Nema ology, 2000, Vol. 2(1), 31-42 Symposium
He e o habdi is,S eine nema and hei bac e ial symbion s —
le hal pa hogens o insec s
Ann M. BURNELL 1,*and S. Pa icia STOCK 2
1Depa men o Biology, Na ional Uni e si y o I eland Maynoo h, Maynoo h, Co. Kilda e, I eland
2Depa men o Nema ology, Uni e si y o Cali o nia Da is, CA 95616-8668,USA
P esen ed a he symposium ‘Biodi e si y in he phylum Nema oda’, Gen , Belgium, 17 Sep embe 1999
Summa y – The en omopa hogenic nema odes (EPN) He e o habdi is and S eine nema oge he wi h hei symbion bac e ia
Pho o habdus and Xeno habdus, espec i ely,a e obliga e and le hal pa asi es o insec s. EPN can p o ide e ec i e biological con ol
o some impo an lepidop e an, dip e an and coleop e an pes s o comme cial c ops and hey a e amenable o la ge-scale cul u e in
liquid e men o s.They a e unique among habdi idsin ha ing a symbio ic ela ionshipwi h an en e ic bac e ium species. The bac e ial
symbion is equi ed o kill he insec hos and o diges he hos issues, he eby p o iding sui able nu ien condi ions o nema ode
g ow h and de elopmen . This e iew desc ibes he gene al biology o EPN and hei symbion s and gi es an o e iew o s udies
o da e on EPN biodi e si y, biogeog aphy and phylogeny. The impe us o esea ch in EPN and hei symbion s has come abou
because o hei biological con ol po en ial, wi h much o he ocus in EPN esea ch ha ing been on applied aspec s ela ing o pes
con ol.Howe e EPN and hei symbion s a e inc easinglybeing iewed as exci ing subjec s o basic esea ch in he a eas o ecology,
biodi e si y, e olu ion, biochemis y, symbiosis and molecula gene ics. Much p og ess has been made o e he pas 20 yea s in ou
unde s anding o he basic biology and gene ics o EPN and hei symbion s. We a e now en e ing a new phase in which he ools
o molecula gene ics a e being inc easingly used o add ess a ange o biological ques ions in EPN esea ch. The knowledge gained
om his endea ou should ensu e ha EPN will become e en mo e e ec i e biopes icidesand should also ensu e ha EPN and hei
symbion s gain p ominence as unique and in insicallyin e es ingbiological sys ems.
Résumé – He e o habdi is, S eine nema e leu s symbio es bac é iens — Pa hogènes mo els des insec es –Les néma odes en o-
mopa hogènes (EPN) He e o habdi is e S eine nema, a ec leu bac é ies symbio es Pho o habdus e Xeno habdus, espec i emen ,
son des pa asi es obligés e mo els des insec es. Les EPN peu en se i à un con ôle biologique de quelques lépidop è es, dip è es
e coléop è es impo an s pou les cul u es comme ciales e ils son éle ables à g ande échelle dans des e men eu s liquides. Ils son
uniques chez les habdi ides pa leu ela ion symbio ique a ec une espèce de bac é ie en é ique. La bac é ie symbio e es nécessai e
pou ue l’insec e hô e e pou digé e les issus de l’hô e, pe me an ainsi des condi ons de nu i ion a o ables à la c oissance e au
dé eloppemen du néma ode. La p ésen e e ue déc i la biologie géné ale des EPN e de leu symbio es e donne un é a des é udes
ac uelles su la biodi e si é, la biogéog aphie e la phylogénie des EPN. L’impulsion donnée aux eche ches su les EPN e leu sym-
bio es p o ien de leu po en iali és pou le con ôle biologique, une g ande pa ie des eche ches su les EPN ayan ai à des aspec s
appliqués en ela ion a ec ce con ôle des pa asi es. Cependan , les EPN e leu symbio es bac é iens son de plus en plus considé és
comme des suje s in é essan s pou la eche che ondamen ale dans les domaines de l’écologie, de la biodi e si é, de l’é olu ion, de la
biochimie, des p ocessus symbio iques e de la géné ique moléculai e. De nomb eux p og ès on é é éalisés ces 20 de niè es années
dans la comp éhension de la biologie e de la géné ique des EPN e de leu symbio es. Nous en ons ac uellemen dans une nou elle
phase oùles moyens de la biologie moléculai e son u ilisés de maniè e c oissan e pou o mule une sé ie de ques ions biologiques
pou la eche che su les EPN. Les connaissances ésul an de ces e o s doi en condui e à é i e que les EPN de iend on des
biopes icides oujou s plus e caces e que les EPN e leu symbio es p end on de l’impo ance en an que sys èmes biologiques
uniques e in insèquemen in é essan s.
Keywo ds – biogeog aphy,en omopa hogenic nema ode, habi a p e e ence,Pho o habdus, phylogeny, symbiosis, Xeno habdus.
The e a e many gene a o nema odes ha pa asi ise in-
sec s ( e iewed by Poina , 1979), howe e esea ch on in-
*Co espondingau ho , e-mail: ann.bu [email protected]
sec pa asi ic nema odes is la gely concen a eda p esen
on wo amilies o habdi id nema odes: he S eine ne-
c
®Koninklijke B ill NV, Leiden, 2000 31
Symposium
ma idaeChi wood& Chi wood,1937 and he He e o hab-
di idae Poina , 1976. These soil-dwelling nema odes a e
obliga e and le hal pa asi es o insec s and a e usually
e e ed o as en omopa hogenic nema odes (EPN). EPN
can p o idee ec i ebiologicalcon olo some impo an
lepidop e an, dip e an and coleop e an pes s o comme -
cial c ops and hey a e amenable o la ge-scale cul u e in
liquid e men o s. EPN a e unique among habdi ids in
ha ing a symbio ic ela ionshipwi h an en e ic bac e ium
species. The bac e ial symbion is equi ed o kill he in-
sec hos and o diges he hos issues, he eby p o iding
sui able nu ien condi ions o nema ode g ow h and de-
elopmen .He e o habdi isand S eine nemaspeciesha e
a global dis ibu ion( e iewed by Hominick e al., 1996).
Species in hese gene a exhibi di e ences in hos ange,
in ec i i y,en i onmen al ole ances and in sui abili y o
comme cialp oduc ionand o mula ionand his has s im-
ula ed many su eys, seeking new s ains and species o
EPN o biocon ol applica ions. Thus he numbe o e-
sea ch publica ions on his g oup o nema odes has in-
c eased d ama ically in ecen yea s and a la ge numbe
o labo a o ies wo ld-wide a e cu en ly engaged in EPN
esea ch.
Gene al biology and li e cycle
The hi d s age daue ju enile (DJ) occu s ee in he
soil and i s ole is o seek ou and in ec an insec la a.
S eine nema gainsen y o he insec la a h oughna u al
openings(mou h, anus and spi acles). In addi ion o hese
modes o en y, He e o habdi isalso gainsen y by ab ad-
ing he in e segmen al memb anes o he insec using a
do sal oo h. Once in he haemocoel o he insec he DJ
eleases cells o a symbion bac e ium ha i ca ies in i s
in es ine. The insec haemolymph p o ides ich medium
o he bac e ial cells and hese begin o g ow, elease ox-
ins and exoenzymes and kill he insec . The insec dies
apidly, usually wi hin 24-48 h. The nema odes esume
de elopmen , moul o he J4 s age and each adul hood
wi hin 2 (S. ca pocapsae) o 3 (H. bac e iopho a) days
when cul u ed in i o in la ae o he g ea e wax mo h
Galle ia mellonella a 23°C (Wang & Bedding, 1996).
Nema ode ep oduc ioncon inueso e wo o h eegene -
a ions un il he nu ien s a us o he cada e de e io a es
whe eupon adul de elopmen is supp essed and DJ ac-
cumula e. These non- eedingin ec i e s ages eme ge in o
he soil whe e hey may su i e o se e al mon hs in he
absence o a sui able hos .
In S eine nema ep oduc ionis amphimic ic.S eine ne-
ma id DJ ma u e o become ei he a male o a emale
and sex de e mina ion appea s o be o he XX/XO ype,
ypical o nema odes (Dix e al., 1994). In He e o hab-
di is by con as , he DJ ma u e o gi e s gene a ion
he maph odi e emales, bu hese emales gi e ise o a
second gene a ion o amphimic ic males and emales and
o sel e ile he maph odi e emales and DJ (Dix e al.,
1992; S auch e al., 1994). The male and emale ka y-
o ypes o He e o habdi is ha e no ye been de e mined,
bu a ailable da a indica e ha sexual pheno ype is en i-
onmen ally de e mined. Wang and Bedding (1996) s ud-
ied he dynamics o popula ionde elopmen o H. bac e-
iopho a and S. ca pocapsae in la ae o G. mellonella,
a e injec ionin o he insec haemocoel o one o wo DJ,
espec i ely. Unde hese condi ions h ee adul gene a-
ions we e p oduced by bo h nema ode species (Fig. 1).
Indi idual H. bac e iopho a he maph odi es laid up o
1000 eggs which de elopedin o second gene a ion males
and emales, bu he s gene a ion he maph odi es also
e ained abou 500 eggs which de eloped in o DJ ia en-
do okia ma icida. Second gene a ion emales also laid
ca six o en eggs which de eloped in o ano he gene -
a ion o adul s, bu hey also e ained ano he 30 eggs
wi hin he nema ode body which de eloped in o DJ ia
endo okiama icida. The hi d gene a ion emales did no
o iposi and all o hei eggs (ca 50 pe emale) de eloped
ia endo okiama icida in o DJ. Fi s and second gene a-
ion S. ca pocapsae we e ound o lay a la ge p opo ion
o hei eggs han do H. bac e iopho a, bu all he eggs
p oduced by hi d gene a ion emales de eloped ia en-
do okia ma icida. Unlike H. bac e iopho a, he ju enile
s ages esul ing om endo okia ma icida in S. ca pocap-
sae did no de elopin o daue ju enilesun il hey had ex-
i ed om he body o he mo he nema ode.
In a ou able nu i i e condi ionsin liquid cul u e sec-
ondgene a ionHe e o habdi isDJ eco e and de elop o
he maph odi es (S auch e al., 1994; Johnigk & Ehle s,
1999). S auch e al. (1994) ha e also shown ha when J1
ju enileswe e s a ed o 24 h in Ringe solu ion40% be-
came he maph odi es, 6.6% became amphimic ic adul s
and 53% became DJ. O he J1 ha de eloped in o he -
maph odi es 90% had gone h ough a p e-daue J2 s age,
which was mo phologically dis inc om hose J2 g ow-
ing in o amphimic ic adul s, and 10% we e eco e ed DJ.
These da a clea ly show he impo ance o nu i ionalsig-
nals in He e o habdi is sex de e mina ion. The ex en o
which second gene a ion DJ eco e in i o and esume
de elopmen has no been de e mined, no has he p o-
32 Nema ology
Symposium
Fig. 1. Popula ion dynamics o A: He e o habdi isbac e iopho aand B: S eine nema ca pocapsaeA24 in a la a o Galle ia mellonella
a e injec ing one o wo DJ pe insec espec i ely. The pie cha s ep esen he numbe o DJ p ogeny ec ui ed om each
gene a ion. ( ): DJ p ogeny ec ui ed om s gene a ion emales; ( ): DJ p ogeny ec ui ed om second gene a ion emales;
( ): DJ p ogeny ec ui ed om hi d gene a ion emales ( om Wang & Bedding, 1996).
po ion o second gene a ion ju eniles which en e he J2
s age; howe e Dix e al. (1992) ha e shown ha he ea ly
second gene a ion adul s which de elop in G. mellonella
a e all amphimic ic.
The symbio ic associa ion
Rhabdi id pa asi es o bo h e eb a es and in e e-
b a es a e conside ed o ha e e ol ed om ee li ingbac-
e ial eeding nema odes(Adamson, 1986). Some habdi-
id nema odes ha e an associa ion wi h soil in e eb a es
o which Sudhaus and Schul e (1988) ha e in oduced
he e m nec omency. The DJ o nec omenic nema odes
en e hei hos by he body openings o a e inges ed by
he hos . The DJ emain quiescen in he hos un il i e en-
ually dies and i s body becomes in aded by sap ophy ic
bac e ia. Then he nema ode DJ esumes de elopmen
and g ow h and ep oduc ion occu s based on he bac e-
ia associa ed wi h he decaying cada e . Sudhaus (1993)
has sugges ed ha He e o habdi isand S eine nema mos
p obably e ol ed om nec omenic nema odes which de-
eloped a symbio ic associa ion wi h an en omopa ho-
genic bac e ium. Such a symbiosis specialised o pa a-
si isinganimalshas no been desc ibedso a o any o he
g oup o nema odes. Howe e he nu i ional in e ac ions
be ween EPN and hei symbion bac e ia bea many simi-
la i ies o he ec osymbiosesbe ween insec s and lamen-
ous ungi (Wilkinson & Hay, 1997). Symbion s associ-
a ed wi h S eine nema a e placed in he genus Xeno hab-
dus (Thomas & Poina , 1979) while he bioluminescen
symbion s associa ed wi h He e o habdi is a e placed in
he genus Pho o habdus (Boema e e al., 1993). Sym-
bion bac e ia o bo h gene a a e mo ile and g am-nega i e
and belong o he En e obac e iaceae. Compa isons o
16S DNA sequences show ha species o Pho o habdus
and Xeno habdus o m a phylogene icallycohe en clus-
e ha di e ged ea ly om he main line o adia ion o
he En e obac e iaceae(Fo s e al., 1997).
When symbion bac e ia a e eleased by he nema ode
in o he insec haemolymph he bac e ial cells begin o
Vol. 2(1), 2000 33
Symposium
g ow and dea h o he insec ensues, ei he om oxaemia
o om sep icemia, depending on he sensi i i y o he
insec and he symbion s ain (Fo s e al., 1997; Boe-
ma e & Gi audan, 1998). Some s ains o Xeno habdus
and Pho o habdus a e highly i ulen : injec ion o less
han en cells o he bac e ium in o he haemocoel may
be su cien o kill a suscep ible insec such as G. mel-
lonella o Manduca sex a (Poina & Thomas, 1967; Fo s
e al., 1997, ench-Cons an & Bowen,1999).When cul-
u ed in liquid medium, bo h gene a o symbion bac e ia
sec e e highly i ulen insec icidal oxinsin o he medium
(Ja e e al., 1997; Bowen e al., 1998). As he bac e ia
en e he s a iona y phase o hei g ow h cycle hey se-
c e e lipase(s), p o ease(s) and se e al b oad spec um an-
ibac e ialand an i ungalan ibio ics( e iewed by Akhu s
& Boema e, 1990; Fo s & Nealson, 1996). The likely
ole o he deg ada i e enzymes is o b eak down he in-
sec issues he eby p o iding a ich ood supply o he
de eloping nema ode. The insec cada e con aining he
apidly expanding popula ion o nema odes and bac e ia
e ains i s shape and does no pu e y, implying a ole o
he an ibio ics p oduced by symbion bac e ia. Howe e ,
Ja oz (1996) ound ela i ely low le els o an ibio ics in
cada e s o G. mellonella in ec ed wi h S. ca pocapsae
and he pos ula ed ha he lack o con amina iono insec
cada e s esul ed om he abili yo he symbion bac e ia
o ou -compe e many o he no mal gu mic o o a o he
insec hos .
Since he majo i y o EPN bac e ial complexes a e e -
ec i e o e a wide ange o insec o de s and he ype
o humo al and cellula de ence eac ions o he hos s
a ies signi can ly o e ha ange (Akhu s , 1993), i is
likely ha bo h he nema odes and he bac e ia u ilise a
a ie y o pa hogenic s a egies. Al hough nema ode i -
ulence s a egies ha e ecei ed less a en ion han hose
o he bac e ium, i is known ha DJ o S. ca pocapsae
and H. bac e iopho a elease p o ease sec e ions which
des oy he an ibac e ial ac o s o accina ed G. mel-
lonella la ae (Gö z e al., 1980; Simoes, 1998). The im-
po ance o he symbio ic in e ac ion in he pa hogenesis
p ocess is clea ly seen in he S. glase i/X. poina ii com-
plex. When G. mellonella la ae we e injec ed wi h ei-
he axenic S. glase i o wi h 1150 cells o X. poina ii,
he insec la ae su i ed. Howe e co-injec ion o 115
X. poina ii cells and one S. glase i DJ killed 75% o he
insec la ae (Akhu s , 1986).
Bo h Xeno habdusandPho o habdusoccu in wo phe-
no ypic o ms. Phase I cells a e la ge han phase II cells
and p oducesigni can lyg ea e amoun so exoenzymes,
oxins,an ibio ics han phase II o ms. Howe e he nema-
ode DJ package and anspo only phase I cells. Phase I
cells a e s o ed in a special esicle in he an e io o he
in es ine in s eine nema ids(Bi d & Akhu s , 1983) while
He e o habdi isdoes no ha e a special esicle bu s o es
he cells o he symbion in he an e io o he in es ine
(Endo & Nickle, 1991). The ole o phase II cells in he
symbio ic associa ion is s ill unclea , as a e he molecula
mechanisms esponsible o his phenomenon.
The e a e no epo s o he isola ion o Xeno habdus
and Pho o habdus om soil and i has been gene ally as-
sumed ha hese bac e ia canno exis in he soil en i on-
men in he absence o hei nema ode associa es. Mo gan
e al. (1997) eleased gene icallyma ked s ains o X. ne-
ma ophila and P. luminescens in o non-s e ile soil mic o-
cosms and hey ound ha he eleased cells declined o
below de ec ion limi s wi hin se en days. Al hough i-
able colony o ming uni s could no be de ec ed a e 7
days, measu emen o adenosine iphospha e (ATP) le -
els sugges ed ha he cells may ha e en e ed in o a do -
man , non-cul u ablebu iable phase. Bleakley and Chen
(1999) epo ed ha P. luminescens was able o su i e
and g ow o e a 30 day pe iod when inocula ed in o s e -
ilised soil o which nu ien amendmen s had been added.
Taxonomic s a us
The amily S eine nema idae Chi wood & Chi wood,
1937 is cu en ly composed o wo gene a: S eine nema
T a assos, 1927 and Neos eine nema Nguyen & Sma ,
1994.The o me genus wi h 25 species desc ibed and he
la e wi h only one species: N. longicu icauda(Table 1).
The amily He e o habdi idaePoina , comp ises only one
genus, He e o habdi is Poina , 1976 wi h H. bac e io-
pho aas he ypespeciesand eigh o he speciesdesc ibed
(Table 2): howe e , he axonomics a us o some o hese
species has been ques ioned (Adams e al., 1998).
EPN species ha e mainly been desc ibed using he Lin-
nean and biological species concep s and mo phologi-
cal/mo phome ic c i e ia and c oss-b eeding es s ha e
been he mos equen lyused ools o hei iden ica ion
(Poina , 1990; Dix e al., 1994; Nguyen & Sma , 1996;
Kaya & S ock, 1997). Addi ionally, a numbe o molec-
ula echniques, including isoenzyme pa e ns (Akhu s ,
1987), o al p o ein pa e ns (Poina & Kozodoi, 1988;
Joyce e al., 1994a), RFLP analysis (Cu an & Web-
s e , 1989; Reid & Hominick, 1993; Joyce e al., 1994b),
RAPDs (Ga dne e al., 1994; Liu & Be y, 1995), sa el-
li e DNA (G enie e al., 1996), genomic DNA sequenc-
34 Nema ology
Symposium
Table 1. The gene a and species o he amily S eine nema idae.
Family S eine nema idaeChi wood & Chi wood, 1937
= Neoaplec anidaeSbole , 1953
Type genus: S eine nema T a assos, 1927
Type species: S eine nema k aussei (S eine , 1923) T a assos,
1927
O he species:
S. abbasi Elawad, Ahmad & Reid, 1997
S. a ena ium (A yukho sky,1967) Wou s, M áÏcek, Ge din &
Bedding, 1982
S. a ne (Bo ien, 1937) Wou s, M áÏcek, Ge din & Bedding,
1982
S. bico nu um Tallosi, Pe e s & Ehle s, 1995
S. ca pocapsae (Weise , 1955) Wou s, M áÏcek, Ge din &
Bedding, 1982
S. cauda um Xu, Wang & Li, 1991
S. ce a opho um Jian, Reid and Hun , 1997
S. cubanum M áÏcek, He nandez & Boema e, 1994
S. el iae (Filipje , 1934) Wou s, M áÏcek, Ge din & Bedding,
1982
S. glase i (S eine , 1929) Wou s, M áÏcek, Ge din & Bedding,
1982
S. in e medium (Poina , 1985) Mamiya, 1988
S. ka ii Wa u u, Hun & Reid, 1997
S. kushidai Mamiya, 1988
S. longicaudum Shen & Wang, 1992
S. mon icolum S ock, Choo & Kaya, 1997
S. neocu illae Nguyen & Sma , 1992
S. o egonense Liu & Be y, 1996
S. pue o icense Roman & Figue oa, 1994
S. a um (Douce , 1986) Mamiya, 1988
S. iob a e Cabanillas, Poina & Rauls on, 1994
S. i e i de Douce & Douce , 1992
S. scap e isci Nguyen & Sma , 1992
S. siamkayai S ock, Somsook & Kaya, 1998
Genus: Neos eine nema Nguyen & Sma , 1994
Type and only species: Neos eine nema longicu icauda
Nguyen & Sma , 1994
ing (Liu e al., 1997;Adams e al., 1998), ha e been used,
no only as diagnos ic ools, bu also o s udy phylogene ic
a ni ies among EPN. Recen ly, Adams (1998) p oposed
ha he mos sui able species concep o use in nema ol-
ogy is an amalgama ion o he phylogene ic and he e o-
lu iona y species concep s. Using He e o habdi is axa as
an example, he delimi ed species o his genus based on
his e olu ion-basedapp oach.
Table 2. The gene a and species o he amily He e o habdi ae.
Family He e o habdi idaePoina , 1976
Type and only genus: He e o habdi isPoina , 1976
Genus He e o habdi isPoina , 1976
=Ch omonema Khan, B ooks & Hi schman, 1976
Type species: He e o habdi isbac e iopho a Poina , 1976
=Ch omonema helio hidis Khan, B ooks & Hi schman, 1976
=H. helio hidis (Khan, B ooks & Hi schman, 1976) Poina ,
Thomas & Hess, 1977
O he species:
H. a gen inensisS ock, 1993
H. b e icaudis Liu, 1994
H. hawaiiensis Ga dne , S ock & Kaya, 1994
H. indica Poina , Ka unaka & Da id, 1992
H. ma ela us Liu & Be y, 1996
=H. hepialius S ock, S ong & Ga dne , 1996
H. megidis Poina , Jackson & Klein, 1988
H. poina i Kakulia & Mikaia, 1997
H. zealandica Poina , 1990
EPN biodi e si y and biogeog aphy
Hominick e al. (1996) p o ided a lis wi h he ge-
og aphic dis ibu ion o desc ibed EPN species a bo h
con inen aland na ionalle el. S eine nema idsha e been
eco ded om all con inen s excep An a c ica (G i n
e al., 1990). Wi hin he genus S eine nema wo species,
S eine nema ca pocapsae and S eine nema el iae, ap-
pea o ha e a global dis ibu ion(Hominick e al., 1996).
The o he S eine nema species seem o ha e a mo e
es ic ed geog aphic dis ibu ion and hei occu ence
has been eco ded only a he con inen al o na ional
le el (Hominick e al., 1996). Howe e , as mo e su -
eys a e pe o med, he known ange o many species
is expec ed o expand. Fo ins ance, S eine nema k aus-
sei o iginally isola ed in he Geggen Moun ains, Wes -
phalia, Ge many (S eine , 1923) has subsequen ly been
isola ed om o he loca ions in Ge many (M áÏcek e
al., 1992; M áÏcek, 1994), and also om o he Eu o-
pean coun ies, such as he Czech Republic, (M áÏcek,
1977), he Ne he lands (Hominick e al., 1995), Swi ze -
land (S eine , 1994), he Uni ed Kingdom (Hominick e
al., 1995), and Spain (Ga cia del Pino & Palomo, 1996),
sugges ing his species has a Palea c ic dis ibu ion.How-
e e , he known geog aphic ange o his species has e-
cen ly been expanded o include No h Ame ica (S ock e
al., 1999b) hus indica ing a Hola c ic dis ibu ion. Sim-
ila ly, S. longicaudum, o iginally isola ed in China, has
Vol. 2(1), 2000 35
Symposium
ecen ly been eco e ed in Ko ea and Wes e n USA, indi-
ca ing a wide geog aphic ange o his species (S ock e
al., unpubl.).
The si ua ion is somewha di e en o he e o habdi-
ids, pa icula ly because ewe species ha e been de-
sc ibed. Fo ins ance, H. bac e iopho a is cu en ly he
wides geog aphically dis ibu ed he e o habdi id, ound
in all Ame icas, Sou he n and Cen al Eu ope, Aus alia
and Eas Asia (China, Japan, Ko ea). H. indica also has a
wide dis ibu ion,occu ing in he opics and sub opics,
ound in sou he n India, S i Lanka, peninsula Malaysia,
Indonesia, No h Aus alia, he Ca ibbean egion, Egyp ,
Kenya and in sub opical and wa m empe a e zones in
Japan. In con as ,H. zealandica, and H. ma ela us appea
o be species wi h a mo e es ic ed geog aphic dis ibu-
ion ha ing been ound only, espec i ely,in New Zealand
(Akhu s , 1987) and in O egon and Cali o nia, USA (Liu
& Be y, 1996a; S ock e al., 1997). The summa y p e-
sen ed in Tables 1 and 2 indica es ha he di e si y o
s eine nema ids is g ea e han ha o he e o habdi ids.
This is also e ec ed in he DNA ela edness s udies dis-
cussed in he nex sec ion.
Al hough a numbe o su eys ha e documen ed habi-
a p e e ence o EPN, he e a e a p esen insu cien and
con adic o yda a o es o co ela ions(Hominicke al.,
1996).Howe e , se e al au ho s (S eine , 1994;Hominick
e al.,1995; S ock e al., 1999; S u han, 1999) ha e ob-
se ed ha some S eine nema species a e associa ed wi h
speci c habi a ypes. Fo example, S. el iae,S. a ne
and S. in e medium ha e been ound mainly in g assland
ecosys ems (Boag e al., 1992; Hominick e al., 1995;
S ock e al., 1999). O he S eine nema species, by con-
as , seem o ha e a wide habi a ange. Fo ins ance,
S. k aussei has been ound in coni e ous and deciduous
o es s (M áÏcek e al., 1999; S eine , 1994; S ock e al.,
2000) and also in g asslands (S u han, 1999). These habi-
a p e e ences may e ec no only he dis ibu ion o
sui able insec hos s, bu also physiological and beha -
iou al needs ha equi e speci c niches(Kaya & Gaugle ,
1993;Hominick e al., 1996).
Wi h espec o He e o habdi idae,in o ma ionon habi-
a speci ci y widely indica es ha some species o his
amily a e p e alen in coas al sandy soils (G i n e al.,
1994; Yoshida e al., 1998; S ock e al., 1999). Howe e ,
o he su eys ha e indica ed ha H. bac e iopho acan be
ound and is widely dis ibu ed in u and weedy habi-
a s (S ua & Gaugle , 1994, S ock e al., 1996). Addi-
ionally, G i n e al. (1999) ound ha he I ish ype o
He e o habdi is, which is es ic ed o he coas al egions
o I eland and B i ain, also occu s in g asslandso Cen al
and No he n Eu ope.
In all hese co ela ions and associa ions wi h habi a ,
i is impo an o bea in mind ha ac o s such as sam-
pling size, seasonali y and spa ial dis ibu ion should be
aken in o accoun when eco ding and in e p e ing da a.
Ano he c i ical aspec ha needs o be ca e ully consid-
e ed is he co ec iden ica ion o he isola es which may
equi e he combina ion o di e en me hods (mo phol-
ogy, c oss-hyb idisa ion, molecula echniques) o a oid
e oneousassump ions.Res ic ion diges iono DNA am-
pli ed by he polyme ase chain eac ion (PCR) om he
DNA ITS space egion is a e y con enien and eliable
means o so ing new uniden ied isola es in o species
g oups. This echnique is no di cul o se up in an ecol-
ogy labo a o y and diagnos ic es ic ion p o les o se -
e al He e o habdi is and S eine nema species ha e been
published (Joyce e al., 1994b; Reid e al., 1997). Su -
cien ma e ial o PCR ampli ca ion can be ob ained om
a single in ec i e ju enileo youngadul and i is no nec-
essa y o ca y ou a DNA ex ac ion. A clea and com-
p ehensi e desc ip ion o he p o ocols o he molecula
cha ac e isa ion o EPN ia RFLP analysis o he DNA
ITS egion is gi en in Hominick e al. (1997). P o ocols
o de e mining biological species by c oss-b eeding a e
gi en in Poina (1967) and Akhu s and Bedding (1978)
o S eine nema spp. and in Dix e al. (1992) o He -
e o habdi isspp.
Fi e species o Xeno habdusha e been desc ibed (see
Table 3). Th ee o hese bac e ial species a e associa ed
wi h a single species o S eine nema bu X. bo ienii is as-
socia ed wi h ou (Akhu s & Boema e, 1988; Fische -
Le Saux e al., 1999a) and X. poina ii wi h wo nema-
ode species (Fische -Le Saux e al., 1999a). The genus
Pho o habdus consis s mos ly o he bac e ial symbion s
o He e o habdi is as well as some non-symbio ic clin-
ical isola es om human wounds (Fa me e al., 1989).
Sequence analysis o he 16S DNA gene o 40 s ains
o P. luminescens including ou clinical samples, indi-
ca ed ha P. luminescens was a he e ogeneousg oup and
also showed ha he clinical samples o med a closely
ela ed sub-clus e (Szallas e al., 1997). Fishe -Le Saux
e al. (1999b) ha e ecen ly e ised he axonomy o he
genus Pho o habdus and p oposed he c ea ion o wo
new species, P. empe a a and P. asymbio ica, and, u he ,
ha P. luminescens be di ided in o h ee subspecies. An
in e es ing ques ion ye o be add essed is he equency
o co-specia ion be ween he nema ode hos s and hei
36 Nema ology
Symposium
Table 3. Desc ibed species o bac e ial symbion s o en o-
mopha hogenic nema odes.
Genus: Xeno habdus Thomas & Poina , 1979
Type species: Xeno habdus nema ophila (Thomas & Poina ,
1979) Akhu s & Boema e, 1988
O he species:
X. piona ii Akhu s & Boema e, 1988
X. bo ienii Akhu s & Boema e, 1988
X. beddingii Akhu s & Boema e, 1988
X. japonica Nishimu a, Hagiwa a, Suzuki & Yamanaka, 1994
Genus: Pho o habdus Boema e, Akhu s & Mou an , 1993
Type species: Pho o habdus luminescens (Thomas and Poina ,
1979) Boema e, Akhu s & Mou an , 1993
P. luminescens luminescens Fische -Le Saux, Vialla d, B unel,
No mand & Boema e, 1999
P. luminescens akhu s ii Fische -Le Saux, Vialla d, B unel,
No mand & Boema e, 1999
P. luminescens laumondii Fische -Le Saux, Vialla d, B unel,
No mand & Boema e, 1999
P. empe a a empe a a Fische -Le Saux, Vialla d, B unel,
No mand & Boema e, 1999
O he species:
P. empe a a Fische -Le Saux, Vialla d, B unel,
No mand & Boema e, 1999
P. asymbio ica Fische -Le Saux, Vialla d, B unel,
No mand & Boema e, 1999
symbion bac e ia and he ex en o ho izon al and e ical
ans e o he symbion among he nema ode lineages.
Phylogene ic s udies o EPN
The e olu iona y ela ionships o EPN we e ou lined
o he s ime by Poina (1981) in his book The na -
u al his o y o nema odes, whe e he specula ed ha S ein-
e nema idae and He e o habdi idae a ose as wo sepa a e
lineages, a oughly he same ime in he mid-Palaeozoic,
some 375 million yea s ago. He also indica ed ha simi-
la i ies in hei mo phology,li e cycles and bac e ial sym-
biosis can be a ibu ed o con e gen e olu ion. Sudhaus
(1993) also concluded ha he simila i ies be ween He -
e o habdi isandS eine nemaa e basedon symplesiomo -
phic cha ac e s and con e gence.Poina (1993) sugges ed
po en ial ances o s o bo h amilies based on a li e a-
u e compila iono mo phological,biological,physiolog-
ical and dis ibu ionale idence.Examiningsimila i ies o
he buccal capsule and male ail mo phology,Poina sug-
ges ed ha he e o habdi ids e ol ed om a ‘Pelliodi is-
like ances o ’ in an a enicolous ma ine en i onmen , and
ha s eine nema idse ol ed om a ‘p o o-Rhabdi onema
ances o ’ in a e es ial en i onmen .
Se e al app oaches, bo h om he molecula and mo -
phologicalpe spec i es, ha e been used o s udy he e o-
lu iona y ela ionships o EPN. Reid (1994) and Reid e
al. (1997) s udied phylogene ic ela ionships o S ein-
e nema idaeand He e o habdi idaebased on RFLP analy-
sis o he DNA epea uni . Fo his s udy, 26 isola es
ep esen ing 11 S eine nema and h ee He e o habdi is
species we e conside ed. Addi ionally, wo habdi oids,
Caeno habdi iselegansand Phasma habdi issp. we e in-
cluded o ou g oup compa isons. The ela ionships be-
ween S eine nemaspecies de e minedby es ic ionmap-
ping, mi o ed (in gene al e ms) hose o he mo pholog-
ical da a. Fo example, S. a ena ium and S. glase i, wo
mo phologically and biologically simila species, we e
clus e ed oge he . This analysis also showed a close ela-
ionshipbe ween S. ca pocapsaeand S. scap e isci, which
was o iginally e e ed o as he U uguay s ain o S. ca -
pocapsae (Nguyen & Sma , 1988). Reid e al. (1994)
also showed ha membe s o he amily He e o habdi i-
dae we e mo e closely ela ed o one ano he han was
he case wi h membe s o he S eine nema idae, he la e
g oup being much mo e he e ogeneous. This s udy also
showed ha he he e o habdi id and s eine nema id gen-
e a in es iga ed we e mo e closely ela ed o each o he
han o he wo o he habdi oidsused o ou g oup com-
pa isons.
O he app oachesha eincludedei he combinedanaly-
ses o mo phologicaland RAPD agmen s (Liu & Be y,
1996b),o nucleo idesequence analyses o a ious DNA
egions such as he 18S DNA sequences (Liu e al.,
1997), he ITS-1 space egion (Adams e al., 1998) and
also he ND4 sequenceso mi ochond ialDNA (Liu e al.,
1999). Liu e al. (1997) in e ed phylogene ic ela ion-
ships among bo h amilies o EPN using sequence da a
om pa o he 18S DNA gene. Se en een isola es o
EPN (12 desc ibed and e undesc ibed species) and six
Rhabdi idae axa ha includedRhabdi ellaaxei,Rhabdi is
spp. and ou Caeno habdi isspecies. As in Reid’s (1994)
s udy, Liu e al. (1997) also ound ha s eine nema ids
had mo e sequencedi e gence han he e o habdi ids.This
obse a ion ag eed wi h he documen ed mo phological,
biological and dis ibu ional e idence. Al hough he e-
la ionships o se e al S eine nema and He e o habdi is
species we e no well suppo ed in hei cladis icanalysis,
nal in e p e a ion o hei phylogene ic s udy indica ed
Vol. 2(1), 2000 37
Symposium
ha S eine nema idae and He e o habdi idae a e wo in-
dependen monophyle icg oups.
Phylogene ic ela ionshipsamong cu en ly ecognised
He e o habdi is species we e s udied by Adams e al.
(1998) based on 18S DNA sequences. The ela ionships
among axa we e well es ablished, bu lack o di e -
gence wi hin h ee lineages o sis e axa (H. ma ela us
+H. hepialius;H. indica +H. hawaiiensis;H. bac e-
iopho a +H. a gen inensis) sugges ed conspecici y. In
suppo o his conclusion, a mo phological e-examina-
ion has al eady led o synonymisa ion o H. ma ela us
and H. hepialius (S ock, 1997). The He e o habdi isphy-
logenyo Liu e al. (1999)based on he ND4 m DNA gene
is b oadly in ag eemen wi h ha p esen ed by Adams e
al. (1998). The s udy o Adams e al. (1998)also indica ed
ha he ou g oup axon Pelliodi is was mo e closely e-
la ed o He e o habdi is han o Caeno habdi isand S ein-
e nema.
Blax e e al. (1998) also in es iga ed he phylogene ic
ela ionships o EPN in hei molecula amewo k o he
phylum Nema oda. Based on he analysis o 18S DNA
sequences, heyconcludedhe e o habdi idsand s eine ne-
ma ids do no sha e a common ances y. Thei s udy indi-
ca ed ha He e o habdi is was associa ed wi h S ongyl-
ida and S eine nema was mo e closely ela ed o Pana-
g olaimidae and S ongyloides. An ongoingphylogene ic
s udy (S ock e al., unpubl.) based on a combined analy-
sis o mo phologicaland molecula cha ac e s (28S DNA
sequences) o 24 S eine nema and h ee He e o habdi is
species, sugges s ha he S eine nema idae cons i u e a
pa aphyle ic g oup, and ha membe s o he amily He -
e o habdi idaeseem o ha e e ol ed wi hin he S eine ne-
ma idae.
This summa y o esea ch on he phylogene ic ela-
ionships o EPN shows he e is con adic o y e idence
on he ela ionships among hese wo amilies o EPN.
While some au ho s indica ed ha he e o habdi ids and
s eine nema idsha e e ol edas wo sepa a e independen
lineages (Liu e al., 1997; Adams e al., 1998; Blax e e
al., 1998), o he s sugges ed hese wo amilies a e ei he
sis e axa (Reid, 1994), o ha e e ol ed oge he (S ock
e al., unpubl.). Incong uencebe ween hese s udies may
be a ibu ed o many causes, including homoplasy, low
esol ing powe o he echniques used, o use o ee
building algo i hms wi h di e en e olu iona y assump-
ions. The e o e, igo ous examina ion o EPN species,
wi h mo phologicaland biologicals udiesandsequencing
o mo e genes, is encou aged o u he assess obus phy-
logene ic ela ionshipsamong his g oup o nema odes.
Concluding ema ks
The impe us o esea ch in EPN and hei symbion s
has come abou because o hei biological con ol po-
en ial, so much o he ocus in EPN esea ch has been
on applied aspec s ela ing o pes con ol (see Gaugle &
Kaya, 1990;Beddinge al. 1993 o e iews on hese op-
ics). Howe e EPN and hei symbion s a e inc easingly
being iewed as an exci ing subjec o basic esea ch in
ecology, biodi e si y,e olu ion,biochemis y and molec-
ula gene ics. The bac e ial symbion s p oduce no el in-
sec icidal oxins, an ibio ics and exoenzymes, bu many
o hese bac e ial species and s ains a e s ill unexplo ed.
The molecula in e ac ions be ween EPN and hei sym-
bion bac e iawhich enable he nema odes o packageand
ansmi he bac e ia a e s ill la gely unknown. EPN be-
long o he same amily as C. elegans whose genome has
been ully sequencedandanno a ed.Like C. elegans, hei
genomesize is small (G enie e al., 1997).EPN also ha e
he ad an age ha hey can be g own in i o on lipid aga
pla es and a e ex emely p oli c. They a e easy o isola e
om soil by bai ing wi h suscep ible insec la ae hus a-
cili a ing s udies in biogeog aphy and habi a p e e ence
(Bedding & Akhu s , 1975). In he 10 yea s since he s
in e na ional mee ing on EPN a Asiloma (see p oceed-
ings edi ed by Gaugle and Kaya, 1990), much p og ess
has been made in ou unde s andingo he basic biology
and gene ics o EPN and hei symbion s. We a e now en-
e ing a new phase in which he ools o molecula gene -
ics a e being inc easingly used o add ess a ange o bio-
logical ques ions in EPN esea ch. The knowledge gained
om his endea ou should ensu e ha EPN will become
e en mo e e ec i e biopes icides and should also ensu e
ha EPN and hei symbion s gain p ominenceas unique
and in insically in e es ing biological sys ems.
Acknowledgemen s
Wo k in he au ho s’ labo a o ies is suppo ed by he
Eu opean Communi y (STD-3 P og amme Con ac s TS3
CT94-0273 and FAIR CT 97-3116, AMB) and by he
Eppley Founda ion o Resea ch (SPS).
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Vol. 2(1), 2000 39
