and Bifidobacterium aemilianum sp. nov., from the carpenter bee (Xylocopa violacea) digestive tract. Systematic and Applied Microbiology, 42(2):205–16, DOI: 10.1016/j.syapm.2018.11.005

This is the final peer-reviewed accepted manuscript of:

Alberoni D, Gaggìa F, Baffoni L, Modesto MM, Biavati B, Di Gioia D (2019) Bifidobacterium xylocopae sp. nov.

and Bifidobacterium aemilianum sp. nov., from the carpenter bee (Xylocopa violacea) digestive tract. Systematic and Applied Microbiology, 42(2):205–16, DOI: 10.1016/j.syapm.2018.11.005

The final published version is available online at: https://doi.org/10.1016/j.syapm.2018.11.005

© 2018. This manuscript version is made available under the Creative Commons Attribution-NonCommercial-NoDerivs (CC BY-NC-ND) 4.0 International License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Bifidobacterium xylocopae sp. nov. and Bifidobacterium aemilianum sp.

nov., from the carpenter bee (Xylocopa violacea) digestive tract

Daniele Alberoni

, Francesca Gaggìa

, Loredana Baffoni

, Monica Marianna Modesto

, Bruno Biavati

, Diana Di Gioia

aDipartimentodiScienzeeTecnologieAgro-Alimentari,UniversityofBologna,VialeFanin44,40127,Bologna,Italy

bInstituteofEarthSystems,DivisionofRuralSciencesFoodSystems,UniversityofMalta,Msida,Malta

Keywords: Newspecies Bifidobacteriumxylocopaesp.nov. Bifidobacteriumaemilianumsp.nov. Phylogenetics Xylocopaviolacea Osmiaspp.

Social bees harbor a community of gut mutualistic bacteria, among which bifidobacteria occupy an important niche. Recently, four novel species have been isolated from guts of different bumblebees, thus allowing to suppose that a core bifidobacterial population may be present in wild solitary bees. To date there is sparse information about bifidobacteria in solitary bees such as Xylocopa and Osmia spp., this study is therefore focused on the isolation and characterization of bifidobacterial strains from soli- tary bees, in particular carpenter bee (Xylocopa violacea), builder bee (Osmia cornuta), and red mason bee (Osmia rufa). Among the isolates from Osmia spp. no new species have been detected whereas among Xylocopa isolates four strains (XV2, XV4, XV10, XV16) belonging to putative new species were found.

Isolated strains are Gram-positive, lactate- and acetate-producing and possess the fructose-6-phosphate phosphoketolase enzyme. Full genome sequencing and genome annotation were performed for XV2 and XV10. Phylogenetic relationships were determined using partial and complete 16S rRNA sequences and hsp60 restriction analysis that confirmed the belonging of the new strains to Bifidobacterium genus and the relatedness of the strains XV2 and XV10 with XV16 and XV4, respectively. Phenotypic tests were performed for the proposed type strains, reference strains and their closest neighbor in the phylogenetic tree. The results support the proposal of two novel species Bifidobacterium xylocopae sp. nov. whose type strain is XV2 (=DSM 104955

= LMG 30142

), reference strain XV16 and Bifidobacterium aemilianum sp.

nov. whose type strain is XV10 (=DSM 104956

= LMG 30143

), reference strain XV4.

Introduction

Bifidobacteriaarecommensalanaerobicbacteriaofthehuman andanimalgut,wheretheyexertimportantfunctionsforthehost.

In-depthstudiesofbifidobacteriatype-straingenomessupportthe hypothesisof co-evolutionwiththehostwithboth DNAacqui- sitionanddeletionevents[47].Thistaxonconstitutesoneofthe mostnumerousgroupsofbeneficialbacteriainthegutcontribut- ingtotheintestinalmicrobiotaindifferentpercentagesdepending onthehostspecies,ageanddiet [69].Differentmoleculeshave beendescribedandcharacterizedtodateasmediatorsoftheBifi- dobacteriumcross-talkwiththehost[57,21,71]andaccountablefor anumberofpositiveeffectsinhostdevelopmentandphysiology [10,64].Asmammals,insectsrelyonamutualisticgutmicrobial community.Insomeinsects,suchasseveralantspecies,themicro-

∗ Correspondingauthor[email protected]

biotaseemstobeacquiredfromthefoodandtheenvironment [18], whereas inhoney bees and bumblebeesit appearsto be morehost-specific[36].Thesespecializedbacteriamayinfluence host nutrition, as they contain genesinvolved in carbohydrate digestion [17], and contribute to host defense and physiology [50,23].Whethergutmicrobesareenvironmentallyacquiredor host-specific,they areextremely important for the hosthealth statusandbifidobacteriarepresentsanimportantguttaxontobe investigatedforitsbeneficialproperties.Bifidobacterialpopulation hasbeencharacterizedinthesixtiesinsomepollinatinginsects, includingthehoneybeesApismelliferaandApiscerana,andisolated strainswereclassifiedasnewspecies:Bifidobacteriumcoryneforme, BifidobacteriumasteroidesandBifidobacteriumindicum[59].Later studies, aimed at the identification of dominant and recurring honeybee-associatedgutmicroorganisms,confirmedbifidobacte- riaasstablecolonizers/inhabitantsofhoneybeegut[58].Honey beesarenottheonlypollinatorsharboringbifidobacteria:inthe lastdecadefournovelspecieshavebeenidentifiedfromBombus spp.gut:Bifidobacteriumbombi,Bifidobacteriumactinocoloniiforme

andBifidobacteriumbohemicumfromthedigestivetractofBom- buslucorumandBifidobacteriumcommunefromBombuslapidarius [30,31,53].However,noinformationisavailableintheliterature onthepresenceofbifidobacteriainsolitarybeessuchasXylocopa spp.,knownascarpenterbeesfortheirabilitytoburrowintohard plantmaterial,andOsmiaspp.,knownasmasonbeesfortheirhabit ofusingmudorother“masonry”productstoconstructtheirnests.

Thesebeesexertahighlyefficientpollinationservice[74].Xylo- copaspeciesareknowntopollinateseveralcrops,suchaslegumes, eggplants,broccolietc.[68,73]andseveralfruitcrops,especially Prunusspp.[73,15],thusassuminggreatvalueforcroppollination strategies.Osmiaspp.,especiallyOsmiacornuta,arealsoofutmost importancefororchardpollination,especiallyforRosaceaefamily plants,likepearandappleandalsoforalmond[42,9].

Thehealthstatusofsolitarybeesiscrucialforthemaintenance ofthepollinationservice.Inthelastcentury,manywildbeepop- ulations,includingsolitarybees,havebecomereducedinnumber andthelossoftheirgeneticdiversitymakesthemmorevulnerable toinfectiousdiseasesandotherstressorssuchaspesticides[20,62].

Moreover,thespreadofparasiticinfectionsfrommanagedbeesto wildbeeshasbeenreportedandrecentlyreviewedbyGraystock etal.[20]andrepresentsapotentialthreatforwildbees’popula- tion.Thegutofinsectsmayharboroneofthelargestreservoirsof ayetunexploredmicrobialdiversity.Adeeperknowledgeofthe gutmicroorganismsofalternativepollinators,suchasOsmiaand Xylocopa,couldbeofgreatimportance,sincetheirhealthstatus dependsonthepresenceandactivityofcommensalmicroorgan- ismsasforanyotheranimal.Novelstrainsmightshowapotential asbeneficialbacteriaforpollinatorinsects,reinforcingattemptsto establishabeneficialbacteriastrategyforbeehealth[6,1,2].This workisthereforeaimedatincreasingtheknowledgeonthepres- enceanddiversityofcultivablebifidobacteriainsolitarybees,in particularinthegeneraXylocopaandOsmia.

Materialsandmethods

Samplescollectionandmicroorganismsisolation

In spring 2016, 2 worker carpenter bees were collected in a flowery meadowin Spilamberto (Modena, Italy),whereas 10 builderand10redmasonbeeswerecollectedina rearingfield locatedinCadriano(Bologna,Italy)andpromptlytransferredto thelaboratory.Allsolitarybeeswereanesthetizedandsacrificed, and theirgut content wasextracted, weighted, seriallydiluted and plated on two different media for bifidobacteria isolation:

Tryptone, Phytone, and Yeast extract (TPY) agar medium [44]

supplementedwithmupirocin(200mgL⁻¹)and deManRogosa Sharpe(MRS)agarmedium(ScharlauChemie,GatoPerez,Spain) supplementedwithfructose(10gL⁻¹),cysteine(1gL⁻¹)[51]and cycloheximide(0.01mgL⁻¹).Plateswereincubated5daysat35^◦C, inbothanaerobicandmicroaerophilicatmospheregenerated in jarswithGasPak^TMEZsystems(Becton,DickinsonCo.,Sparks,MD, USA).Thechoiceofthetwodifferentoxygenconcentrationswas doneconsideringthatoftenBifidobacteriumstrainsisolatedfrom insectsshowa fastergrowthtimeinmicroaerophilicconditions [10]thaninstrictanaerobiosis.

Morphologicallydifferentcolonieswererandomlypicked,re- streaked,checked forpurity and preparedfor cryoconservation at−80^◦C.Foreachisolatedstrain,theanaerobic/microaerophilic growingconditionsweretestedtodefinethebestculturecondition.

DNAisolationfrommicroorganismsandfingerprintinganalyses

Chromosomal DNA was extracted from each isolate with Wizard^® GenomicDNA Purification Kit(Promega, Madison,Wi,

USA).IsolateswerecharacterizedwithtwoPCR-dependentfinger- printingtechniques.RandomlyamplifiedpolymorphicDNA(RAPD) PCRwasperformed withprimerM13 accordingtoAndrighetto et al.[4]. Enterobacterialrepetitiveintergenic consensus(ERIC) sequence PCR wascarried outwithprimers ERIC-1 and ERIC-2 accordingtoVersalovicetal.[72].Obtainedfingerprintingprofile patternswereanalyzedwithGelComparII6.6(AppliedMaths,Kor- trijk,Belgium)usingtheDICEcoefficientandtheUPMGAclustering algorithm.

16SrRNAgenesequencing

DNA amplification of the 16S rRNA gene was performed withprimers27f(5^-AGAGTTTGATCCTGGCTCAG-3^)and1492r(5^- GGTTACCTTGTTACGACT-3^) [37]according toGaggìa etal. [19].

Amplicons were purified and sent to commercial sequencing facility (Eurofins MWG, Ebersberg, Germany). Sequence chro- matogramswereanalyzed,manuallyeditedand classifiedusing BLASTtoolfromNCBI[3].SequencesweredepositedinGeneBank nucleotidedatabase(accessionnumbersMG597261–MG597286, MH043274andMH043277)(Table1).

Whole-genomesequencingandGCcontentofnewlyproposed species

GenomicDNAfromXV2andXV10strainswassequencedonthe IlluminaMiSeqNGSplatformbyBMRGenomicsfacilities(www.

bmr-genomics.it),accordingtoIlluminaprotocolfor NexteraXT DNAlibrarypreparation.Readswereassembledintocontigswith SPAdes3.7accordingtoBankevichetal.[7]andassemblyquality assessedwithQUAST[22].AverageNucleotideIdentity(ANI)values werecalculatedwithPYANI[54],apython3module,usingdifferent methods:ANIb(basedonBLASTalgorithm),ANIm(basedonMUM- meralgorithm)andTETRA(TetranucleotideSignatureFrequency CorrelationCoefficient)asdescribedbyRichterandRosselló-Móra [56].Twoannotationprogramsbasedondifferentalgorithmswere usedfortheidentificationoftheORFsandsubsequentfunctional annotation:(i)PROKKA –rapid prokaryoticgenome annotation software– [61]implemented withHMMERpackage forriboso- malRNAprofileannotation[16]and(ii)BlastKOALA(KEGGtools) [27].Full-lengthsequencesof 16SrRNAofXV2andXV10were obtainedfromthewhole-sequencingresultsandfurtheranalyzed withthewebtoolEMBOSS-Water(EMBL-EBIrelease)thatusesthe Smith-Watermanalgorithmtocalculatethelocalalignmentoftwo sequences.Thisin-depthanalysisallowedtocalculatesimilarity percentagesbetweenXV2,VX10andthe16SrRNAfull-sequences oftheirclosestrelatives(obtainedfromtypestrainsrepository)(the accessionnumbersoffull-16SrRNAsequencesofXV2andXV10are MH043275andMH043276).

EstimationoftheG+CcontentwasperformedbyDSMZIdenti- ficationService(Braunschweig,Germany)accordingtotheDSMZ protocol[11,46,66].

Phylogenetictreesandcoreandpan-genomes

Evolutionary analyses were conducted in MEGA7 [34] and exportedinNewickformat.TheiTOLweb-basedsoftware[38]has beenusedfortheannotationandmanagementofthepublished phylogenetictrees.Partial16SrRNAsequenceswereusedtoverify therelatednessofXV2andXV10withXV16andXV4respectively, thetreewasinferredbyusingtheMaximumLikelihoodmethod [12]basedontheTamura-Neimodel[67].Ontheotherhand,fifty- ninecomplete16SrRNAandhsp60sequenceswereretrievedfrom wholegenomesequencingrepositorydatabaseofNCBI(ftp://ftp.

ncbi.nlm.nih.gov/genomes/refseq/bacteria/)toinferthephylogeny ofthenewspecies.For 16SrRNA,theevolutionaryhistory was

Table1

Identificationofisolatesbasedonpartial16SrRNAgenesequencesandNCBIblasttool.

IsolatedStrain Isolationsource GenBank Microorganism(RDPclassifier) GenBank %identityNCBI

OCN2 Osmiacornuta MG597261 Cutibacteriumacnes NR040847.1 100.00

OCN10 Osmiacornuta MG597263 Cutibacteriumacnes NR040847.1 99.925

OCV2 Osmiacornuta MG597265 Cutibacteriumnamnetense NR151943.1 99.777

OCV7 Osmiacornuta MG597266 Cutibacteriumgranulosum NR118646.1 99.851

OCV18 Osmiacornuta MG597267 Cutibacteriumacnes NR040847.1 99.629

OCV20 Osmiacornuta MG597268 Paenibacilluspeoriae NR117743.1 99.156

OCV21 Osmiacornuta MG597269 Cutibacteriumacnes NR040847.1 99.926

OCV26 Osmiacornuta MG597270 Cutibacteriumgranulosum NR 118646.1 99.851

OCV28 Osmiacornuta MG597271 Bifidobacteriumcoryneforme NR115978.1 99.925

OCV32 Osmiacornuta MG597272 Vagococcusentomophilus NR133886.1 99.927

ORV8 Osmiarufa MG597273 Bifidobacteriumcoryneforme NR115978.1 99.852

ORV20 Osmiarufa MG597274 Cutibacteriumacnes NR040847.1 100.00

ORV27 Osmiarufa MG597276 Bifidobacteriumcoryneforme NR115978.1 99.854

ORV30 Osmiarufa MG597277 Bifidobacteriumcoryneforme NR 115978.1 99.926

XV2 Xylocopaviolacea MG597278 Bifidobacteriumbifidum NR113873.1 95.573

XV2L Xylocopaviolacea MG597279 Lactobacillusbombi NR134065.1 98.763

XV4 Xylocopaviolacea MH043274 Bifidobacteriumcoryneforme NR115978.1 96.935

XV5L Xylocopaviolacea MG597280 Lactobacillusbombi NR134065.1 98.692

XV8L Xylocopaviolacea MG597281 Lactobacillusbombi NR134065.1 98.695

XV10 Xylocopaviolacea MG597282 Bifidobacteriumcoryneforme NR115978.1 96.952

XV11 Xylocopaviolacea MG597283 Bifidobacteriumactinocoloniiforme NR108438.1 99.048

XV13L Xylocopaviolacea MG597284 Lactobacillusapis NR125702.1 96.906

XV16 Xylocopaviolacea MG597285 Bifidobacteriumbifidum NR113873.1 95.997

XV16L Xylocopaviolacea MH043277 Lactobacillusbombi NR134065.1 98.697

XV17L Xylocopaviolacea MG597286 Lactobacillusbombi NR134065.1 98.694

inferredbyusingtheMaximumLikelihoodmethodbasedonthe Tamura-Neimodel[67].Initialtree(s)fortheheuristicsearchwere obtainedbyapplyingtheNeighbor-Joiningmethodtoamatrixof pairwisedistancesestimatedusingtheMaximumCompositeLike- lihood(MCL)approach.AdiscreteGammadistributionwasusedto modelevolutionaryratedifferencesamongsites(5categories(+G, parameter=0.3807)).Theratevariationmodelallowedforsome sitestobeevolutionarilyinvariable([+I],65.2412%sites).Thetree isdrawntoscale,withbranchlengthsmeasuredinthenumberof substitutionspersite.Allpositionswithlessthan95%sitecoverage wereeliminated.Thatis,fewerthan5%alignmentgaps,missing data,andambiguousbases wereallowedatanyposition.There wereatotalof1512positionsinthefinaldataset.Forhsp60genethe MaximumLikelihoodmethodbasedontheGeneralTimeReversible model(adiscreteGammadistributionwasusedtomodelevolu- tionaryratedifferencesamongsites,+G)wasusedaccordingtothe resultobtainedwithMEGA7aftercalculationofthebestmodelon hsp60sequences.

Thebifidobacteriacore-andpan-genomewereanalyzedusing BPGA(BacterialPanGenomeAnalysistool)[14].Forthecore-tree, afterevaluationofcore-geneswithBPGA,theaminoacidsequences of273geneswereconcatenatedandalignedtogeneratethephy- logenetictreeusingtheNeighbor-Joiningmethod.

PCR-RFLP

TherestrictionofXV2andXV10hsp60gene,aswellasofclosely relatedspecies,wasperformedaccordingtoStenicoetal.[65].An in-silicoanalysiswaspreliminarydonetoobtainthetheoretical restrictionprofilesforthespeciesthatwerenotpreviouslyreported inStenicoetal.[65],usingthehsp60sequencesretrievedfromthe GenBankandRefSeqdatabasesandusingWebcutterb2.0(http://

rna.lundberg.gu.se/cutter2/)(TableS1).

Metabolicprofiles,peptidoglycanandfattyacidsanalysis

Enzymaticactivitiesandsubstratefermentationcapabilitiesfor thenovelstrainsand thespecies closelyrelatedtothem(listed in Table 2) wereobtained with API 50 CHL and Rapid ID 32A kits(bioMérieux, Lyon,France) accordingtothemanufacturer’s

instructionswithaslightmodification:10␮L⁻¹ofacysteinester- ilesolution(1gL⁻¹)wereaddedtotheAPI50CHLmedium.API50 CHLinoculatedgallerieswereincubatedinanaerobiosis,exceptfor XV10thatwasincubatedinmicroaerophilicconditions.Catalase andOxidasetestswereperformedaccordingtoModestoetal.[49].

ThecellwallmureincompositionwasdeterminedbyDSMZIdenti- ficationService,accordingtotheprotocoldescribedbySchumann [60].

AnalysesofcellularfattyacidswerecarriedoutatDSMZIdenti- ficationServiceaccordingtoMiller[48]andKuykendalletal.[35]

withminormodifications described intheDSMZ protocolpub- lishedonline.

Hemolyticactivitytestandanalysisonrelatedproteins

HemolyticactivityofXV2andXV10wastestedinthreedifferent media:ColumbiaBloodAgar(BIOLIFE,Milan,Italy),MRSandTPY supplementedwith5%ofdefibrinatedsheepblood.

Sequencerelatedtohemolyticactivityhavebeenidentifiedin thegenomeannotationfileandanUniProtBLASTanalysishasbeen performedsearchingforHemolysinIIIrelatedproteinsinbifidobac- terialspecies.Thefirst47hitsweredownloaded,alignedinMEGA7 withMUSCLEandthebestMLsubstitutionmodelwasevaluated (WAG+I+F)togeneratethetree.

Resultsanddiscussion

Microorganismsisolation,fingerprintingandclusteranalysis

Atotalof33,34and24bifidobacteriaisolateswereobtained fromX.violacea,O.cornutaandO.rufa(synonymOsmiabicornis), respectively.Fiveclusterswereobtainedinfingerprintingprofiles ofmicrobialisolatesfromX.violacea(Fig.S1),andnineclustersin fingerprintingprofilesofisolatesfromO.cornutaandO.rufa(data notshown).Arepresentativestrainforeach clusterwasfurther processed.

Table2

CharacteristicthatdifferentiatetheproposednovelspeciesofBifidobacteriumfromtheclosestrelatives.Strains:(1)Bifidobacteriumsp.XV2;(2)Bifidobacteriumsp.XV16;(3)Bifidobacteriumsp.XV10;(4)Bifidobacteriumsp.XV4;

(5)B.bombiDSM19703^T;(6)B.asteroidesDSM20089^T;(7)B.coryneformeDSM20216^T;(8)B.indicumDSM20214^T;(9)B.actinocoloniiformeDSM22766^T;(10)B.bohemicumDSM22767^T;(11)B.communeLMG28292^T;(12)B.

minimumATCC27538^T;(13)B.subtileDSM20096^T.BiochemicaltestwereperformedusingAPICH50andRapidID32tests(bioMérieux).+,Positive;w,weaklypositive;−,negative;ND,notdetermined;.

Characteristics Sp.nov. Insectcorebifidobacteria Otherbifidobacteriaspecies

XV2 XV16 XV10 XV4 B.bombi B.asteroides B.coryneforme B.indicum B.actinoco-loniiforme B.bohemicum B.commune B.minimum B.subtile

1 2 3 4 5 6 7 8 9 10 11 12 13

Temperaturerangeforgrowth(^◦C) 25–37 25–37 25–37 25–37 10–37 21–42 22–43 22–43 25–37 10–37 10–37 15–35 ND

DNAG+Ccontent(mol%) 61.3 ND 61.9 ND 50.5 59 55 60 52.7 51.2 54.3 61.5 61.5

Growthundermicroaerophilicconditions − + + + − + + + + − + − −

Peptidoglycanstructure A4␣ A4␣ A3␣ A4␣ A4␣ A3␣ A4␣

l-Lys–d-Asp ND l-Lys–l-Ala₂–Gly^a ND ND l-Lys–Gly l-Lys–d-Asp l-Lys–d-Asp ND ND ND l-Lys–l-Ser^a l-Lys–d-Asp Acidproductionfrom

l-Arabinose w w w w w − w + − + − − −

d-Ribose w + + + + + + + w + − − w

d-Xylose − − + + − − − − − w − − −

d-Galactose − − − − + − + w − + + − +

d-Glucose + − + + + + + + + + + + +

d-Fructose + + + + w + + + − − − + +

d-Mannose − − + + w − − w − + − − −

d-Lactose − − − − − − + w − − − − w

d-Sorbitol − − − − − − w − − − − − −

Methyl␣-d-glucopyranoside − − w − w − − w − + + − w

Arbutin w w + w + + w + w + + − −

Aesculin + + + + + + + + + + + − −

Salicin + + + + + + + + + + + − −

Amygdalin − − − − + + + − + − + − −

d-Cellobiose − − − − + + − + + − − − −

d-Maltose − − − − w − + w − w − + +

d-Melibiose − + w − + − + + w + + − +

d-Sucrose + + + + w + + + − − w − +

d-Trehalose − − − − − − − − − − − − −

Inulin − − w − − − − − − − − − −

d-Raffinose − − + + + − + + − + + − +

Starch − − w − − − − − − − − + +

Glycogen − − − − − − − − − − − + +

d-Turanose − − w − − − − w − − − + +

Gentiobiose − − − − + + + + + + − − −

l-Fucose − − − − − − − w − − − − −

PotassiumGluconate − − − − − − − − − w − − −

Potassium5-ketogluconate − − w − − − − − − w − − −

Enzymaticactivity

␣-Galactosidase + + − − + + + + + − + − +

␤-Galactosidase + + + − + + + + + − + − +

␣-Glucosidase − + + + + + + + + + + + +

␤-Glucosidase + + + + + + + + + + + − +

␣-Arabinosidase + + + + + + + + + + + − +

N-Acetyl-␤-glucosaminidase − − + + − − − − + − − − −

␣-Fucosidase + + − − − + − − − − − − −

Alkalinephosphatase − − − − − − − − − + − − +

Prolinearylamidase + + + + + + + + + + − + +

Leucylglycinearylamidase + − w − − + + − − − − + +

Tyrosinearylamidase + + + + + + + + − + + + +

Alaninearylamidase + − w − − + + − − − + + −

Glycinearylamidase + + + + + + + + − + + + +

Histidinearylamidase + + + + + + + + − + + + +

Serinearylamidase + + + − + + + − − − + + +

a␣-Carboxylgroupofd-Glusubstitutedbyglycine.

Fig.1.Averagenucleotideidentity.GraficaloutputofPYANIforaveragenucleotideidentityanalyses(a)ANIb(BLASTalgorithm)(b)ANIm(MUMmeralgorithm).ANIvalues oflessthan0.95arecoloredinblue,showingthatthestrainsaredifferentspecies.RedsquareswithANIvaluesgreaterthan0.95indicatethatthesesstrainsbelongtothe samespecies.ColorintensitiesfadeastheANIvaluesapproach0.95.

16Ssequencingandmicroorganismsidentification

Recentstudiesshowhowgutmicrobial populationof honey bees(A.mellifera)istransmittedhorizontallythroughcontactwith nursebees,freshfacesorhivesurfaces[43],thereforeit canbe defined asa “socially transmittedgutmicrobiota”.On the con- trary,OsmiaandXylocopageneraconsistofsolitarybees,therefore socialtransmission atthetimeof edgingishardlyoccurringas alsoevidenced byLozoetal.[39].Inrecentstudies[39,28]Bifi- dobacteriumspecieswerenotdetectedinmasonbeesalimentary tract.Inourstudy,onthecontrary,isolatesfromOsmiaspp.inTPY agarmediumwereidentifiedasCutibacteriumacnes,Cutibacterium granulosum,B.asteroidesandB.coryneforme(Table1),showinga similaritywithA.melliferabifidobacteriacorespecies.Anacqui- sitionofbifidobacteriafromfoodsources(flowers)thatmightbe occasionallysharedbetweensolitaryandhoneybees[45,33]can behypothesized.NovelspecieswerenotdetectedinOsmiaspp.

gut.

Theanalysesofpartial16S rRNAsequencesofTPYandMRS isolates(Table1)showedthatXV2andXV10strainsfromX.vio- laceadeservedanin-depthanalysisonthewhole16SrRNAgene sequence retrieved from full genome sequencing NCBI reposi- tory files(described in thefollowing paragraph). The in-depth analysisperformedwithEMBOSS-Water(Smith-Watermanalgo- rithm) allowed to compare XV2 and XV10 strains with their closesttypestrains.XV2analysisgaveasimilarityof97.6%with B. actinocoloniiforme DSM 22766^T and of 95.4% with B. aster- oidesPRL2011,B.coryneformeDSM20216^T andB.indicumDSM 20214^T.Asimilarityof95.8%toB.bohemicumDSM22767^T and 95.7%tobothB.communeLMG28292^TandBifidobacteriumsub- tileDSM20096^T wasobtainedforXV10.Thesescoresarebelow the threshold of 98.5% similarity to be clustered in the same species[32]. Otherisolates fromX.violaceawereassignedtoB.

actinocoloniiforme,LactobacillusapisandLactobacillusbombi,allow- ing us to assume a certain affinity for food sources between carpenter bee and Bombus spp. Bifidobacterium sp. XV2 and Bifidobacterium sp. XV10 are described and proposed as novel species.

GCcontentandfullgenomesequencing

ThetypestrainsXV2andXV10showedaG+Ccontentof61.3 and61.9mol%G+C,respectively.AllvalueswerewithintheG+C contentrangeofthegenusBifidobacterium(52–67mol%)[8,29].

ThesequencingoutputofXV2fastqcounted2,634,000reads whereasXV10fastqoutputcounted3,007,626readscorresponding toanesteemedcoverageof350×and400×,respectively.Thereads assemblyoutputconsistedin242contigs(1.88Mbp)forXV2and 241contigs(2.05Mbp)forXV10.AverageNucleotideIdentity(ANI) values,calculated withPYANI[54],arereportedinTableS2a–c.

XV2andXV10showedthehighestidentityvalueswitheachother of79.3%,86.9%and95.4%forANIb,ANImandTETRArespectively.

Thispercentagesarefarbelowthethresholdof96%forANIband ANImand99%forTETRAproposedbyRichterandRosselló-Móra [56]andmirroringtheDDHvalueof60–70%(Fig.1aandb).

Annotation wascarried outwithBlastKOALAthat performs KO(KEGGOrthology)assignmentstocharacterizeindividualgene functionsandreconstructsKEGGpathways,BRITEhierarchiesand KEGGmodulestoinferhigh-levelfunctionsoftheorganismorthe ecosystem[27] (Figs.2 aand b,S2and S3).Rawreads,contigs assemblyandfeaturetablesweredepositedontheNCBIdatabase (accessionnumberPDCH00000000andPDCG00000000).Genome annotationsresultedinthepredictionof1556genesofwhich1412 codingsequences(CDSs),51genesforRNAand93pseudogenes forXV2strain,whereasforXV10straintheannotationresulted in 1719genes ofwhich 1531CDSs, 52 genes forRNA and 136 pseudo genes(TableS3).Among detectedgenes,discussionhas beenfocusedongenesthatmightbeofinterestforthefutureuse ofthestrainsasbeneficialbacteriainanimalfeedinglikeantibiotic resistanceandvitaminproductions.BothstrainspossessORFsfor twodifferentBvitaminpathways(B6andB9)intheirsequences.

Pyridoxin (B6)pathway ORFs areinvolved only in the“salvage pathway”forpyridoxal5^-phosphaterecycling(throughpyridoxal kinase,PdxK).ORFsannotatedinthefolatebiosynthesisalsoregard a“savagepathway”becauseoftheabsenceofenzymesforprecur- sorformation(thepteridineringandthepara-aminobenzoicacid unitpABA). OnlyXV10straincontainsORFs annotatedasbiotin

Fig.2.Kronacharts.(a)Bifidobacteriumsp.XV2and(b)Bifidobacteriumsp.XV10annotatedwithBLASTKOALAandelaboratedwithKronaToolsv2.7[52].

(B7)biosynthesis,however thegenespresent areonlyinvolved inbiotinuptake(i.e.BioY,componentofbiotinECFtransporter).

ConcerningORFsforantibioticresistance,agenefortetracycline resistancehasbeenannotatedboth inXV2andXV10strains(a ribosome protection type tetracycline resistance gene) and 4- fluoroquinoloneresistancemutationsintheparC,parE,gyrAand gyrBgeneshavebeenfound.SomeannotatedORFsforsugaruti- lizationhavebeenchosenforthediscussioninordertocompare phenotypesinvestigatedwithAPItestsandgenotyperesultsand clarifydiscrepanciesoccurredduringthestraincharacterization.

Inthefermentationtests,negativeresultswereobtainedforlac- tosefermentationwhilethepresenceof␤-galactosidaseactivity wasobservedwiththeuseofRAPIDID32A.However,thisappar- entcontradictionisexplainedbytheabsence,inbothstrains,of lacSgene coding for the lactose–galactose permease,as shown ingenomeannotations.Somegenesrelatedtomaltoseandmal- todextrinutilizationarepresentinXV10strainsbutalsoin this casenotallgenesfortheproductionofthemaltose/maltodextrin transporterswereshownandindeedfermentationdidnotoccur withthis carbon source (Table 2). Genes for arabinose utiliza- tionusingAraBAD pathwayare present inboth XV2and XV10 strains.l-arabinoseresiduesarewidelydistributedamongmany heteropolysaccharidesofdifferentplanttissues.InBifidobacterium sp.XV10afullriboseutilizationoperonandribose-5-phosphate isomeraseenzymehavebeenannotated.Moreover,thestrainhas axylosetransportsystempermease(proteinXylH)togetherwith genesoftheXylABpathway,thereforeit iscapableofferment- ingxylose.AsalreadyreportedandfullyannotatedinB.asteroides PRL2011[10],asimplified respiratorymetabolismwasalsoevi- dencedinBifidobacteriumsp.XV2andXV10togetherwithenzymes involvedintheantioxidantactivitysuchascatalaseandsuperoxide dismutase(thelatterpresentonlyinXV2strain).Thisisconsistent

withthedescriptionofthebeegutasanenvironmentwithahigher oxygenconcentrationcomparedtohumansandothermammals.

Hemolyticactivityandanalysisonrelatedproteins

Since the discovery in Bifidobacterium scardovii of a weak hemolyticactivity[24],hemolysisshouldalwaysbecheckedinnew Bifidobacteriumspecies.XV2,XV16,XV10andXV4strainsshowed

␣-hemolyticactivityinallmediaasagreenishdiscolorationthat surroundsbacterialcoloniesinthetestedmedia.Genomeanno- tationwithProkkaaswellastheannotationprovidedbytheNCBI pipelineduringsequencesubmissionhighlightedtwodistinctORFs annotatedasHemolysinandHemolysinIII.Hemolysinsequence isamembraneproteinoftheCCB3/YggTfamily(IPR003425)and UniProtBLASTresultsshoweditspresenceinseveralbifidobacterial strainswithidentityscoresrangingfrom85.6%to57.9%(takinginto considerationonlythefirst47hits).AnYggTproteincharacterized inEscherichiacoliseemstoberelated toosmoticshockprotec- tion[26],evenifthelengthofE.coliYggYproteinis180amino acidswhilebifidobacteriasequencesareabout100aminoacid-long.

ConcerningtheannotationofHemolysinIII,thisisanintegralmem- brane protein component of the AdipoR/Haemolysin-III-related (IPR004254)family. Thisfamily, among others, groups proteins from pathogenic and non-pathogenic bacteria, including pore- formingproteins.Aphylogenetictreehasbeengenerated(Fig.S4) using47sequencesrelatedtoHemolysinIIIsequencefromUniProt databaseandnewlycharacterizedspecies.Thisproteinseemsto haveahighintra-speciesconservation.Proteinsfrombeespecies clusteredtogether,includingXV2,whereasXV10strainclustered with its closest 16S rRNA gene neighbor. Being hemolysin-III proteinpoorlycharacterized,itisnotpossibletoinferacorrela- tionwiththehemolyticactivity.However,acorrelationbetween

hemolyticactivity and iron availability in the intestine can be discussed.Ironisessentialforbothhost[63]andbacterialphys- iologicalprocessesandmicroorganismshavedevelopeddifferent strategiesforironuptakesuchasreductionofferricironwithsub- sequenttransport,ironacquisitionfromhemeoriron-containing proteinsofthehostandproductionofsiderophores[13].Differ- entlyfrommammalsthatrelyonerythrocytestotransportoxygen, insectstransportoxygenviathehemolymph,afluidthatcontains freeproteinsnamedhemocyanins(copperasoxygencarrier).The useofcoppercofactorasoxygenbinderdoesnotexcludethepres- enceinthehemolymphof otherproteinsbindingiron,suchas ferritin, holoferritinand apoferritin. Ferritinaccumulatesinthe smoothendoplasmicreticulumofthemidgutcellsofinsects[25,55]

anditcanbespeculatedthatironcontainedinthesecellscanbe usedbybacteriaonceintestinalcellslyse.Moreover,genomeanno- tationsreportedthepresenceofaferrousirontransportproteinand anirontransporterpermeasebothinBifidobacteriumsp.XV2and XV10.Therefore,afirsthypothesisofthehemolyticphenotypeis thathemolysinscanactinsynergywithotherproteinstouptake ironfromthesurroundingenvironment,includinginsectsgutcells containingferritin.Thispeculiarweakhemolyticactivityisthere- forenotapathogenictrait[41],butitmaybeawaytoimprove ironuptake.Furtherinvestigationsarenecessarytoconfirmthis hypothesis.Asecondhypothesisofthedetectedhemolyticactiv- itycouldberelatedtotheeffectofH2O2producedbySOD,whose sequencewasfoundinXV2genome.However,thisphenotypewas alsofoundintheXV10straineveniftheSODwasnotannotatedby ProkkaorBlastKOALAprograms.

Phylogenetictrees

Twophylogenetictrees,withcomplete(Fig.3)andpartial(Fig.

S5)16SrRNAgenesequenceswereconstructed.Thephylogenetic treewith partial16S rRNA gene sequences confirms the relat- ednessof XV2andXV10withXV16and XV4,respectively.The phylogenetictreewithfulllength16SrRNAgenesincludesfifty- ninesequencesfromtypestrains.SevenBifidobacteriumspecies(B.

aerophilumDSM100689^T;B.aesculapiiDSM26737^T;B.apriCCM 8605^T;B.avesaniiDSM100685^T;B.callitrichidarumDSM103152^T; B.faecaleJCM19861; B.ramosum DSM100688^T)wereexcluded fromthe16SrRNAbasedphylogenetictreeduetoonlypartial16S rRNAsequenceavailable.Therewereatotalof1512basesinthe finaldataset.XV2clusteredclosetoB.actinocoloniiformeintheB.

asteroidesgroupwhileBifidobacteriumsp.XV10clusteredwithB.

subtileDSM20096,abacteriumisolatedfromsewage.Phylogenetic treeconstructedwithhsp60 genes(Fig. 4)confirmedthe relat- ednessofXV2withB.actinocoloniiformeandotherbifidobacteria fromhoneybees.XV10fallsjustoutsidethesameclustershowing aminorrelatednesstoXV2andotherbifidobacteriaisolatedfrom Apoidea.

Coreandpan-genomeanalysis

TheBPGApipelinecalculatessharedgenesafterstepwiseaddi- tionofeach individualgenomeand plotsthetrendascoreand pan-genome profilecurve (data not shown). According to this curve,thebifidobacteriapan-genomecanbeconsideredas“open”, as also recently evidenced by Lugli et al. [40]. Moreover, the pipelinegeneratesaphylogenetictreebasedonpan-matrixdata (Fig.5a).Fig.5bshowsthecore-treecalculatedwith273aminoacid sequences.TobetterevidencetherelationshipamongBifidobac- teriumspecies,nooutgroupwasusedforthepan-genometree.The phylogeneticanalysisofpan-genomehighlighteddistinctgroups (Fig.5a)thatwere,however,notconfirmedinthecore-genome tree.Inthelatter,thepresenceofanoutgroupspeciesrestrictsthe core-genesoftheBifidobacteriumtaxonandprobablyunderesti-

matespeculiaritieslinkedtotheadaptationtodifferentecological nichesincludinghorizontalgenetransferevents[70,71,75].

PCR-RFLP

PCR-RFLPanalysisofhsp60geneallowsa rapidandaccurate identification of common species of the genus Bifidobacterium [5,65].Theinsilicoanalysisshowedtwonewanddistinctrestric- tionprofilesforXV2andXV10confirmingthehighdiscriminating abilityofHaeIIIenzyme.Moreover,restrictionprofileswerecon- firmedonagarosegelforspeciesinboldinTableS1andfortheXV4 andXV16strains.TherestrictionprofilesofXV4andXV16were identicaltothoseofXV10andXV2respectively(datanotshown).

Metabolicprofiles,peptidoglycanandfattyacidsanalysis

Metabolic profiles and fatty acids analysis are reported in Table2. XV2and XV10strainstested negativefor catalaseand oxidaseactivities; however, thegenome annotationunderlined thepresenceofa catalase-relatedcodingsequence bothin XV2 and XV10strains.In XV2asuperoxidedismutaseCDSwasalso evidenced,even ifXV2strainresultedmoreaffectedbyoxygen presencethan XV10.It ispossibletospeculatethat thediverse phenotypes can berelated to differentregulatory mechanisms.

Peptidoglycantype resulteddifferentfor thetwo strainsand is reportedinTable2.Analysesofcellularfattyacidsaresummarized inTable3.

DescriptionofBifidobacteriumxylocopaesp.nov.

Bifidobacterium xylocopae [xy.lo.co’pae. N.L.gen. n. xylocopae of Xylocopa,a wood cutter, thegenusname of theinsect from whichthestrainwasisolated].CellsareGram-positive,non-motile, non-sporulating,F6PPK-positive,catalase-and oxidase-negative, indole-negative.XV2straingrowsinanaerobicconditionsandcan- notsurviveinmicroaerophilicconditions.Colonies,grownonthe surface ofTPY agarplate,are whiteand circular. Thediameter ofeach colony rangesfrom0.5to1.0mm. StrainXV2growsin thetemperaturerange 25–40^◦C;nogrowthoccursat orbelow 20^◦C.CellsgrowinthepHrange4.5–9.0.Optimalconditionsfor growthoccuratpH6.5and35^◦C.FermentationprofilesofB.xylo- copaeXV2showthatitisabletofermentanarrowrangeofmono anddi-saccharides:d-glucose,d-fructoseandd-sucrose.Moreover, thestrainhydrolyzesaesculinandsalicin,whileitdisplaysscarce growthonl-arabinose,d-riboseandarbutin.Positiveenzymatic activity is observed for ␣- and ␤-galactosidase, ␤-glucosidase,

␣-arabinosidase,␣-fucosidase,argininearylamidase,prolineary- lamidase,leucyl-glycinearylamidase,phenylalaninearylamidase, leucine arylamidase, tyrosine arylamidase,alanine arylamidase, glycinearylamidase,histidinearylamidaseandserinearylamidase.

Whereas,␣-glucosidase,N-acetyl-␤-glucosaminidaseandalkaline phosphataseactivitiesarenegative(Table2).Themajorfattyacids identified are palmitic acid, oleic and linoleicacid plus a mix- tureofunresolvedfattyacids(referredtoassummedfeatures7 intherelated Table3).Thepeptidoglycantype isA4␣l-Lys–d- Asp.TheDNAG+Ccontentis61.3%.ThetypestrainXV2(=DSM 104955^T=LMG 30142^T) and the reference strain XV16 (=DSM 106832=LMG30564)wereisolatedfromgutsamplesofcarpenter bees(X.violacea).Theformalproposalofthenewspecies“Bifidobac- teriumxylocopaesp.nov.”isgiveninTableS4withtheTaxonumber TA00367.

Fig.3.Phylogenetictreeon16SrRNAgene.16SrRNAgenecompletesequencesofrecognizedBifidobacteriumspecies,Bifidobacteriumsp.XV2andBifidobacteriumsp.XV10.

Theanalysisinvolved58nucleotidesequenceswiththeMicrococcusluteusNCTC2665^Tstrainasoutgroup.

DescriptionofBifidobacteriumaemilianumsp.nov.

Bifidobacteriumaemilianum[ae.mi.li.a’num.L.neut.adj.aemil- ianum from the Emilia region, referring to the Italian region wherethebacteriumwasfirstisolated].CellsareGram-positive, non-sporulating,F6PPK-positive,catalase-andoxidase-negative, indole-negative.XV10straingrowsinmicroaerophilicconditions butit cannot survivein bothanaerobic andaerobic conditions.

Colonies,grownonthesurfaceofMRSagarplatessupplemented withcysteineandfructose,arewhiteandcircular.Thediameter ofeachcolonyrangesfrom0.2to0.5mm.StrainXV10growsin

thetemperaturerange20–40^◦C;nogrowthoccursbelow20^◦C.

ThestraingrowsinthepHrange5.0–9.0.Optimalconditionsfor growthoccuratpH6.5and35^◦C.FermentationprofilesofB.aemil- ianum XV10 reveal that it is able to ferment a wide range of monoanddi-saccharides:d-ribose,d-xylose,d-glucose,d-fructose d-mannose, d-raffinose and d-sucrose. The strain hydrolyzes aesculin, arbutin and salicin, while it displays scarce growth onl-arabinose,Methyl␣-d-glucopyranoside,d-melibiose,inulin, methadone, d-turanose and potassium 5-ketogluconate. Posi- tiveenzymatic activityis observed for ␤-galactosidase, ␣- and

␤-glucosidase,␣-arabinosidase,N-acetyl-␤-glucosaminidase,argi-

Fig.4.Phylogenetictreeonhsp60gene.hsp60genecompletesequencesofrecognizedBifidobacteriumspecies,Bifidobacteriumsp.XV2andBifidobacteriumsp.XV10.The analysisinvolved58nucleotidesequenceswiththeMicrococcusluteusNCTC2665^Tstrainasoutgroup.

Fig.5.Core-andpan-genome.(a)Phylogenetictreebasedonpan-genomeanalysis.(b)Phylogenetictreebasedontheconcatenationof273coreaminoacidsequencegenes ofXV2,XV10andmembersoftheBifidobacteriumgenus.

Table3

CellularfattyacidprofilesofinsectrelatedBifidobacterium.Strains:(1)Bifidobacteriumsp.XV2;(2)Bifidobacteriumsp.XV10;(3)B.bombiDSM19703^T;(4)B.actinocoloniiforme DSM22766^T;(5)B.bohemicumDSM22767^T.DataarefromthisstudyandapreviousstudyofKilleretal.[30].Relativeconcentrations(%)oftotalfattyacidswerecalculated.

Fattyacid UPACname Commonname 1 2 3 4 5

C10:0 Decanoicacid Capricacid 0.11 0.06 – – –

C12:0 Dodecanoicacid Lauricacid 0.71 0.46 – – –

C14:0 Tetradecanoicacid Myristicacid 1.28 1,43 – 1.97 2.86

C15:0 Pentadecanoicacid Pentadecylicacid – – – – –

Iso-C15:0 13-Methyltetradecanoicacid Methylmyristicacid – – – – –

C16:0 Hexadecanoicacid Palmiticacid 39.68 21.29 7.14 20.17 15.97

C16:1␻9c (7Z)-7-Hexadecenoicacid cis-7-Palmitoleicacid 1.11 0.41 – – –

C17:0 Heptadecanoicacid Margaricacid – – 4.21 2.11 2.56

C17:1␻9c (8Z)-8-Heptadecenoicacid – – 1.10 – – –

Cyclo-C17:0 cis-9,10-Methylene-Hexadecanoicacid – 0.13 – – – –

C18:0 Octadecanoicacid Stearicacid – 3.85 5.91 7.05 6.56

C18:1␻6c cis-12-Oleicacid – 1.19 – – – –

C18:1␻7c cis-Vaccenicacid – 2.58 6.6 – – –

C18:1␻9c (9Z)-9-Octadecenoicacid Oleicacid 19.3 57.92 7.49 9.99 4.69

C18:1␻9cDMA (9Z)-1,1-Dimethoxy-9-Octadecene – – 8.47 – – –

C18:2␻6c (9Z,12Z)-9,12-Octadecadienoicacid Linoleicacid 6.05 – 7.34 – –

C20:0 Icosanoicacid Arachidicacid – – 7.18 3.25 4.62

Iso-C19:0 17-Methylstearicacid – 0.6 0.74 – – –

C22:0 Docosanoicacid Behenicacid – – 5.87 – –

C23:0 Tricosanoicacid Tricosylicacid – – 5.38 – –

C24:0 Tetracosanoicacid Lignocericacid – – 4.68 – –

C15:1␻ⁿc (ⁿZ)-ⁿ-Pentadecenoicacid – – – 2.47 – –

– Summedfeatures3a^a NA 0.49 – ND ND ND

– Summedfeatures3b^a NA – 0.84 ND ND ND

– Summedfeatures7^a NA 26.49 1.12 ND ND ND

– Summedfeatures8^a NA 3.76 – ND ND ND

– Summedfeatures10^a NA – 5.96 ND ND ND

– Summedfeatures12^a NA – 0.66 ND ND ND

aSummedfeaturesaregroupsoftwoormorefattyacidsthatcannotbeseparatedbyGLC(MIDISystem).Summedfeature3acontainedC16:1␻6cand/orC16:1␻7c;

Summedfeature3bcontainedC16:1␻7cand/orC15:0ISO2-OH.Summedfeature7containedC19:0CYCLO␻10cand/orC19:1␻6c;Summedfeature8containedC18:1

␻7cand/orC18:1␻6c;Summedfeature10containedC18:1␻7cand/orunknownECL17.834;Summedfeature12containedunknownECL18.622and/oriso-C19:0.

ninearylamidase,prolinearylamidase,phenylalaninearylamidase, leucine arylamidase, tyrosine arylamidase,glycine arylamidase, histidine arylamidase and serine arylamidase.On thecontrary,

enzymaticactivityis negativefor␣-galactosidase, ␣-fucosidase, alkaline phosphatase, leucyl glycine arylamidase, alanine ary- lamidase(Table 2).Themajorfattyacidsidentifiedarepalmitic