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.
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422 M.R. ENRIGHT, C.T. GRIFFIN:ASSOCIATION BETWEEN PAENIBACILLUS AND HETERORHABDITIS