Class Trematoda:

PHYLOGENETIC RELATIONSHIPS OF THE GENUS PSILOTREMA (P. oschmarini, P.

acutirostris, P. simillimum) BASED ON 28S rDNA SEQUENCES

Atopkin D.M.

Institute of Biology and Soil Sciences, Far Eastern Branch of Russian Academy of Sciences

Class Trematoda:

• 2 Subclasses: Aspidogastrea and Digenea

• 12 Orders

• ~ 90 Families

• ~ 7000 Species

Life cycle of trematodes

Adult wormMiracidium

CercariaAdolescaria

Metacercaria

Hermaphroditegeneration

Parthenogeneticgeneration

Adults eggs

sporocyst mother rediae

daugher rediae cercaria

Inside snail tissue

Species of the genus Psilotrema, Odhner, 1913• Psilotrema acutirostris, Oshmarin, 1963• Psilotrema simillimum (Muhling, 1989) • Psilotrema oschmarini, Besprozvannykh, 2007• Psilotrema breve Oshmarin, 1963• Psilotrema castoris Orlov, 1946• Psilotrema marki Skvorzov, 1934• Psilotrema medioporum Oshmarin, 1963• Psilotrema oligoon (von Linstow, 1887)• Psilotrema pharyngeatum Grabda 1954• Psilotrema spiculigerum (Muhling, 1989)• Psilotrema tuberculatum, (de Filippi, 1857)• Psilotrema zibethicum Varenov, 1965

Molecular genetic investigations, includes species of the family Psilostomatidae

• Psilochasmus oxyurus - Tkach,V., Pawlowski,J. and Mariaux,J. 2000. Phylogenetic analysis of the suborder plagiorchiata (Platyhelminthes, Digenea) based on partial lsrDNA sequences // Int. J. Parasitol. 30 (1), 83-93

• Sphaeridiotrema pseudoglobulus - Moszczynska,A., Locke,S.A., Mclaughlin,J.D., Marcogliese,D.J. and Crease,T.J. Development of primers for the mitochondrial cytochrome c oxidase I gene in digenetic trematodes (Platyhelminthes) illustrates the challenge of barcoding parasitic helminths // Unpublished

• Ribeiroia marini and R. ondatrae - Wilson,W.D., Johnson,P.T., Sutherland,D.R., Mone,H. and Loker,E.S. 2005. A molecular phylogenetic study of the genus Ribeiroia (Digenea): trematodes known to cause limb malformations in amphibians // J. Parasitol. 91 (5), 1040-1045.

Species of the genus Psilotrema• Psilotrema acutirostris, Oshmarin, 1963• Psilotrema simillimum (Muhling, 1989) • Psilotrema oschmarini, Besprozvannykh, 2007• Psilotrema breve Oshmarin, 1963• Psilotrema castoris Orlov, 1946• Psilotrema marki Skvorzov, 1934• Psilotrema medioporum Oshmarin, 1963• Psilotrema oligoon (von Linstow, 1887)• Psilotrema pharyngeatum Grabda 1954• Psilotrema spiculigerum (Muhling, 1989)• Psilotrema tuberculatum, (de Filippi, 1857)• Psilotrema zibethicum Varenov, 1965

Investigated species of the genus Psilotrema

(photo by V.V.Besprozvannykh)

P. oschmarini P. simillimumP. acutirostris~ 1,5 m

m

~ 1,25 mm

~ 0,55 mm

28S rDNA second structure of Gydrodactulus salaris, Monogenea (from

Matejusova and Cunningham, 2004)

D3

D1

D2

D3

D1

D2

D1

D3

D2

D1

D3

D2

D1

In this study D2 and a part of D3 variable donains of 28S rRNA gene are used.

D1-D3 lenght ~ 1200-1500 bp

Aim:

To investigate phylogenetic relationships of the genus Psilotrema and family Psilostomatidae within order Echinostomida using partial sequence of 28S rRNA gene

• Optimizing of DNA extraction and PCR technique for trematodes;

• Sequencing 28S rRNA gene fragment of three Psilotrema species;

• Reconstruction of phylogenetic relationships of the genus Psilotrema and the family Psilostomatidae using current data and data from GenBank.

Tasks:

Material

Adults

Adolescaria

Cercaria

Chicken

isolation

encyst

infecting

after 8 days

• Phenol-chlorophorm extraction with Proteinase K lysis

• Proteinase K lysis with following boiling with Chelex-100

• Boiling with Chelex-100

• DNA extraction with Silica

• Fast DNA extraction kit, Q-Biogene

MethodsDNA extraction

PCR and DNA sequence performed with ABI GeneAmp 2700 thermal cycler and ABI 3130

genetic analyzer in the IBSS FEB RAS

Snail

Families of the order Echinostomida, used in preset study (n – number of

sequences)

• Psilostomatidae (n=11)

• Fasciolidae (n=7)

• Echinostomatidae (n=5)

• Phylpthalmidae (n=2)

• Cyclocoeliidae (n=1)

28S rRNA gene fragment variation within EchinostomidaOverall alignment (with gaps)

Overall alignment (without gaps)

Variable Parsimony-informative

P.oschmarini 759 759 0 0

P.oschmarini+P.simillimum

759 759 14 0

P.oschmarini+P.acutirostris

759 759 13 0

Psilotrema 759 759 14 12

Fasciola 759 759 22 2

Psilostomatidae 760 759 – 760 75 12

Fasciolidae 760 758 - 759 79 24

Echinostomatidae 760 758 64 49

Echinostomida 764 757 – 760 264 181

Nucleotide composition of the 28S r RNA gene fragment of the Psilotrema species

A T(U) G C

P.oschmarini 18.5 26.5 33.0 22.0

P.acutirostris 18.4 26.4 33.2 22.0

P.simillimum 18.4 26.3 33.2 22.1

Psilostomatidae 18.5 26.4 33.1 22.0

Echinostomida 18.2 26.2 33.3 22.4

Genetic p-distances between families of Echinostomida

P.oschmarini

P.acutirostris

P.simillimum

Psilostomatidae

Echinostomatidae

Fasciolidae

Phylopthalmidae

Cyclocoeliidae

Dicrocoeliidae

P.oschmarini 0

P.acutirostris 0.017 0

P.simillimum 0.017 0.001 0

Psilostomatidae

0

Echinostomatidae

0.1 0

Fasciolidae 0.119 0.089 0

Phylopthalmidae

0.125 0.12 0.117 0

Cyclocoeliidae 0.168 0.154 0.15 0.155 0

Dicrocoeliidae 0.177 0.187 0.199 0.193 0.22 0

Fasciola hepatica

Fasciola gigantica

Fascioloides magna

Fasciola jacksoni

Parafasciolopsis fasciolaemorpha

Fasciolopsis buski

Protofasciola robusta

Cyclocoelum mutabile

Eupariphium melis

Isthmiophora hortensis

Echinoparyphium cinctum

Echinostoma paraensei

Echinostoma revolutum

Philopthalmid sp.

Cloacitrema narrabeenensis

Psilochasmus oxyurus

Psilotrema oschmarini

Psilotrema similimum

Psilotrema acutilostris

Dicrocoelium dendriticum

Dicrocoelium hospes

Schistosoma mansoni

Gyrodactylus solaris

Ascaris suum

Ascaris lumbricoides

Notentera ivanovi

Acmaea mitra

Eulepetopsis vitrea100

100

100

99

9099

99

96

5085

80

70

5158

89

75

100

100

100

73

50

Maximum parsimony tree of the order Echinostomida

(tree topology with 50% frequency)

Fasciolidae

Psilostomatidae

Echinostomatidae

Philopthalmidae

Outgroup

- Cyclocoeliidae

Fasciola hepatica

Fasciola gigantica

Fascioloides magna

Fasciola jacksoni


Fasciolopsis buski

Eupariphium melis











Philopthalmid sp.



Dicrocoelium hospes

Schistosoma mansoni

Ascaris suum


Notentera ivanovi

Acmaea mitra

Eulepetopsis vitrea

Gyrodactylus solaris

100

100

99

99

9498

7675

96

92

87

57

57

57

57

55

99

100

100

100

71

0.1

Neighbor-Joining tree

(model TrN) of the order

Echinostomida (tree topology

with 30% frequency)

Fasciolidae

Echinostomatidae

Philopthalmidae

Psilostomatidae

Outgroup

- Cyclocoeliidae

Fasciola hepatica

Fasciola gigantica

Fasciola jacksoni

Fascioloides magna


Fasciolopsis buski

Eupariphium melis










Philopthalmid sp.



Schistosoma mansoni

Ascaris suum


Notentera ivanovi

Eulepetopsis vitrea

100

100100

100

9092

100

100

10099

93

89

76

99

100

100

10098

0.00.10.20.30.40.5

Fasciolidae

Psilostomatidae

Echinostomatidae

Philopthalmidae

Outgroup

UPGMA tree (model TrN) of the

order Echinostomida (tree topology with 20%

frequency)

- Cyclocoeliidae

Psilotrema oschmarini 6







Psilotrema similimum 2


Psilotrema acutilostris 1



Gyrodactylus salaris

98

99

99

99

50 Psilotrema oschmarini 2









Psilotrema acutilostris 1



Gyrodactylus salaris

97

7085

92

0.1

Maximum parsimony tree of the genus

Psilotrema

Neighbor-Joining tree of the genus

Psilotrema

P.oschmarini

P.acutirostris + P.simillimum

P.oschmarini

P.acutirostris + P.simillimum

Conclusions• The present study shows that given the current data,

family Psilostomatidae is monophyletic and possible an ancient taxon for the order Echinostomida;

• Psilotrema acutirostris and Psilotrema simillimum are likely more closely related to each other, then to P. oschmarini; Psilochasmus oxyurus is an ancient taxon relative to studied species of the genus Psilotrema;

• There is no intraspecific variation of the 28S rDNA variable domains between specimens of P. oschmarini, which originated from one miracidium of previous generation; however, further studies required using other molecular markers, including mitochondrial DNA.

THANKS FOR YOUR ATTENTION!

Documents

Class Trematoda: