Use of Random Amplified Polymorphic DNA (RAPD) for generating specific DNA probes for Oxyuroid species (Nematoda)

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Use of Random Amplified Polymorphic DNA (RAPD) for generating specific DNA probes for Oxyuroid species

(Nematoda)

E. Jobet, M. E. Bougnoux, Serge Morand, Colette Rivault, Ann Cloarec, J. P.

Hugot

To cite this version:

E. Jobet, M. E. Bougnoux, Serge Morand, Colette Rivault, Ann Cloarec, et al.. Use of Random Ampli- fied Polymorphic DNA (RAPD) for generating specific DNA probes for Oxyuroid species (Nematoda).

Parasite, EDP Sciences, 1998, 5 (1), pp.47-50. �10.1051/parasite/1998051047�. �hal-01320079�

USE OF RANDOM AMPLIFIED POLYMORPHIC D N A ( R A P D ) FOR GENERATING SPECIFIC D N A PROBES

FOR OXYUROID SPECIES (NEMATODA)

JOBET E.*^,**, BOUGNOUX M.-E.*, MORAND S.**, RIVAULT C.***, CLOAREC A.*** & HUGOT J.-P.****

Summary :

Random amplified DNA markers (RAPD; Williams et al., 1990) were used to obtained specific RAPD fragments characterising different species of oxyuroids. We tested six species of worms parasitizing vertebrates or invertebrates: Passalurus ambiguus Rudolphi, 1819, parasite of Leporids; Syphacia obvelato (Rudolphi, 1802) Seurat, 1916, a parasite of rodents; Blatticola blattete (Graeffe, 1 860) Chilwood, 1932 parasite of the cockroach Blattella germanica; Hammerschmidtiella diesingi (Hammerschmidt, 1 838) Chitwood, 1932 and Thelastoma bulhoesi (Magalhaes, 1990) Travassos, 1929, parasites of the cockroach Periplaneta americana, and an undescribed parasite species of a passalid insect from New Caledonia. Among 15 oligonucleotides tested, nine produced several specific bands allowing the interspecific discrimination.

KEY WORDS : RAPD-PCR, Oxyuroidea, Nematode, taxonomidentification.

INTRODUCTION

Oxyurid n e m a t o d e s are m o n o x e n o u s parasites o f vertebrates and invertebrates (Adamson, 1 9 8 9 ; Morand et al., 1 9 9 6 ) . Their t a x o n o m y and p h y l o g e n e t i c relationships still remain confused, particularly for invertebrate parasites (Adamson, 1 9 8 9 ; Adamson & Van W a e r e b e k e , 1 9 9 2 ) , and w e suspect numerous c a s e s o f synonymy. T h e s e parasites have a h a p l o - d i p l o i d m o d e o f r e p r o d u c t i o n a n d s h o w a female-biased sex-ratio (Adamson, 1 9 8 9 ) . Males are often very small and rare. Moreover, in the c a s e o f c o n g e n e r i c s p e c i e s infecting the s a m e host s p e c i e s , such as Dermoptera oxyuroids (Hugot, 1 9 8 6 ) , tortoises oxyuroids (Petter, 1 9 6 6 ) or s o m e invertebrate oxyuroids

* Laboratoire de Parasitologie-Mycologie, Hôpital Ambroise-Paré, 9, avenue Charles-de-Gaulle, F-92104 Boulogne-Cedex.

** Laboratoire de Biologie Animale (UMR 5555 du CNRS), Centre de Biologie et d'Écologie Tropicale et Méditerranéenne, Université de Perpignan, F-66860 Perpignan Cedex.

*** Laboratoire d'Ethologie (UMR 6552 du CNRS) Université de Rennes 1. Campus de Beaulieu, F-35042 Rennes Cedex.

**** Laboratoire de Biologie parasitaire (URA 114b CNRS), F-75231 Paris Cedex 05.

Correspondence : Edouard Jobet.

Email: Digene@univ-perp.fr - Fax : 04 68 66 22 8 1 .

Résumé : UTILISATION DES R A P D (RANDOM AMPLIFIED POLYMORPHIC D N A ) POUR GÉNÉRER DES MARQUEURS SPÉCIFIQUES D'ESPECES D'OXYURES (NEMATODA)

La technique du RAPD (Random Amplified Polymorphic DNA;

William et al., 1990) a été utilisée pour caractériser différentes espèces d'oxyures. Nous avons testé six espèces d'oxyures de vertébrés ou d'invertébrés : Passalurus ambiguus Rudolphi, 1819, un parasite de léporidés ; Syphacia obvelata (Rudolphi, 1802]

Seurat, 1916, un parasite de rongeurs ; Blatticola blattae (Graeffe, 1 8 6 0 ) Chitwood, 1932, parasite de la blatte Blattella germanica (L) ; Hammerschmidtiella diesingi (Hammerschmidt,

1838) Chitwood, 1932, et Thelastoma bulhoesi (Magalhaes, 1990} Travassos, 1929, parasites de la blatte Periplaneta americana, et une espèce non décrite d'un passalide de Nouvelle- Calédonie. Sur les 15 amorces testées, neuf ont produit plusieurs bandes spécifiques permettant la différenciation interspécifique.

MOTS CLES : RAPD-PCR, Oxyuroidea, Nematoda, identification taxonomique.

(Adamson & Noble, 1 9 9 2 ) , it is difficult to assign a female to its species. More difficulties arise w h e n taxo- n o m i c identification is b a s e d on individuals o f o n e s e x only. T h e use o f molecular technics could h e n c e b e helpful to resolve such t a x o n o m i c p r o b l e m s (Bandi et al., 1 9 9 3 ; C h a c o n et al., 1 9 9 4 ; Andrews et al., 1 9 9 5 ; Humbert & Cabaret, 1995). Our aim was to test the use o f RAPD markers m e t h o d for resolving t a x o n o m i x problems by applying this method to six oxyuroid spe- cies: t w o s p e c i e s from vertebrate hosts and four from invertebrates.

MATERIAL AND METHODS

PARASITE RECOVERY

Passalurus ambiguus ( 1 1 females and 10 males) w a s o b t a i n e d from Oryctolagus cuniculus ( L a b o r a t o r y M N H N ) , Syphacia obvelata (50 females and o n e male) from Mus domesticus (Labo-

r a t o r y M N H N ) , Hammerschmidtiella diesingi ( 2 5 females and nine m a l e s ) and Thelastoma bulhoesi ( 4 3 females and two m a l e s ) from the c o c k r o a c h Per- iplaneta americana (originated from o n e population in Paris), Blatticola blattae ( 9 3 females and 13 m a l e s ) from the c o c k r o a c h Blattella germanica (originated Article available athttp://www.parasite-journal.orgorhttp://dx.doi.org/10.1051/parasite/1998051047

JOBET E., BOUGNOUX M.-E., MORAND S., RIVAULT C , CLOAREC A. & HUGOT J.-P.

from two populations in R e n n e s and o n e population in Paris) and an undescribed s p e c i e s (five females) from a New-Caledonian Passalid.

Parasites w e r e c o l l e c t e d from the c a e c u m o f e a c h host s p e c i e s , carefully w a s h e d in physiological saline and in t a m p o n T E [10 mM TrisHCL pH 8, 1 mM EDTA]

before b e i n g stored and c o n s e r v e d at - 8 0 °C.

For e a c h parasite sample, c e a c u m fluid w e r e recovered and stored as m e n t i o n e d a b o v e .

Males and females n e m a t o d e w e r e identified with c o n f i d e n c e to their m o r p h o l o g i c a l c h a r a c t e r s , a n d according to Basir ( 1 9 5 6 ) and A d a m s o n & van W a e - r e b e k e ( 1 9 9 2 ) .

D N A EXTRACTION

W e u s e d a modified protocol from Barrai et al. ( 1 9 9 6 ) . DNA from c a e c u m sample fluid w a s e x t r a c t e d follo­

wing the s a m e procedure.

P C R AMPLIFICATIONS

Fifteen oligonucleotides w e r e u s e d for the amplifica­

tion o f random DNA markers. Primer s e q u e n c e s were as follows:

RP2 5'-AAGGATCAGA-3' ; V G 1 5'-ACGTATCTGC-3';

MNH1 5'-ACGTCTATGC-3'; R 1 0 8 5'-GTATTGCCCT-3';

RP4-2 5'-CACATGCTTC-3'; R 2 8 5'-ATGGATCCGG-3' ;

Primers RP2 MNH1 R28 OPAXS OPB04 OPGB SB2 OPB11 OPA9

Concentration in MgC12 2,5 3,5 3,5 1,5 3,5 2,5 1,5 2,5 2,5

Species

P. ambiguus 1 050 875 1 050 1 400 400 1 250

620 800 500 320 700

600 150 450

575 390

S. obvelata 1 100 1 050 1 150 950 1 550 850 790 900

900 1 090 650 1 400 800 500 820

550 1 000 400 1 320 550 450 800

450 850 350 1 260 500 680

440 800 925 600

750 625 560

700 570 520

350

H. diesingi 1 700 1 200 1 090 1 250 1 250 1 700 1 900

850 950 950 1 100 600 1 550 800

780 850 900 800 500 1 120 675

680 675 750 575 270 1 050 450

450 625 500 500 600 440

480 450 150 575

300 375 525

T. bulhoesi 1 750 1 950 1 250 1 600 825 1 700

1 600 1 250 1 050 1 300 750 1 500

1 350 875 800 1 200 700 1 100

920 775 660 900 480 1 000

770 650 525 425 850

490 450 375 325 710

475 375 350 450

300

B. blattae 1 100 1 400 700 1 250 1450 1 150 700 950

790 925 550 700 700 910 590 650

580 725 360 450 225 890 510 560

450 350 750 475 500

275 290 400

Undescribed species 1 300 1 600 850 1 000 1 250 900

of a passalid insect 600 1 150 590 600 1 150 775

350 700 520 700

250 575

500 475

Table I. — Specific bands generated by nine primers for each species of oxyuroids (optimal concentration in MgC12, from 1.5 to 3.5 mM, was determined for each primer).

4 8 Mémoire Parasite, 1998, 5, 47-50

SPECIFIC D N A PROBES FOR OXYUROID SPECIES

Figs 1-2. — Interspecific differences among 6 oxyurid species revealed with the R28 primer.

Fig. 2. — Culumn M: marker with a lOOPairBaseLadder molecular weight; column 8: T. bulboesi (7 gravid females); column 9: H. die- singi (10 gravid females); columns 10 and 11: T. bulboesi (10 and 5 gravid females respectively); columns 12, 13 and 14: B. blattae (19, 21 and 2 gravid females respectively).

Fig. 3- — Intraspecific differences among 6 B. blattae ( 6 females) with R28 primer.

Column M: DNA-£co RIplusHin DIII marker with a X molecular weight; columns 1 to 6: 6 different females of B. blattae.

Fig. 4. — Intraspecific differences among 5 B. blattae (5 males) with RP2 primer.

Column M: marker with a lOOPairBaseLadder molecular weight;

columns 1 to 5: 5 different males of B. blattae.

O P A X S 5 ' - A G T G C A C A C C - 3 ' ; O P B 0 4 5 ' - G G A C T G - G A G T - 3 ' ; O P B 1 1 5 ' - G T A G A C C C G T - 3 ' ; S B 1 5 ' - A G G T C C C T G C - 3 ' ; S B 2 5 - T G C A C C C T G C - 3 ' ; O P G B 5'- G A G C C C T C C A - 3 ' ; O P A 9 5 ' - G G G T A A C G C C - 3 ' ; A2 5 ' - T G G T C G C G G C - 3 ' ; NS33 5'-GCCAGCAGCC-3'.

T h e PCR reaction was carried out in a volume o f 25 pi containing 10 uM Tris HC1 pH 8.3, 5 0 m m KC1 1U o f

Taq DNA polymerase ( B o e h r i n g e r M a n n h e i m G m b H , G e r m a n y ) , 2 0 0 uM o f e a c h dNTP (dATP, dCTP, dGTP, d T T P ) , 5 0 p m o l e primer and a final concentration o f MgC12 d e p e n d i n g o f the primer used. From 25 to

50 ng total n e m a t o d e DNA was used as a template for the PCR.

T h e PCR cycle w a s carried for 3 0 s e c o n d s at 9 4 °C, for 3 0 s e c o n d s at 3 6 °C and for 7 5 s e c o n d s at 7 2 °C for a total o f 4 4 c y c l e s followed by an e x t e n s i o n poly- merisation reaction o f five minutes at 72 °C. Amplifi- cation was performed in a Perkin Elmer ( 9 6 0 0 ) thermo cycler.

T h e amplified DNA fragments resulting from PCR were analysed directly o n 1.5 % agarose gels by ethidium bromide staining (0.5 u g / m l ) . T h e gels were run in T B E Fig. 1. — Column M: DNA-Eco RIpIusHin DIII marker with a X mole-

cular weight; column 1: P. ambiguus (5 gravid females); column 2:

P. ambiguus (10 males); column 3: DNA gut fluid of P. ambiguus host; column 4: S. obvelata (4 gravid females); column 5: S. obve- lata (4 non-gravid females); column 6: DNA gut fluid of S. obve- lata host; column 7: Passalidae oxyurids (5 gravid females).

J O B E T E . , B O U G N O U X M . - E . , M O R A N D S., R I V A U L T C , C L O A R E C A . & H U G O T J . - P .

I X ( B i o P r o b e ) at a constant voltage ( 1 2 0 v ) a n d p h o - tographed. T h e m o l e c u l a r sizes o f the fragments w e r e determined using the 100 b p DNA Ladder (Pharmacia B i o t e c h , USA) o r the A,DNA-£coRIplusHindIII ( B o e - rhinger, M a n n h e i m ) as references.

Amplifications w e r e performed twice to asses repro- ducibility a n d blanks w e r e d o n e without DNA tem- plate. T o detect potential contamination o f n e m a t o d e DNA w e performed RAPD e x p e r i m e n t s DNA sample extracted from host digestive fluid.

RESULTS

T

DNA patterns o f host gut fluid control differed c o m - pletely from nematode DNA patterns e x c e p t in the case o f P. ambiguus for w h i c h a n u m b e r o f fragments w e r e similar in both control a n d DNA template.

Interspecific differentiation w a s easily assessed. Nine o f 15 oligonucleotides assayed revealed u n a m b i g u o u s profiles ( T a b l e I ) . Each o f these primers generated pat- terns that w e r e specific o f e a c h n e m a t o d e s p e c i e s . An e x a m p l e is given for primer R 2 8 (Figs 1, 2 ) . Similar results w e r e o b t a i n e d either b y using individual o r p o o l e d n e m a t o d e s .

Intraspecific variability w a s l o w a n d c o n c e r n e d n o n specific bands as s h o w e d in Figures 3 and 4 . Patterns o b t a i n e d b y RAPD m e t h o d for B. blattae from diffe- rent origins w e r e similar.

DISCUSSION

T

Patterns obtained for males and females w e r e identical.

Therefore, this method is appropriate for resolving pro- b l e m s o f t a x o n o m i c identification either in the c a s e o f s y n o n y m y o r w h e n only m e m b e r s o f o n e s e x ( g e n e - raly females) w e r e collected from their hosts (Adamson

& Noble, 1 9 9 2 ) .

W e r e c o r d e d a similarity in patterns g e n e r a t e d b y RAPD b e t w e e n P. ambiguus a n d its host environment ( c o n t r o l ) . W e h y p o t h e s i z e that t h e s e results a r o s e b e c a u s e o f t h e particular b e h a v i o u r o f t h e females o f this s p e c i e s , w h i c h release their eggs into the internal environment. Hence, there is s o m e c h a n c e that w e may

have also amplified also parasite e g g DNA with host gut fluid.

Finally, t h e RAPD m e t h o d revealed g e n e t i c variability a m o n g the six s p e c i e s o f w o r m s studied. This preli- minary m e t h o d o l o g i c a l study should e n a b l e further investigations o f inter a n d intraspecific g e n e t i c varia- bility.

ACKNOWLEDGEMENTS

T

W e thank an a n o n y m o u s referee for its valuable c o m - ments.

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5 0 Mémoire Parasite, 1998, 5, 47-50