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Title南アジアにおける反芻動物の胆管内寄生吸虫Fasciola spp.およびExplanatum explanatumの分子系統に関する研究( 本文(Fulltext) )
Author(s) 林, 慶
Report No.(DoctoralDegree) 博士(獣医学) 甲第480号
Issue Date 2017-03-13
Type 博士論文
Version ETD
URL http://hdl.handle.net/20.500.12099/56194
※この資料の著作権は、各資料の著者・学協会・出版社等に帰属します。
Fasciola spp. Explanatum explanatum
2 0 1 6
Fasciola spp. Explanatum explanatum
Fasciola spp.
2 pepck pold
Fasciola spp.
3 Fasciola spp.
1
5
6
6
10
13
15
26
27
28
29
30
31
35
36
36
39
41
43
4 Explanatum explanatum
48
49
49
51
53
54
61
64
AMOVA
DNA
ITS
MJ network
nad1
PCR
RFLP
analysis of molecular variance
deoxyribonucleotic acid
internal transcribed spacer
median-joining network
nicotinamide adenine dinucleotide dehydrogenase subunit 1
polymerase chain reaction
restriction fragment length polymorphism
1
Fasciola Echinostomida Fasciolidae
Fasciola
Lymnaeidae Fasciola
20 [35]
9,000 [30]
Fasciola Fasciola hepatica Linnaeus, 1758 F. gigantica Cobbold, 1855
F. hepatica F. gigantica
[50] 2
Fasciola hepatica F. gigantica
[41] DNA internal transcribed spacer 1 ITS1
ITS2 [1, 24,
27, 32] DNA NADH dehydrogenase subunit 1 nad1
Fasciola [24]
DNA DNA
F. hepatica
[33] F. hepatica 15
[22] F. gigantica
2
[33]
F. hepatica F. gigantica
aspermic Fasciola sp. [17, 36, 48] ITS1
ITS2 F. hepatica Fh F. gigantica
Fg Fh/Fg
[18, 27] DNA F.
hepatica F. gigantica [2, 24, 25, 27] nad1
F.
hepatica F. gigantica
[24] F. hepatica F.
gigantica [24]
F. hepatica F. gigantica
F. hepatica F. gigantica
[23, 40] F.
hepatica F. gigantica
[23, 40]
[36, 42]
F. gigantica
[36, 42]
F. gigantica
Fasciola gigantica Bos indicus
3
[33] F. gigantica
[6] B. indicus
F. gigantica
Fasciola
Explanatum Echinostomida Paramphistomidae
Fasciola
Gyraulus [42]
Explanatum
[11, 34] Explanatum
Explanatum
E. explanatum Creplin, 1847 E. bathycotyle Fischoeder, 1901 E. anisocotyle
Faust, 1920 3 Explanatum E. explanatum
[8, 44]
[3, 11, 44] E. explanatum
Bubalus bubalis [8, 11, 38, 45]
[7, 39] Explanatum
explanatum DNA ITS2
[21, 28].
E. explanatum DNA nad1
E. explanatum
4
[37] 2
E. explanatum
E. explanatum
Fasciola
E. explanatum
1 Fasciola
2 Fasciola
3 Fasciola
4 E. explanatum
Fasciola
5
1 Fasciola spp.
6
F. gigantica
F. gigantica
B. indicus
[36, 42]
Fasciola
F. gigantica
[36, 42] F. gigantica
Fasciola
.
(1) Fasciola
2014 1 9 Imphal Kohima
7
Gangtok 35
15 1 Fasciola
Imphal 1 1 5 Kohima
1 1 8 Gangtok 1 10 16 157
70%
[48]
High Pure PCR Template Preparation Kit
Roche, Mannheim, Germany DNA 20
(2) PCR
ITS1-F 5’-TTGCGCTGATTACGTCCCTG-3’
ITS1-R 5’-TTGGCTGCGCTCTTCATCGAC-3’ ITS1 Ita 10 5’-
AAGGATGTTGCTTTGTCGTGG-3’ Ita 2 5’-GGAGTACGGTTACATTCACA-3’
nad1 PCR [24] PCR 0.025 U GoTaq
DNA Polymerase Promega, Madison, USA DNA 100 ng/μL
dNTP 0.08 mM 0.5 pM each primer, 0.025 U 5 GoTaq buffer 50
mM Tris-HCl, 50 mM NaCl, 5 mM MgCl2 50 μL 94°C
8
90 94°C 90 55°C 90 72°C 120 30
72°C 10 PCR 1.0%
(3) ITS1 nad1
ITS1 PCR 10U Rsa I Roche buffer enzyme L
Roche 50 μL 37
PCR 1.8%
[18] ITS1 360 bp 100 bp
60 bp 3 Fh 360bp 170 bp 60
bp 3 Fg 360 bp 170bp 100 bp
60 bp 4 Fh/Fg
F. hepatica Fh F. gigantica Fg
Fh Fg Fh/Fg
[5, 17-20, 23-26, 36, 40, 43]
nad1 PCR NucleoSpin® Gel and PCR Clean-up Takara Bio Inc., Otsu,
Japan 535 bp
BigDye Terminator v3.1 Cycle Sequencing Kit Applied
Biosystems, Foster City, USA ABI
9
PRISM 3500 Genetic Analyzer Applied Biosystems
PCR
ATGC ver. 6. 0. 3 Genetyx, Tokyo, Japan GENETYX ver. 10. 0.
2 Genetyx
(4) nad1
Network 4. 6. 1. 2 F. gigantica
nad1 Median-joining MJ network
[4] Fasciola nad1
[20, 24] [19, 25] [23, 40] [26] [5]
[17] [36] [43] Fasciola nad1
MJ network
median vector
(5) nad1
Fasciola
10
DnaSP v5. 1. 0
Hd π [31]
Alrequin 3.5 pairwise fixation index Fst
[9] Fasciola
6 1
F. gigantica 2 A B
(1) ITS1
Imphal 23 Kohima 3 Gangtok
13
118
1 1 118 ITS1
Fg F. gigantica
39 ITS1 Fg 2 1
3 F. gigantica
3
11
(2) nad1
Fasciola 157 nad1 535bp 40
ND1-E1 ND1-E37 37
GenBank accession numbers: LC012895–
LC012931 1 ND1-E1 Imphal Kohima Gangtok 32
[5, 17, 19, 20, 24, 25, 26, 27, 36, 40, 42, 47] ND1-E2 Imphal
1 ND1-E1 1
MJ network
ITS1
39 32 ND1-E1 1 ND1-
E2 6 5 ND1-E3 1 ND1-E21
MJ network F. gigantica
4 6
F. gigantica
nad1
Fasciola sp.
12
ND1-E6 Imphal Kohima Gangtok F. gigantica 45 28.7%
Fasciola
M15
[17] TH13 [5] Bd9 [36] N1
[42] F. gigantica Imphal 30
ND1-E3 M1 [17]
TH15 [5] 4
ND1-E4 ND1-E5 ND1-E3 F. gigantica B
ND1-E3 ND1-E7 ND1-E37
ND1-E6 ND1-E6 F. gigantica
A Fasciola
F. gigantica C A
B 4 F. gigantica C
nad1
(3) nad1
Fasciola gigantica A
0.00366 0.00362
0.00242 0.00255
13
F. gigantica B
0.00170 0.00028
F. gigantica
0.00000 0.00011
Fasciola gigantica Fst
A B
A B
Fst 0.86934 p<0.05
A A Fst 0.02545
p<0.05 Fst
A B
3
F. gigantica 3
DNA nad1 F. gigantica
F. gigantica
14
3
[6, 13] F.
gigantica nad1
F. hepatica F. gigantica
[40]
75.3% 86.0% F.
gigantica [36, 42] F.
gigantica 24.8% 2, 2
[36]
19
Fasciola
6 Fasciola
F. gigantica nad1
A B
15
A
2 A F.
gigantica MJ
network A F. gigantica
4
A F. gigantica
Bos indicus Fasciola
[6] B. indicus F. gigantica
[33] B. indicus A F.
gigantica B
F. gigantica
4 B F.
gigantica
2
16
B F. gigantica
B F. gigantica MJ network
F. gigantica
Fasciola
Fasciola 157
DNA ITS1
F. gigantica
Fasciola
DNA nad1 535 bp
F. gigantica
F. gigantica nad1
17
A B
F. gigantica A
B
Fasciola
18
Fasciola
ITS1
nad1
Accession no.
Imphal
(30)
16 Fg ND1-E1 LC012895
1 Fg ND1-E2 LC012896
16 + Fg ND1-E3 LC012897 F. gigantica
5b Fg ND1-E3 LC012897 Fasciola sp.
1 + Fg ND1-E4 LC012898 F. gigantica
1 + Fg ND1-E5 LC012899 F. gigantica
21 + Fg ND1-E6 LC012900 F. gigantica
4 + Fg ND1-E7 LC012901 F. gigantica
1 + Fg ND1-E8 LC012902 F. gigantica
1 + Fg ND1-E10 LC012904 F. gigantica
1 + Fg ND1-E15 LC012909 F. gigantica
1 + Fg ND1-E16 LC012910 F. gigantica
1 + Fg ND1-E17 LC012911 F. gigantica
1 + Fg ND1-E19 LC012913 F. gigantica
2 + Fg ND1-E20 LC012914 F. gigantica
1 + Fg ND1-E21 LC012915 F. gigantica
1b Fg ND1-E21 LC012915 Fasciola sp.
1 + Fg ND1-E22 LC012916 F. gigantica
1 + Fg ND1-E23 LC012917 F. gigantica
1 + Fg ND1-E27 LC012921 F. gigantica
1 + Fg ND1-E28 LC012922 F. gigantica
1 + Fg ND1-E31 LC012925 F. gigantica
1 + Fg ND1-E32 LC012926 F. gigantica
1 + Fg ND1-E34 LC012928 F. gigantica
1 + Fg ND1-E35 LC012929 F. gigantica
(11)
9 + Fg ND1-E3 LC012897 F. gigantica
11 + Fg ND1-E6 LC012900 F. gigantica
1 + Fg ND1-E9 LC012903 F. gigantica
2 + Fg ND1-E10 LC012904 F. gigantica
1 + Fg ND1-E18 LC012912 F. gigantica
1 + Fg ND1-E24 LC012918 F. gigantica
1 + Fg ND1-E25 LC012919 F. gigantica
3 + Fg ND1-E27 LC012921 F. gigantica
1 + Fg ND1-E36 LC012930 F. gigantica
1 + Fg ND1-E37 LC012931 F. gigantica
19
Fasciola
ITS1 nad1
Accession no.
(5) 3 Fg ND1-E1 LC012895
4 + Fg ND1-E6 LC012900 F. gigantica
1 + Fg ND1-E10 LC012904 F. gigantica
1 + Fg ND1-E11 LC012905 F. gigantica
1 + Fg ND1-E12 LC012906 F. gigantica
1 + Fg ND1-E13 LC012907 F. gigantica
1 + Fg ND1-E20 LC012914 F. gigantica
1 + Fg ND1-E28 LC012922 F. gigantica
1 + Fg ND1-E33 LC012927 F. gigantica
(1) 1 + Fg ND1-E6 LC012900 F. gigantica
1 + Fg ND1-E7 LC012901 F. gigantica
1 + Fg ND1-E14 LC012908 F. gigantica
(4) 13 Fg ND1-E1 LC012895
8 + Fg ND1-E6 LC012900 F. gigantica
1 + Fg ND1-E26 LC012920 F. gigantica
2 + Fg ND1-E29 LC012923 F. gigantica
2 + Fg ND1-E30 LC012924 F. gigantica
157 a "+" , "-" b Fasciola sp.
20
Fasciola
A:
B:
A B
21
Fasciola PCR-RFLP ITS1
1: Milli Q
2: Fh
3: Fg
4: Fh/Fg
M: DNA
5-8: Fasciola Fg
1 2 3 4 M 5 6 7 8 M
500 bp
1,000 bp
360 bp
170 bp
60 bp 100 bp
200 bp
22
Fasciola
F. gigantica Fasciola sp.
23
Fasciola nad1 MJ network
1
1
E1 E37 FspN1 FspN2 N1 N10
Bd1 Bd11 M1 M19 T1 T30
V1 V5 C1 C16 Kor2a Kor2b Fsp2
24
nad1 F. gigantica
N S h Hd ± SD π
F. gigantica A 91 32 32 0.751±0.050 0.00242 20 16 10 0.758±0.101 0.00366 20 15 10 0.832±0.075 0.00362 13 7 6 0.641±0.150 0.00225 B 27 2 3 0.145±0.090 0.00028 67 18 13 0.485±0.076 0.00170
33 1 2 0.061±0.056 0.00011 61 1 2 0.033±0.031 0.00006 127 0 1 0.000±0.000 0.00000 7 0 1 0.000±0.000 0.00000
N: , S: , h: , Hd: , SD: ,
:
25
A0.
0263
4*0.
0254
5*0.
0089
7-0
.012
470.
0025
0-0
.012
00 B
0.86
934*
0.88
145*
0.88
030*
0.93
454*
0.85
475*
0.85
099*
0.84
865*
0.87
363*
-0.0
0119
* (P
<0.0
5)
B A
3na
d1F.
gig
antic
a6
pai
rwis
e fix
atio
n in
dex
Fst
26
pepck pold
Fasciola spp.
27
1 Fasciola
ITS1 157
6
nad1 F. gigantica
Fasciola
[17, 36] ITS1 Fh Fg Fh/Fg
Fasciola 3 [18]
F. gigantica F. hepatica
[17, 36]
DNA phosphoenolpyruvate carboxykinase pepck DNA
polymerase delta pold Fasciola
F. gigantica F. hepatica
[44] F. hepatica Fh
F. gigantica Fg Fh/Fg
Fh/Fg
Fasciola
28
1 Fasciola 157
Fasciola 81 [43] Fasciola 138 [36]
Fasciola 87 [20] 463 DNA
ITS1
nad1 4
pepck Fh-pepck-F 5′-GATTGCACCGTTAGGTTAGC-3′ Fg-
pepck-F 5′-AAAGTTTCTATCCCGAACGAAG-3′ Fcmn-pepck-R 5′-
CGAAAATTATGGCATCAATGGG-3′ pepck multiplex PCR
PCR 0.025 U GoTaq DNA Polymerase Promega, Madison, WI,
USA DNA 100 ng/μL dNTP 0.08 mM Fh-pepck-F
Fg-pepck-F 0.5 pM Fcmn-pepck-R 1.0 pM each primer, 0.025 U 5 GoTaq buffer
50 mM Tris-HCl, 50 mM NaCl, 5 mM MgCl2 50 μL
94°C 90 94°C 90 61°C 90 72°C 120 30
72°C 10 multiplex PCR 1.0%
[44]
pold Fasciola-pold-F1 5′-
GCTAACTTATCTGCTTACACGTGGACA-3′ Fasciola-pold-R1 5′-
ATCGCATTCGATCAAAGCCCTCCCATG-3′ pold PCR
1.0% PCR
0.025 U GoTaq DNA Polymerase Promega DNA 100 ng/μL
dNTP 0.08 mM 0.5 pM each primer, 0.025 U 5 GoTaq buffer
50 mM Tris-HCl, 50 mM NaCl, 5 mM MgCl2 50 μL
29
94°C 90 94°C 90 55°C 90 72°C 120 35
72°C 10 pold PCR 10U
Alu I Roche buffer enzyme L Roche 50μL
37 PCR 1.8%
[44]
pepck 509 bp Fg 241 bp 509 bp
Fh/Fg 5 pold 544 bp
164 bp 136 bp Fg 602 bp
Fh/Fg 6 463 pepck
pold ITS1 nad1
[17, 36, 43] 4
ITS1 97.6%
nad1 100%
1
Fasciola 9 nad1 F.
gigantica [17, 36] 4 9
pepck pold Fg F. gigantica
4 Fasciola 1
ITS1 Fg nad1
4 [36]
pepck pold Fh/Fg
30
4 Fasciola
1 ITS1
Fh/Fg nad1
4 [36] pepck pold Fh/Fg
4
1 nad1 F. gigantica
Fasciola 245 pepck pold
Fg F. gigantica 1
nad1
Fasciola 218 pepck pold Fh/Fg
4 [44]
1 Fasciola 463
pepck pold
nad1
4 Fasciola 463
pepck pold Fasciola
463 pepck PCR
pold 7
31
4
pepck 241bp 509bp 510bp pold
844bp [44]
ITS1
97.6% 11 11
F. gigantica
[48, 49]
Fasciola pepck pold
1 157 6
nad1 F. gigantica
DNA pepck pold F. hepatica
F. gigantica
Fasciola nad1 F. gigantica
pepck pold F. gigantica
pepck pold
pepck
pold Fasciola
32
aIT
S1a
pepc
kpo
ld11
5―
FgFh
/Fg
Fh/F
g4
―Fg
Fh/F
gN
.D.
97―
Fh/F
gFh
/Fg
Fh/F
g
1bFh
/Fg
Fh/F
gFh
/Fg
1bFg
Fh/F
gFh
/Fg
F. g
igan
tica
233
FgFg
FgF.
gig
antic
a3
FgFg
N.D
.F.
gig
antic
a9b
―Fg
FgFg
F. g
igan
tica
463
4Fa
scio
la
N.D
.PC
Ra
1b
1IT
S1na
d1
nad1
a
FhF.
hep
atic
aFg
F. g
igan
tica
Fh/F
gFh
Fg
33
5 multiprex PCR Fasciola pepck
1: Milli Q
2: Fh
3: Fg
4: Fh/Fg
M: DNA
5-6: Fasciola Fg
7-8: Fasciola Fh/Fg
1 2 3 4 M 5 6 7 8
509 bp
241 bp
500 bp
1,000 bp
100 bp
M
34
6 PCR-RFLP Fasciola pold
1: Milli Q
2: Fh
3: Fg
4: Fh/Fg
M: DNA
5, 6, 7, 9: Fasciola Fg
8, 10: Fasciola Fh/Fg
1 2 3 4 M 5 6 7 8 9 10
708 bp
544 bp
164 bp 136 bp
35
Fasciola spp.
36
1 F. gigantica
F. gigantica nad1
A
B F. gigantica
F. gigantica A B
B.
indicus Bos indicus
Delhi Fasciola 2
.
(6) Fasciola
2014 12 11
37
Fasciola 1 1 8
40 70%
[48] High Pure PCR Template Preparation Kit
Roche DNA 20
(7) PCR
DNA ITS1 DNA pepck pold
DNA nad1 PCR ITS1
ITS1-F ITS1-R 1 nad1 Ita 10
Ita 2 1 pepck Fh-pepck-F Fg-pepck-F
Fcmn-pepck-R 2 pold Fasciola-pold-F1
Fasciola-pold-R1 2 PCR 1 2
DNA PCR NucleoSpin® Gel and PCR Clean-up
(8) ITS1 pepck pold nad1
ITS1 pold PCR-RFLP
38
1 2 pepck PCR 1.0%
2
nad1 PCR 535 bp
1 ATGC ver. 6. 0. 3 Genetyx
GENETYX ver. 10. 0. 2 Genetyx
(9) nad1
Network 4. 6. 1. 2 F. gigantica nad1
MJ network [4] Fasciola
nad1 [20, 24] [19, 25] [23,
40] [26] [5] [17] [36] [43]
1 Fasciola nad1
MJ network
median vector
(10) nad1
Delhi F.
39
gigantica A DnaSP
v5. 1. 0 Hd π [31]
(4) ITS1 pepck pold
Delhi 40 39
ITS1 pepck pold F. gigantica
Fg 39 F. gigantica
1
ITS1 pepck pold Fg
F. gigantica 5 [44]
(5) nad1
Fasciola gigantica 40 nad1 535 bp 19
ND1-IN1 ND1-IN18 18
GenBank LC128314–LC128330 LC128751
5 F. gigantica
40
A F. gigantica
ND1-IN1 F. gigantica 15
ND1-M15 [17]
TH13 [5] Fg-ND1-Bd9 [36] Fg-ND1-
N1 [43] F. gigantica F. gigantica
A 7 ND1-IN2
ND1-IN18 ND1-IN1 F. gigantica A
ND1-IN1
(6) nad1
Fasciola gigantica A Delhi
nad1
Hd
6 0.00366
0.00362 Delhi 0.00255
0.00242 0.00255
41
75.3 % [43] 86.0 % [36] F. gigantica
24.8 % 1
Delhi
Delhi
40
F. gigantica
[36]
F. gigantica 1
F. gigantica
[17, 36]
Delhi F. gigantica
F. gigantica A nad1
42
7 A
Delhi 0.00255 0.00242
A F. gigantica
Delhi
0.00366 0.00362
6 Delhi
A F. gigantica
Delhi
F. gigantica
Delhi
B C F. gigantica
F. gigantica
3 A
B. indicus
43
B
7 [5, 17] B Imphal
[45] C
[5, 12, 17, 23, 26, 40]
7 [5, 12]
Bubalus bubalis Fasciola
[14, 15]
C F. gigantica B. bubalis
Fasciola 40
pepck pold 40
F. gigantica nad1
40 A
A F. gigantica
44
F. gigantica
B C F. gigantica
45
5 Delhi F. gigantica
nad1
ITS1 pepck pold Accession no.
15 + Fg Fg Fg ND1-IN1 LC128314
1 + Fg Fg Fg ND1-IN2 LC128315
1 + Fg Fg Fg ND1-IN3 LC128316
1 + Fg Fg Fg ND1-IN4 LC128317
1 + Fg Fg Fg ND1-IN5 LC128318
3 + Fg Fg Fg ND1-IN6 LC128319
3 + Fg Fg Fg ND1-IN7 LC128320
1 + Fg Fg Fg ND1-IN8 LC128321
2 + Fg Fg Fg ND1-IN9 LC128322
1 + Fg Fg Fg ND1-IN10 LC128323
2 + Fg Fg Fg ND1-IN11 LC128324
1 + Fg Fg Fg ND1-IN12 LC128325
1 Fg Fg Fg ND1-IN12 LC128325
2 + Fg Fg Fg ND1-IN13 LC128326
1 + Fg Fg Fg ND1-IN14 LC128327
1 + Fg Fg Fg ND1-IN15 LC128328
1 + Fg Fg Fg ND1-IN16 LC128329
1 + Fg Fg Fg ND1-IN17 LC128330
1 + Fg Fg Fg ND1-IN18 LC128751
Total 40
a "+" , " "
46
7 Fasciola nad1 MJ network
1
1
Delhi IN1 IN17 E1 E37 FspN1
FspN2 N1 N10 Bd1 Bd11 M1 M19
T1 T30 V1 V5 C1 C16 Kor2a
Kor2b Fsp2
47
6 nad1 A F. gigantica
N S h Hd SD
Delhi 40 19 18 0.853±0.052 0.00255
91 32 32 0.751 0.050 0.00242
20 16 10 0.758 0.101 0.00366
20 15 10 0.832 0.075 0.00362
13 7 6 0.641 0.150 0.00225
N: , S: , h: , Hd: , SD: ,
:
48
4 Explanatum explanatum
49
1 3 Fasciola
Fasciola
Fasciola
Fasciola
Eeplanatum explanatum Fasciola
[3, 11, 45]
Eeplanatum explanatum
DNA
[37]
ITS2 E. explanatum
DNA nad1
E. explanatum
(1) Explanatum
Delhi Mumbai Gangtok
Imphal 2014 2 12
30 4 Delhi
Imphal 1 2 Mumbai Gangtok
1 5 70 7 70%
50
High Pure PCR Template Preparation Kit Roche DNA
20
(2) PCR
ITS2-F 5’-TGTGTCGATGAAGAGCGCAG-3’
ITS2-R 5’-TGGTTAGTTTCTTTTCCTCCGC3’ [24] ITS2 Pc-nad1-F1
5′-CAGATTCGGAAGGGGCCTAA-3′ Pc-nad1-R1 5′-
ACGTAGCACGAGCCCAAATA-3′ [37] nad1 PCR
PCR 1 DNA PCR
NucleoSpin® Gel and PCR Clean-up
(3) ITS1 nad1
ITS2 442 bp nad1 657 bp PCR
BigDye
Terminator v3.1 Cycle Sequencing Kit
ABI PRISM 3500 Genetic Analyzer
PCR ATGC ver. 6. 0. 3
Genetyx, Tokyo, Japan GENETYX ver. 10. 0. 2 Genetyx
(4) nad1
E. explanatum nad1 N1
N15 , LC101715–LC101729 E. explanatum Bd1 Bd30
51
, LC101685–LC101714 [37] Paramphistomum cervi KT198987
Fasciola hepatica AF216697 nad1
MEGA version 6.06 gamma Tamura and Nei
[47] 1,000
Network 4. 6. 1. 2 E. explanatum nad1
MJ network [4] E. explanatum nad1
E. explanatum
nad1 [37] MJ
network
median vector
(5) nad1
Delhi Mumbai Gangtok Imphal Bogra
Khulna Sylhet Mymensingh Chitwan
E. explanatum Arlequin ver. 3.5.1.2 [9]
AMOVA [10]
(1) ITS1
70 ITS2 442bp
AB743577 LC101684 LC101682
52
E. explanatum ITS2 [21, 37]
70 E. explanatum
(2) nad1
E. explanatum nad1 60
ND1-IN1 ND1-IN28 28
GenBank LC128866–LC128893 7 ND1-IN1
4 45.7%
7 8 N1
Bd12 ND1-IN7 ND1-
IN13 ND1-IN21 ND1-IN 5 ND1-IN 10 ND1-IN 17
ND1-IN 22 ND1-IN 27
10 [37]
E. explanatum nad1 4
A B C E 9
A C nad1 64.3% 31.4%
B E
7 9 A C
[37]
ND1-IN1 A ND1-IN14 C Buffalo#1
7
(3) nad1
53
AMOVA 3
0.05<p
p<0.05
72.41%
9.31% 8
E. explanatum ITS2
E. explanatum ITS2 100%
[21, 37] E. explanatum ITS2
GenBank
Paramphistomum leydeni 7 ITS2 E.
explanatum
nad1
AMOVA 3
E. explanatum
[36]
E. explanatum
1 3 F. gigantica E.
explanatum nad1 10
1 3 E. explanatum F. gigantica
54
E.
explanatum F. gigantica
F. gigantica
E. explanatum
F. gigantica
E. explanatum
E. explanatum
E. explanatum
Explanatum E. explanatum E. bathycotyle
E. anisocotyle 2
70 nad1
E. bathycotyle E. anisocotyle
4 70
DNA ITS2 442bp
70 E. explanatum
nad1 657bp
55
3 6
A C
AMOVA E.
explanatum
E. explanatum
E.
explanatum E. explanatum
56
7. E. explanatum
( ) nad1
Accession number
Delhi (27) 26 ND1-IN1 LC128866 2 ND1-IN2 LC128867 1 ND1-IN3 LC128868 2 ND1-IN4 LC128869 1 ND1-IN5 LC128870 1 ND1-IN6 LC128871 1 ND1-IN7 LC128872 1 ND1-IN8 LC128873 1 ND1-IN9 LC128874 5 ND1-IN10 LC128875 2 ND1-IN11 LC128876 1 ND1-IN12 LC128877 2 ND1-IN13 LC128878 1 ND1-IN14 LC128879 5 ND1-IN15 LC128880 1 ND1-IN16 LC128881 3 ND1-IN17 LC128882 1 ND1-IN18 LC128883 1 ND1-IN19 LC128884
Mumbai (1) 5 ND1-IN1 LC128866 Gangtok (1) 1 ND1-IN14 LC128879
1 ND1-IN20 LC128885 1 ND1-IN21 LC128886 1 ND1-IN22 LC128887 1 ND1-IN23 LC128888
Imphal (1) 1 ND1-IN1 LC128866 1 ND1-IN24 LC128889 (2) 1 ND1-IN25 LC128890 1 ND1-IN26 LC128891 1 ND1-IN27 LC128892 1 ND1-IN28 LC128893
70
57
8 E. explanatum nad1
58
Fasciola nad1 MJ network
9 E. explanatum nad1
95% 6
E1 E28 N1 N15
Bd1 Bd30
59
10 E. explanatum nad1 MJ network
1
1
E1 E28 N1 N15 Bd1 Bd30
60
Sum
of
squa
res
Var
iatio
nco
mpo
nent
sFi
xatio
nin
dex
221
1.41
41.
5980
7 V
a18
.28
Fct
= 0
.182
79
687
.871
0.81
383
Vb
9.31
Fsc
= 0
.113
91*
147
930.
651
6.33
096
Vc
72.4
1F
st =
0.2
7587
*
155
1229
.936
8.74
286
3D
elhi
Mum
bai
Gan
gtok
Imph
alBo
gra
Khu
lna
Sylh
etM
ymen
sing
hC
hitw
an*
(P<0
.05)
61
Fasciola Explanatum explanatum
Fasciola
F. gigantica
F. hepatica F. gigantica
F. gigantica
F. gigantica
Fasciola
Fasciola
Fasciola E. explanatum
Fasciola pepck pold
Fasciola
pepck pold nad1
F. gigantica
nad1
F. hepatica F. gigantica
62
F. gigantica nad1
A B
Delhi F. gigantica nad1
A
F. gigantica 3
A B C A
B. indicus
B Imphal
Imphal
B C
B. bubalis
E.
explanatum
E. explanatum
E. explanatum
E. explanatum
Fasciola gigantica
E. explanatum
F. gigantica
63
E. explanatum
Fasciola E. explanatum
F.
gigantica nad1 3
E. explanatum
Fasciola E. explanatum
Fasciola E. explanatum
64
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