Specificity of Association between Paenibacillus spp. and the Entomopathogenic Nematodes, Heterorhabditis spp.

Speci ici y o Associa ion be ween Paenibacillus spp. and he En omopa hogenic Nema odes, He e o habdi is spp. M.R. En igh and C.T. G i in Depa men o Biology and Ins i u e o Bioenginee ing and Ag oecology, Na ional Uni e si y o I eland, Maynoo h, Coun y Kilda e, I eland Recei ed: 19 Augus 2003 / Accep ed: 20 No embe 2003 / Online publica ion: 23 Sep embe 2004 Abs ac Endospo e- o ming bac e ia, Paenibacillus spp., ha e ecen ly been isola ed in associa ion wi h insec pa h- ogenic nema odes He e o habdi is spp. Spo angia adhe e o nema ode in ec i e ju eniles (J3) and a e ca ied wi h hem in o insec s. Paenibacillus p oli e a es in he killed insec along wi h He e o habdi is and i s obliga e bac e ial symbion , Pho o habdus, despi e he an ibio ic p oduc ion o he la e . Nema ode in ec i e ju eniles lea e he insec cada e wi h Paenibacillus spo angia a ached. The speci ici y o he ela ionship be ween Paenibacillus and He e o habdi is was in es iga ed. Spo angia o nema ode-associa ed Paenibacillus adhe ed o in ec i e ju eniles (bu no o he s ages) o all He - e o habdi is species es ed, and o in ec i e ju eniles o e eb a e pa asi ic S ongylida species, bu no o a a ie y o o he soil nema odes es ed. Paenibacillus species ha we e no isola ed om nema odes, bu we e phylogene ically close o he nema ode-associa ed s ains, did no adhe e o He e o habdi is, and hey we e also sensi i e o Pho o habdus an ibio ics in i o, whe eas he nema ode-associa ed s ains we e no . Unusual longe i y o he spo angium and esis ance o Pho o habdus an ibio ics may ep esen speci ic adap- a ions o he nema ode-associa ed Paenibacillus s ains o allow hem o coexis wi h and be anspo ed by He e o habdi is. Adap a ion o speci ic He e o habdi is- Pho o habdus s ains is e iden among he h ee nem- a ode-associa ed Paenibacillus s ains (each om a di - e en nema ode s ain). Paenibacillus NEM1a and NEM3 each de eloped bes in cada e s wi h he nem- a ode om which i was isola ed and no a all wi h he nema ode associa e o he o he s ain. Di e ences be- ween nema ode-associa ed Paenibacillus s ains in c oss-compa ibili y wi h he a ious He e o habdi is s ains in cada e s could no be explained by di e - en ial sensi i i y o an ibio ics p oduced by he nema- odes’ Pho o habdus symbion s in i o. In oduc ion En omopa hogenic nema odes o he gene a He e o - habdi is and S eine nema a e le hal pa hogens o insec s and a e used as biological con ol agen s o se e al insec pes s [22]. In ec i e ju eniles (IJs) o hese nema odes a e specialized hi d-s age ju eniles ha ca y in hei in es- ines symbio ic bac e ia belonging o he gene a Pho o - habdus and Xeno habdus, espec i ely [12]. Each nema ode species is no mally associa ed wi h a single species o bac e ial symbion , hough a symbion species may be associa ed wi h mo e han one nema ode species [7]. Following en y in o a sui able insec , he IJ eleases he symbion in o he hemocoel, whe e i causes dea h o he hos insec wi hin 48 h [29]. The bac e ia diges he insec issues [13], and he nema odes eed on he bac- e ia and deg aded insec issue. Pho o habdus and Xe- no habdus p oduce a a ie y o an ibio ics [13] which ha e a b oad spec um o ac i i y [3] and p e en pu e ac ion o he insec cada e [17]. Nema ode ep oduc ion is op imal when hei symbion domina es he mic obio a o he insec cada e [12]. Two o h ee gene a ions o nema odes occu wi hin he hos , depending on a ailable esou ces. As he nu i i e s a us o he cada e declines, he nema odes a es a he IJ s age. Hund eds o housands o such IJs may be p o- duced in a single la ge insec . The non eeding IJs mig a e om he cada e in o soil and sea ch o new hos s [29]. He e o habdi is spp. belong o he o de Rhabdi ida, and hei IJ s age is unc ionally and de elopmen ally analo- gous o he daue ju enile (dispe si e and su i al s age) o some ee-li ing membe s o he o de , including Caeno habdi is elegans [5]. Co espondence o: C.T. G i in; E-mail: [email p o ec ed] 414 DOI: 10.1007/s00248-003-0166-0 dVolume 48, 414–423 (2004) dSp inge Science+Business Media, Inc. 2004 Monoxenici y is a no able ea u e o he He e o - habdi is-Pho o habdus and S eine nema–Xeno habdus complexes [7, 8]. The symbion is selec i ely e ained wi hin he in es ine o he nema ode in ec i e ju enile by an as ye unknown mechanism and, once eleased in o he insec hemocoel, an imic obial compounds p oduced by he symbion minimize he de elopmen o o he bac e ial species in he insec cada e . How- e e , he e ha e been a numbe o epo s o bac e ia o he han he symbion being isola ed om en omo- pa hogenic nema odes, pa icula ly om S eine nema species, and hese a e e iewed in [8]. Some o hese con aminan s a e p obably an a i ac o p olonged labo a o y cul u e in insec s, whe e an ibio ic- esis an con aminan s a e e ained. The na u al occu ence o many con aminan s in S eine nema scap e isci is seen as an excep ion, which diminishes he nema ode’s po en- ial o killing insec s [8]. En omopa hogenic nema ode IJs e ain he mol ed cu icle o he p e ious ju enile s age as a p o ec i e shea h [20]; in S. scap e isci he con aminan s a e ca ied in he in e cu icula space [8]. Fewe nonsymbion bac e ia ha e been epo ed om He e o habdi is.P o idencia e ge i was isola ed om se e al s ains ollowing p olonged labo a o y cul u e [18], whe eas Och obac um spp. we e ound na u ally associa ed wi h one hi d o newly isola ed H. indica om he Ca ibbean [4]. In bo h o hese s udies he nema odes we e su ace s e ilized, bu no deshea hed, p io o isola ion o he bac e ia [4, 18]; hence, he bac e ia we e ca ied ei he in he in es ine o be ween he shea h and he li ing cu icle. We ha e ound a no el endospo e- o ming bac e- ial species, Paenibacillus nema ophilus, na u ally asso- cia ed wi h a s ain o He e o habdi is megidis [10]. The associa ion be ween he bac e ium and his nema ode s ain has been main ained h ough ou ine labo a o y cul u e since hei isola ion om soil om Es onia in 1994 [14]. Spindle-shaped spo angia adhe e o he nema ode IJs and can be ca ied by hem in o an in- sec . P. nema ophilus ep oduces wi hin he dead insec , despi e Pho o habdus-p oduced an ibio ics. Sho ly be- o e IJs eme ge om he hos , spo angia accumula e in he insec cada e . When he IJs eme ge, hey ca y spo angia adhe ing o he cu icle. Simila bac e ia we e na u ally associa ed wi h an uniden i ied He e o habdi is sp. om Geo gia, USA [25], and wi h He e o habdi is indica om sou he n India. Endospo e- o ming bac e- ial isola es om all h ee nema ode sou ces we e iden i ied as Paenibacillus and ound o sha e a highe deg ee o 16S RNA gene sequence iden i y wi h each o he han wi h any o he named species wi hin he genus [10]. The nema ode-associa ed s ains also sha e a high deg ee o pheno ypic and chemo axonomic simila i y and a e easily dis inguished om ela ed species, hough DNA–DNA simila i y s udies e ealed ha hey ep esen a leas wo, and possibily h ee, sepa a e species [10]. Paenibacillus macqua iensis, Pae- nibacillus azo educens, and Paenibacillus bo ealis a e among he species wi h highes sequence simila i y o he nema ode-associa ed s ains. The Paenibacillus–He e o habdi is associa ions a e no able o he endency o he nema ode IJs o clump in wa e (nema odes s ick oge he in la ge c oss-linked g oups). Such clumping in wa e also occu s in o he associa ions whe e bac e ia adhe e o a nema ode (such as compa ible Ra hayibac e –Anguina combina ions), and he deg ee o clumping can be used as a semiquan i a i e measu e o he speci ici y o he adhesion [32]. Paeni- bacillus-induced clumping can be dis inguished om he ose e agg ega ions, in which s o ed He e o habdi is IJs adhe e by hei ails [31], by he adhesion o IJs a mul iple and di e se poin s along hei bodies, and by he onse o clumping immedia ely a e eme gence om a hos . As pa o a b oade s udy in o he associa ion, his wo k examines he speci ici y o he associa ion be- ween He e o habdi is and Paenibacillus a wo le els. A he highe axonomic le el, we ask on he one hand whe he nema ode-associa ed Paenibacillus s ains ad- he e o nema odes o he han He e o habdi is, and on he o he hand whe he o he species o Paenibacillus adhe e o He e o habdi is nema odes. We also compa e he sensi i i y o nema ode and non-nema ode-associ- a ed Paenibacillus species o Pho o habdus an ibio ics in an in i o assay. A a lowe axonomic le el we add ess he speci ici y o associa ion o he a ious nema ode- associa ed Paenibacillus s ains wi h he He e o habdi is s ain om which hey we e isola ed. C oss-compa i- bili y is assessed bo h in e ms o adhesion o nema ode IJs and mul iplica ion in he insec cada e . In an a - emp o explain di e en ial compa ibili y in cada e s, he sensi i i y o nema ode-associa ed Paenibacillus s ains o a ious Pho o habdus spp. is in es iga ed in i o. Me hods Nema odes and Bac e ia. The nema ode species and s ages used a e lis ed in Tables 1 and 2. He e o habdi is and S eine nema spp. we e cul u ed in i o in la e ins a la ae o he wax mo h, Galle ia mellonella, a ei he 20C o 26C depending on he s ain. In ec i e ju eniles we e ha es ed in wa e om he insec cada e s on Whi e aps [21]. The IJs o H. megidis EU17, H. indica LN2, and He e o habdi is Line 1 we e ea ed wi h 0.5% so- dium hypochlo i e o 15 min ( ollowed by washing wi h s e ile dis illed wa e ) o emo e Paenibacillus a ached o he su ace o he IJs. The ea ed IJs we e used o in ec G. mellonella la ae, hus es ablishing uncon amina ed nema ode cul u es. M.R. ENRIGHT, C.T. GRIFFIN:ASSOCIATION BETWEEN PAENIBACILLUS AND HETERORHABDITIS 415 Paenibacillus nema ophilus NEM1a, Paenibacillus sp. NEM2, and Paenibacillus sp. NEM3, isola ed om he nema odes H. megidis EU17, He e o habdi is sp. Line 1, and H. indica LN2, espec i ely [10], we e ou inely main ained on pla es o nu ien aga (Ox- oid) a 30C. Adhesion o Spo angia o Nema odes Adhesion o Paenibacillus nema ophilus NEM1a Spo angia o Va ious Species and S ages o Nema odes. Adhesion o P. nema ophilus NEM1a spo angia o nema odes was de- ec ed bo h by hei abili y o cause clumping o nema- odes in wa e and by mic oscopic examina ion. A Table 1. Adhesion o spo angia o Paenibacillus nema ophilus NEM1a o a a ie y o soil-dwelling nema ode species and s ages, including in ec i e ju eniles (IJs) Nema ode o de and species Nema ode s age es ed Li e his o y Adhesion Rhabdi ida He e o habdi is megidis Enshea hed IJ Insec pa hogen + Exshea hed IJ + Adul s age ) H. downesi Enshea hed IJ Insec pa hogen + H. bac e iopho a Enshea hed IJ Insec pa hogen + H. indica Enshea hed IJ Insec pa hogen + H. a gen iniensis 1 Enshea hed IJ Insec pa hogen + He e o habdi is sp. Line 1 2 Enshea hed IJ Insec pa hogen + S eine nema el iae Enshea hed IJ Insec pa hogen ) S. glase i Enshea hed IJ Insec pa hogen ) S. ca pocapsae Enshea hed IJ Insec pa hogen ) Phasma habdi is he maph odi a 3 Enshea hed IJ Slug pa asi e ) Caeno habdi is elegans 4 Mixed popula ion F ee-li ing ) Daue ju eniles ) C. b iggsae 6 Mixed popula ion F ee-li ing ) Rhabdi is my iophila 6 Mixed popula ion F ee-li ing ) R. blumi 6 Mixed popula ion F ee-li ing ) Te a o habdi is palma um 6 Mixed popula ion F ee-li ing ) Rhabdi ella axei 6 Mixed popula ion F ee-li ing ) Panag ellus edi i us 6 Mixed popula ion F ee-li ing ) P is ionchus paci icus 6 Mixed popula ion F ee-li ing ) Pelliodi is ypica 6 Mixed popula ion F ee-li ing ) Meso habdi is longespiculosa 6 Mixed popula ion F ee-li ing ) Rhabdi oides egina 6 Mixed popula ion F ee-li ing ) S ongylida Haemonchus con o us 5 Enshea hed IJ Ve eb a e pa asi e + Os e agia ci cumcinc a 5 Enshea hed IJ Ve eb a e pa asi e + T ichos ongylus colub i o mis 5 Enshea hed IJ Ve eb a e pa asi e + Coope ia oncopho a 5 Enshea hed IJ Ve eb a e pa asi e + Tylenchida Meloidogyne sp. 7 Second s age ju eniles Plan pa asi e ) Globode a sp. 7 Second s age ju eniles Plan pa asi e ) Aphelenchida Aphelenchus a enae 4 Mixed popula ion F ee-li ing ) Nema odes ob ained om: 1 By on Adams, Uni e si y o Flo ida, USA; 2 Dick Ma i, USDA, Ti on, Geo gia, USA; 3 Mic oBio L d, UK; 4 John B owne, NUI Maynoo h, I eland; 5 Ge ald Cole, Uni e si y o B is ol, UK; 6 Caeno habdi is Gene ics Cen e , Uni e si y o Minneso a, S . Paul, USA; 7 Tes ed by D . Kei h Da ies, IACR, Ro hams ed, England. Table 2. S ains o He e o habdi is used in expe imen s, wi h hei na u al Pho o habdus symbion and associa ed Paenibacillus s ain Nema ode species and s ain Symbion Paenibacillus s ain Geog aphic sou ce He e o habdi is megidis EU17 Pho o habdus empe a a empe a a EU17 NEM1a Es onia H. downesi K122 P. empe a a a K122 —I eland H. bac e iopho a EU222 P. luminescens b EU222 —Hunga y H. bac e iopho a MOL P. luminescens b MOL —Molda ia H. indica LN2 P. luminescens akhu s ii LN2 NEM3 India He e o habdi is sp. c Line 1 Pho o habdus sp. c Line 1 NEM2 Geo gia, USA a Subspecies no named, bu dis inc om P. empe a a empe a a [11]. b Subspecies no iden i ied. c Uniden i ied species. 416 M.R. ENRIGHT, C.T. GRIFFIN:ASSOCIATION BETWEEN PAENIBACILLUS AND HETERORHABDITIS spo angial suspension wi h u bidi y equi alen o McFa land s anda d 3 (bioMe ieux, F ance) was added o an aqueous suspension o nema odes (1–3·10 3 nema- odes/mL) a a a io o 1:200 / , in wells o 25-well pla es. Nema odes used in hese assays we e no su ace s e ilized, o a oid po en ial al e a ion o he su ace cu icle p ope ies o any o he nema ode species o s ages es ed. The expe imen was conduc ed on di e en da es wi h g oups o nema ode species depending on a ail- abili y. He e o habdi is megidis IJs we e included as a posi i e con ol on each da e. S e ile dis illed wa e was added o each nema ode ea men as a nega i e con ol. The e we e h ee eplica es o each ea men . T ea men s we e le o s and o ‡2 h and we e hen examined o clumping o nema odes. A sample o nema odes (‡20 pe eplica e) was hen examined mic oscopically (400·), and he p esence o adhe ing spo angia was no ed. Nema odes we e washed by sedimen a ion in s e ile dis- illed wa e o emo e ee spo angia and a second sample was examined o spo angia. Adhesion o plan pa asi ic nema ode species (Meloidogyne sp. and Globode a sp.) was es ed by K. Da ies (IARC, Ro hams ed, England), using a simila me hod. Numbe s o spo angia adhe ing o nema odes we e no mally no coun ed, excep in he compa ison o en- shea hed and exshea hed IJs. He e o habdi is IJs spon a- neously shed hei shea h du ing s o age. A mix u e o exshea hed and enshea hed in ec i e ju eniles, ob ained by incuba ing H. megidis UK211 IJs in wa e a 20C o 8 weeks, was included in he assay, and he numbe o spo angia adhe ing o 10 indi iduals o each ype pe eplica e was coun ed. Some nema ode species we e es ed as mixed cul- u es, which included a ious de elopmen al s ages. Clumping was sco ed o he mix u e bu indi idual s ages we e sco ed o he p esence o spo angia. Species es ed as mixed popula ions included all he ee-li ing bac i o ous habdi id species ecei ed om he Caeno - habdi is Gene ics Cen e (Minneso a), which we e es ed wi hou u he subcul u e, as s a ed bu iable cul u es. This ensu ed he p esence o a p opo ion o daue ju eniles in hose species ha o m hem. Adhesion o Non-Nema ode-Associa ed Paenibacillus and Bacillus Species o He e o habdi is spp. In ec i e Ju e- niles. Spo ula ed cul u es o nine Paenibacillus and Bacillus species we e es ed o he abili y o clump He e o habdi is spp. IJs. Paenibacillus mace ans DSM 24 T , P. macqua iensis DSM 2 T ,P. polymyxa DSM 36 T ,P. al ei DSM 29 T ,P. azo o ixans DSM 5976 T ,P. pabuli DSM 3036 T , and Bacillus hu ingiensis subsp. galle iae DSM 6110 we e ob ained om he Ge man Collec ion o Mic oo ganisms and Cell Cul u es (DSMZ), B aun- schweig, Ge many. Paenibacillus azo educens DSM 13822 T was ob ained om G. McMullan (Uni e si y o Uls e ), and six P. bo ealis isola es, including he ype s ain DSM 13188 T , we e ob ained om S. Elo (Uni e - si y o Helsinki). P. nema ophilus NEM1a DSM 13559 T se ed as a posi i e con ol. All s ains we e main ained on nu ien aga pla es a 30C excep P. macqua iensis, which was main ained a 20C. Spo e suspensions we e made up o app oxima ely McFa land s anda d 2, and 100 lL was added o 2.9 mL o He e o habdi is IJ sus- pension (3000 IJs) in wells o 25-well pla es. The e we e a leas wo eplica es pe ea men . These we e le a oom empe a u e and sco ed o clumping a in e als o e 24 h. All bac e ial s ains we e es ed wi h H. megidis EU17, and some we e also es ed wi h H. indica LN2. Adhesion o Nema ode-Associa ed Paenibacillus S ains o Va ious He e o habdi is Species and S ains. Adhesion o he h ee nema ode-associa ed Paenibacillus s ains o each o six He e o habdi is species/s ains (H. megidis EU17, H. downesi K122, H. bac e iopho a EU222, H. bac e iopho a MOL, He e o habdi is sp. Line 1, and H. indica LN2) was compa ed. Fo his pu pose, he clumping assay desc ibed abo e was made semiquan i- a i e. Th ee concen a ions o spo angia we e p epa ed o gi e a inal concen a ion o 10, 100, and 1000 spo- angia/IJ when added o he nema ode suspension (4500 IJs in 3 mL wa e ). All combina ions we e se up in duplica e. They we e le a oom empe a u e o 3 h, shaken gen ly o 30 s, and le o s and o a u he 10 min, a e which clumping was semiquan i a i ely assessed on a scale o 0 o 4: 0, no clumping; 1, >20 small clumps wi h nume ous ee nema odes; 2, 10–20 small clumps wi h some ee nema odes; 3, 3–10 medium-sized clumps wi h some ee nema odes; 4, 1–3 la ge clumps wi h ew ee nema odes. C oss-Compa ibili y o Nema ode-Associa ed Paenibacil- lus S ains wi h Va ious He e o habdi is Species and S ains in Insec Cada e s. The compa abili y o a ious He e o habdi is species and s ains wi h each o he h ee nema ode-associa ed Paenibacillus s ains, in allowing hei mul iplica ion and anspo , was in es i- ga ed by in ec ing insec s wi h a ious na u al o no el Paenibacillus–He e o habdi is combina ions and assessing he numbe o nema ode-bo ne Paenibacillus spo angia ca ied om he insec cada e s in which hey had de eloped. Nema ode suspension (1500 IJs/mL) was mixed wi h Paenibacillus spo angial suspension o a known concen- a ion (assessed by hemocy ome e coun s) o gi e a inal a io o 100 spo angia/IJ. Fi e G. mellonella la ae we e exposed o 400 lL o his suspension on il e pape in 50- mm Pe i dishes. Galle ia mellonella we e also exposed o 400 lL o a 1500 IJs/mL suspension o nema odes wi hou added Paenibacillus. In ec ions in ol ing nema ode M.R. ENRIGHT, C.T. GRIFFIN:ASSOCIATION BETWEEN PAENIBACILLUS AND HETERORHABDITIS 417 s ains om empe a e egions (H. megidis EU17, H. downesi K122, H. bac e iopho a MOL, and H. bac e io- pho a EU222) we e incuba ed a 20C, while hose om semi opical/ opical clima es (H. indica LN2 and He - e o habdi is sp. Line1) we e incuba ed a bo h 20C and 26C. The e we e h ee eplica es o each ea men . Cada e s we e placed on Whi e aps and IJs eme ging in o wa e we e assessed o clumping (indica ing ha spo angia had been p oduced in he cada e and we e being ca ied by he nema odes). The e we e h ee expe imen s. Clumping was assessed in each expe imen using a semi-quan i a i e scale. Numbe s o spo angia we e enume a ed in one o he expe imen s. When nema odes ceased o eme ge, all IJs ha es ed om a gi en Whi e ap we e pooled. The numbe o spo angia adhe ing o en indi idual IJs was de e mined unde 400· magni ica ion. The o al yield o IJs was assessed by coun ing eplica e aliquo s o nema ode suspension. To aid coun ing, samples we e ea ed wi h 0.5% sodium hypochlo i e o 15 min o dispe se clumps, hen washed h ee imes by sedimen a ion in wa e . The o al numbe o spo angia ca ied by nema odes om cada e s was calcula ed by mul iplying he o al numbe o IJs ha eme ged by he a e age numbe o spo angia/IJ. Fo combina ions in which no spo angia we e ca ied by IJs, samples o cada e ma e ial we e examined mic oscopi- cally 1 week a e IJ eme gence ceased (>4 weeks pos in- ec ion) o e idence o spo angia. Fo combina ions in ol ing P. nema ophilus NEM1a, he numbe o spo angia emaining in he cada e s was assessed 1 week a e IJs s opped eme ging. The i e cada e s om each Whi e ap we e placed in 45 mL o s e ile dis illed wa e and dis up ed by epea ed o - exing. The numbe o spo angia was de e mined by hemocy ome e coun s o h ee eplica e samples. Sensi i i y o Paenibacillus spp. o Pho o habdus spp. in Vi o. Pho o habdus was isola ed om su ace-s e - ilized [0.4% hyamine 2389 (BDH)] He e o habdi is IJs mace a ed in s e ile dis illed wa e . Pho o habdus spp. exhibi pheno ypic a ia ion [12]; p ima y o m ( he no mal wild- ype o m) was selec ed based on dye ad- so b ion on NBTA aga [2]. Ten lL o o e nigh 30C Pho o habdus nu ien b o h cul u e was inocula ed cen ally on a nu ien aga pla e (glass Pe i dish; 90 mm). Pla es we e incuba ed a 20C o 3 days. Pho o habdus was killed by in e ing he pla es and looding he lids wi h chlo o o m, which was hen al- lowed o e apo a e. Paenibacillus spo angial suspension in s e ile dis illed wa e (equi alen o McFa land s an- da d 3) was s eaked in a s aigh line om he edge o he Pe i dish o he edge o he killed Pho o habdus colony. A e 1 week a 20C, he dis ance be ween isible Paenibacillus g ow h and he Pho o habdus colony edge was measu ed. In he i s expe imen , he h ee nema ode-associ- a ed Paenibacillus s ains (NEM1a, NEM2, and NEM3) and h ee non-nema ode-associa ed species, Paenibacillus polymyxa, P. mace ans, and P. azo educens, we e es ed o sensi i i y o wo Pho o habdus s ains, Pho o habdus empe a a empe a a EU17 and Pho o habdus sp. Line 1 (see Table 2). Each pla e ecei ed ou s eaks, one o which was always Paenibacillus mace ans o check o a ia ion be ween eplica e pla es o a gi en Pho o habdus s ain. Li le a ia ion was ound in his con ol. The o he h ee s eaks we e andomly chosen om among he six Paenibacillus s ains. Each Paenibacillus–Pho o - habdus combina ion was es ed a leas ou imes. Simila expe imen s we e ca ied ou o es he sensi i i y o he h ee nema ode-associa ed Paenibacillus s ains o he Pho o habdus spp. s ains lis ed in Table 2, a 20 and 30C. Each pla e was s eaked wi h NEM1a, NEM2, and NEM3. The e we e i e independen expe - imen s ( ou a 20C and one a 30C) wi h a selec ion o Pho o habdus s ains in each. The e we e a leas i e eplica es o each Pho o habdus–Paenibacillus ea men a 20C, and wo o ou eplica es a 30C. S a is ical Analysis. Da a we e es ed o no - mali y using he Ande son–Da ling me hod, and homo- genei y o a iance was es ed by Ba le ’s me hod. The e ec o shea h p esence on numbe s o spo angia adhe ing o nema ode IJs was es ed using a - es , pai ed by sample, as each sample con ained bo h enshea hed and exshea hed IJs. Compa ison o h ee o mo e ea - men s was by one-way ANOVA; whe e a signi ican ea men e ec was de ec ed, means we e sepa a ed using Tukey’s mul iple ange es (a= 0.05). Da a o numbe s o NEM1a spo angia pe hos we e i s sub- jec ed o squa e oo ans o ma ion o homogenize a iances. P opo ion da a we e subjec ed o angle (a c- sine squa e oo ) ans o ma ion. The K uskal–Wallis es (conduc ed sepa a ely o each Paenibacillus s ain) was used o in es iga e a ia ion in numbe s o spo angia ca ied om di e en nema ode hos s. Co ela ion be- ween pa ame e s was es ed using Pea son’s co ela ion. All es s we e ca ied ou using Mini ab 13.1 (Mini ab Inc., Pennsyl ania) and in each case signi icance was a - ained a P< 0.05. Resul s Adhesion o Spo angia o Nema odes Adhesion o Paenibacillus nema ophilus NEM1a Spo angia o Va ious Species and S ages o Nema odes. Resul s o es s o he adhesion o P. nema ophilus NEM1a spo angia o a ious nema ode species a e shown in Table 1. Addi ion o NEM1a o enshea hed in ec i e ju eniles o all He - e o habdi is spp. es ed esul ed in clumping o hese 418 M.R. ENRIGHT, C.T. GRIFFIN:ASSOCIATION BETWEEN PAENIBACILLUS AND HETERORHABDITIS nema odes, and he spo angia adhe ed along hei en i e su ace. A ew spo angia adhe ed o H. megidis IJs which had los hei shea h (1.0 ± 0.27 spo angia/IJ; mean ± SE), bu o a lesse ex en han o enshea hed indi iduals (17.2 ± 1.31 spo angia/IJ). The di e ence was signi ican (pai ed - es , P= 0.006). The e was no adhesion o H. megidis adul s (he maph odi es, males and emales). Spo angia adhe ed in subs an ial numbe s o enshea hed in ec i e ju eniles o all he animal pa a- si ic S ongylida species es ed bu did no adhe e o o clump enshea hed IJs o S eine nema, he o he genus o en omopa hogenic nema ode. Spo angia ailed o clump o adhe e o any o he ee-li ing bac i o ous habdi id species o any o he a ious o he nema odes es ed. Occasionally, single indi iduals o Meloidogyne sp. and Globode a sp. we e ound wi h a ew spo angia adhe ing. Howe e , he as majo i y o indi iduals we e ee o spo angia and clumping did no occu . Adhesion o Non-Nema ode-Associa ed Paenibacillus and Bacillus Species o He e o habdi is spp. In ec i e Ju e- niles. Nei he Bacillus hu ingiensis no any o he eigh non-nema ode-associa ed species o Paenibacillus caused clumping a He e o habdi is spp. IJs. Adhesion o Nema ode-Associa ed Paenibacillus S ains o Va ious He e o habdi is Species and S ains. All h ee nema ode-associa ed Paenibacillus s ains adhe ed o and clumped all six He e o habdi is s ains a each o he h ee spo angial concen a ions es ed. In each case, he e was an ob ious dec ease in he le el o clumping as he spo angial concen a ion was dec eased, om a sco e o 3 o 4 a he highes concen a ion o 1 ( a ely 2) a he lowes . NEM3 ended o cause sligh ly mo e clumping han he o he wo s ains. This was mos e iden a he highes spo angia/IJ concen a ion (whe e clumping was sco ed 4 o NEM3 and 3 o NEM1a and NEM2) and was obse ed o all o he nema ode s ains es ed. The e was no e idence o di e ences be ween He e o habdi is s ains in he deg ee o clumping caused by any o he nema ode-associa ed Paenibacillus s ains. C oss-Compa ibili y o Nema ode-Associa ed Paenibacil- lus S ains wi h Di e en He e o habdi is Species and S ains in Cada e s. The numbe o P. nema ophilus NEM1a spo angia p oduced a ied depending on he nema ode wi h which i was inocula ed, anging om 0 o 67 ·10 6 spo angia pe G. mellonella cada e (Table 3). The numbe o spo angia ca ied om he cada e s by nema ode IJs was simila ly nema ode-dependen (Fig. 1). The numbe o NEM1a spo angia ca ied by nema ode IJs om he G. mellonella cada e s is co ela ed wi h he numbe emaining wi hin he cada e (Pea son co ela- ion = 0.98, P= 0.003), showing ha measu ing he numbe s o spo angia ca ied by nema odes om di - e en hos cada e s is a good indica ion o he ela i e sui abili y o he hos s o p oduc ion o spo angia. Be- ween 17% and 48% o he NEM1a spo angia p oduced we e ca ied ou o he cada e by eme ging nema odes, wi h a highe p opo ion ca ied whe e ewe spo angia we e p oduced (Table 3). The e we e di e ences be ween he Paenibacillus s ains in hei compa ibili y wi h he di e en nema ode s ains, as judged bo h by he deg ee o clumping o IJs (da a no shown) and numbe s o spo angia ca ied by he IJs (Fig. 1). Fo each o he h ee nema ode-associ- a ed Paenibacillus s ains he numbe o spo angia ca ied om insec cada e s was signi ican ly (K uskall–Wallis, P < 0.05) a ec ed by he nema ode wi h which he insec was inocula ed. NEM2 and NEM3 sha ed a simila spec um o compa ibili y: o bo h s ains, he highes numbe o spo angia was ca ied om he cada e by H. indica LN2 and none we e ca ied by H. megidis EU17 o H. downesi K122. P. nema ophilus NEM1a showed he opposi e end, wi h la ge numbe s o spo angia being ca ied by H. megidis EU17 and H. downesi K122 and none by H. indica LN2 (Fig. 1). Whe e no spo angia we e ca ied by IJs, none we e ound by mic oscopic exami- na ion o he cada e s. Incuba ions we e pe o med a a empe a u e op imal o he nema ode associa e (20 o 26C); when he wa m-adap ed nema odes, H. indica and He e o habdi is sp. Line 1, we e incuba ed a 20C, esul s we e simila o hose a 26C: spo angia we e p oduced by NEM2 and NEM3 bu no by NEM1a. NEM1a and NEM3 we e each ca ied in highes numbe by he nema ode om which hey we e o iginally isola ed, whe eas NEM2 was ca ied in highe numbe by H. indica LN2 ( he na u al associa e o NEM3) han by He e o habdi is sp. Line 1, i s own na u al associa e (Fig. 1). Table 3. The o al numbe o Paenibacillus nema ophilus NEM1a spo angia p oduced pe Galle ia mellonella in ec ed wi h di - e en species o He e o habdi is, and he numbe and p opo - ion o spo angia emaining wi hin he insec cada e a e nema ode eme gence ceased Numbe o spo angia ·10 6 P opo ion o spo angia emaining Nema ode To al 1 In cada e in cada e H. megidis EU17 66.7 a 2 53.3 a 2 0.79 ab 3 H. downesi K122 39.4 ab 32.7 a 0.83 a H. bac e iopho a EU222 13.6 bc 8.4 b 0.62 bc H. bac e iopho a MOL 3.6 c 1.9 b 0.52 c H. indica LN2 0 0 – 1 Spo angia emaining wi hin he cada e + spo angia ca ied by nema ode IJs. 2,3 Wi hin a column, ea men s wi h he same le e a e no signi ican ly di e en (Tukey’s es ; a= 0.05, on squa e- oo 2 o angle 3 ans o med da a). Da a a e un ans o med means. M.R. ENRIGHT, C.T. GRIFFIN:ASSOCIATION BETWEEN PAENIBACILLUS AND HETERORHABDITIS 419 The p esence o Paenibacillus did no a ec he numbe o nema ode IJs p oduced ela i e o he con ol in i e o he six He e o habdi is s ains (da a no shown; ANOVA, P> 0.05 o each nema ode s ain). Yield o H. bac e iopho a MOL a ied be ween ea men s (ANOVA, P< 0.05); he numbe o IJs p oduced in he p esence o Paenibacillus NEM3 (2.6 ·10 5 IJs/cada e ) di e ed (Tukey’s es , a= 0.05) om he con ol (3.5 ·10 5 IJs/ cada e ), whe eas numbe s o NEM1a and NEM2 did no . Sensi i i y o Nema ode-Associa ed Paenibacillus S ains and Rela ed Species o Pho o habdus spp. An i- bio ics. Each o he h ee nema ode-associa ed Pae- nibacillus s ains was inhibi ed o a much lesse ex en han each o he o he h ee species o Paenibacillus es ed (Fig. 2). All h ee nema ode-associa ed s ains had a e - age inhibi ion zones o 0–2 mm, while he o he species had inhibi ion zones anging om 12 o 23 mm. In expe imen s in ol ing addi ional Pho o habdus s ains, he e was li le o no inhibi ion o nema ode- associa ed Paenibacillus (inhibi ion zones no mally <4 mm). The la ges inhibi ion zone (6 mm) was e- co ded o P. nema ophilus NEM1a wi h Pho o habdus luminescens akhu s ii LN2 (wi h which i was no com- pa ible in cada e s). Howe e , NEM1a ended o be he mos sensi i e o he h ee nema ode-associa ed s ains, no ma e which Pho o habdus was used—e en ha wi h which i is na u ally associa ed. Discussion O all he nema ode species es ed, Paenibacillus ne- ma ophilus NEM1a adhe ed only o IJs o He e o habdi is and S ongylida species. Adhesion o he la e p obably e lec s he close phylogene ic ela edness o He e o - habdi is wi h membe s o he S ongylida [6]. Like He - e o habdi is spp., he in ec i e ju enile s age o many S ongylida species e ains he mol ed second-s age cu icle as a shea h. Howe e , possession o a shea h is no he only ac o de e mining whe he spo angia will ad- he e. In ec i e ju eniles o ano he habdi id nema ode, he slug pa asi ic Phasma habdi is he maph odi a, also possess a shea h [34], as do IJs o S eine nema spp. and a numbe o he ee-li ing habdi id species es ed. Howe e , he e was no adhesion o NEM1a spo angia o enshea hed ju eniles o hese nema odes. The lack o adhesion o NEM1a spo angia o species o he o he genus o en omopa hogenic nema odes, S eine nema,is no inconsis en . Despi e sha ing a simila li e his o y, including an associa ion wi h mu ualis ic bac e ia belonging o he En e obac e iaceae, He e o habdi is and S eine nema a e no e y closely ela ed [6, 24] and a e assumed o ha e a i ed a hese simila i ies by con e - gen e olu ion [30]. Figu e 2. Inhibi ion o g ow h in an aga pla e assay o nema ode- associa ed (NEM1a, NEM2, NEM3) and non-nema ode-associa ed Paenibacillus species by Pho o habdus empe a a empe a a EU17 and Pho o habdus sp. Line 1. Da a a e means (±SE) o ou o 11 eplica es pe combina ion. Figu e 1. The numbe o spo angia ca ied by He e o habdi is spp. in ec i e ju eniles pe Galle ia mellonella cada e o each o h ee Paenibacillus s ains: P. nema ophilus NEM1a, Paenibacillus sp. NEM2, and Paenibacillus sp. NEM3. The He e o habdi is species es ed we e H. megidis (H. m.), H. downesi (H. d.), H. bac e iopho a (H. b.), He e o habdi is sp. (He . sp.), and H. indica (H. i.). *No es ed. N deno es he na u al Paenibacillus–He e o habdi is asso- cia ion. Da a a e means (±SE) o h ee eplica es o i e cada e s each. 420 M.R. ENRIGHT, C.T. GRIFFIN:ASSOCIATION BETWEEN PAENIBACILLUS AND HETERORHABDITIS The ac ha all h ee nema ode-associa ed Paeni- bacillus s ains adhe ed o all He e o habdi is species and s ains es ed sugges s a b oad conse a ion o he ele- an su ace moie ies in ol ed in adhesion ac oss he nema ode genus. The genus He e o habdi is is mo pho- logically conse a i e [15] and con ains ela i ely li le phylogene ic di e si y [24]. The possible exis ence o sub le quan i a i e di e ences be ween He e o habdi is and/o Paenibacillus s ains wi h espec o hese su ace moie ies canno be discoun ed, because o he ela i e insensi i i y o he semiquan i a i e clumping assay em- ployed. Howe e , he b oad conse a ion o binding moie ies is u he suppo ed by he inding (unpublished da a) ha spo angia-induced nema ode clumping in all h ee na u al He e o habdi is–Paenibacillus associa ions was inhibi ed by he same ca bohyd a e, N-ace ylneu- aminic acid (sialic acid). This inding may poin o he in ol emen o a sialic acid–binding lec in in spo angial adhesion. Such b oad conse a ion o binding moie ies ac oss he He e o habdi is genus would di e ma kedly om he s ain-speci ic adhesion o he pa asi e Pas eu ia pene ans o i s nema ode hos s [33], and o Ra hayib- ac e spp. o hei nema ode ec o s, Anguina spp. [26]. Nema ode-associa ed Paenibacillus s ains cause no ob ious pa hology o hei He e o habdi is associa es and ha e no e ec s on hei de elopmen , hough adhesion o P. nema ophilus spo angia impedes he dispe sal o IJs [9]. Howe e , ei he he e ec s a e no s ong enough unde na u al condi ions o hese associa ions a e no common enough o exe e olu iona y p essu e on He - e o habdi is o a y hei su ace ecep o s o e ade Pa- enibacillus a achmen . F om he esul s o es s in ol ing a ange o Pa- enibacillus species, i appea s ha he abili y o adhe e o and clump He e o habdi is IJs is a ea u e peculia o he nema ode-associa ed s ains. A mo phological cha ac e is ic sha ed by hese nema ode-associa ed s ains is ha he spo angia emain in ac e en ol- lowing long pe iods o s o age [10], whe eas hose o o he Paenibacillus spp. es ed end o disin eg a e and elease he bac e ial endospo e. Such longe i y o he spo angium is no a common ea u e wi hin he genus Paenibacillus, o in G am-posi i e ae obic endospo e o me s in gene al. The adhesion o he spo angium o ce ain nema ode species may be an impo an ai and could ep esen a speci ic adap a ion allowing hese bac e ia access o a niche dependen on anspo by nema odes. Fo bac e ia o su i e and ep oduce in a He e o - habdi is-in ec ed cada e hey need o ole a e he con- di ions p oduced by he Pho o habdus sp. symbion . S ains o P o idencia e ge i and some (bu no all) o he Och obac um spp. s ains which we e ound associ- a ed wi h He e o habdi is spp. we e esis an o Pho o - habdus an ibio ics [4, 18]. When es ed in i o, he h ee nema ode-associa ed Paenibacillus s ains we e shown o be esis an o Pho o habdus, unlike o he species o Pa- enibacillus including he closely ela ed P. azo educens. This sugges s ha esis ance o an ibio ics by hese nema ode-associa ed s ains has been selec ed o du ing associa ion wi h Pho o habdus. We canno exclude he possibili y ha some u he selec ion occu ed du ing in i o labo a o y cul u e o he associa ions. Howe e , H. megidis EU17 IJs clumped s ongly (indica ing he p es- ence o abundan spo angia) om he ime o hei i s isola ion, sugges ing ha P. nema ophilus NEM1a was al eady esis an be o e labo a o y cul u e. The di e en ial abili y o he a ious s ains o He - e o habdi is (wi h hei associa ed Pho o habdus) o suppo g ow h o he h ee nema ode-associa ed Paeni- bacillus s ains may be pa ially unde s ood in e e ence o Paenibacillus and He e o habdi is/Pho o habdus axo- nomic ela ionships. Paenibacillus NEM2 and NEM3 we e compa ible wi h a simila spec um o He e o - habdi is s ains, di e en om ha o P. nema ophilus NEM1a, and i has been shown ha hese wo s ains sha e highe DNA-DNA simila i y (60%) han ei he sha es wi h NEM1a (bo h 40%) [10]. The close ela - edness be ween H. megidis and H. downesi [1] and ha o hei symbion s (bo h Pho o habdus empe a a) may help explain why bo h species pe o med simila ly in allowing de elopmen o NEM1a bu no o ei he NEM2 o NEM3. C oss-compa ibili y esul s sugges ha , in he- o y, Paenibacillus bac e ia could be exchanged be ween some (bu no all) species o He e o habdi is ollowing di ec o indi ec con ac in soil; o example, all h ee nema ode-associa ed Paenibacillus s ains we e compa i- ble wi h H. bac e iopho a, wi h which Paenibacillus has so a no been ound in he wild. Di e en ial compa ibili y o he nema ode-associa ed Paenibacillus s ains wi h a ious He e o habdi is s ains in cada e s could no be explained by he esul s o in i o assays es ing hei sensi i i y o he nema odes’ Pho o habdus symbion s. In hese assays, all h ee nem- a ode-associa ed s ains we e ela i ely insensi i e o all o he Pho o habdus s ains es ed; hus, di e ences be- ween nema ode-associa ed s ains we e small, bu showed a end o NEM1a o be always he mos , and NEM3 he leas inhibi ed, i espec i e o Pho o habdus s ain used. The ailu e o hese assays o explain he di e en ial compa ibili y in cada e s may be due o seconda y me aboli e p oduc ion by Pho o habdus being impo e ished on aga ela i e o ha in insec cada e s [16]. Al e na i ely, ac o s o he han an imic obials may be esponsible o ende ing some cada e s mo e hos ile han o he s o nonadap ed mic obes; o example, i has been p oposed [19] ha Pho o habdus’s compe i i e g ow h a es a e mo e impo an han an imic obials in supp essing compe ing mic oo ganisms. Al e na i ely, he nema odes hemsel es may play an as-ye -unknown M.R. ENRIGHT, C.T. GRIFFIN:ASSOCIATION BETWEEN PAENIBACILLUS AND HETERORHABDITIS 421 ole in modi ying he cada e en i onmen o mic o- o ganisms. Al hough he bac e ia o which NEM1a and NEM3 a e ep esen a i e we e each eco e ed om soil only once, NEM2 is ep esen a i e o a mo e widesp ead associa ion: spo angia-encumbe ed nema odes we e iso- la ed a ou o he eigh si es sampled in Geo gia [25]. In addi ion o he h ee associa ions included in he p esen s udy, spo angia mo phologically simila o hose o P. nema ophilus a e ou inely ound associa ed wi h H. zealandica and H. indica in Flo ida (pe s. comm., L.W. Duncan). Associa ions be ween He e o habdi is spp. and Paenibacillus simila o hose o his s udy may u n ou o be o widesp ead occu ence. They ha e al eady been ound in bo h empe a e and opical egions, in h ee con inen s (Eu ope, Asia, and No h Ame ica), and in- ol e a leas h ee species o nema ode and wo closely ela ed gene ic clus e s o Paenibacillus [10]. Such asso- cia ions a e no es ic ed o He e o habdi is: a bac e ium which is en a i ely iden i ied as a Paenibacillus close o P. popilliae is equen ly ound adhe ing o IJs o S eine - nema diap epesi, appa en ly in a pho e ic ela ionship (FE Bo ai, LW Duncan, JF P es on, D Dunn. A pho e ic associa ion be ween a pu a i e Paenibacillus sp. and he en omopa hogenic nema ode S eine nema diap epesi, Abs ac , Socie y o Nema ologis s Annual Mee ing 2003). The genus Paenibacillus con ains a numbe o species ha a e associa ed wi h dead insec s in one capaci y o ano he . These include obliga e insec pa hogens such as P. popilliae, P. len imo bus, and P. la ae [28], as well as non-pa hogens o acul a i e pa hogens such as P. apia- us [27] and P. al ei [23]. Al hough he He e o habdi is- associa ed s ains lie phylogene ically close o species wi h no known associa ion wi h insec s, such as P. macqua iensis and P. azo educens [10], hey p o ide an in e es ing addi ion o his lis . As ye he e is no e idence ha nema ode-associa ed Paenibacillus s ains a e en o- mopa hogens, bu only limi ed in es iga ions ha e been ca ied ou [9]. The e idence so a sugges s ha hese Paenibacillus s ains u ilize He e o habdi is o anspo and in oduc ion in o he nu ien - ich en i onmen o an insec cada e . Adap a ions o his way o li e may include he unusual longe i y o he spo angia allowing adhesion o he nema odes, and esis ance o Pho o - habdus an ibio ics. The ac ha he Paenibacillus s ains ended o be mos compa ible wi h hei na u al He - e o habdi is–Pho o habdus associa e indica es ha a de- g ee o adap a ion o local species o s ains has occu ed. Al hough ela i ely ew spo angia a e p oduced pe insec hos (7 ·10 7 o P. nema ophilus NEM1a wi h i s na u al associa e), ac i e dissemina ion o p opagules by ens o housands o mobile in ec i e ju eniles may compensa e o his. Acknowledgmen s We a e g a e ul o Dick Ma i and Pa icia Timpe (USDA-ARS, Ti on, GA), By on Adams (Uni e si y o Flo ida), John B owne (NUI Maynoo h, I eland), Ge ald Cole (Uni e si y o B is ol, England), Mic oBio L d (England), and Caeno habdi is Gene ics Cen e (Uni e - si y o Minneso a) o p o iding us wi h nema ode samples and in o ma ion ega ding hem. Re e ences 1. Adams, BJ, Bu nell, AM, Powe s, TO (1998) A phylogene ic analysis o He e o habdi is (Nema a: Rhabdi idae) based on in e nal ansc ibed space 1 DNA sequence da a. J Nema ol 30: 22–39 2. Akhu s , RJ (1980) Mo phological and unc ional dimo phism in Xeno habdus spp., bac e ia symbio ically associa ed wi h he insec pa hogenic nema odes Neoaplec ana and He e o habdi is. J Gen Mic obiol 121: 303–309 3. Akhu s , RJ (1982) An ibio ic ac i i y o Xeno habdus spp., bac- e ia symbio ically associa ed wi h pa hogenic nema odes o he amilies He e o habdi idae and S eine nema idae. J Gen Mic obiol 128: 3061–3065 4. 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