Class Trematoda:
description
Transcript of 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oxidase I gene in digenetic trematodes (Platyhelminthes) illustrates the challenge of barcoding parasitic helminths // Unpublished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
Parafasciolopsis fasciolaemorpha
Fasciolopsis buski
Eupariphium melis
Isthmiophora hortensis
Echinoparyphium cinctum
Echinostoma paraensei
Echinostoma revolutum
Protofasciola robusta
Cyclocoelum mutabile
Psilochasmus oxyurus
Psilotrema oschmarini
Psilotrema similimum
Psilotrema acutilostris
Philopthalmid sp.
Cloacitrema narrabeenensis
Dicrocoelium dendriticum
Dicrocoelium hospes
Schistosoma mansoni
Ascaris suum
Ascaris lumbricoides
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
Parafasciolopsis fasciolaemorpha
Fasciolopsis buski
Eupariphium melis
Isthmiophora hortensis
Echinoparyphium cinctum
Echinostoma paraensei
Echinostoma revolutum
Protofasciola robusta
Psilochasmus oxyurus
Psilotrema oschmarini
Psilotrema similimum
Psilotrema acutilostris
Philopthalmid sp.
Cloacitrema narrabeenensis
Cyclocoelum mutabile
Schistosoma mansoni
Ascaris suum
Ascaris lumbricoides
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 oschmarini 9
Psilotrema oschmarini 4
Psilotrema oschmarini 1
Psilotrema oschmarini 7
Psilotrema oschmarini 3
Psilotrema oschmarini 2
Psilotrema similimum 2
Psilotrema similimum 1
Psilotrema acutilostris 1
Psilochasmus oxyurus
Dicrocoelium dendriticum
Gyrodactylus salaris
98
99
99
99
50 Psilotrema oschmarini 2
Psilotrema oschmarini 7
Psilotrema oschmarini 6
Psilotrema oschmarini 9
Psilotrema oschmarini 3
Psilotrema oschmarini 4
Psilotrema oschmarini 1
Psilotrema similimum 2
Psilotrema similimum 1
Psilotrema acutilostris 1
Psilochasmus oxyurus
Dicrocoelium dendriticum
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!