Comparison between Apicystis cryptica sp. n. and Apicystis bombi (Arthrogregarida, Apicomplexa): Gregarine parasites that cause fat body hypertrophism in bees

ScienceDirect

EuropeanJournalofProtistology73(2020)125688

Comparison between Apicystis cryptica sp. n. and Apicystis bombi (Arthrogregarida, Apicomplexa): Gregarine parasites that cause fat body hypertrophism in bees

Karel Schoonvaere

, Marleen Brunain

, Femke Baeke

, Michiel De Bruyne

, Riet De Rycke

, Dirk C. de Graaf

aLaboratoryofMolecularEntomologyandBeePathology,DepartmentofBiochemistryandMicrobiology,FacultyofSciences, GhentUniversity,Krijgslaan281BlockS2,9000Ghent,Belgium

bDepartmentforBiomedicalMolecularBiology,GhentUniversity,VIBCenterforInflammationResearch,Ghent,Belgium

cGhentUniversityExpertiseCentreforTransmissionElectronMicroscopyandVIBBioImagingCore,Ghent,Belgium

Received16March2019;receivedinrevisedform31January2020;accepted15February2020 Availableonline21February2020

Abstract

Themoleculardivergence,morphologyandpathologyofacrypticgregarinethatisrelatedtothebeeparasiteApicystisbombi LipaandTriggiani,1996isdescribed.The18SribosomalDNAgenesequenceofthenewgregarinewasequallydissimilarto thatofA.bombiandtheclosestrelatedgenusMattesiaNaville,1930,althoughphylogeneticanalysissupportedacloserrelation toA.bombi.PronounceddivergencewithA.bombiwasfoundintheITS1sequence(69.6%similarity)andsevenprotein-coding genes(nucleotide78.05%andprotein90.2%similarity).ThenewgregarinewasisolatedfromaBombuspascuorumScopoli, 1763femaleandcausedheavyhypertrophismofthefatbodytissueinitshost.Inaddition,infectedcellsofthehypopharyngeal glandtissue,animportantexcretoryorganofthehost,wereobserved.Matureoocystswerenavicularinshapeandcontainedfour sporozoites,similartoA.bombioocysts.Giventhesecharacteristics,weproposedthenameApicystiscrypticasp.n.Detections sofarindicatedthatdistributionandhostspeciesoccupationof Apicystisspp. overlapatleast inEurope,andthathistorical detectionscouldnotdiscriminatebetweenthem.Specificmolecularassaysweredevelopedthatcanbeimplementedinfuture pathogenscreensthataimtodiscriminateApicystisspp.inbees.

©2020TheAuthors.PublishedbyElsevierGmbH.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://

creativecommons.org/licenses/by-nc-nd/4.0/).

Keywords: Apicystis;Beeparasite;Bumblebee;Gregarine;Moleculardetection;Neogregarine

Introduction

ProtozoansoftheorderArthrogregarida(phylumApicom- plexa) are parasites of arthropods (Cavalier-Smith 2014).

Fivespecieshavebeenassociatedwithpathologiesinbees (Hymenoptera; Anthophila clade), of which four septate

∗Correspondingauthor.

E-mailaddress:[email protected](D.C.deGraaf).

gregarinesdescribedfromthehoneybeeApismelliferaLin- naeus,1758(Stejskal1965)andasingleaseptategregarine, alsoreferredtoasaneogregarine,describedfrommultiple bumblebeespeciesinCanada(Liuetal.1974).Uponitsdis- covery,thelattergregarinewasnamedMattesiabombiand two decades laterit was accommodated in the newgenus ApicystisLipaandTriggianiafteritsre-discoveryinEurope (LipaandTriggiani1992,1996).Apicystiswasdifferentiated fromMattesiabythenavicularinsteadofthelemon-shaped

https://doi.org/10.1016/j.ejop.2020.125688

0932-4739/©2020TheAuthors.PublishedbyElsevierGmbH.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

licenses/by-nc-nd/4.0/).

oocysts, the presence of four instead of eight sporozoites peroocystandspecializationof beehosts. Althoughbum- ble beesare consideredto serve as the predominanthosts (Macfarlaneetal.1995),A.bombihasalsobeendetectedin honeybees(Menailetal.2016;Plischuketal.2011;Schulz etal.2019),stinglessbees(Nunes-Silvaetal.2016)andsoli- tarywildbees(Ravoetetal.2014;Tianetal.2018)butno insecthostsotherthanbeessofar.Earlymonitoringstudies assumedacosmopolitandistributionofApicystis(Lipaand Triggiani1996).However,themorerecentdetectionsofA.

bombiinSouth-America(PlischukandLange2009)andAsia (Morimotoetal.2013)haveraisedquestions aboutitstrue nativerange(Maharramovetal.2013;Meeusetal.2011).

TheprevalenceofA.bombidiffersgreatlyacrossstudiesand castes(queens,workersandmales)withaveragereportsof 5–10%in5bumblebees,10–15%inhoneybeesandinvasive bumblebees(JonesandBrown2014;Plischuketal.2011, 2017)andover25%inpopulationsproximoustocommercial pollinatorcustomers(Graystocketal.2014).

GregarinesclassifiedintheformerorderNeogregarinorida arenotablymorevirulentthanotherparasites(Undeenand Vávra1997).Infectionsare generallylimited toaspecific tissue,oftenthefatbodyorMalpighiantubules(Yamanand Radek2017)resultinginproliferationanddestructionofthe infectedtissue.Thelifecycleoftheseparasitesiscomplex andthatofA.bombiismoderatelyresolvedthankstoasin- gleultrastructurestudy(Liuetal.1974).Infectionstartswith theoraluptakeofaviableoocyst(cfr.long-livingspore)and releaseoffoursporozoitesinthemidgutlumen.Thesporo- zoites penetratethe midgut wall andundergo asexualand sexualreproductioninthefatbodycells.Priortosporogony, the gametocyst containstwo oval sporoblasts that quickly developintoyoungoocysts(ValigurováandKoudela2006).

Astheymature,theoocystwallthickens,apairofpolarplugs attheendsoftheoocystsbecomevisibleandthegametocyst membranesultimatelydissociates.Thecycle isrepeatedin thesamehostbyautoinfectionof fatbodycells.Typically, healthyfatbodycellsappearyellowwhileheavilyinfected cellsappearswollenanddistinctlywhiteasaresultofhyper- trophism(LipaandTriggiani1996).Otherthanthefatbody, oocystshavebeendetectedinthemidgutandhindgutbody partsof beesandin asinglecaseinthe spermathecaof a matedbumblebeequeen(Liuetal.1974).Theexacttrans- mission route of A. bombi has not been studied in detail exceptfor Graystock etal.(2015) demonstratinghorizon- taltransmissionviacontaminatedflowers. Inotherstudies, transmissionviatheoral-fecalrouteandingestionofcontam- inatedfoodsourcesordeadbodiesinthenestareassumed (JonesandBrown2014;Schmid-Hempel1998).Theexact mechanism by which mature oocysts re-enter the alimen- tary tract is unknown to date. Experimental studies have demonstratedanegativeeffectofA.bombiinfectiononbee longevity (Graystock et al. 2016; Jones andBrown 2014;

Tianetal.2018)andcolonyfoundationsuccessinbumble bee queens (Jones andBrown 2014; Rutrecht andBrown 2008).

Microscopic examination for the presence of navicular oocysts has been an approachable method for A. bombi detection.Morerecently,moleculartechniqueshavegained popularity as theyare morescalable andversatile (Meeus et al.2010).In metagenomicstudies, the parasite’s subset genetic material(subtranscriptome)isidentified along that ofthehostandotherinhabitants.Inapreviousmetagenomic study,wediscoveredagregarineinthebumblebeeBombus pascuorum that showed considerable DNA sequence dis- similarity (moleculardivergence)withrespect toA.bombi (Schoonvaereetal.2018).Inafollow-upstudy,weaimedat re-samplingtheparasiteformicroscopicanalysisandinves- tigateditspathology,morphologyandgeneticbackground.

Here,wereportourfindingsthattwosimilarspeciesinfect bumblebees.WeproposetoretainthenameApicystisbombi forthespeciesthatmatchesthemajorityofhistoricalreports, whereasthe nameApicystiscrypticasp.n. isproposedfor thespeciesrecentlydiscoveredbyametagenomicsurvey.

Materials and methods

SpecimensofthegenusBombuswerecollectedduringtwo independentsamplingrounds,thefirstin2015andthesec- ondin2017,inBelgium.Thefirstsamplingroundwascarried outatfivelocalitiesaspartofmetagenomicstudiesinwild bees(Schoonvaere etal.2016,2018).Allsamplingevents werecarriedoutduringthemonthsAprilandMayonrainless dayswithfavorabletemperaturehigherthan17^◦C.Sampling permissionsinthesouthernprovincesweregrantedbyRef.

Avenant3déro2014/RS/n^◦18.Followingnettrapping,bees were directlystoredon carboglass andlatertransferredto

−80^◦Cinthelab.In thefirstlocality,oneoutof fivecol- lectedB. pascuorumqueens was infectedwithA.cryptica sp.n.(samplenameA06,dateofsampling14.IV.2015;Triv- ières;decimaldegreecoordinates50.45316,4.134969).Inthe secondlocality,alsooneoutoffivecollectedB.pascuorum queenswasinfectedwithA.crypticasp.n.(A88,21.IV.2015;

Torgny;49.511117,5.482273).Inthethirdlocality,twooutof fivecollectedBombusterrestrisLinnaeus,1758queenswere infectedwithA.bombi(A50;17.IV.2015;Ghent;51.024801, 3.711731andA96;04.V.2015;Ghent;51.023306,3.709452).

Outof10sampledbumblebeespecimensineachofthetwo otherlocalities,nogregarineinfectionswerefound(Moorsel andFrancorchamps).Hostspeciesidentificationwasdoneby barcodingasdescribedbySchoonvaereetal.(2018).Thesec- ondsamplingroundwascarriedoutformicroscopypurposes.

Here,wefocusedonthespeciesB.pascuorumbecauseboth A.bombiandA.crypticasp.n.havebeenfoundinthisbumble bee species inBelgium (Schoonvaere et al. 2018). More- over,weaimedtocollectforagingB.pascuorum(females) inmid-Junetoensureahighgregarineinfectionload,being eitherinfected workersor latequeens thatfailedincolony founding.Beeswerecaptured,euthanizedandprocessedon

Table1. PCRprimersequencesusedinthisstudy.

Name Sequence(5>3) Specific? Reference

ApBF1^a CGTACTGCCCTGAATACTCCAG No Meeusetal.(2010)

ApBR1 TGAAAGCGGCGTATACATGA No Meeusetal.(2010)

ApB143r TGCCACTTTTCTTTGCAGTC No Thisstudy

ApB170f CTTCGGTATAGTTAATTGGTGATCCA Yes Thisstudy

ApB681r CACCTCTAACATTTTATTTTAGCAAGC Yes Thisstudy

ApC500f^b CAACCCCATGCAAGTATCAA No Thisstudy

ApC666r TGCCAATAAAAACAAAGCTCAA Yes Thisstudy

ApUF2^c ATCTGGTTGATCCTGCCAGT No Meeusetal.(2010)

ApUR2 TTTCTCATTCTTCAGATGATTTGG No Meeusetal.(2010)

aApB,Apicystisbombi.

bApC,Apicystiscrypticasp.n.

cApU,universalforgenusApicystis.

thesame daywithoutafreeze-thaw cycle. Thisprocedure allowedustosampleuptoeightbeesperday.Thelocation wasMoorsel(50.953475, 4.108034±100m). Eightspec- imensB01-08werecollectedandprocessedon10.VI.2017 andfivemorespecimensB09-13on15.VI.2017.Specimens usedformicroscopywereB04(healthycontrol),B05,B06, B10andB13.

DNAextraction

DNAextractions from freshmaterial were done by the chelex-proteinaseKmethodstartingfromabeehindlegas describedbySchoonvaereetal.(2018).DNAextractionfrom tissuesdissectedinfixationbuffer(4%formaldehyde,2.5%

glyceraldehyde, 0.1M cacodylate buffer) was done using amodifiedphenol-chloroform-isoamylalcohol(PCI)proto- col.Dissection timeneverexceeded30 min.Fixed tissues werewashedinphosphatebufferedsalinethreetimes.Sam- pleswerehomogenizedbybeat-beatingin0.5mlphosphate bufferedsalineandapproximately70␮lzirconiumbeadsand fivesteelbeadsusingaPrecellysinstrument.Onevolumeof PCIwasaddedtothehomogenate,vortexedandcentrifuged for5min,maxspeedatroomtemperature.Theupperaque- ousphasewasisolatedand1/10volume3Msodiumacetate pH5.2wasadded. Twovolumesice-coldabsoluteethanol wereadded,gentlymixedandsampleswereincubatedfor2 hat−20^◦C.Afterincubation,precipitatedDNAwascen- trifugedfor 30minat4 ^◦C,maxspeedandthe (invisible) pelletwaswashedwith70%ethanol.Allsupernatantethanol wasremovedandthepelletwasallowedtoair-dryfor15min.

Finally,DNAwasre-suspendedin30␮l100mMTris−HCl bufferpH8preheatedto60^◦C.

Moleculardetection

The 18S ribosomal DNA (rDNA) gene was sequenced based on the method described by Meeus et al. (2010).

Briefly,primersusedfortheoverlapamplificationwerefor- ward/reversedApUF2/ApBR1(NeoL)andApBF1/ApUR2

(NeoR).PCR reaction conditions were thesame, with the exception of a proofreading enzyme (Platinum Pfx, Life Technologies). PCR products were submitted to GATC Biotech AG (Constance, Germany) for dideoxy DNA sequencing in both directions. Sequence trace files of NeoL and NeoR products were examined and assembled using SeqTrace(Stucky 2012),the resultant sequencewas GenBankrecordMF998086.1.Sequenceintegritywascon- firmed by BLAST analysis using MF998086.1as a query against the Sequence Read Archive records SRR6148372 and SRR6148369. Sequence alignment was done using MUSCLE(default options)embedded inthe EBImultiple sequencealignmenttoolsportal.Variablesequenceregions were used as a template for specific primer design using the online primer3 tool (Untergasseret al.2012). Specific andhistoricalprimersequencesusedinApicystismolecular detectionare includedinTable1.The PCRreactionscon- tainedfinalconcentrations1mMMgCl2,0.2mMdNTPs,2

␮Mofeachprimer,1unitHotStarTaqPlusDNApolymerase (Qiagen,203605inddH2Oandcolored10xbuffer.Weused 1␮loftemplateDNAsample(seesectionDNAextraction).

ThePCRreactionconditionswere5minat95^◦C,35cycles of30sat94^◦C,30sat55^◦Cand45sat72^◦C,andafinal elongationstepof10minat72^◦C.

Bioinformatics

BLASTn analysis was used against the latest non- redundant (nt), Transcriptome Shotgun Assembly (TSA) orSequenceReadArchivedatabases.Protein-codinggenes were selected after metagenomics profiling described by Schoonvaereetal.(2018).Forsevengenesweobtainedhigh- coverageassembly.Thesewerepreferredoverlow-expressed genesbecause coveragecorrelates withassemblyaccuracy andcompleteness. Fullandabbreviated genenames: heat- shock protein 70 (hsp70), heat-shock protein 90 (hsp90), elongation factor 1 alpha (ef1a), fibrillarin (fib), methion- ineadenosyltransferase(mat),ribosomalproteinS6(rps6), tubulinebeta-chain(tubb).ConservationinthephylumApi- complexa was verified by (t)BLASTn/x analysis and we

confirmed assembly correctness by mapping the original NGSreadstothe transcripts (A.bombi: SRR6148372and SRR6148369;A.crypticasp.n.:SRR1502945).

Phylogeneticanalyses

WefollowedtherevisedclassificationbyCavalier-Smith (2014) with respect to gregarines. In this classification, gregarines of the former order Neogregarinorida were demonstrated to be multiply polyphyletic and all neogre- garine species are accommodated in the broader order Arthrogregarida. Despite of our awareness that a former classificationis still commonlyused, we favorthe revised classificationbecauseitaddsmoleculardivergenceevidence toformersystematiccriteria(speciesmorphology,lifecycle andhostoccupancy). The 18S rDNAlocuswas used as a phylogeneticmarkerbecauseitisthemostwidelyavailable DNAsequence for arthrogregarines inpublic repositories.

Arthrogregarine species of the families Ophryocystidae, Lipotrophidae,Monocystidae,SyncystidaeandGregarinidae were included in the phylogenetic analysis. Species of Cryptosporidiidae(orderCryptogregarida)wereusedasan outgroup. Fasta sequences were retrieved from GenBank and aligned by MUSCLE (default options) in MEGA X (Kumaretal.2018)usingtheMUSCLEalgorithmatdefault parameters.Theoptimalnucleotidesubstitutionmodelwas determinedasTamura3-parametermodel(Tamura1992)and theevolutionaryrelationwas inferredusing theMaximum Likelihood method.The initialtreetopology wasobtained automatically(Neighbor-JoinandBioNJalgorithms)using theMaximum CompositeLikelihood approach.Adiscrete Gamma distributionwas used (+G,4 categories)andgaps or missing data were omitted (complete deletion option).

Confidencelevelswerecalculatedbyapplyingthebootstrap methodand1000replicates.

Lightmicroscopyandoocystmeasurements

Matureoocystsweredistinguishedfromimmatureoocysts by (i) the dissociation of the gametocyst membrane (=

unpairedoocysts)and(ii)the presenceof two densepolar plugsatthe frontandrearsides.Mature oocystsmayalso bedistinguishedby themorerefractivesurface (appearing morebright)thanimmatureoocysts.The samplesusedfor mature oocyst measurements originatedfrom the −80 ^◦C stored mesosoma homogenate of samples A88, A50 and A96(see SampleCollection). Length andwidthof spores weremeasuredusinganinternalcalibratoronaLeicaDMLS instrumentat400×magnification.Photographsweretaken usingaZeissAxioCamERC5 scamera.Oocystsizesare reported as mean ± standarderror. Statistical two-sample t-testswereperformedinRv3.4.2(RCoreTeam2017).

Transmissionelectronmicroscopy

Bees were not frozen or kept alive for longer than 6 h betweencaptureanddissection.Priortodissection,thebee waseuthanizedbydecapitation.Theprosoma,mesosomaand metasomawerecutopenusingasterilepairofscissorsina 1mldropletoffixationbuffer(4%formaldehyde,2.5%glu- taraldehyde, 0.1 M cacodylate buffer). Quickly, six tissue typeswereisolatedusingsterilizedforceps:hypopharyngeal gland(HG)tissuefromtheprosoma,flightmuscle(MU)tis- suefromthemesosomaandthealimentarytract(ALI),fat body(FB),malpighiantubules(MT)andovaries(OV)from themetasoma.Thedissectiontooknolongerthan15minper beeandtheimmersionofinternalorgansinfixationbuffer wasensured duringtheentireprocedure.Individualtissues were transferredtoaseparate,sealed meshcontainer.The remaining tissuewas used for nucleicacid extraction(see DNAextraction).Sampleswereimmersedinfreshfixation buffer,placedinavacuumovenfor30minandleftrotating for3hatroomtemperatureandsubsequentlyrotatingover nightat4^◦C.Afterwashing,sampleswerepostfixedin1%

OsO4withK3Fe(CN)6in0.1Mcacodylatebuffer,pH7.2.

AfterwashinginddH2O,samplesweredehydratedthrough agradedethanolseries,including abulk stainingwith2%

uranylacetateatthe50%ethanolstepfollowedbyembed- ding inSpurr’s resin.To select the areaof intereston the blockandinordertohavealargeroverviewofthe pheno- type,semithinsectionswerefirstcutat0.5␮mandstained withtoluidinblue.Ultrathinsectionsofagoldinterference colorwerecutusing anultra-microtome(LeicaEMUC6), followedbyapost-staininginaLeicaEMAC20for40min inuranylacetateat20^◦Candfor10mininleadstainat20

◦C.SectionswerecollectedonFormvar-coatedcopperslot grids.GridswereviewedwithaJEM1400plustransmission electron microscope(JEOL,Tokyo, Japan)operatingat60 kV.

Results

MoleculardiscriminationofA.bombiandA.

crypticasp.n

PairwisecomparisonofA.bombiandA.crypticasp.n.18S rDNA gene sequencesrevealed only two variable regions with considerable sequence dissimilarity (Fig. 1a, b). All historicalPCRprimersdevelopedfor“Apicystisbombi”diag- nosisfelloutsidetheseregions.Weusedthevariableregions asatemplateforspecificprimerdesign.ForA.bombi,two primersetshadtobedevelopedbeforeaspecificassaywas obtained. The firstset(ApUF2/ApB143r)was notspecific despitea2-ntgapdifferenceinthereverseprimersequence.

Asecondprimerset(ApB170f/ApB681r)specificallyampli- fied a 512 bp sequence (Fig. 1c). For A. cryptica sp. n., primers ApC500f/ApI666r specifically amplified a167 bp

Fig.1. a–c.MoleculardiscriminationofApicystisbombiandApicystiscrypticasp.n.a)OverlapamplificationofA.crypticasp.n.18S ribosomalDNAgenesequenceusing(Meeusetal.,2010)PCRprimersApUF2/ApBR1(NeoL)andApBF1/ApUR2(NeoR)followedby assemblyintoGenBankaccessionMF998086.PCRprimersareindicatedbyarrows,eitherspeciesspecificprimers(fullline)ornon-specific primers(dashedlines).PrimersequencesareincludedinTable1.b)Multiplesequencealignmentofvariableregions1and2ofallApicystis spp.recordsthatweresequencedto date.FN546182,SAMN02870293=A.bombi;MF998086=A.crypticasp.n.;SAMN02870335= undescribedspeciesofMegachilewillughbiella.Specific(fullline)andnon-specific(dashedline)primerbindingsitesareillustratedontop ofthealignment.c)Gel-electrophoresisofspecificPCRassayresultsforA.bombi(ApB170f/ApB681r,firstlane)andA.crypticasp.n.

(ApC500f/ApC666r,thirdlane)infectedbumblebees.ApicystisbombispecificPCRgeneratesa512bpamplicon,A.crypticasp.n.specific PCRgeneratesa167bpamplicon.SecondlaneM=GeneRuler50bpDNAladder(ThermoScientific).

sequence(Fig.1c).Thespecificassayswereusedthrough- out the study to successfully differentiate A. bombi from A. cryptica sp. n. Of the 13 B. pascuorum females col- lected in the second sampling round, one tested positive for A. bombi (B06) andthree tested positive for A. cryp- tica sp. n. (B05, B10, B13). Double infections were not detected.

Divergencebasedon18S,ITS1and protein-codinggenes

Phylogeneticanalysisbasedonthe18SrDNAgenesepa- ratedA.crypticasp.n.fromA.bombidespitealow(52%) bootstrapsupport(Fig.2).Bothgenotypeswere separated fromthe genusMattesiaas asistergroupwith84% boot- strapsupport.In the phylogenetic analysis,also three18S rDNAgenesequencesofgregarineswereincludedthatwere discovered by tBLASTn against public TSA datasets: A.

bombi inBioSampleSAMN02870293 (Euceranigriscens, Apidae),Apicystissp.thatismorerelatedtoA.crypticasp.

n.thantoA.bombiinSAMN02870335(Megachilewillugh- biella,Megachilidae)andafar-branchingGregarinasp.most relatedtoG.diabroticainSAMN02870316 (Hylaeus var- iegatus,Colletidae).GenBankandTSAaccessionnumbers aresummarizedinSupplementalinformation1.Divergence

between A.bombi andA. crypticasp. n. wasmore elabo- rate in the ribosomal ITS1 sequence. The ITS1 part was highly dissimilar and caused a 370 bp sequence of the 18S- ITS1-5.8S to misalign by default BLAST parame- ters.Alignmentwassuccessfulafter applyingminimalgap costs (Existence: 2, Extension: 2). Over the entire query length,A.bombiandA.crypticasp.n.had69%nucleotide identity. Phylogenetic analysis based on thisregion of all available GenBank and TSA records resulted in a clear bifurcationbetweenthespecies(SupplementalFig.1).The TSArecordsofA.crypticasp.n.GHFQ01001441.1(sample A06) and GHFY01001509.1(sample A88)clustered with six GenBank records isolated from bumblebees inWest- Siberia (KY126621, KY126622, KY126625, KY126628, KY126631 andKY126632). Ofthe 120 GenBank records referred to as A. bombi to date, 107 truly belong to A.

bombi,12belongtoA.crypticasp.n.and1Mexicanrecord isambiguous. UnambiguousA.bombi identificationsorig- inate from Eurasia (Ireland, Japan and West-Siberia) and South-America (Argentina, Brazil, ColombiaandMexico) whereasunambiguousA.crypticasp.n. identificationsare currentlylimitedtoBelgiumandWest-Siberia(Supplemental information1).Nexttoribosomalloci,sevenprotein-coding geneswithrelativehighexpressionwerecompared(Table2).

Sequence recordsoriginate fromthe parasite subtranscrip-

Fig.2. PhylogeneticanalysisofApicystisbombiandApicystiscrypticasp.n.basedon18SribosomalDNAgene.Thetreetopologywiththe highestloglikelihood(−5443.83)isshown.Atotalof921positionswereincludedinthefinaldataset.Bootstrapsupportvalues(n=1000)are indicatedinpercentages.Thescalebardenotesthenumberofsubstitutionspersite.ApicystisbombiisclusteredtogetherwithA.crypticasp.

n.(52support)andbothareseparatedfromMattesiaspp.(84support).BioSamplerecordsofbeespeciesofwhichthepublicTranscriptome ShotgunAssembly(TSA)datasetcontainsgregarinesequencesareindicatedbyanasterisk.ThegregarineofEuceranigriscensismostlikely A.bombi,thegregarineofMegachilewillughbiellaismostrelatedtoA.crypticasp.n.andthegregarineofHylaeusvariegatusismostrelated toGregarinacoronata,aseptategregarinedescribedfrombeetles.

Table2. BLASTidentitybetweenApicystisspp.ofribosomalandprotein-codinggenes.

Gene A.bombi A.crypticasp.n. Nucleotide% Protein%

18SrDNA FN546182.1 MF998086.1 95,36 –

ITS1^a KY126621.1 KY126623.1 69,57 –

hsp70 GBPG01002195.1 GHFY01000033.1 78,81 94,63

hsp90 GBPG01002200.1 GHFY01000029.1 77,43 86,78

ef1a GBPG01026098.1 GHFY01011377.1 82,68 96,5

fib GBPG01025281.1 GHFY01015325.1 78,28 92,95

mat GBPG01025316.1 GHFY01014334.1 74,76 83,16

rps6 GBPG01023297.1 GHFY01007309.1 74,37 81,25

tubb GBPG01000440.1 GHFY01003556.1 80,05 96,3

aITS1sequencesareonlyalignablewhengapcostsaresignificantlyreduced(Existence:2,Extension:2).ITS1sequencesinpublicrepositoryrecordsare typicallyflankedbyshortregionsof18Sand5.8Ssequence.

tomes of bee BioSamples SAMN02870293 (cfr. Apicystis bombi)andSAMN07691958(cfr.Apicystiscrypticasp.n.).

Thepairwisenucleotide identitybetweenA.bombi andA.

cryptica sp. n. transcripts ranged from 74.4% for rps6 to 82.7%for ef1a (mean 78.0%). The pairwiseprotein iden- tityoftranslatedopenreadingframesrangedfrom81.25%

forrps6to96.5%foref1a(mean90.2%).

Infectionofthefatbodyandhypopharyngeal gland

The B. pascuorum female with a high A. cryptica sp.

n.infectionload(B05)wasexaminedbylightmicroscopy.

Comparedtothetypicalyellowishfatglobulesinahealthy individual B04, infected fat globules appeared swollen

Fig.3. a–f.LightmicroscopyofhealthyandApicystiscrypticasp.n.infectedfatbodyandhypopharyngealglandcellsinaBombuspascuorum female.a)Evisceratedmetasomaofahealthybumblebee.Sterniteshavebeenremovedtovisualizetheinteriorsideofthetergites.Parietal fatbodyislocatedasauniformlayerdirectlybelowtheexoskeleton.Healthyfatbodyiscolouredyellow.b)400×magnificationofahealthy fatbodyglobulecomposedofthetwoadipocytecelltypes:big-nucleatedoenocytesandvacuolatedtrophocytes.c)Evisceratedmetasomaof adiseasedbumblebee.Theparietalfatbodyisheavilyinfectedandappearsswollenanddullywhiteinsteadofyellow.Notethebeeheart isnolongervisiblebetweentheparietalfatbodylayer.d)400×magnificationofhypertrophiedfatbodycells.Navicularoocystsaretightly packedinsideacell-likesphere.Oocystsarearrayedside-by-sidenearthecellperiphery.Infectedadipocytesaresignificantlyenlarged.e–f) Oocyst-filledacinicellsofthehypopharyngealglandofthesameinfectedbumblebeeasincandd.Infectedacini(arrows)appearedrandomly inbetweentheclusterofhealthyacini.ac,acini;adi,adipocyte;ali,alimentarytract;c,crop;fb,fatbody;h,heart;oe,oenocyte;t,trachea;

tr,trophocyte.Scalebars:a,c=1mm;e=100␮m;b,d,f=25␮m.(Forinterpretationofthereferencestocolourinthisfigurelegend,the readerisreferredtothewebversionofthisarticle).

andwhite (Fig.3a–c). Individualinfectedadipocyteswere densely occupied by mature oocysts (Fig. 3d). The con- toursoftheadipocyteswereonlydistinguishablebymeans of the peripheral row of arrayed oocysts. All adipocytes wereoccupiedbyoocysts,indicatingthatbothoenocyteand trophocytecelltypeswereinfected.Asimilarhypertrophism wasobservedinasmallnumberofHGcells(Fig.3e):infected HG cells were also densely occupied by mature oocysts.

However,the majorityof HGcells appearedhealthy, sug-

gestingthatthistissueisnottheprimaryinfectiontargetof A.crypticasp.n.Nooocystsorotherdevelopmentalstages wereobservedinAL,MT,MUandOVtissues.

Theultrastructureofoocystsarecomparable betweenspecies

Six tissuetypesof bumblebeesinfected withA. bombi (B06) andA.crypticasp. n. (B10)wereexamined for the

Fig.4. TransmissionelectronmicroscopyofApicystisbombiandApicystiscrypticasp.n.oocysts.Apicystisbombiinleft-sidemicrographs andA.crypticasp.n.inright-sidemicrographsfromfatbodytissueofBombuspascuorum(respectivelysamplesB06andB10).a)Longitudinal sectionofanavicularoocystofA.bombi.Otheroocystsarevisibleadjacenttoahealthyadipocytecells.Theoocystshaveanelectron-dense appearance.b)Longitudinalsectionof thenavicularoocystofA.crypticasp.n.Oocystswere notobservedin pairsandappearedless electrondensecomparedtomaturingA.bombioocysts.Nexttohealthyadipocytes,thefatbodytissuealsocontainedsmoothmusclecells.c) Transversesectionoftwooocystsandtworesidualbodiesassociatedinagametocyst.Thegametocystmembraneisreadilyvisible(asterisks).

Eachoocystcontainsfoursporozoitesthatareenclosedbyanelectron-lucentoocystwallofvariablethickness.d)Longitudinalsectionofa matureA.crypticasp.n.oocystscontainingfoursporozoitesarrangedside-by-side.Theupperandlowersporozoitesarecompleteandthe electron-densenucleiaredistinctlystained.Thetwomiddlesporozoitesarebendoutofthevisualplaneandappearintertwined.Theoval polarplugsattheoocystpolesstaineddark.e)Close-upmicrographofanA.bombioocystpolewithdetailedbilayeredcompositionofthe oocystwall.Theinnerbilayerconsistsofathinexternalmembrane(whitearrow)andabroadinternalmembrane(whitearrowhead)directly enclosingthesporozoites.Theouterbilayerhasasimilarstructure,consistingofaverythinexternalmembrane(blackarrows)andabroad internalmembrane(blackarrowheads).Thebroadinternalmembraneoftheouterlayerisaveragely70nmthickandbecomesthickernearthe oocystpoles.Theinnerandouterbilayersareseparatedbyanelectron-lucentspace.f)Close-upofA.crypticasp.n.oocystpole,showingthe thickeningoftheoocystwallbetweentheinternal(blackarrowhead)andexternal(blackarrow)membranesoftheouterbilayer.Theexternal membranestainsverydark.adi,adipocytecells;gc,gametocyst;n,nucleus;oc,oocyst;ow,oocystwall;pp,polarplug;rb,residualbody;sm, smoothmusclecells;sp,sporozoite.Scalebars:a,b=5␮m;c,d=2␮m;e,f=500nm.

presenceofoocystsorotherdevelopmentalstagesbyTEM.

Oocystsof both specieswere navicular inshape andcon- tainedfoursporozoites(Fig.4a,b).Oocystswereobserved inFBtissuebutwereabsentfromHG,AL,MT,MUandOV

tissues.OocystsofA.bombiwereassociatedinpairs(Fig.4c) butthiswasnotobservedforA.crypticasp.n.Oocystsofthe latterappeared lesselectron-dense withthickerwalls indi- catingamoreadvancedstageofmaturation.Thestructureof

theoocystwallwascomparablebetweenthespeciesdespite thedifferenceinmaturation(Fig.4e,f).ForA.bombi,also non-synchronoustransitionofsporoblastsintoyoungoocysts insidethegametocystwereobserved(SupplementalFig.2).

Nofreetrophozoitesweredetectedandnomerogonialstages werefoundinsidefatbodycellsorintheothertissues.

MatureoocystsofA.crypticasp.n.inB.

pascuorumarelongerthanoocystsofA.bombiin B.terrestris

Matureoocystswidthandlengthweremeasuredinfresh and TEM-fixed preparations. Oocyst sizes of historical reportsandthisstudyaresummarizedinTable3.FreshA.

bombimatureoocystsmeasured14.8±0.8␮m×4.5±0.2

␮m(A96andA50,n=47),thesmallestoocystwas13.2× 4.1␮mandthelargestoocystwas16.4×4.8␮m.FreshA.

crypticasp.n.matureoocystsmeasured17.4±0.8␮m×5.3

±0.2␮m(A06andA88,n=51),min.15.9×4.9␮mand max.18.8×6.2␮m.Oocystslongerthan20␮mwerenot encountered.Thefreshoocystsizeswere significantlydif- ferentbetweenspecies(WelchTwoSamplet-test,p<0.05, n=98),althoughitshouldbenotedthatsamplesoriginated fromdifferentbumblebeehostspecies.ForbothA.bombi andA.crypticasp.n.,asmallnumberofangledoocystswere observed.FixedA.bombiimmatureoocystsmeasured13.7

±0.8␮mby2.8±0.2␮m(B06,n=8),min.12.4×2.7

␮mandmax.14.9×2.9␮m.FixedA.crypticasp.n.mature oocystsmeasured17.0±0.9␮m×3.7±0.5␮m(B10,n= 3).

Discussion

Inthisstudy,anewgregarinespeciesisdescribedbasedon morphologicalandgeneticdata.Wedemonstratedthat this parasiteisable toinfect thefatbodycellsof bumblebees andproducesnavicularoocystscontainingfoursporozoites.

Together,thesearethetaxonomicdescriptivefeaturesofthe genusApicystiswhichwasmonotypicuntilnow.Compari- sonofribosomalandprotein-codingDNAsequencesofthe newgregarinetotherespectiveDNAsequencesofApicystis bombirevealedthatthereisconsiderabledivergencebetween thetwo(upto31%nucleotidedissimilarity).Despiteofthis notablemoleculardivergence,oocystmorphologyishighly comparablebetween the species. Forthe newspecies, we proposethenameApicystiscrypticasp.n.Thedelineations ofA.bombiandA.crypticasp.n.isinlinewithrecentdis- coveriesofcrypticbeeparasiteincludingCrithidiamellificae andLotmariapassim(Schwarzetal.2015),C.bombiandC.

expoeki(Schmid-HempelandTognazzo2010),andNosema apisandN.neumanii(Chemurotetal.2017).Allof these delineationdescriptions reliedonmoleculardivergenceby

comparingsequencediversityacrossmultipleloci. Table3.ComparisonofApicystisspp.oocystsizesacrossdifferentstudies. abcStudySpeciesHostspeciesLocationOocystsize(␮m)L:WTissue Liuetal.(1974)Ap?Bombusspp.Canada,N-America16.2×5.4(young)3.00Fatbody,midgut, 21.6−27×5.4(mature)4.50hindgut,spermatheca LipaandTriggiani(1996)Ap?Bombusspp.,ApismelliferaItaly,Europe16.2–21.6×5.63.38Fatbody PlischukandLange(2009)Ap?B.terrestrisArgentina,S-America12.94±0.08×3.36±0.043.85Fatbody Plischuketal.(2011)ApBA.melliferaArgentina,S-America11.31±0.07×3.21±0.033.52Fatbody Plischuketal.(2011)ApBB.terrestrisArgentina,S-America12.94±0.08×3.36±0.043.85Fatbody ThisstudyApBB.terrestrisBelgium,Europe14.8±0.8×4.5±0.23.30Fatbody 13.2−16.4×4.1−6.2 ThisstudyApCB.pascuorumBelgium,Europe17.4±0.8×5.3±0.23.29Fatbody, 15.9−18.8×4.9−6.2hypopharyngealgland aAp?,Apicystissp.;ApB,Apicystisbombi;ApC,Apicystiscrypticasp.n. bMeasurementvaluesareexpressedasmean±SEMorminimum-maximum,allvaluesin␮m.Unlessindicated,sizesareoffresh(unfixed),matureoocysts. cLength:Widthratiowasobtainedbydividingthemeanlengthbythemeanwidth.

The pathology of A. bombi andA. cryptica sp. n. pre- dominantly takesplace in the fatbody tissue of the host.

However,weobservedasmallnumberofacinicellsofthe prosomalHGtissueinfected withA.cryptica sp.n.which isanuncommontissuetargetforgregarines.Speciesinthe relatedgeneraMattesia andOphryocystismainlyreplicate intheFB andMT,respectively(YamanandRadek2017).

OocystsofonedescribedMattesiasp.accumulateinthepro- somaofadultants,butitisunclearifHGcellsaretargeted (Pereira et al. 2002). The few infected HG cells that we observedwerelikelyassociatedwithanadvancedstageof infection.MostoftheFBtissuewasalreadyhypertrophied byA.crypticasp.n.hencetheHGmayserveasasecondary targettissue.Anotherexplanationforthisobservationmay involve the infection of HG cells as part of the parasite’s transmissionstrategy.TheHGisanexcretoryorganthat is involvedinthefeedingofbroodbyyounghoneybeework- ers(KnechtandKaatz1990).Hence,oocyststhatreachthe HGductthroughtheinfectionofacinicellsarepassedonvia glandularsecretionstotheprogenyinthenest,atransmis- sionstrategyalsoreportedforapicornavirus(Bailey1969).

Themaintransmissionrouteofgregarinesisconsideredby defecationandingestionofcontaminatedfoodsources(oral- fecaltransmissionroute)followedbysporozoitepenetration ofthe intestinalepithelium.However,gregarineshavealso been reported in the reproductive system of insects (Dias etal.2017)suggestingthatsomespecieshavethepotential tobetransmittedduringcopulation(venerealtransmission).

Next to hypertrophies in the fat tissue, Liu et al. (1974) reportedmature A.bombi oocystsinspermathecaofbum- blebeequeens.WhetherApicystisspp.oocystsareeffectively passedonbyglandularsecretionsorbyvenerealtransmission awaitsexperimentalconfirmation.

A comprehensive investigation of developmental stages andlifecyclemorphologicaldifferencesbetweenA.bombi and A. cryptica sp. n. fell out of the scope of thisstudy.

However,wefoundgametocystsofA.bombiandconfirmed theobservationsdone byLiuetal.(1974)thatsporoblasts developinpairsinsidethegametocystmembrane.Thegame- tocyststagewasnotfoundinA.crypticasp.n.TEManalysis likelybecausetheinfectionwastoolow.Thefatbodytissue isalarge,scatteredtissueandwemighthavemissedthestart pointof infectionwhichisconsideredtotakeplacelocally inasmallsubsetoftissue.Inordertoobservepotentiallife cycle differences between the species,standardized single infectionsasdonebyGraystocketal.(2016)arenecessary tocomparethedevelopmentalstagesoftheparasites.

Aquickandreliablemorphologicalmarkerwouldbenefit pathogenmonitoring studiesthat intendtodiscriminate A.

bombiandA.crypticasp.n.bylightmicroscopy.However, thenavicularshapeof A.bombi oocystswasacharacteris- ticfeaturethatisnowambiguous.Apotentialmorphological markercouldbeoocystsize,althoughevidencesofarindi- catesthatoocystssizesarehighlyvariableandinfluencedby maturationstage,geographicallocationandhostspecies.Pre- viousstudiesreportedasizevariationofupto60%between

youngandmatureoocysts(LipaandTriggiani1996;Liuetal.

1974; Plischuk andLange 2009, summarized inTable 3).

Also, Plischuk et al. (2011) reported validated A. bombi oocyst sizes in B. terrestris that were 13% smaller than A. bombi oocyst sizes inB. terrestris reportedhere. They alsoobservedsizedifferencesinfunctionofthehostspecies withA.bombi oocystsbeingsmallerinhoneybeesthanin bumble bees. In the presentstudy, A. bombi oocystswere obtained fromB.terrestris individualswhereasA.cryptica sp.n.oocystswereobtainedfromB.pascuorumindividuals.

Although ourmeasurementssuggestthatA.crypticasp.n.

oocystsareslightlylongerthanA.bombi,thesizedifference may be attributedtoone or moreof the above mentioned factors. Intraspecific size variability of Apicystis spp. thus appearscommonandmayimpedetheuseofoocystsizeasa morphologicalmarker.

ThespecificPCRassaysthatweredesignedinthisstudy arecurrentlytheonlyscalablediagnostictoolforA.bombi andA.crypticasp.n.However,thespecificityofapathogen detectionassayonlyreachesasfarasthehithertoknowledge ofunderlyingspeciesdiversity.IfmoreApicystisspeciesare discoveredinthefuture(Fig.2,atleastonemoreApicystissp.

isdetectedintranscriptomedataofMegachilewillughbiella inGermany(Petersetal.2017,unpublisheddata)),itislikely that abandoningthe18SrDNAlocusandreplaceitbyone ormorelessconservedprotein-codinggenes,forexamplein GisderandGenersch(2013),willbeinevitable.Atemporarily solutionmayrelyinadoptingmoresensitivemolecularmeth- odssuchashighresolutionmelting(Higueraetal.2013)or fragmentlengthpolymorphism (Ravoetetal.2015).Alter- natively, an efficient though costly method remains PCR amplificationandSangersequencingoftheribosomalITS1 region.TheITS1sequencebearsahighinterspecificdiver- genceandprovidesadditionalinformationaboutintraspecific haplotypestructuring(Maharramovetal.2013).

Taxonomic summary

SuperphylumAlveolataCavalier-Smith,2010 InfraphylumApicomplexaLevine,1970 ClassGregarinomorpheaGrassé,1953 OrderArthrogregaridaCavalier-Smith,2014 SuperfamilyActinocephaloideaLéger,1892 FamilyLipotrophidaeGrassé,1953

GenusApicystisLipaandTriggiani,1996 SpeciesApicystiscrypticasp.n.

Diagnosis. Molecular detection by PCR and specific primer pairs: forward ApC500f 5- CAACCCCATGCAAGTATCAA and reversed ApC666r 5-TGCCAATAAAAACAAAGCTCAA. GenBank acces- sions: 18S rDNA gene MF998086.1 and ITS1 sequence

KY126621. Oocysts navicular in shape and contain four sporozoites. Mature oocysts17.4 ±0.8 ␮m ×5.3 ± 0.2

␮minfreshextracts(minimalsize15.9␮m×4.9␮mand maximalsize18.8␮m×6.2␮m).Angledoocystspresent butinfrequent.

Etymology. The species-group name cryptica is derived fromtheEnglishnouncrypticmeaningsecret,concealed.It referstothe morphologicalsimilarity toA.bombi andthe need for molecular techniques in order to differentiate it fromthatspecies.

Hosts.ThehostoftheA.crypticasp.n.holotypeisafemale worker bee of Bombus pascuorum. The parasite was first detected ina metagenomic survey in wild-caught B. pas- cuorumqueensinBelgium2015(Schoonvaereetal.2018).

OtherhostspecieswhereA.crypticasp.n.hasbeendetected areB.sicheliiandB.veteranusfromWest-Siberia(GenBank recordsKY126625.1andKY126621-2.1,respectively).

Infection Site. The primary infection site is the fatbody tissuewithoutdiscriminationbetweenoenocyteandtropho- cytecelltypes.Apicystiscrypticasp.n.canalsoinfectacini cellsofthehypopharyngealglandtissue.

Type locality. The localityof the A.cryptica sp. n. holo- type is Moorsel, East-Flanders, Belgium (50^◦5712.5N 4^◦0628.9E). The host bee was foraging on flowers of raspberryRubusfruticosusuponcapture.

Type material. Embedded Spurr resin of the holotype specimenofA.crypticasp.n.infatbodytissue(accession numberNeoFBs11) were deposited inthe L-MEB collec- tion,Ghent,Belgium.Nexttothetypematerial,embedded Apicystisbombiinfectedfatbodytissue(accessionnumbers ApiBFBs2andApiBFBs6)arealsoavailableinthiscollec- tion.

ZooBankregistration.Thenewspecieswasregisteredby LSID: urn:lsid:zoobank.org:act:92BE7D15-9F2A-4E38- 936A-54DAFD527074.

Conclusion

A cryptic parasite species of bees is described based onmorphologicalandmoleculardivergenceevidence.The nameApicystiscrypticasp.n.isproposedandderivesfrom itsambiguousoocystmorphologywithrespecttoApicystis bombi.Despiteofsimilarmorphologybetweenbothspecies, theirmoleculardivergenceispronounced.Phylogenybased onthe18SrDNAgeneconfirmedtheevolutionaryrelation- shipof bothspecies.Interspecific nucleotideidentityis as lowas69% fortheITS1ribosomalsequenceandonaver- age78%forsevenprotein-codinggenes.Wearguethatthe identificationofA.bombiwassystematicallyoverestimated inpreviousstudiesforthreereasons.First,A.bombidetec- tionheavilyreliedonlightmicroscopyandthepresenceof navicular oocysts in crude bee extracts or fatbody tissue

smears. Itturnsoutthat A.crypticasp.n.oocystshavean identical navicularshape,makingit impossibletodiscrim- inatetheonefromthe otherbasedonthisfeature.Second, thePCRprimersusedinmorerecentlydevelopedmolecular detectionassayswerenon-specificastheyboundtoacon- servedregionofthe18SrDNAgene.Hence,A.crypticasp.

n. was equally pickedup bythese assays implicatingthat allmolecularidentificationsofA.bombiwereoverestimated.

Third,preliminaryinsightssuggestthatthedistributionand hostoccupationofA.bombiandA.crypticasp.n.overlap.To date,bothparasiteshavebeenfoundinBombuspascuorum inBelgiumandWest-Siberia.Therefore,implementationof thespecificPCRassaysdevelopedinthisstudywillgivea moreaccurateviewontheprevalence,distributionandhost specificityofA.bombiandA.crypticasp.n.

Author contributions

DDG,MBandKSconceivedthestudy.MBandKScar- ried out dissectionsof bumblebeetissues.KS carried out sampling,lightmicroscopyandmolecularworkthroughout thestudy.FB,MDBandRDRcarriedoutpreparationsand imaging of the transmissionelectronmicroscopy analysis;

KS, RDRand DDG interpreted the results.KS and DDG wrotethemanuscript.Allauthorsrevisedthemanuscriptand approvedthefinalversionofit.

Funding

This study was funded by the Belgian Science Policy (BELSPO;BR/132/A1/BELBEES;Multidisciplinaryassess- ment of BELgian wild BEE decline to adapt mitigation managementpolicy).

CRediT authorship contribution statement

KarelSchoonvaere:Investigation,Visualization,Writing - original draft, Writing - review & editing. Mar- leenBrunain:Conceptualization,Methodology,Resources.

Femke Baeke: Investigation, Resources. Michiel De Bruyne:Investigation,Resources.RietDeRycke:Writing- originaldraft,Validation,Resources,Methodology.DirkC.

deGraaf:Supervision,Conceptualization,Validation.

Acknowledgements

WewishtothanktheGhentUniversityExpertiseCentre forTEM/VIBBioImagingCoreemployeesfortheircontin- uoussupportduringtheworkandtheirvaluableinsightsin ultrastructuremorphology.Wealsothankthetwoanonymous

expertreviewersfortheirhelpfulandconstructivecomments onthemanuscript.

Appendix A. Supplementary data

Supplementary material related to this article can be found,inthe onlineversion,atdoi:https://doi.org/10.1016/

j.ejop.2020.125688.

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