Satellite DNA sequences as taxonomic markers in nematodes of agronomic interest

Techniques

Satellite D N A Sequences as Taxonomic Markers in Nematodes of Agronomic Interest E. G r e n i e r , P C a s t a g n o n e - S e r e n o

The success of alternative crop protection practices against plant-l~irasilic nematodes using host resistance g~'nes depe~Ids fimdamentally upon identification of the species and pathe~yl,~ ~ffectively controlled by these genes. In the same way, biological control of insects by e~tomopathogenic nenu~todes will zvork only if the nematode straias used are indeed active against the pests to be elhnh~ated. For these apphcat:ons, the accurate interspecific and/or bttraspecific identification of nematodes is thus of outstanding importance. Here, Eric Grenier, Philippe Castagnone-Sereno and Pierre Abad discuss the recent use of satellite DNA sequences i~, n~natode taxonomic diag,ostics. Nematode species can generally be identified in a re~able and sensitive fasb2on using morphological analysis. However, this identification is highly clependent on the experience and interpretive skill of the investigator. In species of a~'icultural interest, juvenile identification is partic-alarly important, because it represents the infective stage. This is the rule for mos~ plant-Farasific ne~atodes and for entomopathogenic nematodes, in which juveniles are the only free-living stage present in the soil. The value of recombinant DNA techniques in identifying nematodes has been established since the early 1980s. The first studies were based on m~alysis of restriction fragment length polymorphism (RFLP)L Although RFLP anaiy~.~s allowed clear discn;nination at both interspecific and intraspecific levels, they could not be used routinely because they were time-consuming and required large amounts of DNA. The recent development of the polymerase chain reaction (PCR) technique, which avoids both of these inconveniences, has offered new diagnostic perspectives. Electrophoretic analysis of the amplified regions, either known leg. mitochondrial (mtDNA) or ribosomal (rDNA) sequences2.3] or unknown leg. when using the random amplified polymorphic DNA (RAPD) procedure4-~], has revealed both interspecific and intraspecific variability. However, .Aone of these approaches provides specific unambiguous results useful fcr ~outine diagnostic purposes fie. DNA amplification restricted to a single species) Very recently, repeated element,,: known as satellite DNAs have been isolated from both plantparasitic and entomopathogenic nomatodes, and have proved to be species-specific~ ~. Do these highly repetitive sequence families fulfil the requirements for use in nematode idek~tification? Our present knowledge suggests satellite sequences are aa excellent tool for particular problems of species deterr~ination. Eric C.¢enierPhilippeCastagnone-Serenoand Ph~n~ a~ at the INR/~ Laboratoirede Biolog~ des In,vefLeD~S, 2078.06606Ant]besCedex.Fvar,:e."Tel:+33 493 678 943, Fax: .i.33493 6?8 9S1:,e.maiJ: ~ i m ' a . f r

a n d P. A b a d

Genomic organization and evolution of satellite DNA families Highly repetitive satellite DNAs are a characteristic component of the genome of almost all eukaryotic organisms. These sequences are organized as long arrays of tandemly repeated elements. Each family differ,,; by either the length of the array or its sequence. The repeats are clustered at a few loci per genome, where they are estimated to be present from 103 to 105 timesI°. Despite extensive attempts to elucidate the biological role of satellite DNA, no conclusion has yet been reached concerning any possible function. For years, the most widespread opinion was that satellites simply consisted of 'junk" DNA, lacking any ftmctionu. Although the f'.nction of these sequences remains unclear, they are often found at cenh-omeres, where they may be involved in mitotic and meiotic events, and at other chromosomal locations where they may con~'ibute to chromosomal folding 12. Satellite DNA is most curious with respect to its intraspecific seque~.ce conservation and its special mode of evolution. Studies of such repeated sequences present in closely related species have shown that they often evolve in a concerted manner, resulting in a higher degree of interspecific than intraspecific sequence variation13. Therefore, they might play a role in speciation of organisms with large genomes, with the evolution of large new clusters being responsible for a considerable lack of chromosome homology between species. One case has been reported where satellite DNA may be present in all species of a genus ~4. Other satellite DNA families may be restricted to a specific phylad of related species 15,16 or are amplified to a high copy number in one species but appear only in moderate copy numbers in closely related spe cles " 17• -Thor~ e a-r e a number of cases in which satellite DNAs have been found to be speciesspecific, including nematodegenomes6-9,18, where many DNA families cannot be detected even in sibling species9. All these examples support the theory of a very high rate of evolutionary change in satellite DNAs compared with repeated gene families. Since most satellite DNAs are tandemly arranged in the genome and exhibit a very high degree of sequence homogeneity, they can be easily detected in many cases by restriction analysis of genomic DNA and subsequent electrophoresis. The randomly distributed base changes that may affect restriction ~ites lead to a typical ladder pattern because of some uncleaved sites (Box 1). Satellite D N A as a d i a g n o s t i c marker The application of satellite DNA in taxonomic diagnostics may be illustrated by two examples of agronomic interest from both entomopathogenic and plant-parasitic nematodes. Identification of Steinernema and Heterorhabditis spp. Entomopathogenic nematodes of the genera Parosttology toclov, vol. 13. n:',. I0. •997

Techniques i Box 1. D e t e c t i o n of a Satellite DNA Family by Restriction Analysis of Genomic DNA ....

:I~'on .

.

a .

.

.

.

b .

.

.

.

~

I

Evolution

~onorneHc unit

..~

.

..

.

, .llt

I /

.

.

I

.

,

.

.

, ___~_ units

i

C

etc. I

.

u2n~l I

m i

1

etc.

3 units 2 units 1 unit

,91._. The rnonomer,c un~

In ,'l~egenome of most eukaryotes, satellite DNA is organized as long arrays of tandemly repeated elements (mov,ox'c,e]~) containing a conserved restriction site (a). During evolution, ra,,~.omly distributed base changes may affect these restriction sites (b). Digestion with the appropriate endonuclease wi|; thus theoretically re]ee,se monomers and multimers of the repeated element because of uncleaved sites (c). However, in practice, un!e . there ~ a very high genomic contelit of the saf:ellite, only the monomeric unit can be seen on ethidium bromide-stmned gels after electrophore-3is (d). FoUow-ing purification of the monomer, its use as a probe in a Southern blol experiment will lea~t to a typical ladder pa.P,ern (e).

Steinernema and Heterorhabditis are lethal parasites of insects. Because of their very wide host range, they are being used as biological control agents against a n u m b e r of economically important crop pests. Nematodes act by gaining entry into host insects, usually via natural openings, and kill them within two days. As the various species and strains differ in their effectiveness, rapid a n d accurate identification is necessary for use of these nematodes as biological control agents. Until now, species identification in these two genera has been based mainly on analysis of morphological features and on interbreeding tests. From that point of view, species identification has been a difficult and time-consuming task - especially for Heterorhabditis s p p - even for well-qualified taxonomists. Indeed,

a

b I

DD136 I

m a n y isolates remai,~ unassi;~ned at the species level in both genera. Using species-spedfc sateillte DNAs isolated from S. carpocapsae, H. bacteriophor.~ and H. indicus, it was possible to identify most o~ the unassigned isolates tested so far (Fig. 1). 'Squ.asl',' blot hybridizations (in which a nematode ]s sirap;y squeezed on a nylon membrane; cstab!ished the worldwide predominance of H. bacter;opnura and the fact chat the H. indicus species is restricted ~.o tropical areas with warm climates sg. The tdgh resolution of such hybridizations enableci ,as to develop these satellite DNAs into spedfic nonradioacUve probes (E. Grenier e.~al., unpublished). Idenlification of ~Aeloidogyne spp. Species of the genus Meloh!ogyne are polyphagous and a m o n g the C

HB1

HB1

I I

H. baLteriophora

Breton

S. carpocapsae

HP88

API

-

H. bacteriophora

i

NC

S. glaseri

Coimbatore

T335

S. feltlae

GBF3

I

It. indicus

H. zeal~ndica

HP86

-

Coimbatore GBF3

I

H. indicus

H. zealandica

He !1. megidis He I1. megMis GR 8. krauuei Fig I. Specific identification of entomopathogenic nematode isolates using sacellit(." DNAs isolated frc:m Steinememo carpocapsee, Heterorhabditls bacteriophora and H. ind/cus. Dot blo= were hybridized with: the Hcefll element from $. carpocabsae (Breton strain) (a); the A/ul element from H. ~acter/ophoro (H.~8 strain) (b); and the Alul element from H. indicus (P2H strain) (c). (Adapted, with permission, from Ref. 19,) Pcsrositolo~y To,cloy. vul. 13, .io, l 0, 1997

399

Techniques Fig. 2. Applications of the Styl satelhte D N A from Meloidogyne hal)lo as a specific diagnostic marker: "Squash' blot hybridization with nematode(s) or infected root tissues (a); PCP,-specific amplification from single juveniles using primers deduced from the SWI satellite D N A sequence (b). (Adapted. with permission, from Refs 22. 23.)

u~

o~

o~

<<

<

~

~

~

~

~

i M. hap/a HP 1 M. chitwoodiP.J-D M, d~l~woodiCa M. halola ANZ

most destructive plant-parasitic nematodes. Crop loss d u e to Melaidogyne spp is estimated to be 5%, worldwide ~. In 1980, a n e w species was described, M . chitwoodi, which d a m a g e s potato g r e w n in the temperate climates of north-west USA and Europe 2i. Appropriate pest-management practice requires discrimination of M . chitwoodi fron'~ the sympatric s p e c i e s ~4. hapla, which cioes not sign:ficantly d a m a g e this crop. Meloidogyne hapla is morphologically indistinguishable from M chifwoodi. To oevelop a reliable and rapid diagnostic assay to discriminate between these two species, a 'squash' blot hybridization was designed, using a Styl s~tellite DNA previously cloned from M. hapla 22 as a specific probe. This assay p'e.rmitted u n a m b i g u o u s separation of M. hapla from M . cllitwoodi, with the main advantage of avoiding time-consuming D N A extracUons. Because of the highly repet;ti,,;e nature of satellite DNA, this sequence is able to detect one individual in a simple 'squash' blot hybridization, even in root tissues (Fig. 2.a). Detailed analysis of the variability of the M . hapla satellite D N A also allowed the selection of primer.~ that lead to a specu~c amplification signal in PCR experimentsz~, from a single individual at any developmental stage (Fig. 2b). In these two techniques, positive signals with satellite D N A prove the existence of the particular species, but the absence of a signal obviously cannot be taken as evidem:e of absence of the species, unless appropriate controls are included (ie. specific probes for the other spec.~es). Conclusions The two examples given above demonstrate that satellite DNA sequence~ m a y be powerful tools to show polymort:.hisra between closely related species for which specific markers are needed. Simila~ results have been obtained with other nematodes, such as the phytoparasite Bursaphelenchus xylophilus ~4, and Onchocerca ve,lvuhls, the causal agent of onchocerciasis 400

in h u m a n s 2s. These achievements do not m e a n that satellite D N A analysis guarantees solutions to all identificaUon problems involving closely related species, but satellite markers m a y be particularly useful where there is a need for a rapidly evolving taxonomic marker. Moreover, with the development of nonradioactive labelling or the selection of specific PCR primers, the experimental procedures become easy, safe and rapid to work with, a n d thus suitable for use u n d e r field conditions. As a consequence, satellite D N A markers should prove to be of a great interest for the successful implementation of pestm a n a g e m e n t strategies as well as for ecological and population s t u d i e s References

I Curran, J- el aL (1986) Genolypic differentiation of Meloida~jne populations by detection of restriction fragment length difference in to.*aiDNA.]. Nc;.:aloL 18,83-86 2 Powe,s, TO. and Harris, T.S.(1993)A polymerasechain reaction method for identification of five major Meloidosyne species. ]. Nematal. 25, I-6

3 Vrain, T. et aL (1992) Inlraspecific rDNA restriction fragment polymorphism in the Xiphinema americanmn group. Fundam. Appl. NematoL 85, ]23I-.1237 4 Caswell-Chen,E.P. et al. (1992) Random amplified polymorphi¢ DNA analysis of Helrodera cruclferae and H. schachtii populations.]. Nemalol. 24, 343-351 5 Cas~tgnonL~Sereno,P. el al. (1994) Genetic polymorphism bet~-~.~'~and within Meloidogyne species detected with RAPD markets t.~,ntn;te37, 904-909 6 Piotte,'C.et at ~.199,DCloning and characterizationof two satellite DNAs in the haw-C-valuegenome of the nematode Meloidngyne spp. Q'ae 138.175-180 7 Grenicr, E. et at. (1995) Charecteriza~on of a speciesspecific sat.~i~il~ DNA from the entomr,pathogenic nematodt Steinernema ea;'vocapsae. MoL Biochem.Parasit.q.69, 93-100 8 Grenier,E. el al, (1996)Molecularcharacterizationof two speciesspecific tandemly repealed DNAs from the enlomopaihogenic nematodes Steinenwma and tleterarhabdttis. Mol. Biodu'm. Para~itol. 83, 47-56 ~ Ta~'L,s,S. c~ at. (1993) Cloning and characlerlsationof an highly conserved ~iellile scquence specific for the phyioparasitic ~:ematodeButsaphelenchns xylophilus. G~*w 129,269--273 ll: F,etiz.de,T. (198.6) Satellile DNA, Springer-Vt "lag Porasit¢:lo,~y Today. voL 13. no. I0, 1997

Techniques 11 j~hn, B. and Miklos, C.L.C. (1985) Functional aspects of satellite DNA and heterochrt,matin, ha. I~. Ci/tol.58, l-114 12 Mildos,G.L.G. (1985)in ?dolecularEz~luthma~,Genetics(Mac[ntyre, R.J., ed.), pp 2": -37~,,Plenum 13 Dover, G,A. (1986) ."¢loleculardrive in multigene families: how biological novelties arise, spread and are assimilated. Trends Genct.2,159-165 14 Vignali, R. et at. (1991) T w o dispersed hi~hly repetitive DNA families of Tritnrus vulgarls meridionalls (Amphibia, Urodelal are widely conserved among Salamandridae. Chramosoma lt~, 87-96 15 Cremisl, r,. "et al. (1988) Heterachromafic DNA in Triturus (Amphibia, Urodela). I1. A centromeric satellite DNA. Chromoaoma97, 204-211 16 Tar~, S. et al. (1993) Characterization of :n unusually conserved AInl highly reiterated DNA sequence fasf,ily from the honeybee. Cot,netlcs134,1195-2004 17 Bachmann, L. et al. (1990)Evolution of a telomere associaled satellite DNA sequence in the genorae of Drosophila trisiis and related species. Genetica83, 9-16 18 Meredith, 5.E.O. ct a!. (1989) Cloning and .:haraclerisatlon of an Onchocerca voh,ulus specific DNA seq;.lence. MoL Bh~ch~Tn.

Parasihd.36,1-10 19 Grenier, E. et al. (1996) Use of sl:~'k's-specifie satellite DNA as diasnostic probes in the identification of Steincrnematidae and Heterorlmbditidae entonlopathogenlc nematodes ParasikdokW.113, 487~t89 20 S,asser, I.N. and Carter, C.C. (1985) in An zhl~:anct'dTrvati~, or! Meloidogyne, Vol It. Bialogyand Contr,d (Sasser. I.N. and C,arter, C.C., eds), pp 19--24,North Carolina State Universi~ Graphics 21 Ferris. H. et at (1993)Host status of .';electedcrops to Meloido~yne

chitwoodi. ]. Nemalol.25, 849-857

22 Piette, C. ct al. (1995) Analysis of a ~tellite DNA from l¢leloidogyne hapla arid its use as a dla~aaostic probe. Phyt,~pathology85, 458-462 23 Castab,none~Sereno, p. et al. (1995) Satellite DNA as a larget for PCR-.sped6c deice°don of LI!.e ~|ant-parasltle nematode

Meloidogyne hapla. Cl!rr.Get~et.28, 566-¢,70

24 Ta~;zs, S. t.t al. (1994~ Use of sl~*~'ief.-speeific~tellite DNA from Bu~aphdenchus xylophilus as a dizgnostie probe. Phytol~atholot~/ 84, 294-298 25 Meredith, S.E.O. et al. (1991) Onchocerca volvuluc,: application of the polymerase chain reaction to identification and strain differentiation of the parasite, Exp. l'arasitol.73, 335-344

The ICT Filariasis Test: A Rapid-format Antigen Test for Diagnosis of Bancroftian Filariasis G.J. V V e i l , P.J. L a m m i e

Antigen testing is now recognized as tlw method of choice for detection of W u c h e r e r i a bancrofti infections. Unlike tests that detect microfilariae, antigen tests can be per]ormed with blood collected during ttle day or night. However, existing enzyme-linked imm~;nosorbent assay (ELISA) tests for filarial atttigenen:ia are difficult to perform in the field, attd this has Ihnited their use in endemic countries, h~ this article, Gary Well, PatricL Laramie and N ~ g i Weic.~ reviezo their experience with a n(~v rapM-for,tat filarial a,:tigen test. Thai found that the ICT card test was vert/ easy to perform and that it was comparable with ELlSA for the detection of filarial antigen ;:t ~era from people with microfilar¢'mia. The introduction now of an attti~en test suitable for use in the field is especially timely, in that it may facilitate hnplementation of nezo strategies proposed by the World Health Organization for control and elimination of lymphatic filarhtsis. L y m p h a t i c filariasis is a d e f o r m i n g parasitic disease that affects o v e r 100 million p e o p l e in m o r e than 70 tropical a n d s u b t r o p i c a l countries L2. Most l y m p h a t i c filariasis is c a u s e d b y the mosquito-born,: n e m a t o d e , Wuchereria baJtcwfti. Infections m a y be a s y m p t o m a t i c , b u t often they are associated w i t h acute s y n d r o m e s such as l y m p h a n g i t i s w i t h fever a n d w i t h chronic :'~=P/|. Weft is at Washin~on Unlver~ity Schooi of Mediclnc aud Barnes-JewlshHospital. 2 J6 South Kingshighway.St Lows. MO 631 I0, USA. PatdckJ. Laramie i~ at the Division of Parasit,c Diseases,Centers for DiseaseControl and Prevention.Atlanta, GA 30341-3628, USA. Ni,~i Weiss is at the SwlssTropical Institute. Basle. Switzerland.Tel: + I 314 454 1782, Fax: +1 314 454 5293, e-mail: [email protected],edu

and N. Weiss

c o m p l i c a t i o n s such as h y d r e c e l e s in m e n a n d the m o s t d r e a d e d o u t c o m e of infection, p h y s i c a l l y a n d see:ally d i s a b l i n g e l e p h a n t i a s i s of the extremities. Traditional con~ol presTon-re for l y m p h a t i c filariasis h a v e celled p r i m a r i l y o n a n t i m e a q u i t o m e a s u r e s a n d o n a d m i n i s t r a t i o n of d i e t h y l c a r b a m a z i n e (DEC) thera p y o i fleeted a n d / o r diseased p e r s c n s identified in a c t i v e , creening p r o g r a m s . W h i l e filariasis has been c o n t r o l k : : and e v e n e l i m i n a t e d in s o m e areas w i t h these metr~ods, control efforts in m a n y settings h a v e o~ly stabilized infection rates or lost g r o u n d . H o w ever, n e w tools for d i a g n o s i s a n d t h e r a p ¢ of filariasis a n d n e w control strategies h a v e recently re-energized the s t r u g g l e a g a i n s t filariasis~-3 and, finally, the tide m a y be t u n ' i n g a g a i n s t this difficult disease. D i a g n o s t i c o p t i o n s for b a n c r o f t i a n filariasis Efficient d i a g n o s i s of W, balzcrofti infection is esp e c b : l y i m p o r t a n t as control p r o g r a m s m o v e t o w a r d s the new s t r a t e g y of c o m m u n i t y d i a g n o s i s and repeatPd, a n n u a l m a s s t h e r a p y witi~ singie-dc'~e DEC or c o m b i n a t i o n r e g i m e n s I's. Several m e t h o d s are available for d i a g n o s i s of bancroftian filariasis (Table 1). Clinical d i a g n o s i s is labor-intensive, insensitive and not specific for active infection. Parasitological metho d s for d i a g n o s i s d e p e n d o n detection ef microfilariae (Mr) in p e r i p h e r a l blood. The sensitiv,ty of Mf detection d e p e n d s on the v o l u m e of blo~×l s a m p l e d , the t i m e of blood collection (because of nocturnal periodicity of microfilaremia in m o s t e n d e m i c areas) a n d the skill and dedication of the microscopist. Microscopybased d i a g n o s t i c m e t h o d s 4, w&ile time-honored and 'low-tech', are relatively difficult p r o c e d u r e s in