Mycangia of Ambrosia Beetles Host Communities of Bacteria

12 Pages·2012·1.02 MB·English

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MicrobEcol(2012)64:784–793
DOI10.1007/s00248-012-0055-5
INVERTEBRATE MICROBIOLOGY
Mycangia of Ambrosia Beetles Host Communities of Bacteria
J. Hulcr&N. R.Rountree&S. E. Diamond&
L. L. Stelinski&N.Fierer&R.R. Dunn
Received:4February2012/Accepted:5April2012/Publishedonline:1May2012
#TheAuthor(s)2012.ThisarticleispublishedwithopenaccessatSpringerlink.com
Abstract Theresearchfieldofanimalandplantsymbioses known for transporting symbiotic fungi. We analyze six
is advancing from studying interactions between two spe- beetle species that represent three types of mycangia and
cies to whole communities of associates. High-throughput include several globally distributed species, some with
sequencing of microbial communities supports multiplexed major economic importance (Dendroctonus frontalis, Xyle-
sampling for statistically robust tests of hypotheses about borus affinis, Xyleborus bispinatus–ferrugineus, Xyleborus
symbiotic associations. We focus on ambrosia beetles, the glabratus,Xylosandruscrassiusculus,andXylosandrusger-
increasingly damaging insects primarily associated with manus).Ninety-sixbeetlemycangiayielded1,546bacterial
fungalsymbionts,whichhavealsobeenreportedtosupport phylotypes. Several phylotypes appear to form the core
bacteria. To analyze the diversity, composition, and speci- microbiome of the mycangium. Three Mycoplasma (origi-
ficity of the beetles’ prokaryotic associates, we combine nallythoughtrestrictedtovertebrates),twoBurkholderiales,
global sampling, insect anatomy, 454 sequencing of bacte- andtwoPseudomonadalesarerepeatedlypresentworldwide
rialrDNA,andmultivariatestatisticstoanalyzeprokaryotic inmultiplebeetlespecies.However,nobacterialphylotypes
communities in ambrosia beetle mycangia, organs mostly were universally present, suggesting that ambrosia beetles
are not obligately dependent on bacterial symbionts. The
composition of bacterial communities is structured by the
Electronicsupplementarymaterial Theonlineversionofthisarticle hostbeetlespeciesmorethanbythelocalityoforigin,which
(doi:10.1007/s00248-012-0055-5)containssupplementarymaterial, suggests that more bacteria are vertically transmitted than
whichisavailabletoauthorizedusers. acquired from the environment. The invasive X. glabratus
J.Hulcr and the globally distributed X. crassiusculus have unique
SchoolofForestResourcesandConservation, sets of bacteria, different from species native to North
UniversityofFlorida,
America.Weconcludethatthemycangiumhostsinmultiple
Gainesville,FL32611,USA
verticallytransmittedbacteriasuchasMycoplasma,mostof
: : :
J.Hulcr(*) N.R.Rountree S.E.Diamond R.R.Dunn which are likely facultative commensals orparasites.
DepartmentofBiologyandKeckCenterforBehavioralBiology,
NorthCarolinaStateUniversity,
Raleigh,NC27695,USA
Introduction
e-mail:[email protected]
L.L.Stelinski Thestudyofsymbioses,whetherbetweenhumansandtheir
DepartmentofEntomologyandNematology,
microbiomeorinsectsandtheirsymbionts,hasbeguntosee
CitrusResearchandEducationCenter,UniversityofFlorida,
ashiftfromresearchfocusingonpairsofspeciestowardthe
LakeAlfred,FL33850,USA
studyofsymbioticcommunities.Thisisinpartbecausenew
N.Fierer methods of environmental DNA sampling are revealing
CooperativeInstituteforResearchinEnvironmentalSciences
many previously unnoticed bacterial and fungal associates.
andtheDepartmentofEcologyandEvolutionaryBiology,
Insomesymbioticcommunities,bothcoevolvedsymbionts
UniversityofColorado,
Boulder,CO80309,USA and incidental associates may play important but different
MycangiaofAmbrosiaBeetlesHostCommunitiesofBacteria 785
roles.Onesuchexampleistheambrosiasymbiosisbetween has beenonlymarginallyexplored[12]. It hasbeen noted
wood-boringbeetlesanddiversefungalandmicrobialasso- that bacteria are present in the ambrosia beetle niche [17,
ciates. The beetles are often cited as a textbook example of 18], but their abundance, diversity, composition, and
treediseasevectors[1],yetinsomecases,thediseaseagents transmission in mycangia have not been analyzed.
may be incidental opportunists, rather than the primary Weusedahigh-throughput,culture-independentapproach
symbionts of the beetles [2, 3]. Such complexity of inter- tosearchforand,ifpresent,identifyprokaryotesdirectlyfrom
actions highlights the need to survey the full diversity of the mycangia of five species of fungus-farming beetles. In
microbial associates of ambrosia beetles. Historically, stud- doingso,weaddressedfourquestions:
ies of the ambrosia symbiosis have focused on insect inter-
1. Doambrosiabeetlemycangia,organsoriginallyevolved
actions with the main fungal symbiont, and few addressed
to host fungi, also contain a significant diversity of
the role of other fungi, and even fewer studies exist on the
bacteria?
presence and role of bacteria in this system. Here, we
2. Is there a “core microbiome” associated with ambrosia
expandthefocusofambrosiasymbiosisresearchtoaddress
beetlemycangia?Onehypothesisisthatmycangiaofall
the prokaryotic component. We combine the approaches of
ambrosia beetles, regardless of species, may be domi-
culture-independentmicrobiology,insectanatomy,andmul-
nated by a suite of microbial members shared among
tivariate statistics to analyze bacterial communities inside
samples, the“taxonomic core microbiome” [19]. Alter-
one of the most frequently evolved organs mediating a
natively, associations may vary among different beetle
symbiosis—themycangia ofambrosia beetles.
species orindividuals.
Theambrosiasymbiosisofbeetlesandfungiisoneofthe
3. Ifbacteriaarepresentinmycangiaandtheircommunities
most evolutionarily successful and ecologically important
vary among beetles, to what extent is the community
insect mutualisms [4], with increasing global economic
composition determined by the geographic origin of the
impact[3].Thesymbiosisevolvedinatleast11independent
beetle,andtowhatextentisitthehostbeetlespecies?If
barkbeetleancestors(Curculionidae,Scolytinae,andPlaty-
bacterialcommunitiesaremorespecifictolocalitiesthan
podinae) and involves over 3,500 beetle species and an
to beetle species, they are likely facultative opportunists
unknown but large number of symbiotic wood-decay fungi
from the environment. On the other hand, if bacteria in
[4–6,42,43].Thefungiarecarriedinspecializedmembranous
mycangiadisplay specificity to beetle species regardless
invaginations equipped with secretory glands—mycangia [7,
of locality, they are potentially symbionts transmitted
8].Asmostoftheresearchonambrosiasymbiosisfocusedon
verticallyacrossbeetlegenerations.
mycangial fungi, little is known about bacteria in mycangia.
4. If bacterial communities are specific to beetle species,
Even the question of whether bacteria are present in beetle
are they also more similar between closely related bee-
mycangia is poorly resolved, and whether the bacterial com-
tles then between unrelated beetles? Congruence
munityisshapedbythetypeofmycangium,beetleevolution-
betweenbacterialcommunitysimilarityandbeetlephy-
aryorigin,orgeographicoriginhasneverbeentested.
logeny is expected if bacteria are exclusively transmit-
Presence of bacteria in mycangia has been suggested by
tedvertically within beetle speciesand families.
previous studies [12, 16, 18], but not explicitly tested, as
bacterial associates of beetles are typically studied from By sampling multiple species from nine localities
crushed whole beetles or from externalbeetle surfaces, and from around the world (not all species were collected
the isolated bacteria might not have been inside mycangia. everywhere), we are not only assembling the broadest
In this work, we targeted bacteria specifically within beetle sample of ambrosia beetle symbionts ever collected but
mycangia. This often largeorgan[9]is thefunctional pivot also creating a baseline dataset for further studies on
of the ambrosia symbiosis [5]. Our current understanding global host–symbiont dynamics. This is important for
is that mycangia are the products of millions of years of highly invasive groups such as the ambrosia beetles [3].
mutualistic coevolution between the beetle and fungal Representatives of this symbiotic complex are usually
symbionts. The mycangium provides an environment in not economically important in their native regions but
which exposure to UV light, abrasion, and tree defensive can be devastating in invaded regions [3, 20]. It has
chemicalsarereduced,andwheresymbiontsareprovided not yet been tested whether the increased impact is
nutrients [10]. At the same time, it also excludes most caused by the change of beetle behavior in the invaded
fungi while supporting the few coevolved symbionts regions, or by acquisition of new symbionts (whether
[11]. This selectiveness may hypothetically also affect fungal or bacterial). By analyzing several species across
bacteria. Bacteria are abundant and diverse on the body their global distribution, we are implicitly testing a key
surface and within galleries of ambrosia and bark beetles assumption of the latter hypothesis: Is there evidence
[12–16], but whether they are also present within the for acquisitions of new symbionts in newly colonized
mycangium, or if the mycangium is kept bacteria-free, regions?
786 J.Hulcretal.
Methods supernatant was used in the polymerase chain reaction
(PCR). Amplicons were generated using a universal
To assure sufficient statistical power of our tests, our sam- bacterial/archeal primer pair 515 F and 806R (5′-
pling included six species of fungus-farming beetles, each GTGCCAGCMGCCGCGGTAA-3′ and 5′-GGAC
represented by eight to 28 different individuals, from nine TACVSGGGTATCTAAT-3′ [24]). The primer 515 F
localities around the world (for details, see Table 1). To was appended with a TC linker and a Roche 454 B
include phylogenetically diverse beetle representatives, we pyrosequencing adapter; the 806R primer was appended
includedspeciesfromthreecladesofambrosiabeetles,each with a 12-bp sample-specific barcode sequence, a CA
withitsowntypeofmycangium.Thefirsttypeisaprotho- linker, and a Roche 454 A sequencing adapter. The
racic mycangium in Dendroctonus frontalis, which has a sample-specific, error-correcting barcode allowed for
pairedtubularmycangiumontheinsideofprothoracicpleu- pooling all amplicons into a single pyrosequencing
ral plates; the mycangium opens near the procoxa [10]. run. All samples were amplified in triplicated 25-μL
Dendroctonus represents an independently evolved clade reactions using 5 Prime HotMaster polymerase mix (5
of fungus-farming bark beetles. These beetles inhabit tree Prime, Inc.) under the following cycling conditions—
phloem rather than xylem, and the adults feed primarily on 95°C, 1 min, 33×(95°C, 30 s; 50°C, 1 min; 72°C,
phloem. However, the larvae depend almost entirely on 1 min). Amplicons were purified using the UltraClean
coevolved fungal symbionts for nutrition, thus we include PCR Clean-up kit (MoBio). Concentration of each
itamongambrosiabeetlesinthisstudy.Thesecondtypeisa amplicon was determined using the Quant-iT™ Pico-
mandibular mycangium, the ancestral and most widely dis- Green® dsDNA kit (Invitrogen), and equimolar aliquots
tributed form of mycangium in the ambrosia beetle tribe of all samples were pooled. Pyrosequencing was carried
Xyleborini, and characteristic of the genus Xyleborus [21]. out on a Roche Genome Sequencer FLX system at
Xyleborini are one of the largest and most economically Engencore, University of South Carolina, USA.
importanttribesofambrosiabeetles[3,20].Theopeningof The sequencing output was processed using the pack-
the mandibular mycangium is in the oral cavity. The third age QIIME [25]. All sequences are available upon re-
type,representingadifferentcladewithintheXyleborini,is quest. The output was filtered to contain only sequences
themesonotalmycangium,alargeinternalorganwithopening with lengths >200 and <1,000 bp with an average quality
betweenthepronotumandtheelytralbasis[7].Itappearsto score >25 and no ambiguous characters. Sequences were
haveevolvedonlyoncewithintheXyleboriniandcoincided assigned to samples according to the 12-bp barcode.
with a radiation of a successful clade with many genera, Sequences that were ≥97 % similar were grouped into
includingXylosandrus[21]. phylotypes using the UCLUST method [25], and the
Beetles were obtained either by rearing from colonized taxonomic identity of each phylotype was determined
logs[22]orfromcuptrapsbaitedwithethanol.Beetleswere using the RDP Classifier [26]. Phylotypes occurring
preserved in 95 % ethanol and stored at −80°C. To extract in >30 % samples that were unclassified by RDP were
bacteria from mycangia, we surface-cleaned each beetle by identified individually in NCBI BLAST. To confirm the
vortexing inwaterandethanoland excisedthepart ofeach genus identity of the three common Mycoplasma phylo-
beetlethatcontainedthemycangium.InXyleborusspp.,we types and to rule out possible misclassification in RDP
slicedoffapartoftheheadbetweenthefronsandthejoints and BLAST, we performed a phylogenetic analysis of the
ofmandibles.ThetaxonomicdistinctionbetweenXyleborus placement of our phylotypes in Mycoplasmataceae. We
ferrugineusandXyleborusbispinatusisuncertain[23],thus downloaded 16 S sequences of identified strains of My-
wetreatedrepresentativesofthecomplexasonespecies.In coplasma spp. and representatives of other genera within
Xylosandrus, we excised the content of the mesonotal this family from NCBI-GenBank, aligned them with our
mycangium. In Dendroctonus, we exposed both mycangia sequences using MUSCLE [27] (270 bp alignment), and
ontheinnersidesofthethoraxandsqueezedthecontentout inferred a maximum likelihood phylogeny based on the
with a sterile pin. The environmental control composed of Tamura-Nei model in MEGA5 [28].
two galleries of Xylosandrus germanus were collected dur- For downstream analyses, a distance matrix was derived
ing the same collecting event as the X. germanus beetles. using UNIFRAC [30] which takes into account a phyloge-
Surface of the gallery containing the fungal garden was netic structure of the community. Representative sequences
scraped into a vial and processed in the same way as the from all operational taxonomic units were aligned with
beetle mycangia. MUSCLE [27], and the phylogenetic tree and unifrac dis-
The extracted mycangium was added to 10 μL of Ex-n- tance matrix were produced in QIIME. In order to avoid
Amp™extractionsolution(Sigma-Aldrich),maceratedwith artifacts of PCR, sequencing, and sampling biases [31], we
a pestle, and lysed at 96°C for 10 min. The reaction was do not use bacterial abundance (read counts) in our analy-
deactivated with 10 μL of 3 % BSA; 1 μL of the clear ses; instead, prevalence of a phylotype is calculated as the
MycangiaofAmbrosiaBeetlesHostCommunitiesofBacteria 787
Table1 AmbrosiabeetlesfromwhichmycangiaweredissectedandbacterialDNAsequenced
My cangium Species Locality No. Sum of
beetles sequence
count
Prothoracic USA, Arizona, Flagstaff 4 1857
(native)
Dendroctonus frontalis
USA, Florida, Lake 5 2062
Alfred (native)
Ghana, Ankasa (native) 5 2231
Guyana, Iwokrama 5 2021
(native)
Japan, Okinawa (native) 3 1182
Mandibular
Xyleborus affinis
Papua New Guinea, 3 1365
Madang (native)
USA, Florida, Lake 4 766
Alfred (native)
Xyleborus bispinatus- Ghana, Ankasa (native) 5 2139
Guyana, multiple loc. 2 635
ferrugineus (native)
Panama, Canal zone 3 289
(native)
Papua New Guinea, 1 847
Madang (native)
USA, Florida, Lake 7 4066
Alfred (native)
USA, S. Carolina, Myrtle 5 4114
Xyleborus glabratus Beach (invasive)
USA, Florida (invasive) 5 3411
Ghana (unclear) 5 0
Japan (native) 5 151
Xylosandrus Papua New Guinea 5 90
(unclear)
crassiusculus
Mesonotal USA, S. Carolina, Myrtle 9 1161
Beach (invasive)
USA, Florida (invasive) 4 264
Japan, Okinawa (native) 1 0
USA, Carolinas, Myrtle 3 0
Xylosandrus germanus Beach (invasive)
USA, Massachusetts, 5 0
Harvard Forest (invasive)
Ambient Xylosandrus germanus Japan, Okinawa (native) 2 1191
788 J.Hulcretal.
numberofbeetlesamplesyieldingoneormorereads.Like- similaritythreshold(seethecompletedatasetintheElectronic
wise, sample similarities in multivariate analyses were not supplementarymaterial).
weightedbyabundance.Priortotheanalysis,sampleswere Several phylotypes occurred in many beetle species and
rarefied to 41 reads. Less-strict rarefaction levels were also were broadly distributed geographically (Table 2). Several
tested(122and330readspersample);theydidnotprovide phylotypesofMycoplasma,Burkholderiales,andPseudomo-
greaterexplanatorypowerbutresultedinexclusionofmost nadaleswererecoveredfrommorethanhalfoftheamplified
low-yield mesonotal samples. The structure of the bacterial samples. The genus Mycoplasma was represented by three
communitywasvisualizedusingprincipalcoordinatesanal- frequentlyencounteredphylotypes,includingthemostwide-
ysis based on theUniFracdistances. spread phylotype (present in 75 % of samples). The place-
Tocomparetheeffectofbeetlespeciesandlocalityonthe mentofthethreephylotypesinMycoplasma, asopposedto
composition ofbacterial communities, we performed a per- placement in other Mycoplasmataceae more often reported
mutational multivariate analysis of variance of pairwise frominsecthosts,wasconfirmedusingaphylogeneticanal-
distancesbetweensamples(theAdonisfunctionintheVeg- ysis of the bacterial family (Fig. 1). The second and third
an package in R [29]). The factors beetle species, locality, mostprevalentphylotypesbelongedtounidentifiedBurkhol-
and their interaction were considered fixed factors in the deriales (probably Comamonadaceae, no closer match was
model. Mycangium type was included as a stratifying vari- available in RDP and BLAST) found in 69 % and 61 % of
able, such that permutations were constrained within beetles,respectively.Thelasttwophylotypespresentinmore
mycangium type (the significance of mycangium type was thanhalfofthesampleswerePseudomonadales:oneAcine-
not directly tested, as only one type was sufficiently repli- tobacter and one unidentified Pseudomonadaceae (preva-
cated). To examine pair-wise differences between beetle lence 56 % and 48 %, respectively). Several other
species in their composition of bacterial communities, we phylotypes were found in fewer species but still prevalent
performed Tukey’s honestly significant difference (HSD) withincertainbeetlespecies(Table2).Particularlynotableis
post hoctestsona model ofbacterialcommunity composi- the uniform microbiota of Xyleborus glabratus, where four
tion as a function of beetle species identity (using an anal- bacterialphylotypesweresharedbyallbeetleindividualsand
ysis of multivariate homogeneity of group variances; data thesamephylotypeswereinfrequentorabsentinotherbeetle
were pooled across beetlelocality and mycangium type). species.
To test whether related beetle species have more similar The bacterial community as a whole differed strongly
bacterial communities than unrelated beetles, we tested the among beetle species (Fig. 2; F 04.298, R200.26;
5,47
congruence between a beetle phylogeny and a dendrogram p<0.001) and less strongly but still significantly among
ofsimilaritybetweenbacterialcommunitiessummedwithin regions (F 02.13, R200.18; p<0.001). The prokaryot-
7,47
species. The beetle phylogeny was derived from concate- ic communities of X. crassiusculus and X. glabratus are
natedsequencesofcytochrome-oxidaseImtDNAand28S significantly different from several other beetle species
rDNA of each species from our previous datasets [32] and (Tukey’s HSD post hoc test; Table 3). Similarities between
from NCBI Genbank, using maximum likelihood with the bacterial communities did not correspond to beetle phyloge-
Tamura-Nei model ofevolutioninMEGA [28]. Records of neticrelationships(Manteltestp00.149,Fig.3).Interestingly,
bacterialphylotypesweresummedupwithinbeetlespecies, both specieswithmesonotalmycangia,X.crassiusculusand
distance matrix obtained with unweighted UniFrac, and X.germanus,repeatedlyyieldedverypoorampliconsofbac-
summarized in a dendrogram using the UPGMA algorithm terial16S(averagenumbersofreads,60and0,respectively).
in QIIME. Jack-knife support for nodes in the dendrogram ThetwoenvironmentalsamplesfromgalleriesofX.germanus
were derived from repeating this process on successively yielded a bacterial community as diverse as samples from
rarefied matrices from 1,666 reads (the lowest read count, mostmycangia,butthecommunitywassignificantlydifferent
Xylosandrus crassiusculus)to833reads (50%ofdata) per fromallmycangialcommunities.
beetle species. The significance of congruence between the
two matrices was assessed using Mantel test in PopTools
[33]with1,000iterations.X.germanuswasnotincludedin Discussion
most tests, asit didnot yield any bacterial reads.
Although mycangiaare usually considered tohave evolved
to house and disperse the fungi on which ambrosia beetles
Results depend, they also host several regularly occurring bacterial
phylotypes. Representatives of Mycoplasma, Burkholder-
Wesuccessfullyamplifiedbacterial16Sin66outoftheinitial iales, and Pseudomonadales in particular are widespread in
96beetles.Inthetotalof29,842readsofsufficientquality,we mycangia of multiple beetle species from diverse parts of
distinguished 1,546 bacterial phylotypes using a 97 % the world. These appear to be true inhabitants of beetle
MycangiaofAmbrosiaBeetlesHostCommunitiesofBacteria 789
Table2 Dominantphylotypesofbacterialcommunitiesinambrosiabeetlemycangia(highertaxon,order,andgenus,whereavailable)
Beetle Bacterialphylotypespresentinoverhalfofsamples Prevalence
D.frontalis Betaproteobacteria;Burkholderiales 0.86
Gammaproteobacteria;Pseudomonadales;Pseudomonadaceae 0.71
Mollicutes,Mycoplasma320 0.71
Mollicutes,Mycoplasma661 0.71
Gammaproteobacteria 0.57
X.affinis Mollicutes,Mycoplasma320 0.79
Betaproteobacteria;Burkholderiales;Comamonadaceae 0.68
Mollicutes,Mycoplasma1441 0.63
Betaproteobacteria;Burkholderiales 0.58
Gammaproteobacteria;Enterobacteriaceae 0.53
X.bispinatus–ferrugineus Betaproteobacteria;Burkholderiales;Comamonadaceae 0.88
Mollicutes,Mycoplasma320 0.76
Betaproteobacteria;Burkholderiales 0.71
Gammaproteobacteria;Pseudomonadales;Acinetobacter 0.53
Mollicutes,Mycoplasma661 0.53
X.glabratus Alphaproteobacteria;Rickettsiales;Rickettsia 1
Bacteroidetes;Sphingobacteriales;Sphingobacterium 1
Gammaproteobacteria;Pseudomonadales;Acinetobacter 1
Gammaproteobacteria;Xanthomonadales;Stenotrophomonas 1
Alphaproteobacteria;Caulobacterales;Caulobacter 0.9
Betaproteobacteria;Burkholderiales;Alcaligenaceae 0.9
Gammaproteobacteria;Pseudomonadales;Pseudomonadaceae 0.8
Gammaproteobacteria;Xanthomonadales;Stenotrophomonas 0.8
Actinobacteria;Actinomycetales;Tsukamurella 0.7
Betaproteobacteria;Burkholderiales 0.7
Actinobacteria;Actinobacteria;Actinomycetales 0.6
BacteroidetesFlavobacteriales;Chryseobacterium 0.6
Bacteroidetes;Sphingobacteriales;Sphingobacterium 0.6
Bacteroidetes;Sphingobacteriales;Sphingobacterium 0.6
Betaproteobacteria;Burkholderiales;Achromobacter 0.6
Betaproteobacteria;Burkholderiales;Comamonadaceae 0.6
Betaproteobacteria;Burkholderiales;Delftia 0.6
Gammaproteobacteria;Enterobacteriales;Enterobacteriaceae 0.6
Gammaproteobacteria;Pseudomonadales;Moraxellaceae 0.6
Gammaproteobacteria;Pseudomonadales;Pseudomonadaceae 0.6
Mollicutes,Mycoplasma320 0.6
X.crassiusculus Mollicutes,Mycoplasma320 0.78
Betaproteobacteria;Burkholderiales 0.67
Gammaproteobacteria;Pseudomonadales;Acinetobacter 0.56
Gammaproteobacteria;Xanthomonadales;Stenotrophomonas 0.56
Mollicutes,Mycoplasma1441 0.56
X.germanusmycangium Nobacterialsequencesamplified
X.germanus,fungalgardensfromtwogalleries Alphaproteobacteria 1
Alphaproteobacteria;Rhizobiales 1
Alphaproteobacteria;Rhizobiales;Brucellaceae 1
Bacteroidetes;Sphingobacteriales;Mucilaginibacter 1
Bacteroidetes;Sphingobacteriales;Sphingobacterium 1
Bacteroidetes;Sphingobacteriales;Sphingobacterium 1
Betaproteobacteria;Burkholderiales;Alcaligenaceae 1
790 J.Hulcretal.
Table2 (continued)
Beetle Bacterialphylotypespresentinoverhalfofsamples Prevalence
Betaproteobacteria;Burkholderiales;Herbaspirillum 1
Gammaproteobacteria;Pseudomonadales;Pseudomonadaceae 1
Gammaproteobacteria;Xanthomonadales;Dyella 1
Proteobacteria 1
Onlyphylotypessequencedfromoverhalfofsamplesoftherespectivespeciesarelisted.Prevalence:Proportionofsampleswherethephylotype
wasdetected.Notethesignificantlydifferentcompositionofthegallerysample
mycangia, rather than environmental or laboratory contam- Reports of Comamonadaceae from bodies of insects are
inants: The two control samples of fungus gardens sur- rare. Several strains have been isolated from insects but not
rounding the beetles were dominated by completely from groups relevant to wood-boring beetles [35–37]. Pseu-
different phylotypes; for example, no Mycoplasma was domonadaceae are reportedmorecommonly. The mostrele-
present. vantisarecordofseveralphylotypesofAcinetobacterinthe
The two most common groups of associates of ambrosia gut of the bark beetle Dendrocotnus valens [38]. The 16 S
beetleshavenever(Mycoplasma)oronlyrarely(Comamona- sequenceofone phylotype correspondstotheAcinetobacter
daceae) been recovered from insect hosts. This is the first phylotype detected in our samples as the fourth most wide-
record of Mycoplasma spp. as stable associates of insects. spreadphylotype(only1.5%divergent;otherreportedstrains
Our discovery is at odds with the definition of the genus as are not similar). Burkholderiaceaehavebeenpreviously iso-
parasites of vertebrates [34] and raises questions about the latedfromcrushedwholebodiesofanotherDendroctonus,D.
broaderbiologyofthegroup.TheconfamilialgenusEntomo- valens[15],andPseudomonadaceaefromDendroctonuspon-
plasma has been isolated from insects, but the phylotypes derosae[14],but,inbothcases,the isolatesrepresented dif-
from ambrosia beetles do not fall into this clade (Fig. 3). ferent16Sphylotypesthanthosereportedhere.
Importantly,theMycoplasmaspp.fromambrosiabeetlesare Sincebeetlespeciesisastrongerpredictorforthebacterial
alsonotamonophyleticclade,suggestingthatseveralMyco- community composition than locality, we suggest that more
plasmacladescolonizedambrosiabeetlesindependently. bacterial phylotypes in beetle mycangia are vertically
Figure1 ThethreemostwidespreadMycoplasmataceaefromambro-
siabeetlemycangiaaremorecloselyrelatedtoMycoplasmaspp.from Figure2 Principalcoordinatesanalysis(PCoA)ofbacterialassemb-
vertebratesthantoconfamilialgeneramoreoftenreportedfrominver- lagesfromambrosiabeetlemycangiashowsthesignificantspecificity
tebrates. They are also not monophyletic, which suggests repeated ofbacterialcommunitycompositiontocertainbeetlespecies.Clustered
colonizationofambrosiabeetlesbythisgroupofbacteria.Maximum are samples from X. glabratus, X. crassiusculus, D. frontalis, and
likelihood phylogeny was reconstructed in MEGA [28] based onthe samplesfromthetwogalleriesofX.germanus.Notclusteredarethe
Tamura-Nei model; numbers at nodes represent bootstrap support. widelydispersedsamplesfromX.affinisandX.bispinatus/ferrugineus.
Sequences of identified Mycoplasmataceae were obtained from Firsttwoaxesareshown,explaining9.04%and7.41%ofvariation.
NCBI-Genbank. The analysis is strictly for taxonomic purposes and The PCoA is based on UniFrac distances unweighted by phylotype
didnotattempttoreconstructtheactualevolutionoftheclade abundances
MycangiaofAmbrosiaBeetlesHostCommunitiesofBacteria 791
Table 3 Pair-wisedifferencesinbacterialcommunitycompositionbe- regardless of the locality. These phylotypes in particular de-
tween beetle species resulting from post hoc analyses (Tukey’s HSD
serveadditionalresearchtobetterunderstandtheirroleinthe
followingtheanalysisofmultivariatehomogeneityofgroupvariances)
ambrosia symbiotic complex. It is also worth noting that
Comparison Difference pvalue mycangiaarecomplexorgansandmaynothavebeensampled
(adjusted) completely.Wesampledthelumenofthemycangium,which
iswherethemainmassofsymbiontsresides,butwedidnot
X.affinis–D.frontalis 0.009608 0.998436
attempttoexploretheultrastructureofitswallsreported,for
X.bispinatus/ferrugineus–D.frontalis 0.002645 0.999997
example,inD.frontalis[41].
X.bispinatus/ferrugineus–X.affinis −0.00696 0.998571
Theconclusionthatambrosiabeetlestendtoretainspecies-
X.glabratus–D.frontalis −0.06972 0.081577
specific associatesin differentregionsof theworld is further
X.glabratus–X.affinis −0.07933 0.002512
corroborated by the unique bacterial communities in the
X.glabratus–X.bispinatus/ −0.07236 0.007324
mycangiaofX.crassiusculusandX.glabratus(Table3).Both
ferrugineus
X.crassiusculus–D.frontalis −0.06102 0.210561 species are non-native invaders in North America, and their
X.crassiusculus–X.affinis −0.07062 0.019012 bacterialcommunitiesdiffersignificantlyfromthoseofnative
X.crassiusculus–X.bispinatus/ −0.06366 0.045119 xyleborinebeetles.ParticularlyintriguingisX.glabratus,since
ferrugineus itsentirenon-nativepopulationlikelyoriginatedfromasingle
X.crassiusculus–X.glabratus 0.008703 0.998985 recentintroduction [20, 39]. The bacterialcommunity within
X.germanusgallery–D.frontalis −0.25128 1.02E-06 itsmycangiaisunusuallyuniform(severalphylotypesoccurin
X.germanusgallery–X.affinis −0.26088 5.11E-08 every sample) and dissimilar from bacterial communities in
X.germanusgallery–X.bispinatus/ −0.25392 1.02E-07 nativeNorthAmericanspeciesofXyleboruscollectedfromthe
ferrugineus
sametrees.Thisinvasivebeetlethusdoesnotappeartohave
X.germanusgallery–X.glabratus −0.18156 0.000205
adopted the bacteria of native beetles with which it is now
X.germanusgallery–X. −0.19026 0.000132
sympatric. An interesting question we cannot resolve here is
crassiusculus
whether bacterial communities of X. glabratus mycangium
pvaluesareadjustedformultiplecomparisons.Inboldaresignificantly havechangedrelativetoitsancestorsinitsnativerange.
differentspeciespairs
We have repeatedly failed to amplify bacterial 16 S
rDNA from mesonotal mycangia of both species of the
transmitted than sampled from the environment. Given their genus Xylosandrus. We are unable to distinguish whether
prevalence (Table 2) and their apparent absence outside of this reflects a true absence of bacteria in the mycangia, or
beetle mycangia, several of the widespread phylotypes we the presence of strong PCR inhibitors. It is however not a
found have the potential to be stable associates of ambrosia result of low quality of the samples. The two control sam-
beetles. Based on our present knowledge, we cannot deter- ples from fungal gardens surrounding two different X. ger-
mine the ecological role of the bacterial associates. The role manus were collected directly from the gallery around the
could be anything from mutualists of the beetles to their beetlesandprocessedidentically,yettheyyieldedrichcom-
parasites,orevenendosymbiontsoftheambrosiafungi(i.e., munitiesof bacteria.
Burkholderiales,[40]).Inmostcases,thebeetlesseemunlike- Noneofthecommonphylotypesdetectedherehavebeen
ly to obligately depend on the bacteria, since even the most previously reported from bark and ambrosia beetles. One
prevalentbacterialphylotypesinthebeetlesdidnottendtobe reason may be that the majority of previous studies used
universallypresentwithinahostspecies.However,anexcep- culturing approaches. Here, we show that the most regular
tioninthisregardmaybethefourspecies-specificassociates associatesbelongtogroupsthataredifficultorimpossibleto
of X. glabratus recovered from all samples of this beetle culture, such as Mycoplasma or Rickettsia [34]. Lastly, we
alsoshowthatfocusonspecificorgans,suchasthemycan-
gium, allows refined inference of symbiotic associations,
andwerecommendthatfutureresearchoninsectsymbioses
takes insectanatomy into account.
Tosummarizeourfindingsonthecommunityecologyof
the mycangial microbiome, it appears that (1) bacterial
communitiesaremorespecies-specificthanlocality-specific,
however, (2) the core microbiome is nevertheless shared
Figure3 Similaritybetweenbacterialcommunitiesinmycangiadoes acrossbeetlespecies,and(3)thesimilarityofbacterialcom-
notcorrespondtothebeetlephylogenybutdistinguishes theconifer-
munitiesdoesnotreflectphylogeneticrelatednessoftheirhost
inhabiting D. frontalis.Numbers on thesimilarity dendrogram corre-
beetles. This suggests that, although beetles tend to retain
spondtojack-knifesupportfornodes;nodeswith<50%supportwere
collapsed distinctcommunitieseveninnewlycolonizedregionsandin