Heterorhabditis, Steinernema and their bacterial symbionts - lethal pathogens of insects

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 ica 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 ica 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 ied 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 conspecici 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). Re e ences ADAMS, B.J. (1998). 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