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.(Doctoral Degree) 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。
༡
༡ࢪ࠾ࡅࡿⱄື≀ࡢ⫹⟶ෆᐤ⏕྾
Fasciola spp.࠾ࡼࡧ Explanatum explanatum ࡢศᏊ⣔⤫
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2 0 1 6 ᖺ
ᯘ
ᒱ
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Ꮫ
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㐃 ྜ ⋇ ་ Ꮫ ◊ ✲ ⛉
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ᒾ ᡭ Ꮫ 㸧
༡
༡ࢪ࠾ࡅࡿⱄື≀ࡢ⫹⟶ෆᐤ⏕྾
Fasciola spp.࠾ࡼࡧ Explanatum explanatum ࡢศᏊ⣔⤫
㛵ࡍࡿ◊✲
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㡫 ᗎᩥ ➨㸯❶ ࣥࢻᮾ㒊࠾ࡅࡿFasciola spp.ࡢศᏊ⣔⤫ゎᯒ 㸯㸬ㅖゝ 㸰㸬ᮦᩱ᪉ἲ 㸱㸬⤖ᯝ 㸲㸬⪃ᐹ 㸳㸬ᑠᣓ ➨2 ❶ ᪂つࡢ✀ྠᐃ࣐࣮࣮࢝pepck ࠾ࡼࡧ pold ࢆ⏝࠸ࡓ ༡ࢪ⏘ Fasciola spp.ࡢྠᐃ 㸯㸬ㅖゝ 㸰㸬ᮦᩱ᪉ἲ 㸱㸬⤖ᯝ 㸲㸬⪃ᐹ 㸳㸬ᑠᣓ ➨3 ❶ ࣥࢻす㒊ࡢࢹ࣮ࣜ࠾ࡅࡿ Fasciola spp.ࡢศᏊ⣔⤫ゎᯒ 㸯㸬ㅖゝ 㸰㸬ᮦᩱ᪉ἲ 㸱㸬⤖ᯝ 㸲㸬⪃ᐹ 㸳㸬ᑠᣓ 1 5 6 6 10 13 15 26 27 28 29 30 31 35 36 36 39 41 43➨4 ❶ ࣥࢻ࠾ࡅࡿ Explanatum explanatum ࡢศᏊ⣔⤫ゎᯒ 㸯㸬ㅖゝ 㸰㸬ᮦᩱ᪉ἲ 㸱㸬⤖ᯝ 㸲㸬⪃ᐹ 㸳㸬ᑠᣓ ⥲ᣓ ཧ⪃ᩥ⊩ 48 49 49 51 53 54 61 64
␎ㄒ⾲
AMOVA DNA ITS MJ network nad1 PCR RFLPanalysis of molecular variance
deoxyribonucleotic acid
internal transcribed spacer
median-joining network
nicotinamide adenine dinucleotide dehydrogenase subunit 1
polymerase chain reaction
1
ᗎᩥ
Fasciola ᒓࡣᲲཱྀ྾┠㸦Echinostomida㸧ࠊ⫢⻄⛉㸦Fasciolidae㸧ศ㢮ࡉࢀࡿ྾ ࡛࠶ࡿࠋFasciola ᒓࡣⱄື≀ࢆ⤊ᐟࡋࠊࡑࡢ⫹⟶ෆᐤ⏕ࡍࡿࠋ୰㛫ᐟ ࡣLymnaeidae ⛉ࡢῐỈ᳇ᕳ㈅࡛࠶ࡿࠋFasciola ᒓࡀ⤊ᐟឤᰁࡍࡿࡇ࡛ᘬࡁ㉳ࡇ ࡉࢀࡿ⫢⻄ࡣࠊᐟ࡛࠶ࡿⱄᐙ␆ࡢங㔞ཬࡧయ㔜ࡢῶᑡࡢཎᅉ࡞ࡿࡇࡽࠊ ⋇་Ꮫⓗᴟࡵ࡚㔜せ࡞ᐤ⏕ᝈ࡛࠶ࡿࠋᮏࡼࡗ࡚ᘬࡁ㉳ࡇࡉࢀࡿ⤒῭ⓗᦆኻ ࡣࠊୡ⏺୰࡛ᖺ㛫20 ൨ࢻࣝୖࡿぢ✚ࡶࡽࢀ࡚࠸ࡿ [35]ࠋ ࡲࡓࠊᮏࡣே⋇ඹ㏻ ឤᰁ࡛࠶ࡾࠊୡ⏺୰࡛⣙9,000 ேࡀឤᰁࡢࣜࢫࢡࡀ࠶ࡿࡉࢀ࡚࠸ࡿ [30]ࠋ Fasciola ᒓࡣࠊFasciola hepatica Linnaeus, 1758 F. gigantica Cobbold, 1855 ࡀ▱ࡽ ࢀ࡚࠾ࡾࠊF. hepatica ࡣ࣮ࣚࣟࢵࣃࠊ࣓ࣜ࢝࠾ࡼࡧ࢜ࢭࢽࠊF. giganticaࡣࢪ࠾ࡼࡧࣇࣜ࢝ศᕸࡍࡿ [50]ࠋࡇࢀࡽ 2 ✀ࡣ㈓⢭ᄞෆ⮬㌟ࡀ⏘⏕ࡋࡓ⢭
Ꮚࡀ‶ࡋ࡚࠾ࡾ㸦᭷⢭Ꮚᆺ㸧ࠊཷ⢭ࡼࡿ᭷ᛶ⏕Ṫࡼࡗ࡚ḟୡ௦ࢆ⏘⏕ࡍࡿࠋ
Fasciola hepatica F. gigantica ࡣྂⓗࡣᙧែᏛⓗ≉ᚩᇶ࡙࠸࡚㆑ูࡉࢀ࡚ࡁࡓ ࡀࠊᙧែᏛⓗ≉ᚩࡣᗈ⠊࡞✀ෆኚ␗ࡀㄆࡵࡽࢀࡿࡇࡽࠊᚲࡎࡋࡶṇ☜࡞✀ྠᐃ
ࡣ࡛ࡁ࡞࠸ [41]ࠋ㏆ᖺࠊ᰾ࣜ࣎ࢯ࣮࣒ DNA ࡢ internal transcribed spacer 1㸦ITS1㸧 ࠾
ࡼࡧ ITS2 㡿ᇦࡢሷᇶ㓄ิࡀศᏊ࣐࣮࣮࢝ࡋ࡚୧✀ࡢ㆑ู⏝࠸ࡽࢀ࡚࠸ࡿ [1, 24,
27, 32]ࠋࡲࡓࠊ࣑ࢺࢥࣥࢻࣜ DNA ࡢ NADH dehydrogenase subunit 1㸦nad1㸧㡿ᇦࡢ
ሷᇶ㓄ิࢆ⏝࠸࡚Fasciola ᒓࡢศᏊ⣔⤫ࡢゎᯒࡶ⾜ࢃࢀ࡚࠸ࡿ [24]ࠋ࣑ࢺࢥࣥࢻࣜ DNA ࡣ᰾ࣜ࣎ࢯ࣮࣒ DNA ẚሷᇶ⨨ࢆ✚ࡋࡸࡍ࠸ࡓࡵࠊ✀ෆࡸᴟࡵ࡚㏆⦕࡞ ✀㛫ࡢ⣔⤫ゎᯒ㐺ࡋࡓ㡿ᇦ࡛࠶ࡿࠋ㏆ᖺࡢศᏊᏛⓗ◊✲ࡼࡾࠊF. hepatica ࡣす ࢪࢆ㉳※ࡋࠊࣄࢶࢪࢆዲ㐺ᐟࡋ࡚࣮ࣚࣟࢵࣃࡸ୰ᅜᗈࡀࡗࡓ⪃࠼ࡽ ࢀ࡚࠸ࡿ [33]ࠋࡲࡓࠊ࣮ࣚࣟࢵࣃࡢ F. hepatica ಶయ⩌ࡣ 15 ୡ⣖௨㝆ࠊᐙ␆ࡢ⛣ື కࡗ࡚᪂ୡ⏺࠾ࡼࡧ࢜ࢭࢽࡶศᕸࢆᣑࡋࡓ [22]ࠋ୍᪉ࠊF. gigantica ࡣࣇࣜ
2
࢝ࢆ㉳※ࡋࠊ࢘ࢩࡸࢫࢠࣗ࢘ࢆዲ㐺ᐟࡋ࡚ࢪ࣭ࣇࣜ࢝ᗈࡀࡗࡓ⪃
࠼ࡽࢀ࡚࠸ࡿ [33]ࠋ
᭷⢭ᏊᆺࡢF. hepatica F. gigantica ຍ࠼ࠊ㈓⢭ᄞෆṇᖖ࡞⢭Ꮚࢆᣢࡓࡎ㸦↓⢭
Ꮚᆺ㸧ࠊ༢ⅭⓎ⏕㸦㞤ᛶⓎ⏕㸧ࡼࡾ⦾Ṫࢆ⾜ࡗ࡚࠸ࡿ⪃࠼ࡽࢀࡿ༢Ⅽ⏕Ṫᆺ⫢⻄
㸦aspermic Fasciola sp.㸧ࡀࢪྛᅜศᕸࡍࡿ [17, 36, 48]ࠋ༢Ⅽ⏕Ṫᆺ⫢⻄ࡣ ITS1
࠾ࡼࡧ ITS2 㡿ᇦࡢሷᇶ㓄ิࡀ F. hepatica ୍⮴ࡍࡿ Fh ᆺࠊF. gigantica ୍⮴ࡍ
ࡿ Fg ᆺࠊࡉࡽ୧✀୍⮴ࡋࡓሷᇶ㓄ิࢆేࡏᣢࡘ Fh/Fg ᆺ㸦࣊ࢸࣟᆺ㸧ࡀᏑᅾࡍ
ࡿ [18, 27]ࠋ᰾ DNA ࡣ୧ぶࡽ㑇ఏሗࢆཷࡅ⥅ࡄࡇࡽࠊ༢Ⅽ⏕Ṫᆺ⫢⻄ࡣ F.
hepatica F. gigantica ࡢ㞧ᏊᏞ࡛࠶ࡿ⪃࠼ࡽࢀࡓ [2, 24, 25, 27]ࠋࡲࡓࠊnad1 㡿 ᇦࡢሷᇶ㓄ิࢆ⏝࠸ࡓศᏊ⣔⤫ゎᯒࡼࡾࠊ༢Ⅽ⏕Ṫᆺ⫢⻄ࡢࣁࣉࣟࢱࣉࡣ F. hepatica ࡢࣁࣉࣟࢢ࣮ࣝࣉᒓࡍࡿࡶࡢࠊF. gigantica ࡢࢢ࣮ࣝࣉᒓࡍࡿࡶࡢࡢ୧ ᪉ࡀᏑᅾࡍࡿࡇࡀ᫂ࡽ࡞ࡗࡓ [24]ࠋࡇࢀࡣࠊ༢Ⅽ⏕Ṫᆺ⫢⻄ࡀ F. hepatica F. gigantica ࡢ㸰ࡘࡢẕ⣔ᏊᏞ⏤᮶ࡍࡿ㞟ᅋ࡛࠶ࡿࡇࢆ♧ࡋ࡚࠸ࡿ [24]ࠋ༢Ⅽ⏕Ṫᆺ ⫢⻄ࡣࢪྛᅜ࡛ሗ࿌ࡉࢀ࡚࠸ࡿࡀࠊ୰ᅜࡣF. hepatica F. gigantica ࡢ୧✀ࡀศ ᕸࡋ࡚࠸ࡿୖࠊ༢Ⅽ⏕Ṫᆺ⫢⻄ࡣẕ⣔ࡀF. hepatica ࡛࠶ࡿ㞟ᅋࠊẕ⣔ࡀ F. gigantica ࡛࠶ࡿ㞟ᅋࡀ࠸ࡎࢀࡶศᕸࡋ࡚࠸ࡿ [23, 40]ࠋࡇࡢࡇࡽࠊ༢Ⅽ⏕Ṫᆺ⫢⻄ࡣ F. hepatica F. gigantica ࡢ㞧ᏊᏞࡋ࡚୰ᅜ࡛Ⓨ⏕ࡋࠊࢪㅖᅜศᕸࢆᣑࡋࡓ ⪃࠼ࡽࢀࡓ [23, 40]ࠋࡲࡓࠊ༢Ⅽ⏕Ṫᆺ⫢⻄ࡣ㑇ఏⓗከᵝᛶࡀᴟࡵ࡚ஈࡋ࠸ࡇ ࡽࠊᐟ࡛࠶ࡿᐙ␆ࡢேⅭⓗ࡞⛣ືࡶࠊẚ㍑ⓗ▷㛫ಶయᩘࢆᛴ⃭ቑຍࡉ ࡏࡓ⪃࠼ࡽࢀ࡚࠸ࡿ [36, 42]ࠋ ༡ࢪ㸦ࢿࣃ࣮ࣝ࠾ࡼࡧࣂࣥࢢࣛࢹࢩࣗ㸧ࡣF. gigantica ༢Ⅽ⏕Ṫᆺ⫢⻄ࡀ ศᕸࡋ࡚࠸ࡿ[36, 42]ࠋࡇࢀࡲ࡛ࠊ༢Ⅽ⏕Ṫᆺ⫢⻄ࡘ࠸࡚ࡣከࡃࡢሗ࿌ࡀ࠶ࡿࡀࠊ༡ ࢪ࠾ࡅࡿ F. gigantica ࡢ㉳※ᣑᩓ⤒㊰ࡘ࠸࡚ࡢሗ࿌ࡣ㝈ᐃⓗ࡛࠶ࡿࠋ
3 ࡇࡀ▱ࡽࢀ࡚࠸ࡿ [33]ࠋࢮࣈ࣮∵ࡣࢪࡢ⇕ᖏᆅᇦ࠾࠸࡚ F. gigantica ࡢ㔜せ ࡞⤊ᐟ࡛࠶ࡾࠊࣥࢲࢫ㇂࡛ᐙ␆ࡉࢀࡓᚋࠊࢪ࠾ࡼࡧࣇࣜ࢝ᗈࡀࡗࡓ ⪃࠼ࡽࢀ࡚࠸ࡿ [6]ࠋࡇࡢࡓࡵࠊB. indicus ࡢேⅭⓗ࡞⛣ືࡀࠊࡇࢀࡽࡢᆅᇦ࠾ࡅ ࡿF. gigantica ࡢศᕸᣑ㛵ಀࡋ࡚࠸ࡿྍ⬟ᛶࡀ࠶ࡿࠋࡋࡋ࡞ࡀࡽࠊ༡ࢪ࠾ ࡅࡿFasciola ᒓࡢศᏊᏛⓗሗࡣ㝈ࡽࢀ࡚࠾ࡾࠊヲ⣽࡞ศᏊ⣔⤫Ꮫⓗゎᯒࢆ⾜࠺ࡣ ༑ศ࡛ࡣ࡞ࡗࡓࠋ Explanatum ᒓࡣᲲཱྀ྾┠㸦Echinostomida㸧ࠊཱྀ྾⛉㸦Paramphistomidae㸧 ศ㢮ࡉࢀࡿ྾࡛࠶ࡾࠊᡂࡣⱄື≀ࡢ⫹⟶ෆᐤ⏕ࡍࡿࠋ⏕ά⎔ࡣ Fasciola ᒓ㢮ఝࡋ࡚࠾ࡾࠊ୰㛫ᐟࡣ Gyraulus ᒓࡢ㈅㢮࡛࠶ࡿ [42]ࠋⱄື≀ࡢ⫹⟶ෆ ᐤ⏕ࡋࡓ Explanatum ᒓࡢᡂࡣᚋ྾┙࡛⫹⟶ୖ⓶྾╔ࡍࡿࡇ࡛ࠊ⫹⟶ࡢ⫗ⱆ⭘ ᛶኚࡸ⫹⟶ቨࡢ⫧ཌࢆᘬࡁ㉳ࡇࡍ [11, 34]ࠋࡑࡢࡓࡵࠊExplanatum ᒓឤᰁࡣᐙ␆ࡢ Ⓨ⫱㜼ᐖࡸங㔞ࡢῶᑡࢆᘬࡁ㉳ࡇࡋࠊ␆⏘ᴗ⤒῭ᦆኻࢆ࠼࡚࠸ࡿࠋExplanatum ᒓ
ࡣE. explanatum㸦Creplin, 1847㸧ࠊE. bathycotyle㸦Fischoeder, 1901㸧࠾ࡼࡧ E. anisocotyle
㸦Faust, 1920㸧ࡢ 3 ✀ࡀ▱ࡽࢀ࡚࠸ࡿࠋExplanatum ᒓࡢᶍᘧ✀࡛࠶ࡿ E. explanatum ࡣ
ࢪ࣭ࣇࣜ࢝ᆅᇦศᕸࡋ࡚࠾ࡾ [8, 44]ࠊ≉ࣥࢻள㝣ࡢⱄᐙ␆ࡽ ࡣ୍⯡ⓗ᳨ฟࡉࢀࡿ [3, 11, 44]ࠋࣥࢻ࠾࠸࡚ࠊE. explanatum ࡣᗈᇦศᕸࡋ࡚ ࠾ࡾࠊ≉ࢪࢫࢠࣗ࢘㸦Bubalus bubalis㸧ࡽ୍⯡ⓗ᳨ฟࡉࢀࡿ [8, 11, 38, 45]ࠋ ཱྀ྾ࡢᙧែᏛⓗྠᐃࡣᡂࡢ⤌⧊ᶆᮏࢆ⏝࠸ࡓᙧែほᐹࡀྍḞ࡛࠶ࡿࠋࡋ ࡋࠊᶆᮏసᡂከࡃࡢ㛫ປຊࢆせࡋࠊࡉࡽᙧែ㛵ࡍࡿ㧗ᗘ࡞⇍⦎ࡀᚲせ࡛࠶ ࡿࠋࡑࡢࡓࡵࠊ㏆ᖺࡣศᏊᏛⓗྠᐃἲࡢ㛤Ⓨࡀ⾜ࢃࢀ࡚࠸ࡿ [7, 39]ࠋExplanatum
explanatum ࢆྵࡴཱྀ྾ྛ✀࠾࠸࡚ࠊ᰾ࣜ࣎ࢯ࣮࣒ DNA ࡢ ITS2 㡿ᇦࡢሷᇶ㓄ิ ࡀ✀ྠᐃ࣐࣮࣮࢝ࡋ࡚㛤Ⓨࡉࢀࠊ⏝ࡉࢀ࡚࠸ࡿ [21, 28]. ࡲࡓࠊ༡ࢪࡢࢿࣃ࣮
ࣝ࠾ࡼࡧࣂࣥࢢࣛࢹࢩࣗ࠾ࡅࡿ E. explanatum ࡢ࣑ࢺࢥࣥࢻࣜ DNA ࡢ nad1 㡿ᇦ
4 ከᵝ࡛࠶ࡿࡇࡀ▱ࡽࢀ࡚࠸ࡿ [37]ࠋࡋࡋ࡞ࡀࡽࠊࡇࢀࡽ 2 ᅜࡢ㛫⨨ࡍࡿ ࣥࢻ࠾࠸࡚ E. explanatum ࡢศᏊᏛⓗሗࡣሗ࿌ࡉࢀ࡚࠾ࡽࡎࠊ༡ࢪ࠾ࡅࡿ E. explanatum ࡢヲ⣽࡞ศᏊ⣔⤫Ꮫⓗゎᯒࡣࡇࢀࡲ࡛⾜ࢃࢀ࡚࠸࡞ࡗࡓࠋ ᮏ◊✲࡛ࡣࠊ༡ࢪ࠾ࡅࡿⱄື≀ࡢ୍⯡ⓗ࡞⫹⟶ෆᐤ⏕྾࡛࠶ࡿFasciola ᒓ࠾ࡼࡧ E. explanatum ࡘ࠸࡚ศᏊ⣔⤫ゎᯒࢆ⾜࠺ࡓࡵࠊศᏊᏛⓗሗࡀ㊊ࡋ࡚ ࠸ࡓࣥࢻ࠾ࡅࡿಶయ⩌ࢆศᏊᏛⓗゎᯒࡋࠊࡑࡢᣑᩓᐟື≀ࡢ㛵ಀࡘ࠸ ᳨࡚ドࢆ⾜ࡗࡓࠋ➨1 ❶࡛ࡣࣥࢻᮾ㒊࠾ࡅࡿ Fasciola ᒓࡢศᏊ⣔⤫Ꮫⓗゎᯒࢆ ⾜ࡗࡓࠋ➨2 ❶࡛ࡣ㏆ᖺሗ࿌ࡉࢀࡓ᪂ࡓ࡞ศᏊ࣐࣮࣮࢝ࢆ⏝࠸ࠊ༡ࢪ⏘ Fasciola ᒓࡢྠᐃࢆ⾜ࡗࡓࠋ➨3 ❶࡛ࡣࠊࣥࢻす㒊࠾ࡅࡿ Fasciola ᒓࡢศᏊ⣔⤫Ꮫⓗ ゎᯒࢆ⾜ࡗࡓࠋࡉࡽ➨ 4 ❶࡛ࡣࠊࣥࢻ⏘ E. explanatum ࡢศᏊ⣔⤫ゎᯒࢆ⾜࠸ࠊ Fasciola ᒓࡢẚ㍑ࢆ⾜ࡗࡓࠋ
5
6 㸯㸬ㅖゝ ࡇࢀࡲ࡛ࠊ༡ࢪࡢࢿࣃ࣮ࣝ࠾ࡼࡧࣂࣥࢢࣛࢹࢩࣗ࠾࠸࡚ࡣF. gigantica ࡢ ศᏊᏛⓗ◊✲ࡢሗ࿌ࡀ࠶ࡾࠊࡇࢀࡽࡢᅜࡣ༡ࢪ≉᭷⪃࠼ࡽࢀࡿF. gigantica ಶయ⩌ࡀศᕸࡋࠊࡑࡢศᕸᣑࡣᐙ␆ࡉࢀࡓࢮࣈ࣮∵㸦B. indicus㸧ࡢேⅭⓗ࡞⛣ ືࡀ㛵ࡋࡓ⪃࠼ࡽࢀ࡚࠸ࡿ [36, 42]ࠋࡋࡋ࡞ࡀࡽࠊ༡ࢪ㝣ࡢ㒊ศࢆ༨ ࡵࡿࣥࢻ࠾ࡅࡿFasciola ᒓࡢศᏊᏛⓗゎᯒࡣࢇ⾜ࢃࢀ࡚࠾ࡽࡎࠊ༡ࢪ ࠾ࡅࡿF. gigantica ಶయ⩌ࡢ⣔⤫㛵ಀࡣࡇࢀࡲ࡛༑ศゎ᫂ࡉࢀ࡞ࡗࡓࠋࡲࡓࠊࢿ ࣃ࣮ࣝ࠾ࡼࡧࣂࣥࢢࣛࢹࢩࣗࡣ༢Ⅽ⏕Ṫᆺ⫢⻄ࡶศᕸࡋ࡚࠾ࡾࠊ୰ᅜ࡛Ⓨ⏕ࡋࡓ༢ Ⅽ⏕Ṫᆺ⫢⻄ࡀ༡ࢪࡶศᕸࢆᣑࡋ࡚࠸ࡿࡇࡀ♧၀ࡉࢀࡓࡀࠊࡑࡢᣑᩓ⤒㊰ ࡣ࡛᫂࠶ࡗࡓ [36, 42]ࠋࡑࡇ࡛ᮏ❶࡛ࡣࠊ༡ࢪ࠾ࡅࡿ F. gigantica ࠾ࡼࡧ༢Ⅽ ⏕Ṫᆺ⫢⻄ࡢᣑᩓ⤒㊰ࢆ᫂ࡽࡍࡿࡇࢆ┠ⓗࡋࠊࢿࣃ࣮ࣝࠊࣂࣥࢢࣛࢹࢩࣗ࠾ ࡼࡧ୰ᅜᅜቃࢆ᥋ࡍࡿᆅᇦ࡛࠶ࡿࣥࢻᮾ㒊࠾ࡅࡿ Fasciola ᒓࡢྠᐃ࠾ࡼࡧ ศᏊ⣔⤫ゎᯒࢆ⾜ࡗࡓࠋ 㸰. ᮦᩱ᪉ἲ (1) ࣥࢻᮾ㒊⏘ Fasciola ᒓ྾ࡢ᥇ྲྀฎ⌮ 2014 ᖺ 1 ᭶ࡽ 9 ᭶ࣥࢻᮾ㒊ࡢࣥࣃ࣮ࣝ㸦Imphal㸧ᕷࠊࢥࣄ࣐㸦Kohima㸧
7 ᕷ࠾ࡼࡧ࢞ࣥࢺࢡ㸦Gangtok㸧ᕷࡢ࣐࣮ࢣࢵࢺ࠾࠸࡚ࠊࢮࣈ࣮∵ 35 㢌ࠊࢪࢫ ࢠࣗ࢘ 15 㢌࠾ࡼࡧࣖࢠ 1 㢌ࡢ⫹⟶ࡼࡾ Fasciola ᒓ྾ࡢᡂࢆ᥇ྲྀࡋࠊࡑࢀࡽࡢ࠺ ࡕImphal ᕷ⏤᮶ࡢࡶࡢࡣᐟ 1 㢌ࡘࡁ 1 ࡽ 5 యࠊKohima ᕷ⏤᮶ࡢࡶࡢࡣᐟ 1 㢌ࡘࡁ 1 ࡽ 8 యࠊGangtok ⏤᮶ࡢࡶࡢࡣᐟ 1 㢌ࡘࡁ 10 ࡽ 16 ༉ࠊィ 157 యࢆゎᯒ౪ࡋࡓࠋయࡣ70%࢚ࢱࣀ࣮࡛ࣝᅛᐃࡋࠊᒾᡭᏛᐤ⏕Ꮫ◊✲ᐊ㐠 ᦙࡋࡓࠋᐇయ㢧ᚤ㙾ୗ࡛ྛయࡽ㈓⢭ᄞࢆྲྀࡾฟࡋࠊᖖἲᚑࡗ࡚࣐࣊ࢺ࢟ࢩ࣭ࣜࣥ ࣮࣑࢝ࣥࡼࡾᰁⰍࡋࡓᚋࠊගᏛ㢧ᚤ㙾ࢆ⏝࠸࡚⢭Ꮚࡢ᭷↓ࢆほᐹࡋࡓ [48]ࠋࡲࡓࠊ
⏕Ṫჾᐁࢆྵࡲ࡞࠸యᚋ㒊ࡢ⤌⧊ࡼࡾࠊHigh Pure PCR Template Preparation Kit
㸦Roche, Mannheim, Germany㸧ࢆ⏝࠸࡚ྛయࡢ DNA ࡀᢳฟࡉࢀࠊ⏝ࡲ࡛㸫20Υ
࡛ಖᏑࡉࢀࡓࠋ
(2) ྛ㡿ᇦࡢ PCR
ྛయࡘ࠸࡚ࠊࣉ࣐࣮ࣛITS1-F㸦5’-TTGCGCTGATTACGTCCCTG-3’㸧࠾ࡼࡧ
ITS1-R㸦5’-TTGGCTGCGCTCTTCATCGAC-3’㸧ࢆ⏝࠸࡚ ITS1 㡿ᇦࢆࠊIta
10㸦5’-AAGGATGTTGCTTTGTCGTGG-3’㸧࠾ࡼࡧ Ita 2㸦5’-GGAGTACGGTTACATTCACA-3’㸧
ࢆ⏝࠸࡚nad1 㡿ᇦࢆࠊࡑࢀࡒࢀ PCR ࡼࡾቑᖜࡋࡓ [24]ࠋPCR ࡣ 0.025 U ࡢ GoTaq
DNA Polymerase㸦Promega, Madison, USA 㸧ࠊయ⏤᮶ࡢ DNA 㸦⣙ 100 ng/μL㸧ࠊྛ
dNTP㸦0.08 mM㸧ࠊྛࣉ࣐࣮ࣛ㸦0.5 pM㸧ࠊ each primer, 0.025 U 5GoTaq buffer㸦50
8
90 ⛊ࡢ⇕ኚᛶᚋࠊ94°C 90 ⛊࣭55°C 90 ⛊࣭72°C 120 ⛊ࡢࢧࢡࣝࢆ 30 ᅇ⾜࠸ࠊ᭱ᚋ
72°C 10 ศࡢఙ㛗ᛂࢆຍࡋࡓࠋPCR ቑᖜ⏘≀ࡣ 1.0%࣮࢞ࣟࢫࢤࣝࢆ⏝࠸࡚
㟁ẼὋືࡼࡗ࡚┠ⓗ㡿ᇦࡢቑᖜࢆ☜ㄆࡉࢀࡓࠋ
(3) ITS1 㡿ᇦࡢ㑇ఏᏊᆺ࠾ࡼࡧ nad1 㡿ᇦࡢሷᇶ㓄ิࡢゎᯒ
ITS1 ࡢ PCR ቑᖜ⏘≀ࡣ 10U ࡢไ㝈㓝⣲ Rsa I㸦Roche㸧࠾ࡼࡧ buffer enzyme L
㸦Roche㸧ࢆຍ࠼ࠊ㔞 50 μL ࡢᛂᾮࢆㄪᩚࡋࡓࠋᛂᾮࢆ 37Υ୍࡛ᬌࣥ࢟ࣗ࣋ ࣮ࢩࣙࣥࡍࡿࡇ࡛ PCR ⏘≀ࢆ᩿∦ࡋࠊ1.8%࣮࢞ࣟࢫࢤࣝࢆ⏝࠸ࡓ㟁ẼὋື ࡼࡗ࡚ࣂࣥࢻࣃࢱ࣮ࣥࢆ☜ㄆࡋࡓ [18]ࠋITS1 㡿ᇦࡢ㑇ఏᏊᆺ࡛ࡣ⣙ 360 bpࠊ⣙ 100 bp ࠾ࡼࡧ⣙60 bp 3 ᮏࡢࣂࣥࢻࡀ᳨ฟࡉࢀࡿ Fh ᆺࠊ⣙ 360bpࠊ⣙ 170 bp ࠾ࡼࡧ⣙ 60 bp 3 ᮏࡢࣂࣥࢻࡀ᳨ฟࡉࢀࡿ Fg ᆺࠊ࠾ࡼࡧ⣙ 360 bpࠊ⣙ 170bpࠊ⣙ 100 bp ࠾ࡼࡧ ⣙60 bp 4 ᮏࡢࣂࣥࢻࡀ᳨ฟࡉࢀࡿ Fh/Fg ᆺࡀᏑᅾࡍࡿࠋࡇࢀࡽࡢ㑇ఏᏊᆺࡢ࠺ࡕࠊ ᭷⢭Ꮚᆺ࡛࠶ࡿF. hepatica ࡣ Fh ᆺࢆࠊF. gigantica ࡣ Fg ᆺࢆ♧ࡍࠋ୍᪉࡛ࠊ↓⢭Ꮚᆺ ࡛࠶ࡿ༢Ⅽ⏕Ṫᆺ⫢⻄ࡣFh ᆺࠊFg ᆺ࠾ࡼࡧ Fh/Fg ᆺࢆ♧ࡍయࡀࡑࢀࡒࢀሗ࿌ࡉ ࢀ࡚࠸ࡿ [5, 17-20, 23-26, 36, 40, 43]ࠋ
nad1 㡿ᇦࡢ PCR ቑᖜ⏘≀ࡣ NucleoSpin® Gel and PCR Clean-up㸦Takara Bio Inc., Otsu, Japan㸧ࢆ⏝࠸࡚⢭〇ᚋࠊሷᇶ㓄ิ㸦535 bp㸧ࢆࠊࢲࣞࢡࢺࢩ࣮ࢡ࢚ࣥࢫἲࢆ⏝࠸࡚
ゎᯒࡋࡓࠋࢩ࣮ࢡ࢚ࣥࢫᛂࡣBigDye Terminator v3.1 Cycle Sequencing Kit㸦Applied
9
PRISM 3500 Genetic Analyzer㸦Applied Biosystems㸧ࢆ⏝࠸࡚ሷᇶ㓄ิࡢゎᯒࢆ⾜ࡗࡓࠋ
ࢩ࣮ࢡ࢚ࣥࢫᛂࡣ PCR ᛂྠ୍ࡢࣉ࣐࣮ࣛࢆ⏝࠸ࡓࠋᚓࡽࢀࡓሷᇶ㓄ิࡣ
ATGC ver. 6. 0. 3㸦Genetyx, Tokyo, Japan㸧ࢆ⏝࠸࡚ࢭࣥࣈࣝᚋࠊGENETYX ver. 10. 0.
2 㸦Genetyx㸧ࢆ⏝࠸࡚࣓ࣛࣥࢺࡋࠊࣁࣉࣟࢱࣉࢆ㆑ูࡋࡓࠋ
(4) nad1 㡿ᇦࡢሷᇶ㓄ิᇶ࡙ࡃࢿࢵࢺ࣮࣡ࢡࡢసᡂ
Network 4. 6. 1. 2 ࢆ⏝࠸࡚ࠊࣥࢻᮾ㒊⏘ࡶࢪྛᅜ⏘ F. gigantica ࠾ࡼ
ࡧ༢Ⅽ⏕Ṫᆺ⫢⻄ࡢnad1 ࣁࣉࣟࢱࣉᇶ࡙࠸ࡓ Median-joining 㸦MJ㸧network ࢆస
ᡂࡋࡓ [4]ࠋᮏ❶࡛ゎᯒࡋࡓࣥࢻᮾ㒊⏘ Fasciola ᒓࡢ nad1 ࣁࣉࣟࢱࣉࡑࡢฟ ⌧㢖ᗘຍ࠼ࠊ᪥ᮏ [20, 24]ࠊ㡑ᅜ [19, 25]ࠊ୰ᅜ [23, 40]ࠊ࣋ࢺࢼ࣒ [26]ࠊࢱ [5]ࠊ ࣑࣐࣮ࣕࣥ [17]ࠊࣂࣥࢢࣛࢹࢩࣗ [36] ࠾ࡼࡧࢿࣃ࣮ࣝ [43] ⏘ Fasciola ᒓࡢ nad1 ࣁ ࣉࣟࢱࣉࡑࡢฟ⌧㢖ᗘࢆཧ↷ࡋ࡚⏝࠸ࡓࠋ࡞࠾ࠊᮏ❶࡛ࡣMJ network ࠾࠸࡚ ࡍ࡚ࡢࣁࣉࣟࢱࣉࢆ┤᥋㐃⤖ࡉࡏࡿࡇࡣ࡛ࡁ࡞ࡗࡓࠋࡇࡢࡓࡵࠊ┤᥋㐃⤖ࡉ ࡏࡿࡇࡀ࡛ࡁ࡞࠸ࣁࣉࣟࢱࣉ㛫ࢆ㐃⤖ࡉࡏࡿࡓࡵࡢ௬ࣁࣉࣟࢱࣉࡋ࡚ median vector ࢆ⏝࠸ࡓࠋ (5) nad1 㡿ᇦ࠾ࡅࡿ㑇ఏⓗከᵝᛶࡢゎᯒ ࣥࢻᮾ⏘ࠊ࣑࣐࣮ࣕࣥ⏘ࠊࣂࣥࢢࣛࢹࢩࣗ⏘࠾ࡼࡧࢿࣃ࣮ࣝ⏘Fasciola ᒓࡢྛ
10
ಶయ⩌࠾ࡅࡿ㑇ఏⓗከᵝᛶࢆホ౯ࡍࡿࡓࡵࠊDnaSP v5. 1. 0 ࢆ⏝࠸࡚ࣁࣉࣟࢱࣉ
ከᵝᗘ㸦Hd㸧࠾ࡼࡧሷᇶከᵝᗘ㸦π㸧ࢆ⟬ฟࡋࡓ [31]ࠋ ࡲࡓࠊྛಶయ⩌㛫࠾ࡅࡿ㑇
ఏⓗศࡢ⛬ᗘࢆホ౯ࡍࡿࡓࡵࠊAlrequin 3.5 ࢆ⏝࠸࡚ pairwise fixation index 㸦Fst ್㸧
ࢆ⟬ฟࡋࡓ [9]ࠋࡇࢀࡽࡢゎᯒ࠾࠸࡚ࠊࣥࢻᮾ㒊⏘ Fasciola ᒓࡢ࠺ࡕࠊ☜ᐇ࡞ ✀ྠᐃࢆ⾜࠺ࡇࡀ࡛ࡁ࡞ࡗࡓ6 యࡢሷᇶ㓄ิࡣ㝖እࡉࢀ࡚࠸ࡿ㸦⾲ 1㸧ࠋࡲࡓࠊ ࣥࢻ⏘࠾ࡼࡧ࣑࣐࣮ࣕࣥ⏘F. gigantica ࡣ 2 ࡘࡢࣁࣉࣟࢢ࣮ࣝࣉ㸦A ࠾ࡼࡧ B㸧ศ ࡅ࡚ゎᯒࢆ⾜ࡗࡓࠋ 㸱㸬⤖ᯝ (1) ㈓⢭ᄞෆ⢭Ꮚࡢ᭷↓ ITS1 ᆺࡢゎᯒ⤖ᯝ
Imphal ᕷࡽᚓࡽࢀࡓ 23 యࠊKohima ᕷࡽᚓࡽࢀࡓ 3 య࠾ࡼࡧ Gangtok ᕷ
ࡽᚓࡽࢀࡓ 13 యࡣ㈓⢭ᄞෆṇᖖ࡞⢭Ꮚࡀほᐹࡉࢀࡎࠊ↓⢭Ꮚᆺࡢ༢Ⅽ⏕Ṫᆺ⫢ ⻄ุᐃࡉࢀࡓࠋ୍᪉ࠊṧࡿ 118 యࡢ㈓⢭ᄞෆࡣᡂ⇍ࡋࡓ⢭Ꮚࡀ‶ࡋ࡚࠾ࡾࠊ ᭷⢭Ꮚᆺุᐃࡉࢀࡓ㸦ᅗ1 ࠾ࡼࡧ⾲ 1㸧ࠋ᭷⢭Ꮚᆺࡢయࡣ 118 య࡚ࡀ ITS1 ᆺ ࠾࠸࡚Fg ᆺࢆ♧ࡋࡓࡇࡽࠊF. gigantica ࡛࠶ࡿ⪃࠼ࡽࢀࡓࠋ୍᪉ࠊ༢Ⅽ⏕Ṫ ᆺ⫢⻄39 యࡶ ITS1 ᆺ࠾࠸࡚ࠊࡍ࡚ Fg ᆺࢆ♧ࡋࡓ㸦ᅗ 2 ࠾ࡼࡧ⾲ 1㸧ࠋᮏ◊✲ ࡚యࡢ᥇㞟ࢆ⾜ࡗࡓࣥࢻᮾ㒊ࡢ 3 ᆅᇦࡽࡣࠊ࠸ࡎࢀࡶ F. gigantica ࠾ࡼࡧ ༢Ⅽ⏕Ṫᆺ⫢⻄ࡀ᳨ฟࡉࢀࡓ㸦ᅗ3㸧ࠋ
11
(2) nad1 㡿ᇦࡢゎᯒ⤖ᯝ
ࣥࢻᮾ㒊⏘Fasciola ᒓ 157 యࡢ nad1 㡿ᇦࡢሷᇶ㓄ิ㸦535bp㸧ࡽࡣࠊ40 ࡢ
ሷᇶኚ␗ᗙࡀ☜ㄆࡉࢀࠊND1-E1 ᆺࡽ ND1-E37 ᆺࡲ࡛ࡢ 37 ࣁࣉࣟࢱࣉࡀ㆑ู
ࡉࢀࡓࠋࡇࢀࡽࡢ㓄ิሗࡣ GenBank Ⓩ㘓ࡉࢀࡓ㸦accession numbers: LC012895–
LC012931㸧㸦⾲ 1㸧ࠋND1-E1 ᆺࡣ Imphal ᕷࠊKohima ᕷ࠾ࡼࡧ Gangtok ᕷ⏤᮶ࡢ 32
య࠾࠸࡚☜ㄆࡉࢀࠊ㏆㞄ࢪㅖᅜࡽሗ࿌ࡢ࠶ࡿ༢Ⅽ⏕Ṫᆺ⫢⻄ࡢࣁࣉࣟࢱࣉ ྠ୍࡛࠶ࡗࡓ[5, 17, 19, 20, 24, 25, 26, 27, 36, 40, 42, 47]ࠋࡲࡓࠊND1-E2 ᆺࡣ Imphal ⏤᮶ࡢ1 య࠾࠸࡚☜ㄆࡉࢀࡓ᪂つࣁࣉࣟࢱࣉ࡛࠶ࡾࠊND1-E1 ᆺ 1 ሷᇶ⨨ ࡢ㛵ಀ࡛࠶ࡗࡓࠋࡇࢀࡽࡢࣁࣉࣟࢱࣉࡣMJ network ࠾࠸࡚ࠊ࠸ࡎࢀࡶ༢Ⅽ⏕Ṫᆺ ⫢⻄ࡢࣁࣉࣟࢢ࣮ࣝࣉྵࡲࢀࡓࠋ㈓⢭ᄞෆ⢭Ꮚࡢ᭷↓࠾ࡼࡧ ITS1 㑇ఏᏊᆺᇶ࡙ ࠸࡚༢Ⅽ⏕Ṫᆺ⫢⻄⪃࠼ࡽࢀࡓ39 యࡢ࠺ࡕࠊ32 యࡀ E1 ᆺࠊ1 యࡀ ND1-E2 ᆺࢆ♧ࡋࡓࠋṧࡾ 6 యࡢ࠺ࡕࠊ5 యࡣ ND1-E3 ᆺࠊ1 యࡣ ND1-ND1-E21 ᆺࢆࡑࢀ ࡒࢀ♧ࡋࠊࡇࢀࡽࡢࣁࣉࣟࢱࣉࡣ࠸ࡎࢀࡶMJ network ࠾࠸࡚ F. gigantica ࡢࣁࣉ ࣟࢢ࣮ࣝࣉᒓࡋࡓ㸦ᅗ4㸧ࠋࡑࡢࡓࡵࠊࡇࡢ 6 యࡣ༢Ⅽ⏕Ṫᆺ⫢⻄࡛ࡣ࡞ࡃࠊຍ㱋 ࡼࡿ⢭Ꮚᙧᡂ⬟ࡢᾘኻࡸࠊ᥋ࡼࡿ⢭Ꮚᨺฟ࡞ࡢ⌮⏤ࡼࡾ㈓⢭ᄞෆ⢭Ꮚࢆ ᭷ࡋ࡚࠸࡞ࡗࡓ F. gigantica ࡛࠶ࡿྍ⬟ᛶࡀ⪃࠼ࡽࢀࡓࡀࠊ㈓⢭ᄞෆ⢭Ꮚࡢ᭷↓ nad1 ࡢࣁࣉࣟࢱࣉࡀ୍⮴ࡋ࡞ࡗࡓࡇࡽࠊṇ☜࡞✀ྠᐃࢆ⾜࠺ࡇࡣ࡛ࡁࡎࠊ Fasciola sp.ࡋࡓࠋ
12
ND1-E6 ᆺࡣ Imphal ᕷࠊKohima ᕷ࠾ࡼࡧ Gangtok ᕷࡢ F. gigantica 45 య㸦28.7%㸧
࡛☜ㄆࡉࢀࠊࣥࢻᮾ㒊⏘Fasciola ᒓࡢ࠺ࡕࠊࡶࡗࡶฟ⌧㢖ᗘ㧗࠸ࣁࣉࣟࢱ ࣉ࡛࠶ࡗࡓࠋࡇࡢࣁࣉࣟࢱࣉࡣࡇࢀࡲ࡛࣑࣐࣮ࣕࣥ㸦ࣁࣉࣟࢱࣉྡ㸸M15 ᆺ㸧 [17]ࠊࢱ㸦TH13 ᆺ㸧 [5]ࠊࣂࣥࢢࣛࢹࢩࣗ㸦Bd9 ᆺ㸧[36] ࠾ࡼࡧࢿࣃ࣮ࣝ㸦N1 ᆺ㸧 [42] ࡽ᳨ฟࡉࢀࡓ F. gigantica ࡢࣁࣉࣟࢱࣉྠ୍࡛࠶ࡗࡓࠋImphal ᕷ⏤᮶ࡢ 30 యࡣࠊND1-E3 ᆺࢆ♧ࡋ࡚࠾ࡾࠊࡇࡢࣁࣉࣟࢱࣉࡣ࣑࣐࣮ࣕࣥ㸦M1 ᆺ㸧 [17] ࠾ ࡼࡧࢱ㸦TH15 ᆺ㸧[5] ࠾࠸࡚ሗ࿌ࡉࢀࡓࣁࣉࣟࢱࣉྠ୍࡛࠶ࡗࡓ㸦ᅗ 4㸧ࠋ
ND1-E4 ᆺ࠾ࡼࡧ ND1-E5 ᆺࡣ ND1-E3 ᆺࡶ F. gigantica ࣁࣉࣟࢢ࣮ࣝࣉ B ྵ
ࡲࢀ࡚࠾ࡾࠊND1-E3 ᆺࡢ⾨ᫍࣁࣉࣟࢱࣉࡔࡗࡓࠋྠᵝࠊND1-E7 ᆺࡽ ND1-E37
ᆺࡣND1-E6 ᆺࡢ⾨ᫍࣁࣉࣟࢱࣉ࡛࠶ࡾࠊND1-E6 ᆺࡶ F. gigantica ࣁࣉࣟࢢ
࣮ࣝࣉA ྵࡲࢀࡓࠋࡲࡓࠊ୰ᅜࠊࢱࠊ࣋ࢺࢼ࣒⏘ Fasciola ᒓࡽ᳨ฟࡉࢀࡓ୍㒊 ࡢࣁࣉࣟࢱࣉࡣF. gigantica ࣁࣉࣟࢢ࣮ࣝࣉ C ᒓࡋ࡚࠾ࡾࠊࣁࣉࣟࢢ࣮ࣝࣉ A ࠾ ࡼࡧ B ࡣ༊ูࡉࢀࡓ㸦ᅗ 4㸧ࠋࣥࢻᮾ㒊ࡽࡣࠊF. gigantica ࣁࣉࣟࢢ࣮ࣝࣉ C ᒓࡍࡿnad1 ࣁࣉࣟࢱࣉࡣ᳨ฟࡉࢀ࡞ࡗࡓࠋ (3) nad1 㡿ᇦ࠾ࡅࡿ㑇ఏⓗከᵝᛶ Fasciola gigantica ࣁࣉࣟࢢ࣮ࣝࣉ A ࠾ࡅࡿሷᇶከᵝᗘ㸦ȧ㸧ࡣࢿࣃ࣮ࣝಶయ⩌ 㸦0.00366㸧࠾ࡼࡧࣂࣥࢢࣛࢹࢩࣗಶయ⩌㸦0.00362㸧࠾࠸࡚㧗್ࢆ♧ࡋࠊࣥࢻᮾ 㒊ࡢಶయ⩌㸦0.00242㸧ࡸ࣑࣐࣮ࣕࣥಶయ⩌㸦0.00255㸧࠾࠸࡚ప್ࢆ♧ࡋࡓࠋࡲࡓࠊ
13 F. gigantica ࣁࣉࣟࢢ࣮ࣝࣉ B ࠾ࡅࡿሷᇶከᵝᗘࡣ࣑࣐࣮ࣕࣥಶయ⩌࠾࠸࡚㧗್ 㸦0.00170㸧ࢆ♧ࡋࠊࣥࢻᮾ㒊ࡢಶయ⩌࠾࠸࡚ప್㸦0.00028㸧ࢆ♧ࡋࡓࠋ୍᪉ࠊ ༢Ⅽ⏕Ṫᆺ⫢⻄ࡢሷᇶከᵝᗘࡣࠊ࠸ࡎࢀࡢಶయ⩌ࡶ F. gigantica ẚ࡚ⴭࡋࡃప್ 㸦0.00000 ࡽ 0.00011㸧࡛࠶ࡗࡓࠋ Fasciola gigantica ࠾ࡼࡧ༢Ⅽ⏕Ṫᆺ⫢⻄ࡢྛಶయ⩌㛫࠾ࡅࡿ Fst ್ࡣࣁࣉࣟࢢࣝ ࣮ࣉ A ࠾ࡼࡧ B 㛫࡛ࡣ㧗್ࢆ♧ࡋࡓࡢᑐࡋࠊࣁࣉࣟࢢ࣮ࣝࣉෆ࡛ࡣ࠸ࡎࢀࡶప್ ࢆ♧ࡋࡓࠋ࠼ࡤࠊࣥࢻᮾ㒊ࡢࣁࣉࣟࢢ࣮ࣝࣉ A ಶయ⩌ࣁࣉࣟࢢ࣮ࣝࣉ B ಶ య⩌㛫ࡢFst ್ࡀ 0.86934㸦p<0.05㸧㧗್࡛࠶ࡿࡢᑐࡋࠊࣥࢻᮾ㒊ࡢࣁࣉࣟࢢ ࣮ࣝࣉ A ಶయ⩌ࣂࣥࢢࣛࢹࢩࣗࡢࣁࣉࣟࢢ࣮ࣝࣉ A ಶయ⩌㛫ࡢ Fst ್ࡣ 0.02545 㸦p<0.05㸧ప್࡛࠶ࡗࡓࠋFst ್ࡣಶయ⩌㛫ࡢ┦ᑐⓗ࡞㑇ఏⓗ㊥㞳ࢆ⾲ࡋ࡚࠾ࡾࠊᩘ ್ࡀࡁ࠸㑇ఏⓗศࡢ⛬ᗘࡀࡁ࠸ࡇࢆ♧ࡋ࡚࠸ࡿࠋࡍ࡞ࢃࡕࠊࣁࣉࣟࢢࣝ ࣮ࣉ A B ࡢಶయ⩌㛫ࡣࡁ࡞㑇ఏⓗศࡀㄆࡵࡽࢀࡿ୍᪉࡛ࠊࣁࣉࣟࢢ࣮ࣝࣉ ෆ࠾ࡅࡿྛᅜ㛫ࡢ㞟ᅋࡣ㑇ఏⓗ㏆⦕࡛࠶ࡿࡇࢆ♧၀ࡋࡓ㸦⾲3㸧ࠋ 㸲㸬⪃ᐹ ࣥࢻᮾ㒊ࡽࡣ F. gigantica ࠾ࡼࡧ༢Ⅽ⏕Ṫᆺ⫢⻄ࡀ᳨ฟࡉࢀࡓ㸦ᅗ 3㸧ࠋ࣑ࢺ
ࢥࣥࢻࣜDNA ࡛࠶ࡿ nad1 ࡢሷᇶከᵝᗘ㸦S㸧ࡣࠊ༢Ⅽ⏕Ṫᆺ⫢⻄ࡼࡾ F. gigantica ࠾࠸࡚㧗್ࢆ♧ࡋࡓࡇࡽࠊ༢Ⅽ⏕Ṫᆺ⫢⻄ẚ F. gigantica ࡢ㑇ఏⓗከᵝᛶ
14 ࡀ㧗࠸ࡇࡀ♧၀ࡉࢀࡓ㸦⾲3㸧ࠋ୍⯡ⓗࠊྂ࠸ಶయ⩌㑇ఏⓗከᵝᛶᐩࡴࡇ ࡀ▱ࡽࢀ࡚࠾ࡾ [6, 13]ࠊࣥࢻᮾ㒊ࡣ༢Ⅽ⏕Ṫᆺ⫢⻄ࡀᏑᅾࡍࡿ௨๓ࡼࡾࠊF. gigantica ࡀᏑᅾࡋ࡚࠸ࡓ⪃࠼ࡽࢀࡓࠋࣥࢻᮾ㒊⏘༢Ⅽ⏕Ṫᆺ⫢⻄ࡢ nad1 ࣁࣉ ࣟࢱࣉࡢࢇࡣࡢࢪྛᅜྠᵝ୰ᅜ⏘༢Ⅽ⏕Ṫᆺ⫢⻄ྠ୍࡛࠶ࡾࠊ F. hepatica F. gigantica ࡢ㞧ᏊᏞࡋ࡚Ⓨ⏕ࡋࡓ༢Ⅽ⏕Ṫᆺ⫢⻄ࡀࠊᐙ␆➼ࡢ⛣ື కࡗ࡚ࣥࢻᮾ㒊ࡶศᕸࢆᣑࡋࡓࡇࡀ♧၀ࡉࢀࡓ [40]ࠋ༢Ⅽ⏕Ṫᆺ⫢⻄ࡣ ࢿࣃ࣮ࣝ㸦75.3%㸧ࡸࣂࣥࢢࣛࢹࢩࣗ㸦86.0%㸧࠸ࡗࡓࡢ༡ࢪࡢᅜ࡛ࡣ F. gigantica ẚ࡚ඃໃ࡛࠶ࡿࡢᑐࡋ [36, 42]ࠊࣥࢻᮾ㒊࠾ࡅࡿ᳨ฟ⋡ࡣ F. gigantica ẚపࡗࡓ㸦24.8%㸧㸦ᅗ 2, ⾲ 2㸧ࠋࢿࣃ࣮ࣝ࠾ࡼࡧࣂࣥࢢࣛࢹࢩࣗࡣ ࣥࢻᩥᅪࢆᙧᡂࡋࠊṔྐⓗ࣭ᩥⓗᙉࡃ⤖ࡧࡘ࠸࡚ࡁࡓ୍᪉࡛ [36]ࠊࣥࢻᮾ 㒊ࡣ 19 ୡ⣖ࡲ࡛ࡇࢀࡽࡢᅜࡢ⤖ࡧࡘࡁࡣࢇ࡞ࡗࡓࠋࡑࡢࡓࡵࠊࣥࢻ ᮾ㒊ᆅᇦࠊࢿࣃ࣮ࣝ࠾ࡼࡧࣂࣥࢢࣛࢹࢩࣗࡢⱄᐙ␆ࡢேⅭⓗὶࡣࡲࢀ࡛࠶ࡗ ࡓ⪃࠼ࡽࢀࡿࠋᮏ௬ㄝࢆ᳨ドࡍࡿࡓࡵࠊࢿࣃ࣮ࣝࡸࣂࣥࢢࣛࢹࢩࣗᩥⓗ⤖ࡧࡘ ࡁࡢᙉࡗࡓࣥࢻᮏᅵࡢFasciola ᒓࡘ࠸࡚ࠊศᏊ⣔⤫Ꮫⓗゎᯒࢆ⾜࠺ᚲせࡀ࠶ࡿ ⪃࠼ࡽࢀࡓࠋࡲࡓࠊᮏ❶࡛ࡣ6 యࡢ Fasciola ᒓࡀ✀ྠᐃ⮳ࡽ࡞ࡗࡓࡇࡽࠊ ㈓⢭ᄞෆ⢭Ꮚࡢ᭷↓ᙳ㡪ࢆཷࡅ࡞࠸ࠊ᪂ࡓ࡞✀ྠᐃ࣐࣮࣮࢝ࡢ☜❧ࡀᚲせ࡛࠶ࡿ ⪃࠼ࡽࢀࡓࠋ ࣥࢻᮾ㒊ࡽ᳨ฟࡉࢀࡓF. gigantica ࡣ nad1 ࡢࣁࣉࣟࢱࣉᇶ࡙࠸࡚ࠊ༡ ࢪ࡛ඃໃ࡞ࣁࣉࣟࢢ࣮ࣝࣉ A ࠊᮾ༡ࢪ࡛ඃໃ࡞ࣁࣉࣟࢢ࣮ࣝࣉ B ศ㢮ࡉ
15 ࢀࡓࠋ⯆῝࠸ࡇࠊࣁࣉࣟࢢ࣮ࣝࣉA ᒓࡍࡿಶయ⩌ࡢሷᇶከᵝᗘ㸦S㸧ࡣす ⨨ࡍࡿᅜ࠾࠸࡚㧗ࡃࠊᮾ⾜ࡃࡋࡓࡀࡗ࡚పࡃ࡞ࡿഴྥࡀ࠶ࡗࡓࠋࡍ࡞ࢃࡕࠊ ᭱ࡶす⨨ࡍࡿࢿࣃ࣮ࣝࡢಶయ⩌ࡀ᭱ࡶ㧗್ࢆ♧ࡋࠊ᭱ࡶᮾ⨨ࡍࡿ࣑࣐࣮ࣕࣥ ࡢಶయ⩌ࡀ᭱ࡶప್ࢆ♧ࡋࡓ㸦⾲ 2㸧ࠋࡇࡢ⤖ᯝࡣࠊࣁࣉࣟࢢ࣮ࣝࣉ A ᒓࡍࡿ F. gigantica ಶయ⩌ࡀࠊすࡽᮾศᕸࢆᗈࡆ࡚࠸ࡗࡓྍ⬟ᛶࢆ♧၀ࡋ࡚࠸ࡿࠋࡲࡓࠊMJ network ࠾࠸࡚ࠊࣁࣉࣟࢢ࣮ࣝࣉ A ࡢ F. gigantica ಶయ⩌ࡣ࣎ࢺࣝࢿࢵࢡຠᯝࡸᛴ ⃭࡞ಶయᩘቑຍࢆ⤒㦂ࡋࡓಶయ⩌≉ᚩⓗ࡞ᫍᙧࡢࢿࢵࢺ࣮࣡ࢡࢆᙧᡂࡋࡓ㸦ᅗ 4㸧ࠋ ࡼࡗ࡚ࠊࣁࣉࣟࢢ࣮ࣝࣉA ࡢ F. gigantica ಶయ⩌ࡀẚ㍑ⓗ▷㛫ಶయᩘࢆᛴ⃭ቑ ຍࡉࡏࡓྍ⬟ᛶࡀ♧၀ࡉࢀࠊࡑࡢཎᅉࡋ࡚ࠊᐟ࡛࠶ࡿⱄື≀ࡢᐙ␆࠾ࡼࡧಶ
యᩘቑຍࡀ⪃࠼ࡽࢀࡓࠋBos indicus ࡣ Fasciola ᒓࡢせ࡞ᐟ࡛࠶ࡾࠊࣥࢻள㝣
ࡢす➃⨨ࡍࡿࣥࢲࢫ㇂࡛ᐙ␆ࡉࢀࡓᚋࠊேⅭⓗࡑࡢศᕸࢆᣑࡋࡓ⪃࠼ ࡽࢀ࡚࠾ࡾࠊࣥࢻள㝣ࡢす㒊࠾࠸࡚᭱ࡶ㑇ఏⓗከᵝᛶᐩࡴࡇࡀ▱ࡽࢀ࡚࠸ ࡿ [6]ࠋࡲࡓࠊ⌧ᅾࡢ B. indicus ࡢୡ⏺ⓗ࡞ศᕸࡣ F. gigantica ࡢศᕸᇦ୍⮴ࡋ࡚࠾ ࡾ [33]ࠊࡇࡢࡇࡽࠊB. indicus ࡢேⅭⓗ⛣ືࡀࣁࣉࣟࢢ࣮ࣝࣉ A ᒓࡍࡿ F. gigantica ࡢศᕸᇦࡢᣑᐤࡋࡓྍ⬟ᛶࡀ⪃࠼ࡽࢀࡓࠋ୍᪉࡛ࠊࣁࣉࣟࢢ࣮ࣝࣉ B ᒓࡍࡿ F. gigantica ಶయ⩌ࡣࣥࢻᮾ㒊ࠊ࣑࣐࣮ࣕࣥ࠾ࡼࡧࢱศᕸࡋ࡚࠸ࡿ ࡀࠊࡑࡢ୰ᚰࡣ࣑࣐࣮࡛ࣕࣥ࠶ࡿ⪃࠼ࡽࢀࡿ㸦ᅗ4㸧ࠋࣁࣉࣟࢢ࣮ࣝࣉ B ᒓࡍࡿ F. gigantica ಶయ⩌ࡢሷᇶከᵝᗘ㸦S㸧ࡣす⨨ࡍࡿࣥࢻᮾ㒊ࡢಶయ⩌ẚࠊᮾ ⨨ࡍࡿ࣑࣐࣮ࣕࣥࡢಶయ⩌ࡀ㧗್ࢆ♧ࡋࡓ㸦⾲2㸧ࠋࡇࡢ⤖ᯝࡣࠊࣁࣉࣟࢢ࣮ࣝࣉ
16 B ᒓࡍࡿ F. gigantica ಶయ⩌ࡀࠊ࣑࣐࣮ࣕࣥࡽࣥࢻᮾ㒊ศᕸࢆᗈࡆ࡚࠸ࡗࡓ ྍ⬟ᛶࢆ♧၀ࡋ࡚࠸ࡿࠋࣁࣉࣟࢢ࣮ࣝࣉB ࡢ F. gigantica ಶయ⩌ࡶ MJ network ࠾࠸ ࡚ᫍᙧࡢࢿࢵࢺ࣮࣡ࢡࢆᙧᡂࡋ࡚࠾ࡾࠊࡑࡢᣑᩓࡶఱࡽࡢᐙ␆ࡀ㛵ࡋࡓ⪃࠼ ࡽࢀࡓࠋᚋࡣࠊ༡ࢪ࠾ࡅࡿF. gigantica ࡢᣑᩓࡢヲ⣽ࢆ᫂ࡽࡍࡿࡓࡵࠊ ࣥࢻᮾ㒊㞄᥋ࡍࡿᆅᇦࡸ␗࡞ࡿᐟ⏤᮶ࡍࡿ Fasciola ᒓࢆゎᯒࡍࡿࡇࡀᚲ せ࡛࠶ࡿ⪃࠼ࡽࢀࡓࠋ 㸳㸬ᑠᣓ ࣥࢻᮾ㒊ࡢ࢘ࢩࠊࢫࢠࣗ࢘࠾ࡼࡧࣖࢠࡽ᥇ྲྀࡉࢀࡓFasciola ᒓ 157 య ࡘ࠸࡚ࠊ㈓⢭ᄞෆ⢭Ꮚࡢ᭷↓࠾ࡼࡧ᰾ࣜ࣎ࢯ࣮࣒DNA ࡢ ITS1 㡿ᇦࡢ㑇ఏᏊᆺᇶ࡙ ࠸࡚✀ྠᐃࢆ⾜ࡗࡓࠋࡑࡢ⤖ᯝࠊࡇࢀࡽࡢᆅᇦࡣF. gigantica ࠾ࡼࡧ༢Ⅽ⏕Ṫᆺ⫢⻄ ࡀศᕸࡋ࡚࠸ࡿࡇࡀ᫂ࡽ࡞ࡗࡓࠋḟࠊFasciola ᒓࡢ⣔⤫ゎᯒ᭱ࡶࡼࡃ⏝࠸ ࡽࢀࡿ࣑ࢺࢥࣥࢻࣜDNA ࡢ nad1 㡿ᇦࡢሷᇶ㓄ิ㸦535 bp㸧ࢆࠊࢲࣞࢡࢺࢩ࣮ࢣ ࣥࢫἲࢆ⏝࠸࡚Ỵᐃࡋࡓࠋࡑࡢ⤖ᯝࠊࣥࢻᮾ㒊ศᕸࡍࡿ༢Ⅽ⏕Ṫᆺ⫢⻄ࡢࣁࣉ ࣟࢱࣉࡣ㏆㞄ࡢࢪㅖᅜศᕸࡍࡿಶయ⩌ྠ୍ࡶࡋࡃࡣ୍ሷᇶ⨨࡛࠶ࡾࠊ୰ ᅜ࡛Ⓨ⏕ࡋࡓ༢Ⅽ⏕Ṫᆺ⫢⻄ࡀࣥࢻᮾ㒊ࡲ࡛ศᕸࢆᣑࡋ࡚࠸ࡿࡇࡀ♧၀ࡉ ࢀࡓࠋࡋࡋ࡞ࡀࡽࠊ༢Ⅽ⏕Ṫᆺ⫢⻄ࡢྜࡣ㏆㞄ㅖᅜẚ࡚పࡃࠊF. gigantica ࡀ ඃໃ࡛࠶ࡗࡓࠋF. gigantica ࡢ nad1 ࣁࣉࣟࢱࣉࡣ༡ࢪ࡛ඃໃ࡞ࣁࣉࣟࢢ࣮ࣝࣉ
17 A ࠊᮾ༡ࢪ࡛ඃໃ࡞ࣁࣉࣟࢢ࣮ࣝࣉ B ศ㢮ࡉࢀࡓࠋྛᅜࡢಶయ⩌ࡢሷᇶከᵝ ᗘࢆẚ㍑ࡋࡓ⤖ᯝࠊF. gigantica ࣁࣉࣟࢢ࣮ࣝࣉ A ࡣࢮࣈ࣮∵ࡢ⛣ືకࡗ࡚ࣥࢻ ள㝣ࡢすࡽᮾศᕸࢆᣑࡋࡓࢢ࣮ࣝࣉ࡛࠶ࡿྍ⬟ᛶࡀ♧၀ࡉࢀࡓࠋ୍᪉ࠊࣁ ࣉࣟࢢ࣮ࣝࣉ B ࡶᐙ␆ࡢ⛣ືకࡗ࡚࣑࣐࣮ࣕࣥࡽࣥࢻᮾ㒊ࡶࡓࡽࡉࢀࡓ ྍ⬟ᛶࡀ♧၀ࡉࢀࡓࠋࡲࡓࠊᮏ❶࡛ࡣ✀ྠᐃ⮳ࡽ࡞ࡗࡓFasciola ᒓࡀᏑᅾࡋࡓࡇ ࡽࠊ㈓⢭ᄞෆ⢭Ꮚࡢ᭷↓ᙳ㡪ࢆཷࡅ࡞࠸ࠊ᪂ࡓ࡞✀ྠᐃ࣐࣮࣮࢝ࡢ☜❧ࡀᚲせ ࡛࠶ࡿ⪃࠼ࡽࢀࡓࠋ
18 ⾲㸯㸬ࣥࢻᮾ㒊ࡽ᥇ྲྀࡉࢀࡓFasciolaᒓయࡢゎᯒ⤖ᯝ ᥇㞟ᆅ ᐟ 㸦㢌ᩘ㸧 యᩘ ㈓⢭ᄞ ෆ⢭Ꮚ ITS1 ࣑ࢺࢥࣥࢻࣜnad1 ✀ ࣁࣉࣟࢱࣉ Accession no. Imphal ᕷ 䝊䝤䞊∵ (30) 16 䠉㻌 Fg ND1-E1 LC012895 ༢Ⅽ⏕Ṫᆺ⫢⻄ 㻌 1 䠉㻌 Fg ND1-E2 LC012896 ༢Ⅽ⏕Ṫᆺ⫢⻄ 㻌 㻌 16 + Fg ND1-E3 LC012897 F. gigantica 㻌 㻌 5b 䠉㻌 Fg ND1-E3 LC012897 Fasciola sp. 㻌 㻌 1 + Fg ND1-E4 LC012898 F. gigantica 㻌 㻌 1 + Fg ND1-E5 LC012899 F. gigantica 㻌 㻌 21 + Fg ND1-E6 LC012900 F. gigantica 㻌 㻌 4 + Fg ND1-E7 LC012901 F. gigantica 㻌 㻌 1 + Fg ND1-E8 LC012902 F. gigantica 㻌 㻌 1 + Fg ND1-E10 LC012904 F. gigantica 㻌 㻌 1 + Fg ND1-E15 LC012909 F. gigantica 㻌 㻌 1 + Fg ND1-E16 LC012910 F. gigantica 㻌 㻌 1 + Fg ND1-E17 LC012911 F. gigantica 㻌 㻌 1 + Fg ND1-E19 LC012913 F. gigantica 㻌 㻌 2 + Fg ND1-E20 LC012914 F. gigantica 㻌 㻌 1 + Fg ND1-E21 LC012915 F. gigantica 㻌 㻌 1b 䠉㻌 Fg ND1-E21 LC012915 Fasciola sp. 㻌 㻌 1 + Fg ND1-E22 LC012916 F. gigantica 㻌 㻌 1 + Fg ND1-E23 LC012917 F. gigantica 㻌 㻌 1 + Fg ND1-E27 LC012921 F. gigantica 㻌 㻌 1 + Fg ND1-E28 LC012922 F. gigantica 㻌 㻌 1 + Fg ND1-E31 LC012925 F. gigantica 㻌 㻌 1 + Fg ND1-E32 LC012926 F. gigantica 㻌 㻌 1 + Fg ND1-E34 LC012928 F. gigantica 㻌 㻌 1 + Fg ND1-E35 LC012929 F. gigantica 㻌 䜰䝆䜰㻌 䝇䜲䜼䝳䜴 (11) 9 + Fg ND1-E3 LC012897 F. gigantica 㻌 11 + Fg ND1-E6 LC012900 F. gigantica 㻌 1 + Fg ND1-E9 LC012903 F. gigantica 㻌 㻌 2 + Fg ND1-E10 LC012904 F. gigantica 㻌 㻌 1 + Fg ND1-E18 LC012912 F. gigantica 㻌 㻌 1 + Fg ND1-E24 LC012918 F. gigantica 㻌 㻌 1 + Fg ND1-E25 LC012919 F. gigantica 㻌 㻌 3 + Fg ND1-E27 LC012921 F. gigantica 㻌 㻌 1 + Fg ND1-E36 LC012930 F. gigantica 㻌 㻌 1 + Fg ND1-E37 LC012931 F. gigantica
19 ⾲㸯㸬ࣥࢻᮾ㒊ࡽ᥇ྲྀࡉࢀࡓFasciolaᒓࡢゎᯒ⤖ᯝ㸦⥆ࡁ㸧 ᥇㞟ᆅ ᐟ 㸦㢌ᩘ㸧 యᩘ ㈓⢭ᄞ ෆ⢭Ꮚ ITS1 ࣑ࢺࢥࣥࢻࣜnad1 ✀ ࣁࣉࣟࢱࣉ Accession no. 䝁䝠䝬㻌 䝊䝤䞊∵ (5) 3 䠉㻌 Fg ND1-E1 LC012895 ༢Ⅽ⏕Ṫᆺ⫢⻄ 㻌 4 + Fg ND1-E6 LC012900 F. gigantica 㻌 㻌 1 + Fg ND1-E10 LC012904 F. gigantica 㻌 㻌 1 + Fg ND1-E11 LC012905 F. gigantica 㻌 㻌 1 + Fg ND1-E12 LC012906 F. gigantica 㻌 㻌 1 + Fg ND1-E13 LC012907 F. gigantica 㻌 㻌 1 + Fg ND1-E20 LC012914 F. gigantica 㻌 㻌 1 + Fg ND1-E28 LC012922 F. gigantica 㻌 㻌 1 + Fg ND1-E33 LC012927 F. gigantica 㻌 䝲䜼 (1) 1 + Fg ND1-E6 LC012900 F. gigantica 㻌 㻌 1 + Fg ND1-E7 LC012901 F. gigantica 㻌 㻌 1 + Fg ND1-E14 LC012908 F. gigantica 䜺䞁䝖䜽㻌 䜰䝆䜰䝇䜲 䜼䝳䜴 (4) 13 䠉㻌 Fg ND1-E1 LC012895 ༢Ⅽ⏕Ṫᆺ⫢⻄ 㻌 8 + Fg ND1-E6 LC012900 F. gigantica 㻌 㻌 1 + Fg ND1-E26 LC012920 F. gigantica 㻌 㻌 2 + Fg ND1-E29 LC012923 F. gigantica 㻌 㻌 2 + Fg ND1-E30 LC012924 F. gigantica ྜィ㻌 㻌 157 㻌 㻌 㻌 㻌 㻌 a "+" ᭷⢭Ꮚᆺ, "-" ↓⢭Ꮚᆺ b 㞟ᅋ㑇ఏᏛⓗゎᯒࡽࡣ㝖࠸ࡓయ㸦Fasciola sp.㸧
20 ᅗ㸯㸬ࣥࢻᮾ㒊⏘Fasciola ᒓయࡢ㈓⢭ᄞ࠾ࡅࡿ⢭Ꮚࡢ᭷↓ A: ᭷⢭Ꮚᆺ B: ↓⢭Ꮚᆺ ▮㢌ࡣ㈓⢭ᄞࡢ⨨ࢆ♧ࡍ
A
B
21 ᅗ㸰㸬ࣥࢻᮾ㒊⏘Fasciola ᒓࡢ PCR-RFLP ἲࡼࡿ ITS1 ࡢ㑇ఏᏊᆺ 1: Milli Q 2: Fh ᆺࢥࣥࢺ࣮ࣟࣝ 3: Fg ᆺࢥࣥࢺ࣮ࣟࣝ 4: Fh/Fg ᆺ㸦࣊ࢸࣟᆺ㸧ࢥࣥࢺ࣮ࣟࣝ M: DNA ࣛࢲ࣮ 5-8: ࣥࢻᮾ㒊⏘Fasciolaᒓయ㸦࡚Fg ᆺ㸧 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
ᅗ㸱㸬ࣥࢻᮾ㒊⏘Fasciola ᒓࡢศᕸ
㟷ࡣF. giganticaࠊ㉥ࡣ༢Ⅽ⏕Ṫᆺ⫢⻄ࠊ⥳ࡣFasciola sp.ࢆ♧ࡍࠋ ࢢࣛࣇෆࡢᩘᏐࡣయᩘࢆ⾲ࡋࠊࢢࣛࣇࡢࡁࡉࡣయᩘࢆᫎࡋ࡚࠸ࡿࠋ
23
ᅗ㸲㸬ࢪ⏘Fasciola ᒓࡢ nad1 㡿ᇦࡢሷᇶ㓄ิᇶ࡙ࡃ MJ network
ྛࡢࡁࡉࡣయᩘࢆᫎࡋ࡚࠾ࡾࠊෆࣁࣉࣟࢱࣉࢥ࣮ࢻ࠾ࡼࡧయᩘࢆグࡋࡓࠋ యᩘࡢグ㍕ࡀ࡞࠸ࣁࣉࣟࢱࣉࡣ 1 య࡛࠶ࡿࡇࢆ♧ࡍࠋࡲࡓࠊᯞୖࡢᩘᏐࡣሷᇶ⨨ ᩘࢆ⾲ࡋ࡚࠾ࡾࠊᩘᏐࡀ࡞࠸ࡶࡢࡣ 1 ሷᇶ⨨࡛࠶ࡿࡇࢆ♧ࡍࠋࣁࣉࣟࢱࣉࢥ࣮ࢻࡣࡑ ࢀࡒࢀࠊࣥࢻᮾ㒊㸦E1 ࡽ E37㸧ࠊࢿࣃ࣮ࣝ㸦FspN1ࠊFspN2 ࠾ࡼࡧ N1 ࡽ N10㸧ࠊ ࣂࣥࢢࣛࢹࢩࣗ㸦Bd1 ࡽ Bd11㸧ࠊ࣑࣐࣮ࣕࣥ㸦M1 ࡽ M19㸧ࠊࢱ㸦T1 ࡽ T30㸧ࠊ࣋ࢺ ࢼ࣒㸦V1 ࡽ V5㸧ࠊ୰ᅜ㸦C1 ࡽ C16㸧ࠊ㡑ᅜ㸦Kor2a ࠾ࡼࡧ Kor2b㸧࠾ࡼࡧ᪥ᮏ㸦Fsp2㸧 ࡽ᳨ฟࡉࢀࡓࣁࣉࣟࢱࣉࢆ♧ࡍࠋ
24 ⾲㸰㸬nad1㡿ᇦࡢሷᇶ㓄ิᇶ࡙࠸ࡓࠊF. gigantica࠾ࡼࡧ༢Ⅽ⏕Ṫᆺ⫢⻄ࡢྛಶయ⩌࠾ ࡅࡿ㑇ఏⓗከᵝᛶ ✀ ࣁࣉࣟࢢ࣮ࣝࣉ ಶయ⩌ N S h Hd ± SD π F. gigantica ࣁࣉࣟࢢ࣮ࣝࣉ A ࣥࢻᮾ㒊 91 32 32 0.751±0.050 0.00242 ࢿࣃ࣮ࣝ 20 16 10 0.758±0.101 0.00366 ࣂࣥࢢࣛࢹࢩࣗ 20 15 10 0.832±0.075 0.00362 ࣑࣐࣮ࣕࣥ 13 7 6 0.641±0.150 0.00225 ࣁࣉࣟࢢ࣮ࣝࣉ B ࣥࢻᮾ㒊 27 2 3 0.145±0.090 0.00028 ࣑࣐࣮ࣕࣥ 67 18 13 0.485±0.076 0.00170 ༢Ⅽ⏕Ṫᆺ⫢⻄ ࣥࢻᮾ㒊 33 1 2 0.061±0.056 0.00011 ࢿࣃ࣮ࣝ 61 1 2 0.033±0.031 0.00006 ࣂࣥࢢࣛࢹࢩࣗ 127 0 1 0.000±0.000 0.00000 ࣑࣐࣮ࣕࣥ 7 0 1 0.000±0.000 0.00000 N: యᩘ, S: ከᆺࢧࢺᩘ, h: ࣁࣉࣟࢱࣉᩘ, Hd: ࣁࣉࣟࢱࣉከᵝᗘ, SD: ᶆ‽೫ᕪ, ȧ: ሷᇶከᵝᗘ
25 ࣥࢻᮾ㒊 ࢿࣃ࣮ࣝ ࣂࣥࢢࣛࢹࢩࣗ ࣑࣐࣮ࣕࣥ ࣥࢻᮾ㒊 ࣑࣐࣮ࣕࣥ 䝝䝥 䝻 䜾 䝹 䞊 䝥 A ࣥࢻᮾ㒊 䠉 ࢿࣃ࣮ࣝ 0. 02634* 䠉 ࣂࣥࢢࣛࢹࢩࣗ 0. 02545* 0. 00897 䠉 ࣑࣐࣮ࣕࣥ -0 .01247 0. 00250 -0 .01200 䠉 ࣁࣉࣟࢢ࣮ࣝࣉ B ࣥࢻᮾ㒊 0. 86934* 0. 88145* 0. 88030* 0. 93454* 䠉 ࣑࣐࣮ࣕࣥ 0. 85475* 0. 85099* 0. 84865* 0. 87363* -0 .00119 䠉 * ᭷ពᕪ䛒 䜚 ( P <0 .05) ಶయ⩌ ࣁࣉࣟࢢ࣮ࣝࣉ B ࣁࣉࣟࢢ࣮ࣝࣉ A ⾲ 3 䠊 nad1 㑇ఏᏊ䛾ሷᇶ㓄ิ䛻ᇶ䛵䛔䛯䚸 F . gi gant ic a 䛾 6 ಶయ⩌㛫䛻䛚䛡 䜛 p air w is e f ix atio n in de x䠄 F st ್䠅 䝝䝥 䝻 䜾 䝹 䞊 䝥
26
➨㸰❶ ᪂つࡢ✀ྠᐃ࣐࣮࣮࢝
pepck ࠾ࡼࡧ pold ࢆ⏝࠸ࡓ
༡ࢪ⏘
Fasciola spp.ࡢྠᐃ
27 㸯㸬⫼ᬒ 㸰㸬 ➨1 ❶࡛ࡣࠊࣥࢻᮾ㒊⏘ Fasciola ᒓయࢆࠊᚑ᮶ࡢ㈓⢭ᄞෆ⢭Ꮚࡢ᭷↓࠾ࡼࡧ ITS1 ࡢ㑇ఏᏊᆺᇶ࡙࠸࡚✀ྠᐃࢆ⾜ࡗࡓࠋࡋࡋ࡞ࡀࡽࠊゎᯒ⏝࠸ࡓ 157 యࡢ ࠺ࡕ6 య࠾࠸࡚ࠊ㈓⢭ᄞෆᡂ⇍⢭Ꮚࡀㄆࡵࡽࢀ࡞࠸↓⢭Ꮚᆺ࡛࠶ࡿࡶ㛵ࢃࡽ ࡎࠊnad1 ࣁࣉࣟࢱࣉࡀ F. gigantica ࣁࣉࣟࢢ࣮ࣝࣉ㸦᭷⢭Ꮚᆺ㸧ᒓࡋࠊࡇࢀࡽࡢ యࢆྠᐃࡍࡿࡇࡀ࡛ࡁ࡞ࡗࡓࠋࡲࡓࠊࣥࢻ㏆㞄ࡢࣂࣥࢢࣛࢹࢩࣗ࠾ࡼࡧ࣑ࣕ ࣐࣮ࣥࡢFasciola ᒓయ࠾࠸࡚ࡶࠊࡇࡢᚑ᮶ἲ࡛ࡣྠᐃ࡛ࡁ࡞࠸యࡀሗ࿌ࡉࢀࡓ [17, 36]ࠋ༢Ⅽ⏕Ṫᆺ⫢⻄࠾ࡅࡿ ITS1 ࡢ㑇ఏᏊᆺࡣ Fh ᆺࠊFg ᆺ࠾ࡼࡧ Fh/Fg ᆺ 㸦࣊ࢸࣟᆺ㸧ࢆ♧ࡍయࡀᏑᅾࡍࡿࡓࡵࠊ㈓⢭ᄞෆ⢭Ꮚࡢሗ⤌ࡳྜࢃࡏ࡞ࡅࢀࡤࠊ Fasciola ᒓ 3 ✀ࢆ㆑ูࡍࡿࡇࡣ࡛ࡁ࡞࠸ [18]ࠋࡇࡢࡓࡵࠊఱࡽࡢཎᅉࡼࡾ㈓⢭ ᄞෆ⢭Ꮚࢆ᭷ࡋ࡞࠸ F. gigantica ࡸ F. hepatica ࢆᚑ᮶ἲ࡛㆑ูࡍࡿࡇࡣᅔ㞴࡛࠶ ࡗࡓ [17, 36]ࠋ
㏆ᖺࠊ᰾DNA ࡢ phosphoenolpyruvate carboxykinase 㑇ఏᏊ㸦pepck㸧㡿ᇦ࠾ࡼࡧ DNA
polymerase delta 㑇ఏᏊ㸦pold㸧㡿ᇦ࠾ࡅࡿሷᇶ㓄ิࢆ⏝࠸ࡓ Fasciola ᒓయࡢ㆑ู
ἲࡀ㛤Ⓨࡉࢀࠊ᭷⢭ᏊᆺࡢF. gigantica F. hepaticaࠊ↓⢭Ꮚᆺࡢ༢Ⅽ⏕Ṫᆺ⫢⻄ࡢ㆑ ูࡀྍ⬟࡞ࡗࡓ [44]ࠋࡇࢀࡽࡢ᪂つ࣐࣮࣮࢝㑇ఏᏊࡣ࠸ࡎࢀࡶࠊF. hepatica ࡣ Fh ᆺࠊF. gigantica ࡣ Fg ᆺࠊ༢Ⅽ⏕Ṫᆺ⫢⻄ࡣ Fh/Fg ᆺ㸦࣊ࢸࣟᆺ㸧ࡢ㑇ఏᏊᆺࢆ♧ࡍࠋ ࡲࡓࠊ༢Ⅽ⏕Ṫᆺ⫢⻄ࡣࡇࢀࡽࡢ㆑ูἲ࠾࠸࡚Fh/Fg ᆺࡢࡳࢆ♧ࡍࡇࡽࠊ㈓⢭ ᄞෆ⢭Ꮚࡢ᭷↓ࡢሗࢆᚲせࡋ࡞࠸ࠊ᪂つ✀ྠᐃ࣐࣮࣮࢝ࡋ࡚ᮇᚅࡉࢀࡓࠋࡑࡇ ࡛ࠊࡇࢀࡽࡢ㑇ఏᏊ㡿ᇦࢆ⏝࠸࡚ࠊࡇࢀࡲ࡛ゎᯒࡋࡓ༡ࢪ⏘Fasciola ᒓయࡢ ྠᐃࢆヨࡳࡓࠋ
28 㸰㸬ᮦᩱ᪉ἲ ➨1 ❶࡛ゎᯒࡋࡓࣥࢻᮾ㒊⏘ Fasciola ᒓ 157 య࠾ࡼࡧࠊඛ⾜◊✲࡛⏝࠸ࡽࢀ ࡓࢿࣃ࣮ࣝ⏘Fasciola ᒓ 81 య [43]ࠊࣂࣥࢢࣛࢹࢩࣗ⏘ Fasciola ᒓ 138 య[36] ࠾ ࡼࡧ࣑࣐࣮ࣕࣥ⏘ Fasciola ᒓ 87 య [20] ࡢィ 463 యࡢ DNA ࢆゎᯒ⏝࠸ࡓࠋ ඛ⾜◊✲࡚ゎᯒࡉࢀࡓࡇࢀࡽࡢయࡢ㈓⢭ᄞෆ⢭Ꮚࡢ᭷↓ࠊITS1 ࡢ㑇ఏᏊᆺ࠾ࡼ ࡧnad1 ࡢࣁࣉࣟࢱࣉࡣ⾲ 4 ♧ࡋࡓࠋ pepck ᆺࡢゎᯒࡣࣉ࣐࣮ࣛFh-pepck-F㸦GATTGCACCGTTAGGTTAGC-3′㸧ࠊFg-pepck-F 㸦 AAAGTTTCTATCCCGAACGAAG-3′ 㸧 ࠾ ࡼ ࡧ Fcmn-pepck-R 㸦
5′-CGAAAATTATGGCATCAATGGG-3′㸧ࢆ⏝࠸࡚ pepck 㡿ᇦࢆ multiplex PCR ἲࡼࡾቑ
ᖜࡋࡓࠋPCR ࡢᛂᾮࡣ 0.025 U ࡢ GoTaq DNA Polymerase㸦Promega, Madison, WI,
USA 㸧ࠊయ⏤᮶ࡢ DNA 㸦⣙ 100 ng/μL㸧ࠊྛ dNTP㸦0.08 mM㸧ࠊFh-pepck-F ࠾ࡼ
ࡧFg-pepck-F㸦0.5 pM㸧ࠊFcmn-pepck-R㸦1.0 pM㸧ࠊ each primer, 0.025 U 5GoTaq buffer
㸦50 mM Tris-HCl, 50 mM NaCl, 5 mM MgCl2㸧ࢆຍ࠼㔞50 μL ࡛⾜ࡗࡓࠋᛂ᮲௳ࡣ 94°C 90 ⛊ࡢ⇕ኚᛶᚋࠊ94°C 90 ⛊࣭61°C 90 ⛊࣭72°C 120 ⛊ࡢࢧࢡࣝࢆ 30 ᅇ⾜࠸ࠊ ᭱ᚋ72°C 10 ศࡢఙ㛗ᛂࢆຍࡋࡓࠋmultiplex PCR ࡼࡿቑᖜ⏘≀ࢆࠊ1.0%࢞ ࣮ࣟࢫࢤࣝࢆ⏝࠸࡚㟁ẼὋືࡋࠊࣂࣥࢻࣃࢱ࣮ࣥࢆ☜ㄆࡋࡓ [44]ࠋ pold ᆺ ࡢ ゎ ᯒ ࡣ ࡲ ࡎ ࠊ ࣉ ࣛ ࣐ ࣮ Fasciola-pold-F1 㸦 5′-GCTAACTTATCTGCTTACACGTGGACA-3′ 㸧 ࠾ ࡼ ࡧ Fasciola-pold-R1 㸦 5′-ATCGCATTCGATCAAAGCCCTCCCATG-3′㸧ࢆ⏝࠸࡚ pold 㡿ᇦࢆ PCR ࡼࡾቑᖜࡋࠊ 1.0%࣮࢞ࣟࢫࢤࣝࢆ⏝࠸࡚㟁ẼὋືࡼࡗ࡚┠ⓗ㡿ᇦࡢቑᖜࢆ☜ㄆࡋࡓࠋPCR ᮲௳
ࡣ0.025 U ࡢ GoTaq DNA Polymerase㸦Promega㸧ࠊయ⏤᮶ࡢ DNA 㸦⣙ 100 ng/μL㸧ࠊ
ྛdNTP㸦0.08 mM㸧ࠊྛࣉ࣐࣮ࣛ㸦0.5 pM㸧ࠊ each primer, 0.025 U 5GoTaq buffer
29
ࡣ 94°C 90 ⛊ࡢ⇕ኚᛶᚋࠊ94°C 90 ⛊࣭55°C 90 ⛊࣭72°C 120 ⛊ࡢࢧࢡࣝࢆ 35 ᅇ⾜
࠸ࠊ᭱ᚋ72°C 10 ศࡢఙ㛗ᛂࢆຍࡋࡓࠋpold 㡿ᇦࡢ PCR ⏘≀ࡣ 10U ࡢไ㝈㓝
⣲Alu I㸦Roche㸧࠾ࡼࡧ buffer enzyme L㸦Roche㸧ࢆຍ࠼ࠊ㔞 50μL ࡢᛂᾮࢆㄪᩚ
ࡋࡓࠋᛂᾮࢆ37Υ୍࡛ᬌ࣮ࣥ࢟ࣗ࣋ࢩࣙࣥࡍࡿࡇ࡛ PCR ⏘≀ࢆ᩿∦ࡋࠊ1.8% ࣮࢞ࣟࢫࢤࣝࢆ⏝࠸ࡓ㟁ẼὋືࡼࡗ࡚ࣂࣥࢻࣃࢱ࣮ࣥࢆ☜ㄆࡋࡓ [44]ࠋ 㸱㸬⤖ᯝ pepck 㑇ఏᏊᆺ࡛ࡣ 509 bp ࣂࣥࢻࡀ᳨ฟࡉࢀࡿ Fg ᆺࠊ241 bp ࠾ࡼࡧ 509 bp ࣂࣥࢻࡀ᳨ฟࡉࢀࡿFh/Fg ᆺ᳨ฟࡉࢀࡓ㸦ᅗ 5㸧ࠋࡲࡓࠊpold ࡢ㑇ఏᏊᆺ࡛ࡣ 544 bpࠊ 164 bp ࠾ࡼࡧ 136 bp ࣂࣥࢻࡀ᳨ฟࡉࢀࡿ Fg ᆺࠊࡑࢀࡽࡢࣂࣥࢻຍ࠼ 602 bp ࣂࣥࢻࡀ᳨ฟࡉࢀࡿ Fh/Fg ᆺ᳨ฟࡉࢀࡓ㸦ᅗ 6㸧ࠋゎᯒࡋࡓ 463 య࠾࠸࡚ࠊpepck
࠾ࡼࡧpold ࡢ㑇ఏᏊᆺࡢ⤖ᯝࠊࡉࡽ㈓⢭ᄞෆ⢭Ꮚࡢ᭷↓ࠊITS1 㑇ఏᏊᆺ࠾ࡼࡧ nad1
ࣁࣉࣟࢱࣉࡀᒓࡍࡿࣁࣉࣟࢢ࣮ࣝࣉࡢ⤖ᯝ [17, 36, 43] ࢆ⾲ 4 ♧ࡋࡓࠋ㈓⢭ᄞෆ ⢭Ꮚࡢ᭷↓ ITS1 㑇ఏᏊᆺᇶ࡙ࡃᚑ᮶ࡢྠᐃἲ᪂つ㆑ูἲࡢ⤖ᯝࡣ 97.6%୍⮴ ࡋࡓࠋ୍᪉࡛ࠊnad1 ࣁࣉࣟࢱࣉ᪂つ㆑ูἲࡢ⤖ᯝࡣ 100%୍⮴ࡋࡓࠋ ➨1 ❶࠾ࡼࡧඛ⾜◊✲࠾࠸࡚ࠊࣥࢻᮾ㒊ࠊࣂࣥࢢࣛࢹࢩࣗ࠾ࡼࡧ࣑࣐࣮ࣕࣥ ⏘ Fasciola ᒓ 9 యࡣ↓⢭Ꮚᆺ࡛࠶ࡗࡓࡶ㛵ࢃࡽࡎࠊnad1 ࣁࣉࣟࢱࣉࡣ F. gigantica ࡢࣁࣉࣟࢢ࣮ࣝࣉᒓࡋ࡚࠸ࡓ [17, 36]㸦⾲ 4㸧ࠋᮏ◊✲࡛ࡣࠊࡇࢀࡽ 9 య
ࡢ pepck ࠾ࡼࡧ pold ࡢ㑇ఏᏊᆺࡣ࠸ࡎࢀࡶ Fg ᆺࢆ♧ࡋ࡚࠸ࡓࡇࡽࠊF. gigantica
࡛࠶ࡿ⪃࠼ࡽࢀࡓ㸦⾲4㸧ࠋࡲࡓࠊඛ⾜◊✲࠾࠸࡚ࣂࣥࢢࣛࢹࢩࣗ⏘ Fasciola ᒓ 1
యࡣ᭷⢭Ꮚᆺ࡛࠶ࡾࠊITS1 ࡀ Fg ᆺࢆ♧ࡋࡓࡶ㛵ࢃࡽࡎࠊnad1 ࣁࣉࣟࢱࣉࡣ༢
Ⅽ⏕Ṫᆺ⫢⻄ࡢࣁࣉࣟࢢ࣮ࣝࣉᒓࡋ࡚࠸ࡓ㸦⾲ 4㸧[36]ࠋᮏ◊✲࡛ࡣࠊࡇࡢయࡢ
30 ⫢⻄࡛࠶ࡿ⪃࠼ࡽࢀࡓ㸦⾲4㸧ࠋྠᵝࠊඛ⾜◊✲࠾࠸࡚ࣂࣥࢢࣛࢹࢩࣗ⏘ Fasciola ᒓ 1 యࡣ᭷⢭Ꮚᆺ࡛࠶ࡗࡓࡶ㛵ࢃࡽࡎࠊITS1 ࡀ༢Ⅽ⏕Ṫᆺ⫢⻄ࡢ≉ᚩ࡛࠶ࡿ Fh/Fg ᆺࢆ♧ࡋࠊnad1 ࣁࣉࣟࢱࣉࡶ༢Ⅽ⏕Ṫᆺ⫢⻄ࡢࣁࣉࣟࢢ࣮ࣝࣉᒓࡋ࡚࠸ࡓ 㸦⾲4㸧[36]ࠋᮏ◊✲࡛ࡣࠊࡇࡢయࡢ pepck ࠾ࡼࡧ pold ࡢ㑇ఏᏊᆺࡣ࠸ࡎࢀࡶ Fh/Fg ᆺࢆ♧ࡋ࡚࠾ࡾࠊ༢Ⅽ⏕Ṫᆺ⫢⻄࡛࠶ࡿ⪃࠼ࡽࢀࡓ㸦⾲4㸧ࠋ ➨1 ❶࠾ࡼࡧඛ⾜◊✲࠾࠸࡚ nad1 ࡢࣁࣉࣟࢱࣉࡀ F. gigantica ࣁࣉࣟࢢ࣮ࣝࣉ
ᒓࡋࡓ Fasciola ᒓ 245 యࡣ࠸ࡎࢀࡶᮏ❶࠾࠸࡚ pepck ࡶࡋࡃࡣ pold ࡢ㑇ఏᏊ
ᆺࡀFg ᆺࢆ♧ࡋ࡚࠾ࡾࠊF. gigantica ࡛࠶ࡿࡇࡀ☜ㄆࡉࢀࡓࠋྠᵝࠊ➨ 1 ❶࠾ࡼ
ࡧඛ⾜◊✲࠾࠸࡚ nad1 ࡢࣁࣉࣟࢱࣉࡀ༢Ⅽ⏕Ṫᆺ⫢⻄ࣁࣉࣟࢢ࣮ࣝࣉᒓࡋࡓ Fasciola ᒓ 218 యࡣ࠸ࡎࢀࡶᮏ❶࠾࠸࡚ pepck ࡶࡋࡃࡣ pold ࡢ㑇ఏᏊᆺࡀ Fh/Fg ᆺࢆ♧ࡋ࡚࠾ࡾࠊ༢Ⅽ⏕Ṫᆺ⫢⻄࡛࠶ࡿࡇࡀ☜ㄆࡉࢀࡓ㸦⾲4㸧[44]ࠋ 㸲㸬⪃ᐹ ᮏ❶࡛ࡣ➨ 1 ❶࠾ࡼࡧඛ⾜◊✲࠾࠸࡚⏝࠸ࡽࢀࡓ Fasciola ᒓ 463 యࡘ࠸࡚ࠊ pepck ࠾ࡼࡧ pold ࡢ㑇ఏᏊᆺᇶ࡙࠸ࡓ᪂つ㆑ูἲࢆ⏝࠸࡚ྠᐃࢆ⾜ࡗࡓࠋࡑࡢ⤖ ᯝࠊゎᯒ⏝࠸ࡓ࡚ࡢయ࠾࠸࡚ࠊnad1 ࡢࣁࣉࣟࢢ࣮ࣝࣉ᪂つ㆑ูἲࡢ⤖ᯝࡀ ୍⮴ࡋࡓ㸦⾲4㸧ࠋࡇࡢࡇࡽࠊFasciola ᒓ 463 యࡣṇ☜ྠᐃࡉࢀࡓ⪃࠼ࡽࢀ ࡓࠋࡲࡓࠊࡇࢀࡽࡢ㑇ఏᏊᆺᇶ࡙࠸ࡓゎᯒἲࡣࠊ㈓⢭ᄞෆ⢭Ꮚࡢሗࢆᚲせࡋ࡞ ࠸ࡇࡽࠊ㈓⢭ᄞࢆᙧែᏛⓗゎᯒ࡛ࡁ࡞࠸యࡘ࠸࡚ࡶ᭷ຠ࡛࠶ࡿࠋ௨ୖࡢࡇ
ࡽࠊpepck ࠾ࡼࡧ pold 㡿ᇦᇶ࡙ࡃ✀ྠᐃࡣࠊFasciola ᒓ࠾࠸࡚᭷⏝࡛࠶ࡿࡇ
ࡀ♧၀ࡉࢀࡓࠋ
ᮏ❶࡛ゎᯒ⏝࠸ࡓ463 య࠾࠸࡚ࠊpepck 㡿ᇦࡣ࡚ࡢయ࡛ PCR ࡢቑᖜ⏘≀
31 㸦⾲4㸧ࠋࡇࢀࡣࠊࡇࢀࡽࡢ㡿ᇦࡀࢩࣥࢢࣝࢥࣆ࣮ࡢ㑇ఏᏊ㡿ᇦ࡛࠶ࡿࡇຍ࠼ࠊቑ ᖜ㛗ࡀpepck 㡿ᇦࡣ 241bp ࠾ࡼࡧ 509bp ࡶࡋࡃࡣ 510bp ẚ㍑ⓗ▷࠸ࡢᑐࡋࠊpold 㡿ᇦࡣ844bp ẚ㍑ⓗ㛗࠸ࡇࡀཎᅉ࡛࠶ࡿྍ⬟ᛶࡀ⪃࠼ࡽࢀࡓ [44]ࠋ ㈓⢭ᄞෆ⢭Ꮚࡢ᭷↓ ITS1 㑇ఏᏊᆺᇶ࡙ࡃᚑ᮶ࡢྠᐃἲ᪂つ㆑ูἲࡢ⤖ᯝࡢ ୍⮴⋡ࡣ 97.6%࡛࠶ࡾࠊ11 య࠾࠸୍࡚⮴ࡋ࡞ࡗࡓࠋࡇࢀࡽ 11 యࡣຍ㱋ࡸ ᥋ᚋ࡞ࡢ⌮⏤ࡼࡾ㈓⢭ᄞෆ⢭Ꮚࢆ᭷ࡋ࡚࠸࡞ࡗࡓ F. gigantica ࡸࠊ⢭Ꮚᙧᡂ ⬟ࢆኻࡗ࡚࠸࡞࠸༢Ⅽ⏕Ṫᆺ⫢⻄࡛࠶ࡗࡓ⪃࠼ࡽࢀࡓ [48, 49]ࠋ௨ୖࡢࡇࡽࠊ
ᚋࡢFasciola ᒓࡢ✀ྠᐃࡣࠊpepck ࠾ࡼࡧ pold 㑇ఏᏊᆺᇶ࡙࠸࡚⾜࠺ᚲせࡀ࠶ࡿ
ࡇࡀᙉࡃ♧၀ࡉࢀࡓࠋ 㸳㸬ᑠᣓ ➨1 ❶࠾࠸࡚ゎᯒࡉࢀࡓࣥࢻᮾ㒊⏘⫢⻄ 157 యࡢ࠺ࡕࠊ6 యࡣ㈓⢭ᄞෆ ⢭Ꮚࢆ᭷ࡋ࡞࠸↓⢭Ꮚᆺࡶ㛵ࢃࡽࡎࠊnad1 ࡢࣁࣉࣟࢱࣉࡀ F. gigantica ࢡ࣮ࣞ ࢻᒓࡋ࡚࠸ࡓࡓࡵࠊᚑ᮶ࡢ᪉ἲ࡛ࡣṇ☜࡞✀ྠᐃࡀᅔ㞴࡛࠶ࡗࡓࠋࡇࡢࡼ࠺࡞య ࡣ㞄ᅜࡢࣂࣥࢢࣛࢹࢩࣗ࠾ࡼࡧ࣑࣐࣮࡛ࣕࣥࡶሗ࿌ࡉࢀ࡚࠾ࡾࠊࡼࡾ☜ᐇ࡞✀ྠᐃἲ
ࡀᮃࡲࢀ࡚࠸ࡓࠋ㏆ᖺࠊ᰾DNA ࡢ pepck ࠾ࡼࡧ pold ࡢ㑇ఏᏊᆺࢆ⏝࠸ࠊF. hepaticaࠊ
F. gigantica ࠾ࡼࡧ༢Ⅽ⏕Ṫᆺ⫢⻄ࢆ㆑ูࡍࡿ᪉ἲࡀሗ࿌ࡉࢀࡓࠋࡑࡇ࡛ࠊᮏ✀࡛ࡣࠊ ࡇࢀࡽࡢ✀ྠᐃ࣐࣮࣮࢝ࢆ⏝࠸ࠊࣥࢻᮾ㒊ࠊࣂࣥࢢࣛࢹࢩࣗ࠾ࡼࡧ࣑࣐࣮ࣕࣥࡢ
Fasciola ᒓయࡢ✀ྠᐃࢆヨࡳࡓࠋゎᯒࡢ⤖ᯝࠊnad1 ࣁࣉࣟࢱࣉࡀ F. gigantica ࣁࣉ ࣟࢢ࣮ࣝࣉᒓࡍࡿ࡚ࡢయࡣ pepck ࡶࡋࡃࡣ pold 㑇ఏᏊᆺࡶ F. gigantica ᆺࢆ♧
ࡋࠊ༢Ⅽ⏕Ṫᆺ⫢⻄ࣁࣉࣟࢢ࣮ࣝࣉᒓࡍࡿ࡚ࡢయࡣpepck ࡶࡋࡃࡣ pold 㑇ఏᏊ
ᆺࡀ༢Ⅽ⏕Ṫᆺ⫢⻄࡛࠶ࡿࡇࢆ♧ࡍ࣊ࢸࣟᆺࢆ♧ࡋࡓࠋࡇࡢࡇࡽࠊpepck ࠾ࡼ
32 ㈓⢭ᄞෆ⢭Ꮚ a IT S1 a pe pc k pol d ༢Ⅽ⏕Ṫᆺ⫢⻄ 115 ― Fg Fh /F g Fh /Fg ༢Ⅽ⏕Ṫᆺ⫢⻄ 4 ― Fg Fh /F g N .D . ༢Ⅽ⏕Ṫᆺ⫢⻄ 97 ― Fh /F g Fh /Fg Fh /F g ༢Ⅽ⏕Ṫᆺ⫢⻄ 1 b 㸩 Fh /F g Fh /Fg Fh /F g ༢Ⅽ⏕Ṫᆺ⫢⻄ 1 b 㸩 Fg Fh /F g Fh /Fg ༢Ⅽ⏕Ṫᆺ⫢⻄ F . gi gant ic a 233 㸩 Fg Fg Fg F . gi gant ic a 3 㸩 Fg Fg N .D . F . gi gant ic a 9b ― Fg Fg Fg F . gi gant ic a ྜィ 463 ⾲ 4 㸬ࣥࢻᮾ㒊ࠊࢿࣃ࣮ࣝࠊࣂࣥࢢࣛࢹࢩࣗࠊ࣑࣐࣮ࣕࣥ࠾ࡅࡿ Fa sc io la ᒓࡢゎᯒ⤖ᯝ N. D. ࡣ PC R ࡼࡿቑᖜ⏘≀ࢆᚓࡽࢀ࡞ࡗࡓࡇࢆ♧ࡍࠋ a ➨ 1 ❶࠾ࡼࡧඛ⾜◊✲ࡼࡾゎᯒࡉࢀࡓࠋ b ➨ 1 ❶࠾ࡼࡧඛ⾜◊✲࠾࠸࡚ࠊ㈓⢭ᄞෆ⢭Ꮚࡢ᭷↓ࠊ IT S1 㑇ఏᏊᆺ࠾ࡼࡧ nad1 ࣁࣉࣟࢱࣉࡢ⤖ᯝ ▩┪ࡀㄆࡵࡽࢀࡓయࠋ ✀ యᩘ ᚑ᮶ἲ ᪂つ㆑ูἲ nad1 ࣁࣉࣟࢢ࣮ࣝࣉ a Fh ࡣ F . he pat ic a ᆺࠊ Fg ࡣ F . gi gant ic a ᆺࠊ Fh /F gࡣ Fh Fg ࢆేࡏᣢࡘ࣊ࢸࣟᆺ࡛࠶ࡿࡇࢆ♧ࡍࠋ
33
ᅗ5㸬 multiprex PCR ἲࡼࡿ Fasciola ᒓయࡢ pepck 㑇ఏᏊᆺ
1: Milli Q 2: Fh ᆺࢥࣥࢺ࣮ࣟࣝ 3: Fg ᆺࢥࣥࢺ࣮ࣟࣝ 4: Fh/Fg ᆺ㸦࣊ࢸࣟᆺ㸧ࢥࣥࢺ࣮ࣟࣝ M: DNA ࣛࢲ࣮ 5-6: ࢪ⏘Fasciolaᒓయ㸦Fg ᆺ㸧 7-8: ࢪ⏘Fasciolaᒓయ㸦Fh/Fg ᆺ㸧 1 2 3 4 M 5 6 7 8 509 bp 241 bp 500 bp 1,000 bp 100 bp M
34 ᅗ6㸬 PCR-RFLP ἲࡼࡿ Fasciola ᒓయࡢ pold 㑇ఏᏊᆺ 1: Milli Q 2: Fh ᆺࢥࣥࢺ࣮ࣟࣝ 3: Fg ᆺࢥࣥࢺ࣮ࣟࣝ 4: Fh/Fg ᆺ㸦࣊ࢸࣟᆺ㸧ࢥࣥࢺ࣮ࣟࣝ M: DNA ࣛࢲ࣮ 5, 6, 7, 9: ࢪ⏘Fasciolaᒓయ㸦Fg ᆺ㸧 8, 10: ࢪ⏘Fasciolaᒓయ㸦Fh/Fg ᆺ㸧 1 2 3 4 M 5 6 7 8 9 10 708 bp 544 bp 164 bp 136 bp
35
36 㸯㸬⫼ᬒ ➨ 1 ❶࠾࠸࡚ࠊࣥࢻᮾ㒊ࡣ F. gigantica ࠾ࡼࡧ༢Ⅽ⏕Ṫᆺ⫢⻄ࡀศᕸࡍࡿࡇ ࡀ᫂ࡽ࡞ࡾࠊ୰ᅜ࡛Ⓨ⏕ࡋࡓ༢Ⅽ⏕Ṫᆺ⫢⻄ࡀࣥࢻᮾ㒊ࡲ࡛ศᕸࢆᣑࡋ ࡚࠸ࡿࡇࡀ♧၀ࡉࢀࡓ ࠋࡲࡓࠊࣥࢻᮾ㒊ศᕸࡍࡿ F. gigantica ࡢ nad1 ࣁࣉ ࣟࢱࣉࡣ༡ࢪ࡛ඃໃ࡞ࣁࣉࣟࢢ࣮ࣝࣉ A ࠊᮾ༡ࢪ࡛ඃໃ࡞ࣁࣉࣟࢢࣝ ࣮ࣉB ศ㢮ࡉࢀࡓࠋྛᅜ࠾ࡅࡿ F. gigantica ಶయ⩌ࡢሷᇶከᵝᗘࢆẚ㍑ࡋࡓ⤖ᯝࠊ F. gigantica ࣁࣉࣟࢢ࣮ࣝࣉ A ࡣࣥࢻள㝣ࡢすࡽᮾࠊࣁࣉࣟࢢ࣮ࣝࣉ B ࡣᮾ ༡ࢪࡽࣥࢻᮾ㒊ศᕸࢆᣑࡋࡓࢢ࣮ࣝࣉ࡛࠶ࡾࠊࡑࡢศᕸᇦᣑࡣB.
indicus ࡢேⅭⓗ࡞⛣ືࡀ㛵ࡋ࡚࠸ࡿྍ⬟ᛶࡀ♧၀ࡉࢀࡓࠋBos indicus ࡣࣥࢻள 㝣ࡢす㒊⨨ࡍࡿࣛࣥ㧗ཎࡢᮾ➃ࡽࣃ࢟ࢫࢱࣥ㒊ࡅ࡚ࡢࣥࢲࢫ ㇂࡛ᐙ␆ࡉࢀࡓ⪃࠼ࡽࢀ࡚࠸ࡿࠋࡑࡇ࡛ࠊᮏ❶࡛ࡣࣥࢲࢫ㇂㏆࠸ࣥࢻ す㒊ࡢࢹ࣮ࣜ㸦Delhi㸧ᕷ࡛ศ㞳ࡉࢀࡓ Fasciola ᒓయࢆ➨ 2 ❶᳨࡛ドࡋࡓゎᯒἲࢆ⏝ ࠸࡚ྠᐃࡋࠊ㏆㞄ᆅᇦࡢಶయ⩌ࡢ㛫ࡢศᏊ⣔⤫ゎᯒࢆ⾜ࡗࡓࠋ 㸰. ᮦᩱ᪉ἲ (6) ࢹ࣮ࣜ⏘ Fasciola ᒓࡢ᥇ྲྀฎ⌮ 2014 ᖺ 12 ᭶ࣥࢻす㒊ࡢࢹ࣮ࣜᕷࡢᒕ␆ሙ࠾࠸࡚ࠊࢪࢫࢠࣗ࢘ 11 㢌
37
ࡢ⫹⟶ࡼࡾFasciola ᒓ྾ࡢᡂࢆ᥇ྲྀࡋࠊࡑࢀࡽࡢ࠺ࡕᐟ 1 㢌ࡘࡁ 1 ࡽ 8
యࠊィ 40 యࢆゎᯒ౪ࡋࡓࠋయࡣ 70%࢚ࢱࣀ࣮࡛ࣝᅛᐃࡋࠊᒾᡭᏛᐤ⏕Ꮫ
◊✲ᐊ㐠ᦙࡋࡓࠋᐇయ㢧ᚤ㙾ୗ࡛ྛయࡽ㈓⢭ᄞࢆྲྀࡾฟࡋࠊᖖἲᚑࡗ࡚࣐࣊
ࢺ࢟ࢩ࣭࣮࣑ࣜࣥ࢝ࣥࡼࡾᰁⰍࡋࡓᚋࠊගᏛ㢧ᚤ㙾ࢆ⏝࠸࡚⢭Ꮚࡢ᭷↓ࢆほᐹࡋࡓ
[48]ࠋࡲࡓࠊ⏕Ṫჾᐁࢆྵࡲ࡞࠸యᑿ㒊ࡼࡾࠊHigh Pure PCR Template Preparation Kit
㸦Roche㸧ࢆ⏝࠸࡚ྛయࡢ DNA ࡀᢳฟࡉࢀࠊ⏝ࡲ࡛㸫20Υ࡛ಖᏑࡉࢀࡓࠋ
(7) ྛ㡿ᇦࡢ PCR
ྛయࡘ࠸࡚ࠊ᰾ࣜ࣎ࢯ࣮࣒DNA ࡢ ITS1 㡿ᇦࠊ᰾ DNA ࡢ pepck 㡿ᇦ࠾ࡼࡧ pold
㡿ᇦࠊ࣑ࢺࢥࣥࢻࣜDNA ࡢ nad1 㡿ᇦࢆ PCR ࡛ቑᖜࡋࡓࠋITS1 㡿ᇦࡢቑᖜࡣࣉ
࣐࣮ࣛITS1-F ࠾ࡼࡧ ITS1-R㸦➨ 1 ❶ཧ↷㸧ࠊnad1 㡿ᇦࡢቑᖜࡣࣉ࣐࣮ࣛIta 10
࠾ࡼࡧ Ita 2㸦➨ 1 ❶ཧ↷㸧ࠊpepck 㡿ᇦࡢቑᖜࡣࣉ࣐࣮ࣛFh-pepck-FࠊFg-pepck-F
࠾ࡼࡧ Fcmn-pepck-R㸦➨ 2 ❶ཧ↷㸧ࠊpold 㡿ᇦࡢቑᖜࡣࣉ࣐࣮ࣛFasciola-pold-F1
࠾ࡼࡧ Fasciola-pold-R1㸦➨ 2 ❶ཧ↷㸧ࢆ⏝࠸ࡓࠋྛ PCR ࡢ᮲௳ࡣ➨ 1 ❶࠾ࡼࡧ➨ 2
❶ྠᵝ⾜ࡗࡓࠋྛDNA 㡿ᇦࡢ PCR ⏘≀ࡣ NucleoSpin® Gel and PCR Clean-up ࢆ
⏝࠸࡚⢭〇ࡋࡓࠋ
(8) ITS1 㡿ᇦࠊpepck 㡿ᇦࠊpold 㡿ᇦࡢ㑇ఏᏊᆺ࠾ࡼࡧ nad1 㡿ᇦࡢሷᇶ㓄ิࡢゎᯒ
38
ࡓ㸦➨1 ❶࠾ࡼࡧ➨ 2 ❶ཧ↷㸧ࠋpepck 㡿ᇦࡢ PCR ⏘≀ࡣ 1.0%࣮࢞ࣟࢫࢤࣝࢆ⏝࠸
࡚㟁ẼὋືࡋࠊࣂࣥࢻࣃࢱ࣮ࣥࡼࡗ࡚㑇ఏᏊᆺࢆỴᐃࡋࡓ㸦➨ 2 ❶ཧ↷㸧ࠋࡲࡓࠊ
nad1 㡿ᇦࡢ PCR ቑᖜ⏘≀ࡢሷᇶ㓄ิ㸦535 bp㸧ࢆࠊࢲࣞࢡࢺࢩ࣮ࢡ࢚ࣥࢫἲࢆ⏝ ࠸࡚ゎᯒࡋࡓ㸦➨1 ❶ཧ↷㸧ࠋᚓࡽࢀࡓሷᇶ㓄ิࡣ ATGC ver. 6. 0. 3㸦Genetyx㸧ࢆ⏝࠸
࡚ࢭࣥࣈࣝᚋࠊGENETYX ver. 10. 0. 2 㸦Genetyx㸧ࢆ⏝࠸࡚࣓ࣛࣥࢺࡋࠊࣁࣉ
ࣟࢱࣉࢆ㆑ูࡋࡓࠋ
(9) nad1 㡿ᇦࡢሷᇶ㓄ิᇶ࡙ࡃࢿࢵࢺ࣮࣡ࢡࡢసᡂ
Network 4. 6. 1. 2 ࢆ⏝࠸࡚ࠊࢪ⏘ F. gigantica ࠾ࡼࡧ༢Ⅽ⏕Ṫᆺ⫢⻄ࡢ nad1 ࣁࣉ
ࣟࢱࣉᇶ࡙࠸ࡓMJ network ࢆసᡂࡋࡓ [4]ࠋᮏ❶࡛ゎᯒࡋࡓࢹ࣮ࣜ⏘ Fasciola ᒓ ࡢnad1 ࣁࣉࣟࢱࣉࡑࡢฟ⌧㢖ᗘຍ࠼ࠊ᪥ᮏ [20, 24]ࠊ㡑ᅜ [19, 25]ࠊ୰ᅜ [23, 40]ࠊ࣋ࢺࢼ࣒ [26]ࠊࢱ [5]ࠊ࣑࣐࣮ࣕࣥ [17]ࠊࣂࣥࢢࣛࢹࢩࣗ [36]ࠊࢿࣃ࣮ࣝ [43] ࠾ࡼࡧࣥࢻᮾ㒊㸦➨ 1 ❶㸧⏘ Fasciola ᒓࡢ nad1 ࣁࣉࣟࢱࣉࡑࡢฟ⌧㢖ᗘࢆ ཧ↷ࡋ࡚⏝࠸ࡓࠋ࡞࠾ࠊᮏ❶࡛ࡣMJ network ࠾࠸࡚ࡍ࡚ࡢࣁࣉࣟࢱࣉࢆ┤᥋ 㐃⤖ࡉࡏࡿࡇࡣ࡛ࡁ࡞ࡗࡓࠋࡇࡢࡓࡵࠊ┤᥋㐃⤖ࡉࡏࡿࡇࡀ࡛ࡁ࡞࠸ࣁࣉࣟࢱ ࣉ㛫ࢆ㐃⤖ࡉࡏࡿࡓࡵࡢ௬ࣁࣉࣟࢱࣉࡋ࡚median vector ࢆ⏝࠸ࡓࠋ (10) nad1 㡿ᇦ࠾ࡅࡿ㑇ఏⓗከᵝᛶࡢゎᯒ Delhi ᕷࠊࣥࢻᮾ⏘ࠊ࣑࣐࣮ࣕࣥ⏘ࠊࣂࣥࢢࣛࢹࢩࣗ⏘࠾ࡼࡧࢿࣃ࣮ࣝ⏘ F.
39
gigantica ࣁࣉࣟࢢ࣮ࣝࣉ A ࡢྛಶయ⩌࠾ࡅࡿ㑇ఏⓗከᵝᛶࢆホ౯ࡍࡿࡓࡵࠊDnaSP v5. 1. 0 ࢆ⏝࠸࡚ࣁࣉࣟࢱࣉከᵝᗘ㸦Hd㸧࠾ࡼࡧሷᇶከᵝᗘ㸦π㸧ࢆ⟬ฟࡋࡓ [31]ࠋ
㸱㸬⤖ᯝ
(4) ㈓⢭ᄞෆ⢭Ꮚࡢ᭷↓ ITS1 㡿ᇦࠊpepck 㡿ᇦࠊpold 㡿ᇦࡢ㑇ఏᏊᆺࡢゎᯒ⤖ᯝ
Delhi ᕷࡽᚓࡽࢀࡓ 40 యࡢ࠺ࡕࠊ39 యࡣ㈓⢭ᄞෆṇᖖ࡞⢭Ꮚࡀほᐹࡉࢀࠊ
᭷⢭ᏊᆺุᐃࡉࢀࡓࠋࡉࡽࠊITS1 㡿ᇦࠊpepck 㡿ᇦ࠾ࡼࡧ pold 㡿ᇦࡣ F. gigantica
࡛࠶ࡿࡇࢆ♧ࡍ Fg ᆺࢆ♧ࡋࡓࡇࡽࠊࡇࢀࡽ 39 యࡣ F. gigantica ࡛࠶ࡿ⪃
࠼ࡽࢀࡓࠋ୍᪉ࠊṧࡿ1 యࡣ㈓⢭ᄞෆṇᖖ࡞⢭Ꮚࡀほᐹࡉࢀࡎࠊ↓⢭Ꮚᆺุᐃ
ࡉࢀࡓࡀࠊITS1 㡿ᇦࠊpepck 㡿ᇦ࠾ࡼࡧ pold 㡿ᇦࡀ Fg ᆺࢆ♧ࡋࡓࡇࡽࠊࡇࡢ
యࡶF. gigantica ࡛࠶ࡿ⪃࠼ࡽࢀࡓ㸦⾲ 5㸧[44]ࠋ༢Ⅽ⏕Ṫᆺ⫢⻄ࡣ᳨ฟࡉࢀ࡞ࡗ
ࡓࠋ
(5) nad1 㡿ᇦࡢゎᯒ⤖ᯝ
Fasciola gigantica 40 యࡢ nad1 㡿ᇦࡢሷᇶ㓄ิ㸦535 bp㸧ࡽࡣࠊ19 ࡢሷᇶኚ␗
ᗙࡀ☜ㄆࡉࢀࠊND1-IN1 ᆺࡽ ND1-IN18 ᆺࡲ࡛ࡢ 18 ࣁࣉࣟࢱࣉࡀ㆑ูࡉࢀࡓࠋ
ࡇࢀࡽࡢ㓄ิሗࡣ GenBank Ⓩ㘓ࡉࢀࡓ㸦LC128314–LC128330 ࠾ࡼࡧ LC128751㸧
40 ࣉ A ྵࡲࢀࠊࡑࡢࡢ F. gigantica ࣁࣉࣟࢢ࣮ࣝࣉࡸ༢Ⅽ⏕Ṫᆺ⫢⻄ࡢࣁࣉࣟࢢࣝ ࣮ࣉᒓࡍࡿࣁࣉࣟࢱࣉࡣ᳨ฟࡉࢀ࡞ࡗࡓࠋ ND1-IN1 ᆺࢆ♧ࡋࡓ F. gigantica ࡣ 15 య࡛࠶ࡾࠊࡶࡗࡶฟ⌧㢖ᗘࡀ㧗࠸ࣁࣉࣟ ࢱࣉ࡛࠶ࡗࡓࠋࡇࡢࣁࣉࣟࣖࣉࡣࡇࢀࡲ࡛࣑࣐࣮ࣕࣥ㸦ND1-M15 ᆺ㸧 [17]ࠊ ࢱ㸦TH13 ᆺ㸧 [5]ࠊࣂࣥࢢࣛࢹࢩࣗ㸦Fg-ND1-Bd9 ᆺ㸧[36]࠾ࡼࡧࢿࣃ࣮ࣝ㸦Fg-ND1-N1 ᆺ㸧[43] ࡽ᳨ฟࡉࢀࡓ F. gigantica ࡢࣁࣉࣟࢱࣉྠ୍࡛࠶ࡾࠊF. gigantica ࣁ ࣉࣟࢢ࣮ࣝࣉA ࡢጞ⪅ࣁࣉࣟࢱࣉ࡛࠶ࡿ⪃࠼ࡽࢀࡓ㸦ᅗ 7㸧ࠋࡲࡓࠊND1-IN2 ᆺ
ࡽND1-IN18 ᆺࡣ ND1-IN1 ᆺࡶ F. gigantica ࣁࣉࣟࢢ࣮ࣝࣉ A ྵࡲࢀ࡚࠾
ࡾࠊND1-IN1 ᆺࡢ⾨ᫍࣁࣉࣟࢱࣉ࡛࠶ࡗࡓࠋ
(6) nad1 㡿ᇦ࠾ࡅࡿ㑇ఏⓗከᵝᛶ
Fasciola gigantica ࡢࣁࣉࣟࢢ࣮ࣝࣉ A ᒓࡍࡿ Delhi ᕷࠊࣥࢻᮾ㒊ࠊ࣑࣐ࣕࣥ ࣮ࠊࣂࣥࢢࣛࢹࢩࣗ࠾ࡼࡧࢿࣃ࣮ࣝ⏘ࡢྛಶయ⩌࠾࠸࡚ࠊnad1 㡿ᇦࡢሷᇶ㓄ิࡑ ࡢฟ⌧㢖ᗘᇶ࡙ࡁࠊࣁࣉࣟࢱࣉከᵝᗘ㸦Hd㸧࠾ࡼࡧሷᇶከᵝᗘ㸦ȧ㸧ࡀ⟬ฟࡉࢀ ࡓ㸦⾲ 6㸧ࠋሷᇶከᵝᗘࡣࢿࣃ࣮ࣝಶయ⩌㸦0.00366㸧࠾ࡼࡧࣂࣥࢢࣛࢹࢩࣗಶయ⩌ 㸦0.00362㸧࠾࠸࡚㧗್ࢆ♧ࡋࡓࠋDelhi ಶయ⩌㸦0.00255㸧ࡣࣥࢻள㝣ᮾ➃ࡢ ࣥࢻᮾ㒊ಶయ⩌㸦0.00242㸧ࡸ࣑࣐࣮ࣕࣥಶయ⩌㸦0.00255㸧ẚ㧗್ࢆ♧ࡋࡓࡶࡢ ࡢࠊࢿࣃ࣮ࣝಶయ⩌ࡸࣂࣥࢢࣛࢹࢩࣗಶయ⩌ẚప್࡛࠶ࡗࡓࠋ
41 㸲㸬⪃ᐹ ༢Ⅽ⏕Ṫᆺ⫢⻄ࡣࣥࢻᮾ㒊ࠊࢿࣃ࣮ࣝ࠾ࡼࡧࣂࣥࢢࣛࢹࢩࣗࡽ᳨ฟࡉࢀࠊࡑ ࡢ᳨ฟ⋡ࡣࢿࣃ࣮ࣝ㸦75.3 %㸧[43] ࡸࣂࣥࢢࣛࢹࢩࣗ㸦86.0 %㸧[36] ࡛ࡣ F. gigantica ẚ࡚ඃໃ࡛࠶ࡿࡢᑐࡋࠊࣥࢻᮾ㒊࡛ࡣຎໃ㸦24.8 %㸧࡛࠶ࡗࡓ㸦➨ 1 ❶㸧ࠋ ᮏ◊✲࠾࠸࡚ࠊ༢Ⅽ⏕Ṫᆺ⫢⻄ࡣࣥࢻす㒊ࡢ Delhi ᕷࡽࡣࡃ᳨ฟࡉࢀ࡞ ࡗࡓࠋࡇࡢࡇࡣࠊ୰ᅜ࡛Ⓨ⏕ࡋࡓ༢Ⅽ⏕Ṫᆺ⫢⻄ࡀࠊࣥࢻࡢᗈ⠊ᅖศᕸࢆᣑ ࡋ࡚࠸࡞࠸ࡇࢆ♧၀ࡋ࡚࠸ࡿࠋࡋࡋࠊᮏ◊✲࡛ゎᯒ⏝࠸ࡓయࡣ Delhi ᕷ㝈 ࡽࢀ࡚࠾ࡾࠊゎᯒᩘࡶ 40 య༑ศ࡛ࡣ࡞࠸ࡓࡵࠊᚋࡣゎᯒᆅᇦయᩘࢆቑࡸ ࡋ᳨࡚ウࡍࡿᚲせࡀ࠶ࡿࠋ༢Ⅽ⏕Ṫᆺ⫢⻄ࡣ F. gigantica ẚ⏕Ṫຊࡀ㧗࠸⪃࠼ ࡽࢀ࡚࠸ࡿࡓࡵ [36]ࠊᚋࡣࣥࢻ࠾ࡅࡿ༢Ⅽ⏕Ṫᆺ⫢⻄ࡢศᕸࡘ࠸࡚ὀព῝ࡃ ┘どࡍࡿᚲせࡀ࠶ࡿ⪃࠼ࡽࢀࡓࠋ ᮏ◊✲࡛ࡣࠊF. gigantica 1 యࡣ㈓⢭ᄞෆ⢭Ꮚࡀࡳࡽࢀࡎࠊ↓⢭Ꮚᆺุᐃࡉࢀ ࡓࠋࡇࢀࡲ࡛ࡶ↓⢭ᏊᆺࡢF. gigantica ࡣࣥࢻᮾ㒊ࡸࣂࣥࢢࣛࢹࢩࣗࠊ࣑࣐ࣕࣥ ࣮࡛ࡶⓎぢࡉࢀ࡚࠸ࡿ [17, 36]ࠋ㈓⢭ᄞ⢭ᏊࡀᏑᅾࡋ࡞࠸ཎᅉࡣయࡢຍ㱋࡞ ࡼࡿ⢭Ꮚᙧᡂ⬟ࡢᾘኻࡸࠊ᥋ࡼࡿ⢭Ꮚᨺฟ࡞ࡀ⪃࠼ࡽࢀࡿࡀࠊᮏ◊✲࡛ࡣ᫂ ࡽ࡛ࡁ࡞ࡗࡓࠋ Delhi ᕷࡢ F. gigantica ࡣࣥࢻᮾ㒊ࠊࢿࣃ࣮ࣝ࠾ࡼࡧࣂࣥࢢࣛࢹࢩࣗ࠸ࡗࡓ༡ ࢪᆅᇦࡢ F. gigantica ࡛ඃໃ࡞ࣁࣉࣟࢢ࣮ࣝࣉ A ศ㢮ࡉࢀࡿ nad1 ࣁࣉࣟࢱ
42 ࣉࢆ♧ࡋࡓ㸦ᅗ7㸧ࠋࡉࡽࠊࣁࣉࣟࢢ࣮ࣝࣉ A ಶయ⩌࠾ࡅࡿሷᇶከᵝᗘࡘ࠸࡚ࠊ ࣥࢻす㒊ࡢDelhi ᕷࡢಶయ⩌㸦0.00255㸧ࡣࣥࢻᮾ㒊ࡢಶయ⩌㸦0.00242㸧ẚ ࡚㧗࠸್ࢆ♧ࡋࡓࡇࡽࠊࣁࣉࣟࢢ࣮ࣝࣉ A ࡢ F. gigantica ಶయ⩌ࡣࠊࣥࢻᅜ ෆ࠾࠸࡚ࡣすࡽᮾศᕸࢆᣑࡋࡓྍ⬟ᛶࡀ♧၀ࡉࢀࡓࠋ୍᪉ࠊDelhi ᕷࡣࢿ ࣃ࣮ࣝࡸࣂࣥࢢࣛࢹࢩࣗࡼࡾࡶす᪉⨨ࡋ࡚࠸ࡿࡶ㛵ࢃࡽࡎࠊࡑࡢሷᇶከᵝᗘࡣ ࢿࣃ࣮ࣝಶయ⩌㸦0.00366㸧ࡸࣂࣥࢢࣛࢹࢩࣗ㸦0.00362㸧ಶయ⩌ẚ࡚ప್ࢆ♧ࡋࡓ 㸦⾲6㸧ࠋࡇࢀࡣࠊす᪉⨨ࡍࡿ Delhi ᕷࡢಶయ⩌ࡼࡾࡶࠊᮾ᪉⨨ࡍࡿࢿࣃ࣮ࣝ ࡸࣂࣥࢢࣛࢹࢩࣗࡢಶయ⩌ࡢ᪉ࡀ㑇ఏⓗከᵝ࡛࠶ࡿࡇࢆ♧၀ࡋ࡚࠾ࡾࠊࣁࣉࣟࢢ ࣮ࣝࣉ A ࡢ F. gigantica ಶయ⩌ࡀࣥࢻள㝣す㒊ࢆ㉳※ࡍࡿ࠸࠺ࠊᙜึࡢ௬ ㄝࡣᑐࡢ⤖ᯝࢆ♧ࡋࡓࠋᮏ◊✲࡛ࡣࠊᗈ࡞ࣥࢻᮏᅵ࠾ࡅࡿゎᯒᆅⅬࡀ୍ᆅ Ⅼ㸦Delhi ᕷ㸧㝈ࡽࢀ࡚࠸ࡓࡇࡸࠊゎᯒ⏝࠸ࡓయᩘࡀྛᆅᇦ࠾࠸࡚ࡁࡃ ␗࡞ࡗ࡚࠸ࡿࡇࡀゎ㔘ࢆᅔ㞴ࡋࡓཎᅉ࡛࠶ࡿྍ⬟ᛶࡀ⪃࠼ࡽࢀࡓࠋ༡ࢪ࠾ ࡅࡿ F. gigantica ࡢᣑᩓ⤒㊰ࢆ⌮ゎࡍࡿࡓࡵࠊᚋࡣࣥࢻᮏᅵࢆྵࡴ༡ࢪㅖᅜ ࠾ࡅࡿゎᯒᆅⅬࡸయᩘࢆቑࡸࡍᚲせࡀ࠶ࡿ⪃࠼ࡽࢀࡓࠋࡲࡓࠊDelhi ᕷࡽࡣ ࣁࣉࣟࢢ࣮ࣝࣉB ࠾ࡼࡧ C ࡢ F. gigantica ಶయ⩌ࡣ᳨ฟࡉࢀ࡞ࡗࡓࠋ ௨ୖࡢࡇࡽࠊ༡ࢪࡽᮾ༡ࢪࡅ࡚ศᕸࡍࡿ F. gigantica ࡣᆅᇦ ᛶ㛵㐃ࡋࡓ3 ࡘࡢࣁࣉࣟࢢ࣮ࣝࣉࡀ㆑ูࡉࢀࡿࡇࡀ♧ࡉࢀࡓࠋࣁࣉࣟࢢ࣮ࣝࣉ A ࡣ༡ࢪ㸦ࣥࢻࠊࢿࣃ࣮ࣝࠊࣂࣥࢢࣛࢹࢩࣗ㸧ࢆ୰ᚰศᕸࡍࡿ㞟ᅋ࡛࠶ࡾࠊࡑ ࡢศᕸࡣᐙ␆ࡉࢀࡓ B. indicus ࡢேⅭⓗ⛣ືࡀ㛵ࡋࡓྍ⬟ᛶࡀ⪃࠼ࡽࢀࡓࠋࣁ
43 ࣉࣟࢢ࣮ࣝࣉB ࡣ࣑࣐࣮ࣕࣥࢆ୰ᚰࣥࢻᮾ㒊ࡸࢱศᕸࡍࡿ㞟ᅋ࡛࠶ࡿ㸦ᅗ 7㸧[5, 17]ࠋࣥࢻᮾ㒊࠾࠸࡚ࣁࣉࣟࢢ࣮ࣝࣉ B ࡢయࡀ᳨ฟࡉࢀࡓࡢࡣ Imphal ᕷࡔࡅ࡛࠶ࡗࡓࡀࠊྠᕷࡣṔྐⓗ࣑࣐࣮ࣕࣥࡢᙳ㡪ࢆᙉࡃཷࡅࡓᆅᇦ࡛࠶ࡾࠊᐙ␆ ࡢ⛣ືࡶᐜ࡛᫆࠶ࡗࡓ⪃࠼ࡽࢀࡿ [45]ࠋࡲࡓࠊࣁࣉࣟࢢ࣮ࣝࣉ C ࡣᮾ༡ࢪ࠾ࡼ ࡧ୰ᅜศᕸࡍࡿ㞟ᅋ࡛࠶ࡾ[5, 12, 17, 23, 26, 40]ࠊ㑇ఏⓗከᵝᛶࡀ᭱ࡶ㧗࠸ࢱࡀ㉳ ※࡛࠶ࡿ⪃࠼ࡽࢀࡿ㸦ᅗ7㸧[5, 12]ࠋᮾ༡ࢪࡢᐙ␆ࡋ࡚㔜せ࡞ࢪࢫࢠࣗ
࢘㸦Bubalus bubalis㸧ࡣ Fasciola ᒓࡢせ࡞ᐟ࡛࠶ࡾࠊࢱ㒊࡛ᐙ␆ࡉࢀࡓᚋࠊ
ேⅭⓗᮾ༡ࢪ࠾ࡼࡧ୰ᅜศᕸࢆᣑࡋࡓ⪃࠼ࡽࢀ࡚࠸ࡿ [14, 15]ࠋࡇࡢࡇ ࡽࠊࣁࣉࣟࢢ࣮ࣝࣉC ࡢ F. gigantica ಶయ⩌ࡣࢱࢆ㉳※ࡋࠊB. bubalis ࡢேⅭ ⓗ⛣ືకࡗ࡚ࠊࡑࡢศᕸᇦࢆᣑࡋࡓྍ⬟ᛶࡀ⪃࠼ࡽࢀࡓࠋ 㸳㸬ᑠᣓ ࣥࢻす㒊ࡢࢹ࣮ࣜᕷ࠾࠸࡚ࢫࢠࣗ࢘ࡽ᥇ྲྀࡉࢀࡓ Fasciola ᒓ 40 య ࡘ࠸࡚ࠊpepck ࠾ࡼࡧ pold ࡢ㑇ఏᏊᆺᇶ࡙࠸࡚✀ྠᐃࢆ⾜ࡗࡓࠋࡑࡢ⤖ᯝࠊ40 య ࡣ࡚F. gigantica ࡛࠶ࡿྠᐃࡉࢀࠊ༢Ⅽ⏕Ṫᆺ⫢⻄ࡣ᳨ฟࡉࢀ࡞ࡗࡓࠋḟ nad1 ࡢሷᇶ㓄ิࢆỴᐃࡋࡓ⤖ᯝࠊ40 యࡢࣁࣉࣟࢱࣉࡣ࡚ࣁࣉࣟࢢ࣮ࣝࣉ A ศ㢮 ࡉࢀࡓࠋ༡ࢪࡢྛᆅᇦࡢయࡶሷᇶከᵝᗘࢆẚ㍑ࡋࡓ⤖ᯝࠊࣁࣉࣟࢢ࣮ࣝ ࣉA ᒓࡍࡿ F. gigantica ࡣࣥࢻࡢすࡽᮾࡑࡢศᕸᇦࢆᣑࡋࡓྍ⬟ᛶࡀ♧
44
၀ࡉࢀࡓࠋࡋࡋ࡞ࡀࡽࠊ༡ࢪᅵ࠾ࡅࡿ F. gigantica ࡢᣑᩓ⤒㊰ࢆ᫂ࡽ
ࡍࡿࡣ⮳ࡽ࡞ࡗࡓࠋࡲࡓࠊࣁࣉࣟࢢ࣮ࣝࣉB ࠾ࡼࡧ C ᒓࡍࡿ F. gigantica ࡣ᳨
45 ⾲5㸬ࣥࢻ Delhi ᕷࡽ᥇ྲྀࡉࢀࡓF. giganticaయࡢゎᯒ⤖ᯝ యᩘ ㈓⢭ᄞෆ ⢭Ꮚ 㑇ఏᏊᆺ ࣑ࢺࢥࣥࢻࣜnad1
ITS1 pepck pold ࣁࣉࣟࢱࣉ Accession no. 㻌 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 䠉㻌 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 㻌 㻌 㻌 㻌 㻌 㻌 㻌 㻌 㻌 㻌 㻌 㻌 a "+" ᭷⢭Ꮚᆺ, "㸫" ↓⢭Ꮚᆺ
46
ᅗ7㸬ࢪ⏘Fasciola ᒓࡢ nad1 㡿ᇦࡢሷᇶ㓄ิᇶ࡙ࡃ MJ network
ྛࡢࡁࡉࡣయᩘࢆᫎࡋ࡚࠾ࡾࠊෆࣁࣉࣟࢱࣉࢥ࣮ࢻ࠾ࡼࡧయᩘࢆグࡋࡓࠋ యᩘࡢグ㍕ࡀ࡞࠸ࣁࣉࣟࢱࣉࡣ 1 య࡛࠶ࡿࡇࢆ♧ࡍࠋࡲࡓࠊᯞୖࡢᩘᏐࡣሷᇶ⨨ ᩘࢆ⾲ࡋ࡚࠾ࡾࠊᩘᏐࡀ࡞࠸ࡶࡢࡣ 1 ሷᇶ⨨࡛࠶ࡿࡇࢆ♧ࡍࠋࣁࣉࣟࢱࣉࢥ࣮ࢻࡣࡑ ࢀࡒࢀࠊࣥࢻDelhi ᕷ㸦IN1 ࡽ IN17㸧ࠊࣥࢻᮾ㒊㸦E1 ࡽ E37㸧ࠊࢿࣃ࣮ࣝ㸦FspN1ࠊ FspN2 ࠾ࡼࡧ N1 ࡽ N10㸧ࠊࣂࣥࢢࣛࢹࢩࣗ㸦Bd1 ࡽ Bd11㸧ࠊ࣑࣐࣮ࣕࣥ㸦M1 ࡽ M19㸧ࠊ ࢱ㸦T1 ࡽ T30㸧ࠊ࣋ࢺࢼ࣒㸦V1 ࡽ V5㸧ࠊ୰ᅜ㸦C1 ࡽ C16㸧ࠊ㡑ᅜ㸦Kor2a ࠾ࡼࡧ Kor2b㸧࠾ࡼࡧ᪥ᮏ㸦Fsp2㸧ࡽ᳨ฟࡉࢀࡓࣁࣉࣟࢱࣉࢆ♧ࡍࠋ
47 ⾲6㸬nad1㡿ᇦࡢሷᇶ㓄ิᇶ࡙࠸ࡓࠊࣁࣉࣟࢢ࣮ࣝࣉA ࡢF. giganticaಶయ⩌࠾ࡅࡿ 㑇ఏⓗከᵝᛶ ಶయ⩌ N S h Hd s SD ȧ ࣥࢻDelhi ᕷ 40 19 18 0.853±0.052 0.00255 ࣥࢻᮾ㒊 91 32 32 0.751s0.050 0.00242 ࢿࣃ࣮ࣝ 20 16 10 0.758s0.101 0.00366 ࣂࣥࢢࣛࢹࢩࣗ 20 15 10 0.832s0.075 0.00362 ࣑࣐࣮ࣕࣥ 13 7 6 0.641s0.150 0.00225 N: యᩘ, S: ከᆺࢧࢺᩘ, h: ࣁࣉࣟࢱࣉᩘ, Hd: ࣁࣉࣟࢱࣉከᵝᗘ, SD: ᶆ‽೫ᕪ, ȧ: ሷᇶከᵝᗘ
48
49 㸯㸬⫼ᬒ ➨1 ❶ࡽ➨ 3 ❶࡛ࡣࠊ༡ࢪࡢ Fasciola ᒓಶయ⩌ࢆศᏊ⣔⤫Ꮫⓗゎᯒࡋࠊᐙ ␆ࡢ⛣ືࡀ Fasciola ᒓࡢศᕸᣑᐤࡋ࡚࠸ࡿྍ⬟ᛶࢆぢฟࡋࡓࠋࡋࡋ࡞ࡀࡽࠊ ࡇࡢࡼ࠺࡞ᐙ␆ᐤ⏕ࡢඹఏᦙ⌧㇟ࡀࠊ༡ࢪ࠾ࡅࡿⱄື≀ࡢᐤ⏕ᬑ 㐢ⓗ࡞⌧㇟࡞ࡢࠊFasciola ᒓ≉᭷ࡢ⌧㇟࡞ࡢ࠸࠺ၥࡀ⏕ࡌࡓࠋࡑࡇ࡛ࠊ Fasciola ᒓ㢮ఝࡋࡓ⏕ά⎔ࢆᣢࡘࠊⱄື≀ࡢᐤ⏕ࡘ࠸࡚ࠊྠᵝศᏊ⣔⤫ゎ ᯒࢆ⾜࠺ࡇࡋࡓࠋEeplanatum explanatum ࡣ Fasciola ᒓྠᵝࠊ༡ࢪ࠾࠸୍࡚
⯡ⓗ᳨ฟࡉࢀࡿཱྀ྾㢮ࡢ୍✀࡛࠶ࡾࠊⱄື≀ࡢ⫹⟶ෆᐤ⏕ࡍࡿ [3, 11, 45]ࠋ
Eeplanatum explanatum ࡢศᏊᏛⓗሗࡣ㝈ࡽࢀ࡚࠾ࡾࠊ✀ෆࡢ⣔⤫ゎᯒ⏝࠸ࡿ࣑ࢺ ࢥࣥࢻࣜ DNA 㡿ᇦࡣ༡ࢪࡢࢿࣃ࣮ࣝ࠾ࡼࡧࣂࣥࢢࣛࢹࢩࣗࡢయ࡛ࡢࡳሗ࿌
ࡉࢀ࡚࠸ࡿ [37]ࠋࡑࡇ࡛ᮏ❶࡛ࡣࠊࡲࡎࣥࢻ࠾࠸࡚ⱄᐙ␆ࡢ⫹⟶ࡽ᥇ྲྀࡉࢀ
ࡓయࢆ ITS2 ࡢሷᇶ㓄ิᇶ࡙ࡁࠊE. explanatum ࡛࠶ࡿྠᐃࡋࡓࠋࡑࡢୖ࡛ࠊ
ࣥࢻࠊࢿࣃ࣮ࣝ࠾ࡼࡧࣂࣥࢢࣛࢹࢩࣗಶయ⩌ࡢ࣑ࢺࢥࣥࢻࣜ DNA ࡢ nad1 㡿ᇦࢆ ⏝࠸ࠊ༡ࢪᆅᇦ࠾ࡅࡿE. explanatum ಶయ⩌ࡢศᏊ⣔⤫ゎᯒࢆ⾜ࡗࡓࠋ 㸰㸬ᮦᩱ᪉ἲ (1) ࣥࢻ⏘ Explanatum ᒓ྾ࡢ᥇ྲྀฎ⌮ ࣥࢻࡢࢹ࣮ࣜᕷ㸦Delhi㸧ࠊ࣒ࣥࣂᕷ㸦Mumbai㸧ࠊ࢞ࣥࢺࢡᕷ㸦Gangtok㸧࠾ࡼࡧ ࣥࣃ࣮ࣝᕷ㸦Imphal㸧࠾࠸࡚ࠊ2014 ᖺ 2 ᭶ࡽ 12 ᭶ࡅ࡚ࠊࢪࢫࢠࣗ ࢘ 30 㢌࠾ࡼࡧࢮࣈ࣮∵ 4 㢌ࡢ⫹⟶ෆࡽయࢆ᥇ྲྀࡋࠊࡑࢀࡽࡢ࠺ࡕ Delhi ᕷ࠾ࡼ
ࡧImphal ᕷ⏤᮶ࡢࡶࡢࡣᐟ 1 㢌ࡘࡁ 2 యࠊMumbai ᕷ࠾ࡼࡧ Gangtok ᕷ⏤᮶ࡢ
50
࣮࡛ࣝᅛᐃࡋࠊᒾᡭᏛᐤ⏕Ꮫ◊✲ᐊ㐠ᦙࡋࡓࠋ⏕Ṫჾᐁࢆྵࡲ࡞࠸యᚋ㒊ࡢ
⤌⧊ࡼࡾࠊHigh Pure PCR Template Preparation Kit㸦Roche㸧ࢆ⏝࠸࡚ྛయࡢ DNA
ࡀᢳฟࡉࢀࠊ⏝ࡲ࡛㸫20Υ࡛ಖᏑࡉࢀࡓࠋ
(2) ྛ㡿ᇦࡢ PCR
ྛయࡘ࠸࡚ࠊࣉ࣐࣮ࣛITS2-F㸦5’-TGTGTCGATGAAGAGCGCAG-3’㸧࠾ࡼࡧ
ITS2-R㸦5’-TGGTTAGTTTCTTTTCCTCCGC3’㸧[24]ࢆ⏝࠸࡚ ITS2 㡿ᇦࢆࠊPc-nad1-F1
㸦 5′-CAGATTCGGAAGGGGCCTAA-3′ 㸧 ࠾ ࡼ ࡧ Pc-nad1-R1 㸦
5′-ACGTAGCACGAGCCCAAATA-3′㸧[37] ࢆ⏝࠸࡚ nad1 㡿ᇦࢆࠊࡑࢀࡒࢀ PCR ࡼࡾ
ቑᖜࡋࡓࠋPCR ࡢ᮲௳ࡣ➨ 1 ❶ྠᵝ⾜ࡗࡓࠋྛ DNA 㡿ᇦࡢ PCR ⏘≀ࡣ
NucleoSpin® Gel and PCR Clean-up ࢆ⏝࠸࡚⢭〇ࡋࡓࠋ
(3) ITS1 㡿ᇦࡢ㑇ఏᏊᆺ࠾ࡼࡧ nad1 㡿ᇦࡢሷᇶ㓄ิࡢゎᯒ
ྛయࡢITS2 㡿ᇦ㸦442 bp㸧࠾ࡼࡧ nad1 㡿ᇦ㸦657 bp㸧ࡢ PCR ቑᖜ⏘≀ࡢሷᇶ㓄
ิࢆࠊࢲࣞࢡࢺࢩ࣮ࢡ࢚ࣥࢫἲࢆ⏝࠸࡚ゎᯒࡋࡓࠋࢩ࣮ࢡ࢚ࣥࢫᛂࡣ BigDye
Terminator v3.1 Cycle Sequencing Kit ࢆ⏝࠸ࠊࣉࣟࢺࢥ࣮ࣝᚑࡗ࡚ᛂ⁐ᾮࢆㄪᩚࡋࠊ
ABI PRISM 3500 Genetic Analyzer ࢆ⏝࠸࡚ሷᇶ㓄ิࡢゎᯒࢆ⾜ࡗࡓࠋࢩ࣮ࢡ࢚ࣥࢫ
ᛂࡣPCR ᛂྠ୍ࡢࣉ࣐࣮ࣛࢆ⏝࠸ࡓࠋᚓࡽࢀࡓሷᇶ㓄ิࡣ ATGC ver. 6. 0. 3
㸦Genetyx, Tokyo, Japan㸧ࢆ⏝࠸࡚ࢭࣥࣈࣝᚋࠊGENETYX ver. 10. 0. 2 㸦Genetyx㸧
ࢆ⏝࠸࡚࣓ࣛࣥࢺࡋࠊࣁࣉࣟࢱࣉࢆ㆑ูࡋࡓࠋ
(4) nad1 㡿ᇦࡢሷᇶ㓄ิᇶ࡙ࡃ⣔⤫ᶞ࠾ࡼࡧࢿࢵࢺ࣮࣡ࢡࡢసᡂ
ࣥࢻ⏘E. explanatum ࡢ nad1 ࣁࣉࣟࢱࣉຍ࠼ࠊ㞄᥋ࡍࡿࢿࣃ࣮ࣝ㸦N1 ᆺࡽ
