feed mainly in the nares inthe mainly feed which are forced by larger siblings siblings larger by are forced

flycatcher, do not not do flycatcher, likely to be significantly significantly be to likely ). Whethe ).

larvae et al.

2011 , which has , which r this is r this ) but was) but P. downsi

in ent, ent,

host host . This article isThis article by protected copyright. All rights reserved. members the which of of of Proteobacteria, assembly adiverse with associated predominantly w flies causing myiasis insects and other with comparisons larvalmicrobiomes, adultand of significance the functional on a 1) suggest (Figure microbiota intheir differences The observed aresaprophagic. while theadults host, of and tissues theblood on feed aslarvae different, vastly stage (e.g. life ofeach needs nutritional specific the that complements microbes of community responsive a andmaintain harbor to adaptive be would it Accordingly, stage. developmental byeach niches time both in adults and larvae between separation isthe in insects development holometabolous of the advantages of One them. in overcoming communities microbial the role for adults,and and by larvae challenges faced ecological differential stagesof life between inthe microbiome Variation on effects Life stage investigation further merits and death chick to early addition microbiome in theof host composition the to be due ( insome years ofsome birds inthe nests pupae average than smaller ( parasite and intensity prevalence, in trends divergent already given ofinterest are significant, and may be interactions host this for of consequences 2b).The (Figure components common tree finch groundand of nests infesting theof larvae Accepted Article Dudaniec While future analysis of the transcriptomes of the ofthe transcriptomes ofthe analysis While future et al. et Be . har

2007; 2007; et al. P. downsiP.

ith similar nutritional behavior are instructive. In our study ourstudy In areinstructive. behavior nutritional ith similar Kleindorfer Kleindorfer 200 9 ; Aharon ; Aharon

microbiome - et al. induced mortality among Darwin’s finch species and their hybrids hybrids their and species finch amongDarwin’s mortality induced etal.

2014b; Kleindorfer Kleindorfer 2014b;

2013

). In ).

and space, and the utilization of different trophic ofdifferent trophic theutilization and and space, Philornis Philornis

specie Philornis et al. s

flies are probably det areprobably flies , who have varied diets with many many varieddietswith have , who , the nutrition of larvae and adults is andadults oflarvae the nutrition ,

n affinity to these different these different diets. affinity to n unpublished

microbiota may shed further light light further mayshed microbiota - parasite coparasite Kleindorfer Kleindorfer

data - evolutionary evolutionary larvae et al ermined by the bythe ermined ). For example, example, For P. downsi P. .

2014a were were ) may ) may

This article isThis article by protected copyright. All rights reserved. Acceptedfinch populations of microbiome etal. (Zurek bacteria of absence inthe normally and develop substrates food to utilize of larvae Thus, development. needs and 2012). bacterial cells, cyclorhaphous system of The digestive for Implications Philornis of histories preferences and reflect thefeeding similarly findings our that webelieve but atrend, such are samples adults. Our the of behavior feeding stem opportunistic from that community commensal the species in of abasiccore around reflects probably oscillations This Japan, central Article of diets adult similarity in fleshvs (decaying thelarvaldiets in reflect thedifference (e.g. similar isremarkably genera two these microbiota of theseveral of microbiota the larval c gutbacterial ( adults while stage, thelarval detected in ( similar shift related closely ofthe stages differentdevelopment of microbiome Enterococcaceae. and as Lactobacillaceae were classified which most of Fir mainly females harbored adult Conversely, most prevalent. the were Enterobacteriaceae

2001; Romero Romero 2001;

Accordingly, the absence or imbalance of this resource may severely hamper thei severelyhamper may resource this of imbalance absence or the Accordingly, ibid

flies weassayed. ). Blowflies belonging to the genus to the belonging Blowflies ). Wei

ommunity following the transition from larvae to adults (Singh (Singh toadults larvae from transition the following ommunity Wei indicating that bacteria are a principal source of food for these flies flies forthese source food of aprincipal bacteriaare indicating that Philornis P. downsi P.

et al. (e.g., Jurkevitch 2011; Zindel 2011; Jurkevitch (e.g., et al et al.

2013), whereby whereby 2013), .

could adversely affect larval development and alleviate the pressure on thepressure alleviate development and larval affect adversely could (2014)

2006). 2006). Theref Lucilia

management

found a spatial and seasonal variation in the microbial community. thecommunity. microbial in variation seasonal aspatialand found

and and Macrococcus Lucilia Philornis

dipterans Bacteroides ore, a control strategy based on disruption of the larval thelarval of disruption based on strategy acontrol ore, m

species studied differs from of that from differs studied species uscids such as housefly, hornfly and stable fly are unable areunable fly stable hornflyand ashousefly, such uscids . In another study of adult of study . Inanother Lucilia et al ,

Lactococcus is physiologically adapted for lysing and and digesting for lysing adapted physiologically , . 2011) Koukoulia

(Calliphoridae) also exhibit a significant shift in shift in exhibit asignificant also (Calliphoridae) .

Lactoba blood and living tissues, respectively), and and respectively), tissues, living and blood .

While our study found no immediate immediate foundno study our While , and , and

, and , and Kurthia cillus, Acetobacter Musca domestica Musca Schineria

not extensive enough to track to enough extensive not An analysis ofthe An analysis Lucilia

predominated in the in predominated

( et al. Ignatzschineria Philornis

sp. blowflies in in blowflies sp. et al. et

(Terra 2015).

). This may may ). This revealed a revealed , the adult adult , the 2000; Perotti 2000; r nutritional nutritional r

& micutes, Although Although

Ferreira ) were ) were

This article isThis article by protected copyright. All rights reserved. al. (Robacker sources food to identify 2013), attractants asoviposition theyserve where in flies, and specifically in communication long forthe option of large numbers of adult stages and to both larval bacteria Ami ofreleased m competitiveness the sexual andimproving inrearing highly important competitive isasexually product (SIT) Technique Insect the Sterile of enemies Philornis used to be thisof genus members for s ourlarval of some detected in (e.g. causing Forexample, Islands. Galápagos birds threatened forprotecting solution be a could and investigation, of avenue is apossible cycle provisioning the nest during birds to adult food appropriate together with ap similar adults. A (2014) Recently, available. are bacteria using avenues other several disruption, target such for

Acceptedan provide femaleswould for attractant bacterial effective and a specific Developing 2010). Article etal.

protected Galápagos finch nestlings by providing insecticide treated nesting material to material nesting treated insecticide providing by nestlings finch Galápagos protected In all spheres, ba In all spheres, long and Currently, immediate ( Cha Cha

2010; Gavriel 2010; P. downsi P. O’Callaghan O’Callaghan et al. P. downsi

insects (e.g insects - proach, whereby a disruptive or entomopathogenic species of bacteria is offered offered is bacteria of species entomopathogenic or adisruptive whereby proach,

term management of this invasive species species invasive this of management term 2016) and importation andimportation 2016)

(Bulgarella (Bulgarella cteria are immensely important. important. immensely are cteria et al. et al. et al.

in the in the ., Hoyt ., Hoyt

2011 1996 amples. the genus the genus

et al. sterile for eradicating the fly the eradicating for laboratory with the aim of evaluating the feasibility of SIT asan SIT of thefeasibility evaluating theaim of with laboratory ). A more detailed understanding of the contribution of specific ofspecific the contribution of understanding more detailed ). A ) and muscid muscid and ) etal. as biological control agents agents control biological as

- 2015). term solutions include development of attractants for adult adult for attractants of development include term solutions

A further avenue of investigation is investigation of A furtheravenue male. In tephritid fruit flies targeted by SIT, bacteria are SIT, bacteria by targeted tephritid fruit flies male. In et al. et

P. downsi 1971; Dillon Dillon 1971; Serratia of of

Efforts are also underway to evaluate the potential of potential the to evaluate underway are also Efforts biological control biological

1998) flies (Lys flies , which contains which contains ,

will enable us to support current efforts torear efforts current tosupport us will enable ,

and to med to and . & A SIT program is an industrial process whose whose process industrial isan program A SIT

Bacteria are important components of of components important are Bacteria Dillon 2004; Reviewed by Davis by Davis Reviewed 2004; Dillon y k et al. et al.

(Lahautte (Lahautte agents agents against against iate sexual encounters (Sharon (Sharon encounters iate sexual

2002; Romero Romero 2002; (Lam (Lam known pathogens of myasis of pathogens known using specialized natural natural specialized using pecies from pecies from et al. et et al. P. downsi P. to evaluate potential the to evaluate

2016 2007; Zheng Zheng 2007; et al. ). .

ales (e.g. (e.g. ales

Philornis

2006 Knutie Knutie et al. etal. ) , was , was

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This article isThis article by protected copyright. All rights reserved. Accepted mgm4751500.3 repository Bacterial Data accessibility interactions downsi P. of coevolution the implications for have wereport findings strategies. The control inform effective to required is knowledge base thethat growing to important component an parasiteadds this of structure Peters Darwin’s species of various ( birds land Galápagos Article of management diet. and niche foraging in differ host speciesthat divergent ecologically microbiomebetween larval differences in significant with stage, larval and parasitic larvae) avi the the of in microbiome variation diets. typical and niche foraging in differ host speciesthat divergent ecologically between differences stage, significant larval the for and parasiticlarvae) vs. fly stage (adult parasiticfly, avian the of microbiome inthe findsvariation study Our Conclusion (see research a future focusof the willbe and finches, Darwin's from ofparasitism inburden alleviating the tool important &

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currently receives the largest research and conservation attention for attention conservation and largestresearch receivesthe currently , Cunning finches (e.g. (e.g. finches et al.

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Dudaniec 2016 Dudaniec an parasitic fly, fly, an parasitic Fessl et al. be evident

a similar approach based on yeast). yeast). on based approach a similar et al.

2017

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was deposited at the MG was atthe deposited O’Connor -

specific trajectories and specific trajectories , according to life stage (adult fly vs. vs. tolife fly stage(adult , according

et al. P. downsi P.

The impact,The ecolog

2010a; Koop 2010a; Koop , according to to life , according Our study finds study Our

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2016 ; This article isThis article by protected copyright. All rights reserved. M Quiroga M, Bulgarella I Bisson Ben A, Behar Ben Z, Y, Pasternak Aharon References physiology. and anatomy the Ga number contribution is This Foundation. Darwin Charles tothe Trust) Helmsley Charitable and HarryB. M. TheLeona by awarded grant (witha Foundation t from funding by supported was PC PC Di Park National the Galápagos by granted study was this toconduct Permission Acknowledgements Ben

Accepted Article- - 23 10 - Ami E, Yuval B, Jurkevitch E (2010) Manipulation of the microbiota of mass of theof microbiota Manipulation (2010) E Jurkevitch B, Yuval Ami E, - - 16: Strategic Research into Impacts of Impacts into 16: StrategicResearch Parasite, theInvasive 15: Controlof lápagos Islands lápagos and host specificity of the parasitoid the parasitoid of and host specificity Ecology an ecological, biogeographic, bacteriaacross soil plumage and performance. sexual flies fruit Mediterranean 251 pp. TA), K,Miller Bourtzis commu bacterial ontogeny. during Phylogenetic - A, Marra PP, Burtt Jr EH, Sikaroodi M, Gillevet PM (2007) A molecular comparison of of comparison Amolecular (2007) Gillevet PM M, Sikaroodi Jr EH, Burtt PP, A, Marra

- Yosef M, Lauzon CR, Yuval B, Jurkevich E (2009) Structure and function of the the of function and Structure (2009) E Jurkevich B, Yuval CR, Lauzon M, Yosef

54 , 65

, metabolic, and taxonomic diversities shape diversities taxonomic , and metabolic, . We We - 81. A, Boulton R, Ramirez Boulton A, nity associated with the Mediterranean fruit fly. In: In: fly. fruit Mediterranean the with associated nity

Applied and Environmental M Environmental and Applied

thank I thank - Isme Journal Isme Yosef M Cerati he Galápagos Conservancy and the International Community Community theInternational and Conservancy Galápagos he .

Rozenboim and B and Rozenboim , Behar A, Lauzon C, Yuval B, Jurkevitch E Yuval B, Jurkevitch C, Lauzon Behar A, tis capitata - 271. CRC press, Boca Raton. CRC271. press, Boca

4 Philornis downsi Philornis , 28 Conura annulifera Conura Philornis downsi downsi Philornis

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Perlman for their insights regarding avian regarding their insights Perlman for icrobiology 2148 , Causton CE, , Causton

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(2013) (2013) - reared reared ( 2017 ). This work work ).This ; Project: ) Microbial Microbial .

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(eds. (eds. This article isThis article by protected copyright. All rights reserved. Accepted P UrsellLK, JC, Clemente and Thecharacterization KA(2015) Scoles KL, GA,Brayton KA,GallCA,Mason Clayton AP Hendry EA, JA,Cooper Chaves PF AE,Lahuatte Mieles Cha DH, BJ Sinclair Peck SB, CE, Causton Article Reviewof MP (2014) Lincango CE, Causton parasite avian Managementthe of W(2013) Tapia F, C, Cunninghame Causton Bulgarell ISSN: 1390 p downsi the the America Society Entomological inmainlandEcuador. Islands, Galápagos the to parasiteinvasive nest avian pp. 167 pp. ermethrin. health: An integrative v integrative An health: andersoni tick, wood Mountain Rocky the of microbiome bacterial the of manipulation of radiation Ecology Chemical a 121 the of conservation Islands of agent control biological potential Galápagos ttractant a M, Quiroga MA, Brito Vera GA Brito MA, Quiroga a M,

in in - - 143. 173. 173. n - . ests of 6526. 6526.

Annals of the Entomological Society ofAmerica Society theEntomological of Annals Technical report No 1 report Technical

s for s for . GNPS, GCREG, CDF and GC. Puerto Ayora, PuertoAyora, GC. and CDF GCREG, GNPS,

Parasites &Parasites Vectors Islands: a collaborative and strategic action plan. plan. strategicaction and acollaborative Islands: Darwin's finches.

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arfrey LW, Knight R (2012) The impact of the gut microbiota onhuman gut microbiota (2012) the impact Rof Knight The LW, arfrey 42 Galápagos ,

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nvasive birds, with an with birds, Philornis downsi downsi Philornis , 1258 c – - hemical 250. 250. 636. 636. Annals of the Entomological Society of of America Society of the Entomological Annals ) . Genomic variation at the tips of the adaptive oftheadaptive tips at the variation . Genomic

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This article isThis article by protected copyright. All rights reserved. AcceptedS ( Tebbich MS, Fessl B,Couri insects of microbiota gut The Moran NA(2013) P, Engel and sensitivity UCHIMEimproves R(2011) C,Knight JC,Quince Clemente RC, HaasBJ, Edgar ofand abundance Distribution S (2012) Tebbich S, Kleindorfer E, FesslM, B,Nemeth Dvorak parasiticf the Effects of (2006) S Kleindorfer RY, Dudaniec intensitythe of in variation interspecific and Interannual (2007) S Kleindorfer FesslB, RY, Dudaniec Article microorganisms. resident of function and diversity Multiorganismal insects: AE(2015) Douglas interactions. Nonpathogenic insects: gut bacteriaof The VM (2004) RJ, Dillon Dillon Microbial JK (2013) HofstetterTomberlin RW, TL, Crippen Davis TS, Long N(2017) Greco G. La Young C, B,Sevilla F, Fessl Cunninghame CJB, Lowenberg de Carvalho MS, Couri Microbiology Reviews Microbiology chimera detection. of speed declini for evidence Galápagos: on CruzIsland, Santa birds other land and finches Darwin’s birds. on Muscidae) parasitic fly, ReviewE of Annual Entomology of s 20 Report the to save ( Diptera: emiochemicals. emiochemicals. ng populations. populations. ng

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49 Journal of Chemical Ecology ofChemical Journal , 71 GNPD, CGREG, CDF and GC; Puerto Ayora, Galapagos, Ecuador. Galapagos, Ayora, GC; Puerto and CGREG, CDF GNPD, ntomology Emu endangered endangered Zootaxa - 92.

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Bioinformatics 46 , 13

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This article isThis article by protected copyright. All rights reserved. Accepted AY Rangrez MS, AK,Dharne Gupta reaction: polymerasechain the Deconstructing A(2015) R, Naqib Venkatramanan GreenSJ, i somethe bacteria of on diet ofoccurrence Influence G (2002) Glünder Gavri KL Mason GA, Scoles Gall CAR,K.A., Myiasis. O(2012) Lupi F, Francesconi ArticleCE ( Causton GE, Fessl B,Heimpel RP HG, Young Fessl B,Young Thelife (2006) S BJ,Kleindorfer Fessl B,Sinclair el S, Jurkevitch E, Gazit Y, Yuval B (2011) Bacterially enriched diet improves sexual sexual improves enriched diet Bacterially B (2011) GazitY,Yuval E, Jurkevitch el S, Galápagos Journal of of Journal ureiclastica mismatches. a degeneracies primer associatedwith bias correcting and understanding birds. pet and 564 flies. fruit male Mediterranean sterile of performance andersoni Netherlands. The Dordrecht, Springer, In: challenges. Galápagos Sciences Biological Isabela Island. on finch mangrove the survival. nestling its impacts on and Darwin's finches parasitizing - 573.

ticks influences pathogen susceptibility. susceptibility. pathogen ticksinfluences Islands (Diptera, Muscidae). Muscidae). (Diptera, Islands Systematic and Evolutionary Microbiology Evolutionary and Systematic Islands: colonization history, adaptations to novel ecosystems, and conservation ecosystems,conservation and tonovel history, adaptations colonization Islands:

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(2010) How to save the rarest Darwin's finch from extinction: from finch extinction: Darwin's rarest to save the (2010) How - 1030. Clinical Microbiology Reviews Microbiology Clinical , et al. , et ) Invasion nestavian para an ) Invasion of Galápagos

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Philosophical Transactions of the Royal Society B of theRoyal Transactions Philosophical

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This article isThis article by protected copyright. All rights reserved. Accepted Host RY(2016) Dudaniec S, Kleindorfer Love thy RY(2009) Dudaniec S, Kleindorfer H, Winkler T, Chapman S, Kleindorfer Darwin's in clutchof sizevariation Theecology (2007) S Kleindorfer nest during increases load bacterial Plumage R(2012) Mänd TilgarV, M, Mägi P, Saag P, Kilgas ArticleRussell JA, BER, Rubin Kautz S, combating horse: Trojan Riding the E(2011) Jurkevitch rarebiosphere wrinklesthe in out the Ironing (2010) ML HG,Sogin Morrison DM, Welch Huse SM, an of Production AP (1971) GO,Mulcock Osborne CP, Hoyt J Popovici BL, Montgomery AA, Hoffmann 10.1186/s40850 flyparasite introduced Sociobiology tree finches. Darwin’s in intensity ectoparasite size affect Research finch ( small ofDarwin’s ground populations fuliginosa inapasserine bi building M Environmental d to bacterial uncover methods traditional with Pyrosequencing 454 biotechnology Microbial clustering. improved OTU through b Aedes acteria.

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. Sulloway FJ (2006) Adaptive divergence in contiguous contiguous in Adaptive divergence (2006) Sulloway FJ -

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Applied and Environmental M Environmental and Applied Causton CE (2016) Rearing CE (2016) Causton Philornis downsi Philornis , et al. , et

(2014a) Species collapse via hybridization in hybridization via Species collapse (2014a) on in Darwin’s finches: experimental and historical historical and experimental finches: Darwin’s in on Journal of Applied Ecology Applied of Journal

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- , Fitt BDL, Atkins SD, Walters DR, Daniell TJ (2010) Pathogenesis, parasitism and and parasitism Pathogenesis, TJ (2010) Walters DR,Daniell SD, Atkins BDL, Fitt , N

- Stomoxys calcitrans eto P (2008) The structure of the parasite the of structure The P (2008) eto 0001

320 Serratia marcescens, Aeromonas Aeromonas marcescens, Serratia ) 34 Handbook of of Handbook

Computational Computational - behavior , 1647 Tymensen L, Selinger L (2002) Comparsion of selected growth media for of growth Comparsion selected (2002) SelingerL L, Tymensen

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- - - This article isThis article by protected copyright. All rights reserved. Accepted P Yilmaz Pruesse E, Quast C, (201 S Kleindorfer Peters KJ, treefinches zone:Darwin’s ahybrid in behavior Divergentforaging (2015) S Kleindorfer Peters KJ, T,Kalischuk Lysyk M, Perotti Darwin's (2014) S J, Kleindorfer JA,Robertson O'Connor Article(2010 S RY,Kleindorfer JA,Dudaniec O’Connor (2010 S J, Kleindorfer JA,Robertson O'Connor (2010 S J,Kleindorfer FJ,Robertson JA,Sulloway O'Connor of ThePathogenicity (1996) T Jackson D, Baird T, Nelson M, Garnham M, O'Callaghan improved data processing and web and data processing improved spp.) ( TreeFinches Darwin’s of hybrids detect cannot surveys Acoustical 2013): ( larvae. conspecific and manure cattle immature s Ecology islands. between habitats elevational two contrasting finch: small ground finches. Darwin's of ( finch medium Darwin's tree endangered inthe critically mortality nestling causeof primary 27. s Camarhynchus pauper Camarhynchus Camarhynchus ignal ignal to trains

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, et al. , et - ) Avian population trends in trendsin population ) Avian Tymensen L, Yanke L, Selinger L (2001) Growth and survival of survival and Growth L (2001) L,Yanke Selinger L, Tymensen ).

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(2013) The SILVA ribosomal RNA gene database project: databaseproject: RNAgene SILVA ribosomal The (2013) , 588

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n Apis atural - e ffects ffects 81 , This article isThis article by protected copyright. All rights reserved. Accepted(2 S Weiss B,Aksoy and bacterialcommunities in variations Seasonal (2014) Y Tanji K, R,Miyanaga Ishida Wei T, J, Hu Wei T, P,N Ivanova Luginbuhl F, Warnecke N AK,Bongio Ghosh SB, Wang Article fromdifferent Bacteria the isolated K(2006) Marialigeti AK, G,Borsodi Hell Kovacs EM, E, Toth In: insect of digestion. physiology evolutionary and Molecular CL(2012) Ferreira Terra WR, Fessl M, Dvora B, Taborsky S, Tebbich X Liu ZhangM, Z, Su Parasitology B and Microbiology antibiotic domestica antibiotic awood of microbiota America of States mosquitoes. from vector :Sarcophagidae). (Diptera larval stagesand developmental 93 pestmanagement for integrated nutrition and bioecology variability. spatial seasonal to and Adaptations Darwin's finches: m l arvae and arvae and onoazide onoazide - 119 Miyanaga K, Tanji Y (2013) Comparative analysis of bacterial community and ofbacterialcommunity analysis Comparative K, Y(2013) Tanji Miyanaga , CRC Press,Boca - - ). resistant strains associated with green bottle flies (Diptera: Calliphoridae). Calliphoridae). (Diptera: bottleflies green with associated resistant strains ( housefly the of developmental stages different in resistant strains n r Applied Microbiology and and B Microbiology Applied ewly eveals eveals 011) Microbiome influences on insect host vector competence. competence. vector on insect host Microbiome influences 011)

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tional Taxonomic Units (O Units tional Taxonomic - 48 r m egulated egulated , 864 Scientific Reports Scientific ediate O (2016) Microbiome changes through through Microbiomechanges O (2016) .

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This article isThis article by protected copyright. All rights reserved. 1. 17.15 insect larvae; females vs. larval Figure 2: Samples ( are bars) (shaded larval generacomprising bacterial Figure 1: Actinobacteria Accepted Article

).

( dominated omnivorous tree finches omnivorous dominated n

Similar microbiomes Similar

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are abbreviated as in as abbreviated are N of abundance the relative depicting representation map Heat

20) and adult ( andadult 20) on - metric Multi metric ) or triple dashed (Ba dashed ) triple or coded

stress stress

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fully shaded ( fully shaded Dimensional Dimensional

cluster closer to each to each other. closer cluster 5) microbiomes 5) T = 18.04 = able 1 and 1and able

( n c

= teriodetes). Non teriodetes).

20) and adult ( adult and 20) ) or or ) S Proteobacteria caling (NMDS) , cluster (b) and .

Samples were grouped according weregrouped Samples host host

strictly strictly ed diet

according to similarity to according n - identified bacteria appear as empty bars asempty bacteriaappear identified

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seed dominated omnivorous omnivorous dominated seed ordinations ordinations

, 5) 5) Samp dashed ( dashed P .

downsi le names are abbreviated as in T asin names areabbreviated le Firmicutes representing similarities among among similarities representing

warbler finches warbler microbiomes the 30 .

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most dominant most dominant double dashed dashed double

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(a) . Identified Identified ;

life stage

ground finches ground stress

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( able able . adult adult

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, This article isThis article by protected copyright. All rights reserved. is Floreana) additionally specified. MTF named the to host according 99% ofthebacterial diversityineach sample. (OTUs) wereaffiliated to based genera sequence on97% identity. Analyses covered over adult( 20) and Table 1: 16 MTF FL / 16 15 MTF FL / 15 STF SC / 14 13 STF FL / 13 STF FL / 12 20 WF / SC / WF 20 SC / WF 19 SC / WF 18 SC / WF 17 SGF FL / 10 11 STF FL / 11 25 AF SC / 25 AF SC / 24 AF SC / 23 AF SC / 22 AF SC / 21 SGF SC / 5 SGF SC / 4 SGF SC / 3 SGF SC / 2 SGF SC / 1 9 SGF FL / 9 SGF FL / 8 SGF FL / 7 SGF FL / 6 Accepted Article Sample –

mediumWF finch, tree

instar female adult adult Sample collection data, Sample collectiondata, sequencing parameters anddiversity indices oflarval ( 2nd 2nd 2nd 3rd 3rd 3rd 3rd 3rd 3rd 3rd 3rd 3rd 3rd 3rd 3rd 3rd 3rd 3rd 3rd 3rd n Camarhynchus Camarhynchus Camarhynchus

Certhidea fuliginosa Geospiza Geospiza Species = 5) = 5) parvulus olivacea pauper MTF WF SGF STF --- P. downsi Host and vegetative and Seeds, insects Seeds,insects Insects and and Insects vegetative resources resources Insects Diet ---

parasitized (SGF

– microbiomes Taxonomic examined. Operational Units

warbler finch). site(SC Collection warbler

Location Santa Cruz Santa Cruz Santa Cruz Santa Cruz Santa Cruz Santa Cruz Santa Cruz Santa Cruz Santa Cruz Santa Cruz Santa Cruz Santa Cruz Santa Cruz Santa Cruz Santa Cruz Santa Floreana Floreana Floreana Floreana Floreana Floreana Floreana Floreana Floreana Floreana Collection Adults Larvae Mar 2006 Mar 2006 Mar 2006 Mar 2006 Mar 2006 Mar 2006 Mar 2006 Mar 2006 Mar Mar 2006 Mar 2006 Mar 2006 Mar 2006 Mar Apr 2006 Apr Feb2014 Feb2014 Feb2014 Feb2014 Feb2014 Feb2015 Feb2015 Feb2015 Feb2014 2006 Apr Feb2014 2006 Apr Date

– adult fliesAll samples, excluding (AF)are

small finch, STF ground Reads 35856 45255 17646 48855 55343 40087 11171 39114 37137 60003 51818 62013 41226 46698 31033 39638 19366 26841 31128 34761 27245 18437 10272 51906 24242

Sequencing and diversity indices diversity and Sequencing Genera 40 36 37 33 30 30 38 48 36 26 31 31 29 31 34 31 34 24 32 33 23 27 27 22 23 Evenness 0.469 0.339 0.322 0.403 0.301 0.221 0.294 0.427 0.222 0.303 0.369 0.348 0.471 0.466 0.316 0.389 0.117 0.474 0.476 0.159 0.451 0.299 0.223 0.343 0.51 –

Santa Cruz, FL Santa Cruz, Shannon - 1.729 1.213 1.163 1.408 1.025 0.751 1.974 1.529 0.724 1.039 1.268 1.172 1.618 1.642 1.086 1.372 0.372 1.644 1.664 0.498 1.486 0.987 0.691 1.075

1.07 small finch, tree Simpson 0.6851 0.5974 0.4699 0.6042 0.426 0.3509 0.5267 0.7459 0.7064 0.3529 0.4484 0.6063 0.5981 0.6749 0.7438 0.5496 0.6452 0.1447 0.7552 0.7597 0.2265 0.6807 0.4307 0.3684 0.5552 -

n

= This article isThis article by protected copyright. All rights reserved. Accepted Article