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GPLF Dourdan mai 2005
Molecular Markers,
Tools for Taxonomy, and Phylogneny
of Plants and Insects Trypanosomatids
Michel DolletCirad
Montpellier France
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GPLF Dourdan mai 2005
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
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GPLF Dourdan mai 2005
Trypanosomatids associted with Coconut wilt
« Hartrot » (1976)
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+ 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)
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Plant Trypanosomatids
Subkingdom
Protozoa
Order
Kinetoplastidae
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GPLF Dourdan mai 2005
Family : Trypanosomatidae
Genera:
In Animal, Man :Trypanosoma, Leishmania
In Insects :Leptomonas, Herpetomonas, Crithidia, Blastocrithidia
In Plants : « Phytomonas »(Donovan 1910)
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GPLF Dourdan mai 2005
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
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GPLF Dourdan mai 2005
Plant surface, flowers, nectar,pollen, fruits, seeds :
Huge traffic !
lepidopterous
blow fly
bug
beettle
locust
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GPLF Dourdan mai 2005
Crithidia bombi can be horizontally
transmitted through the shared used of flowers
Durrer and Schmid-Hempel 1994.
Echium vulgare
Bombus sp
Crithidia bombi
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GPLF Dourdan mai 2005
« 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)
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GPLF Dourdan mai 2005
« 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)
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GPLF Dourdan mai 2005
Leptomonas seymouriDysdercus suturellusPyrrhocoridae
(Cotton stainer, orange, eggplant, guava, okra, rose - pest)
Leptomonas collosoma
Gerris dissortisGerridae (Water strider)
Herpetomonas samuelpessoaiZelus leucogrammusReduvidae (predator)
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GPLF Dourdan mai 2005
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
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5 larval stages
Total length of the cycle : 3 to 4 months
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GPLF Dourdan mai 2005
Transmission ElectronMicroscopy
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GPLF Dourdan mai 2005
in vitro cultures of latex, fruit
and phloem Phytomonas
Mang. sp
Tom.sp
Berg. Br
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GPLF Dourdan mai 2005
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GPLF Dourdan mai 2005
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GPLF Dourdan mai 2005
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)
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GPLF Dourdan mai 2005
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GPLF Dourdan mai 2005
- 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
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GPLF Dourdan mai 2005
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
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GPLF Dourdan mai 2005
Kinetoplastic DNA
primers designed around
CSB3, allow specific
amplification of
half size minicircles.
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GPLF Dourdan mai 2005
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
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GPLF Dourdan mai 2005
SL RNA
Other sets of primers
were designed, to
perform
multiplex PCR
able to recognize
group 1 or group 2 in
one PCR run
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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
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GPLF Dourdan mai 2005
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)
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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
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GPLF Dourdan mai 2005
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
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GPLF Dourdan mai 2005
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
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GPLF Dourdan mai 2005
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
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« 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 ?
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Rat/Dog Trypano « X »
The phylogenetic trees
reported herein
surprisingly affiliate
the rodent/canine
isolate with the genus
Herpetomonas. »
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« 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
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Ornidia obesa hosts :
Morales and Köhler 2004
Casearia grandiflora is pollinated by Ornidia obesa
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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 ?
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CrithidiaBlasthocritidiaHerpetomonasLeptomonas
Plant surface-flower- Fruit/ seed Tryp.
Latex Trypanosomatids
?
Plant intraphloemic-pathogenictrypanosomes
(Systemic infection)
Conclusion
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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
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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.