tools for taxonomy, and phylogneny of plants and insects...

GPLF Dourdan mai 2005

Molecular Markers,

Tools for Taxonomy, and Phylogneny

of Plants and Insects Trypanosomatids

Michel DolletCirad

Montpellier France


AtAt first, first, trypanosomatids weretrypanosomatids were discovered in the discovered in the laticiferouslaticiferous tubes of latex bearing plantstubes of latex bearing plants

(Lafont 1909)(Lafont 1909)

Euphorbiaceae AsclepiadiaceaeApocynaceae


Trypanosomatids associted with Coconut wilt

« Hartrot » (1976)


+ Coffee phloem necrosisSurinam (1930-50)

Red ginger wilt (1990)

Phloem restricted trypanosomatids are specificallyassociated with wilts in latin America and in the Caribbean

Oil palm : Marchitez (1977)


Plant Trypanosomatids

Subkingdom

Protozoa

Order

Kinetoplastidae


Family : Trypanosomatidae

Genera:

In Animal, Man :Trypanosoma, Leishmania

In Insects :Leptomonas, Herpetomonas, Crithidia, Blastocrithidia

In Plants : « Phytomonas »(Donovan 1910)


Do really all trypanosomatids found in plants belong to a single genus : « Phytomonas spp »?

No...« monoxemous

insect trypanosomatids »...

have been isolated from tomatoes

and other fruits or seeds.

Leptomonas, Herpetomonas,

Crithidia


Plant surface, flowers, nectar,pollen, fruits, seeds :

Huge traffic !

lepidopterous

blow fly

bug

beettle

locust


Crithidia bombi can be horizontally

transmitted through the shared used of flowers

Durrer and Schmid-Hempel 1994.

Echium vulgare

Bombus sp

Crithidia bombi


« Herpetomonas samuelpessoai camargoi .subp. in the flower of the squash »

Fiorini et al. 2001.

« A trypanosomatid was isolated from the sap of petals and petiolesof Cucurbita moschata flower »

Zelus sp, Reduviidae(predator &Tomato!) (H. samuelpessoai)


« insect trypanosomatids multiplying in tomato fruits »Conchon et al 1989.

C. luciliae. thermophylaZelus leucogrammusReduvidae (predator) .

And Zelus sp. : tomato, Solanaceae

Crithidia acanthocephaliAcanthocephala femorataCoreidae (predator)

« Plant juices »

Crithidia fasciculataCulex pipiensDipter, Culicidae

(Birds, mammals)


Leptomonas seymouriDysdercus suturellusPyrrhocoridae

(Cotton stainer, orange, eggplant, guava, okra, rose - pest)

Leptomonas collosoma

Gerris dissortisGerridae (Water strider)

Herpetomonas samuelpessoaiZelus leucogrammusReduvidae (predator)


Trypanosomatids associated with hartrotand marchitez are transmitted by pentatomid bugs of the genus Lincus Stal.

Hartrot (coconut) :L. croupius

L. tumidifrons

L. lamelliger

L. lobuliger

L. vandoesburgi

Marchitez (oil palm ):

L. lethifer

L. tumidifrons

L. lethifer. Oil palm. Ecuador


5 larval stages

Total length of the cycle : 3 to 4 months


Transmission ElectronMicroscopy


in vitro cultures of latex, fruit

and phloem Phytomonas

Mang. sp

Tom.sp

Berg. Br


Leptomonas mirabilisCynomyopsis cadaverinaDiptera: Calliphoridae

Several are nectar feeders

« Insect 1 »(T177 TCC/USP)

Edessinae sp.Pentatomidae

« Insect 2 »(T103 TCC/USP)

Dysdercus sp.Pyrrhocoridae(Cotton stainer, orange pest)


- Kinetoplastic DNA

- Ribosomal DNA

Small Subunit (18S)

Internal Transcribed spacers (ITS 1&2)

5S

- Splice Leader RNA gene (mini-exon)

Sequences used for molecular markers


Mbo

CR2

CSB 1 2 3

CR1

MboPst Pst

Cloning and sequencing of a hartrot isolate was obtained.

Kinetoplastic DNA : minicircles

1 60 Ps1GH GTATGGGCGT TCTGC-AAAA TTTGCGATTT TTATGCA-TA ACTCCCGTTC ATAATT-TG- Ps1GS

.......... .....A.... A...A-.AAA .C.....-.. .......... ......- Hart1

.......... ...C.----- -----.T... .G..A..G.. .......... .A..A.A.. CritF ...G...... .....G.... -.C.A-C... .G..A..GG. .A........ .A..A.-C. WalL. ...G...... ..AT.G.TTT .GG.T.TG.C AC..A..GG. .T-....... .A.T..TG.G CritO ...G...... ..AA.GTTTT .CG.-.GG.C -...A..GAC TT....C..A .C.T..T-.G CSB 1 CSB 2 61 122 Ps1GH CCATTTTTGG CATTTTTTCA CTCGGAGG-- ----GGGTTT TTGAGGGGTT GGTATAATAT AA Ps1GS TGT....... .G........ ........-- ----...... .......... .......... .. Hart1 .GG.....T. ---------- G.G...T.-- ----...... ..T....... .......... .. CritF TG......TC A.....---- ---.....CA AACT...GA. ..CC...... ...G..G... TT Wall. G..AAA.CAC G.....CGG- --.C..TTT- --CGT.--G. ..TC...... ...G..TA.. .. CritO .......A.C GG...GCGG. --..A.A--- --CCT..CG. ...GA..... ...G.T.A.. .G CSB3


Kinetoplastic DNA

primers designed around

CSB3, allow specific

amplification of

half size minicircles.


Splice Leader RNA

The SL RNA generepeat provides definedsequences with differentselection pressure

Probe 1

Probe 2

The exon, highlyconserved

The intron, lessconserved

A spacer, highly variable


SL RNA

Other sets of primers

were designed, to

perform

multiplex PCR

able to recognize

group 1 or group 2 in

one PCR run


5S rRNA

5S ribosomal RNA gene repeats are relativelyconserved in kinetoplastids

Primers were designed in a conserved block withinthe transcribed region

trimer

dimer

monomer 5S

Non-transcribedspacer


5S rRNA5 3

28 79 hi.gu ACAGGCTCAG [- - -] CCCTGAGTGC CGTACTCTCA AACGCACTC hi.vz .......C.. [- - -] .......... .....T.... ..C..A...

Man.sp .......T.. [- - -] .......... .T....C... .T.AT.C

Tom.sp .......T.. [- - -] .......... .T....C... .T.AT.C

Mand.br.......T.. [- - -] .......... .T....C... .T.GT.C Hi.su.......T.. [- - -] .......... .T....C... .T.AT.C

Hart.13.......T.. [- - -] .......... ......C... ..CAA..... Hart. 2.......T.. [- - -] .......... ......C... ..CAA.....

Berg.br.......T.. [- - -] ....A..... ......C... .T.TAA.A.TC Lima.pe.......T.. [- - -] ....A..... ......C... .T.A.ATA..C

EM1.fr.......T.. [- - -] ....A..... ......C... .T.ACACTCA Hi.se.......T.. [- - -] ....A..... ......C... .TGACACTCA

Ascl.Vz.......T.. [- - -] ....A..... ......G..T C.AA...C.

Latex and fruit isolates can be closely related(Spacer : much more variable)


rRNA Gene : 16S-23S /Internal Transcribed Spacers

18S(ssu) 5.8S Large su 1 LS 2 NTS

28S

r RNA locus contains between the small sub-unit 18S and

the large subunit, 2 variable sequences ITS 1 and ITS 2

ITS1 ITS2

It was possible to amplify this region with 2

primers located between the Ssu and the Lsu


rRNA Gene : 16S-23S / ITS5.8 S 5S 3 End ITS-2

GroupLima T T T T G G A T C C A T . C C T TPs1G T T T T G G nd T C C A T . C C T T

E.M.1 T C T T G G D T C C A T . T C G TE.hi.Sen T C T G C G D T C C A T . T C G T

Mar 1 T T C T G G H T C C A T G T C T CMar 6 T T C T G G H T C C A T G A C T CHart 1 T T C T G G H T C C A T G T C T C

Aca.Sur C T A C G G nd T C C A T . . C T CE.hi.Sur C T A C G G B T C C A T . . C T C

Tom.Sp T T C T G G C T C C G T . . C T CE.hi.Vz T T C T G G G T C C G T . . C T CE.hy.Gu T T C T G G G T C C A T . . C T C

H. musc. T T T C . G HerpetomonasL. mira. T T A C . G LeptomonasC. onco. C T T T . G Crithidia

The ITS sequences confirm the 5S resultsand show that the tomato isolate from Spainoriginates in latin America


5.8 SSSU

ITS. 1

[TA]

Lima

Groupor isolateSpecificmicrosat.

[TC]

Microsat.

Absent

[TC]

H

TG]

H

[TG]

[TA]

A

[CA]

[TC]

A ; D ; F ; G ; I

Ble. Br

Rhab. Sur

[TTC]

A ; D

[TC]

[CA]

A ; D ; G ; H

+ Ascl. ;Tom Sp

[TA]

[TG]

A ; D ; G + Tom.Sp

ITS-2

5. 8 S

+1

+3

+4

+5

A ; D ; G ; I

Tom.Sp

C ; F ; Aca

- 3

- 2

- 4-1

- 5- 6

[TC]

- 7

- 8

[TG]

- 9

- 10

A ; B

+E.hi Se.; EM1

E.heter. Vz; Tom Sp.

+2

LSU

Tom. Sp

Tom. Sp

E.hi. Se

E.hi. Se H H A

There is a variabilty in the Microsatellites located in ITS 1 & 2

among the different groups of trypanosomatids

Some microsatellites are specific markers for one group or

one given isolate inside a defined group.

rRNA Gene : ITS Microsatellites


5S

Maximum likelihood tree

RAPD Zym

LimaPs1GBerg

E.hi.SenE.M.1

Mar6Hart1E.hi.Sur

E.het.Vz

E.hi.Ind

Mar1

Rhab.SurAca.Br

Bleph.Sur

Tom.SpH. muscarumL. mirabilisC. oncopeltiE.hi.Vz

Mand.sc.BrMani.Br

E.hy.GuAscl.cu.Vz

10097

86

98

99

100

10099

100

9498

76

100

100

100

69 72

53 A A*

ndnd

D D }H H }B B } B ndB

ndndB nd

nd CF F

GG

nd I

}nd

ndnd

nd

*nd

ndnd

nd

C C


« The discovery and preliminary characterization of a novel

trypanosomatid parasite from Rattus norvegicus and stray dogs

from Alexandria, Egypt. »Morsy et al. 1998. Annals Trop. Med. Parasitol.82: 437-444.

Plant trypanosomes («Phytomonas»),« Insect trypanosomes »

Only in plant and insects ?


Rat/Dog Trypano « X »

The phylogenetic trees

reported herein

surprisingly affiliate

the rodent/canine

isolate with the genus

Herpetomonas. »


« Infection of mouse dermal fibroblasts by themonoxenous trypanosomatid protozoaCrithidia deani and Herpetomonas roitmani »

Santos et al 2004. J. Eukaryot. Microbiol.51:570-574

Zelus sp.C.deani

Ornidia obesaDiptera : Syrphidae

H. roitmani


Ornidia obesa hosts :

Morales and Köhler 2004

Casearia grandiflora is pollinated by Ornidia obesa


Diffuse cutaneous infection caused by a presumed monoxenoustrypanosomatid in a patient infected with HIV

Dedet et al 1995. Trans. R. Soc. Trop. Med. Hyg. 89: 644-646

HIV co-infection with a currently non pathogenic flagellate.Jimenez et al. 1996. The lancet.347: 264-65

« Immunocompromised patients could be vulnerable to other

currently non-human trypanosomatids »

« Plant and insect trypanosomatids »in mammals ?in man ?


CrithidiaBlasthocritidiaHerpetomonasLeptomonas

Plant surface-flower- Fruit/ seed Tryp.

Latex Trypanosomatids

?

Plant intraphloemic-pathogenictrypanosomes

(Systemic infection)

Conclusion

GPLF Dourdan mai 2005GPLF Dourdan mai 2005

The name «Phytomonas » does not reflect the large diversity of trypanosomatids found in Plants

There is at least one homogenous groupthat could be explicitly defined by

a single species name that differs from all other groups :

Conclusion

Molecular markers + isoenzymes, serology, in vitro culture

Phloem restricted trypanosomatids, - transmitted by pentotomids bugs «Phloemicola »- associated with wilts- in latin America and the Caribbean


In collaboration with:

Theo Baltz, and Sylvie Marché, Bordeaux 2 University, Fr

Michel Tibayrenc, Anne Laure BanyulsIRD, Montpellier, Fr

David Campbell and Nancy Sturm,UCLA, USA

Daniel Gargani, Emmanuelle MullerCIRAD Montpellier, Fr

Jean Charles Ahomadegbe and Guy Riou,IGR, Villejuif, Fr

Manuel Sanchez Moreno, Granada University, Sp.

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