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A tɛ i n’a fɔ kolotugudaw, a bɛ miiri kosɛbɛ ko fɛnɲɛnamaw tɛ ni cɛya-ɲini-stɛroyi ɔrimɔni ye.Anopheles gambiae kɔnɔ, a bɛ i n’a fɔ ecdysone steroid 20-hydroxyecdysone (20E) jiginna walasa ka kɔnɔw falenni kɔlɔsi ni musow ye a labɛn2 ani ka furuɲɔgɔnya waati dɔ bila mɔgɔ la ni cɛw ye cɛnimusoya kɛ.Kabini kɔnɔw ka yiriwali ani furuɲɔgɔnma ye bangeko jogo nafamaw ye, ka faamuyali sɔrɔ cogo min na Anopheles soso musomanw bɛ nin ɔrimɔni taamasiɲɛw fara ɲɔgɔn kan, o bɛ se ka sumayabana kɛlɛli porogaramu kura dilanni nɔgɔya.Yan, an b’a jira ko nin bangeko baara ninnu bɛ labɛn ni cɛya ni musoya furakisɛw ye minnu tɛ kelen ye. 3-dehydro-20E (3D20E), min bɛ bangebagaw lakana ni muso ka cɛnimusoya tali dabila ye cɛnimusoya jiginni kɔfɛ ani ka baara kɛ ni defosforilasi ye.Kɛrɛnkɛrɛnnenya la, 3D20E jiginni fana ye bangekɔlɔsi fɛɛrɛw jirali bila sen kan minnu bɛ kɔnɔw falenni sabati Plasmodium banakisɛ sɔrɔtuma na, o bɛ muso banakisɛw ka kɛnɛya sabati.20E min bɔra muso la, o tɛ cɛnimusoya lawuli jaabi, nka a bɛ a to furuɲɔgɔnma kelen-kelen bɛɛ ka kɔnɔw da 20E-inhibiting kinases kɔfɛ.
Sumayabana sɔrɔli ni a fagali bɛ ka caya tuguni4 ka da fɛnɲɛnamafagalanw tali caya kan Anopheles sosow la, olu minnu ye hadamadenw ka sumayabana banakisɛw sɔrɔbaga kelenpe ye.O soso ninnu ka furusiri ɲɛnamaya kɛcogo ye laɲini ye min bɛ mɔgɔ sama kɛrɛnkɛrɛnnenya la sumayabana kɛlɛli fɛɛrɛ kuraw kama bawo musow bɛ furu siɲɛ kelen dɔrɔn ka nin furuɲɔgɔnma kelen in kɛ sterili ye, o bɛna se ka kɛ sababu ye ka sosow hakɛ dɔgɔya foro kɔnɔ.
Musow bɛ kɛ cɛnimusoya dɛsɛbagatɔw ye u kɛlen kɔ ka stɛroyi ɔrimɔni caman sɔrɔ cɛw fɛ.Sɛgɛsɛgɛliw y’a jira ko gɛlɛya min bɛ furuɲɔgɔnya wɛrɛw lase mɔgɔ ma, o ye 20-hydroxyecdysone (20E) ye, o ye stɛroyi ɔrimɔni ye min lakodɔnnen don kosɛbɛ i n’a fɔ sɔgɔsɔgɔninjɛ cycle regulateur in the larval stage.Cɛw ka se ka 20E dilan ani k’a bila a la, o jiginna kɛrɛnkɛrɛnnenya la san Anopheles suguya minnu ye Cellia7 suguya fitinin dɔ ye, o min bɛ jɛnsɛn Afiriki kɔnɔ ani sumayabana fɛnɲɛnamafagalan faratilenw bɛ a la, Anopheles gambiae fana sen bɛ o la.O ye ko ye min ka kan ka kɔlɔsi kɛrɛnkɛrɛnnenya la bawo o suguya ninnu na, musow fana bɛ 20E bɔ joli dumuni kelen-kelen bɛɛ kɔfɛ, ani 20E bɛ oogenesis cycle ɲɛminɛ (aw ye sɛbɛn ɲɛ 8 lajɛ).Nka, fɛn caman tɛ dɔn musow bɛ don ɲɔgɔn na cogo min na taamasiɲɛw bɛ bɔ ɛkidizɔni sɔrɔyɔrɔ fila danfaralenw na (cɛ jiginni ni joli baloli) k’a sɔrɔ u ma u yɛrɛ ka furuɲɔgɔnya seko tiɲɛ.Tiɲɛ na, ni 20E min bɛ bɔ musow fɛ, o bɛ cɛnimusoya muɲunbaliya bila mɔgɔ la, o bɛna kɛ sababu ye ka bangekɔlɔsi kɛ npogotiginin balobagaw la, o ye kɛwale ye min ka ca kosɛbɛ nin soso ninnu na
Ɲɛfɔli min bɛ se ka kɛ, o ye ko A. gambiae cɛw bɛ cɛ kɛrɛnkɛrɛnnen ecdysone sɛmɛntiyalen dɔ wuli, o min bɛ taamasiɲɛw ka cakɛda dɔ baara muso ka bangekɔlɔsi fɛɛrɛw la, o bɛ na ni furuɲɔgɔnma basigibaliya ye.Nka, hali ni kolotugudaw bɛ ni stɛroyi ɔrimɔni caman ye, i n’a fɔ ɔrimɔni stɛrozɛni ni andorojɛni (a lajɛra ref. 9 kɔnɔ), an ka dɔnniya la, stɛroyi minnu bɛ kɛ ni andorojɛni ye, olu ma dɔn san kɔnɔ fɛnɲɛnɛmaw.
An y’a daminɛ ka stɛroyi ɔrimɔni hakɛ dɔn A. gambiae cɛma cɛkɔrɔba dɛmɛnan (MAG) la ka stɛroyiw ɲini minnu bɛ se ka fɛn caman sɛmɛntiya.Ka baara kɛ ni jilama sɛgɛsɛgɛli kɛcogo ɲuman ye min faralen don ni tandem mass spectrometry (HPLC-MS/MS) ye sanni ka fɛɛrɛ kɛrɛnkɛrɛnnen min kɛ ka tɛmɛ, an ye ɛkidizɔni (E) ni 20E sɔrɔ nin farikolo yɔrɔ in na, o min ye o sɛmɛntiya ka tɛmɛ result.Nka, sɛgɛsɛgɛli kɛra ka caya ni stɛroyi fosifɔrilenw ye minnu bɛ oksidan sɔrɔ, minnu bɛ bɛn formula 3-dehydro-20E-22-phosphate (3D20E22P)12 ma (Ja 1).A suguya wɛrɛw ye 3-dehydro-20E (3D20E) ani 20E-22-phosphate (20E22P) ye.The HPLC-MS/MS signality of 3D20E22P tun ka bon ni a cogoya defosforilasi ye, n’o ye 3D20E ye, ani hakɛ saba tun ka bon ni E ni 20E ta ye (Ja 1).Hali ni farikolo yɔrɔ wɛrɛw la ani bangekɔlɔsi fɛɛrɛ duguma (LRT; Extended Data Fig. 1a).An ye ecdys fana sɛgɛsɛgɛ cɛw la minnu datugulen don kokura (<1 tile 1) ani musow ni 3D20E ni 3D20E22P sɔrɔlenw MAG dɔrɔn de kɔnɔ; E, 20E ani 20E22P tun bɛ cɛ ni muso fila bɛɛ la (Kunnafoni janyalenw ja 1b).Nin kunnafoniw b’a jira ko A. gambiae cɛkɔrɔba balikuw bɛ ɔrimɔni caman caman dilan u ka MAGw kɔnɔ minnu tɛ dilan musow fɛ.
MAG ni muso LRT (minnu kɔnɔ, atria, seminal vesicles, ani parovarium) tigɛra ka bɔ cɛ npogotigi tile 4 (tile 4) la ani muso npogotigininw ni muso furulenw na (0,5, 3, ani 12 hpm).Ecdysone min bɛ o farikolo yɔrɔw la, o sɛgɛsɛgɛra ni HPLC-MS/MS ye (kɛrɛnkɛrɛnnenya la ± sem; t-test unpaired, fan fila, nkalon sɔrɔli hakɛ (FDR) latilenna, a tɛ nafa sɔrɔ 3D20E: sanga 3 ni sanga 0,035, P = 0,0015; Lɛrɛ 3 ni sanga 0,5, P = 0,25;lɛrɛ 12 ni sanga 3, P = 0,0032;Lɛrɛ 12 ni sanga 0,5, P = 0,015).Dɔnniyaw bɔra biologique replicates saba la.Ecdysone kelen-kelen bɛɛ ka nafa bonya yɔrɔ jatera ka normalize ni soso hakɛ ye.Ecdysone bɛ jira ni kulɛri ye nin cogo la: E, green 20E, oranjɛ ye; 20E22P, ɲɛ jɛman; 3D20E, bulu ye; 3D20E22P, pink.Inset bɛ dɔ fara sɛgɛsɛgɛli kan y-axis kan walasa ka ecdysone hakɛ dɔgɔyali jira.
Walasa ka sɛgɛsɛgɛli kɛ ni 3D20E22P ni 3D20E bɛ wuli ka taa ɲɔgɔn fɛ furuɲɔgɔnma waati, an ye muso LRTw tigɛtigɛ waati caman na furuɲɔgɔnma kɔfɛ.Hali ni ɛkidizɔni ma sɔrɔ npogotigininw na, an ye 3D20E22P hakɛ caman kɔlɔsi LRT kɔnɔ furuɲɔgɔnma kɔfɛ dɔrɔn (0,5 h furuɲɔgɔnma kɔfɛ, hpm), o bɛ dɔgɔya waati kɔnɔ, k’a sɔrɔ 3D20E hakɛ cayara kosɛbɛ (Ja 1).An kɛlen ka baara kɛ ni 3D20E ye min dilannen don ni kemikɛli ye i n’a fɔ sariya, an y’a ye ko nin ɔrimɔni stɛroyi hakɛ min bɛ furuɲɔgɔnma LRTw la, o hakɛ ka ca ni 20E ye a dɔgɔyalenba la siɲɛ 100 (Jaabi janyalenw Tablo 1).O cogo la, 3D20E22P ye cɛ ka ɛkdisɔnba ye min bɛ wuli ka taa muso LRT la furuɲɔgɔnma waati, ani a cogoya min tɛ fosifori la, n’o ye 3D20E ye, o bɛ caya kosɛbɛ furuɲɔgɔnma kɔfɛ dɔɔnin.O b’a jira ko jɔyɔrɔba bɛ ɛkidizɔni laban in na muso ka furuɲɔgɔnma kɔfɛ ɲɛnamayako la.
ARN sinsinni (ARN-seq) kunnafonidilan kura bɔlen kɔfɛ (Ja 2a), ka baara kɛ ni biyoinformatiki pipeline ye min dilannen don ka kɛɲɛ ni mɔgɔw sago ye, an ye ecdysone kinase (EcK), ecdysone oxidase (EO) ɲini, ani ecdysone encoding 20E-modified phosphatase gene.EPP) bɛ jira bangekɔlɔsi fɛɛrɛw la.An ye EPP jamu kandida kelen ni fila dɔn EcK jamu minnu bɛ se ka kɛ (EcK1 ani EcK2), nka u ma se ka EO jamu kandida ɲuman sɔrɔ.Kɛrɛnkɛrɛnnenya la, EPP jamu kelen-kelen bɛɛ jirala hakɛ caman na (kɛmɛsarada la 98,9nan) Gambia MAGw la nka a ma jira muso LRTw la (Ja 2b), o bɛ danfara don an jigi la kabini 3D20E22P defosforilasi kɛra muso farikolo yɔrɔ in na.O de kama, an dalen b’a la ko cɛ EPP bɛ se ka wuli furuɲɔgɔnma waati.Tiɲɛ na, an ye in vivo stable isotope labeling kɛ ka muso ka farikolojɔlifɛn datugu furuɲɔgɔnma kɔfɛ, o ye anzimu ye min dɔnna MS fɛ muso ka kɔnɔbara la (Ja 2c ani Tablo Supplémentaire 1).EPP sɔrɔli MAG kɔnɔ ani muso LRT furulen (nka a tɛ npogotigi ye) fana dafara ni farikolojɔlifɛn kɛrɛnkɛrɛnnenw ye (Ja 2d).
a, Bioinformatique pipeline min dilannen don ka kɛɲɛ ni mɔgɔw sago ye walasa ka cɛ ni muso kelen-kelen bɛɛ ka bangekɔlɔsi fɛɛrɛw ɲini walasa ka EcKw, EOw ani EPPw kofɔ.Jatew minnu bɛ jiribolo kɛrɛfɛ, olu bɛ cɛ ni muso kandidaw hakɛ jira sen kelen-kelen bɛɛ la.Nin sɛgɛsɛgɛli in ye EPP jamu kelen (EPP) ani EcK jamu kelen (EcK1) jira minnu bɛ jira cɛw la, ani EcK jamu kelen (EcK2) min bɛ jira cɛ ni muso fila bɛɛ la nka a tɛ EO jamu kandida di.b, Heatmap min bɛ jamu kandida jirali suma npogotiginin (V) ni furuɲɔgɔnma (M) Anopheles gambiae ni Anopheles albicans tissues.Spca, fertilization MAGw, glandes accessoires cɛw la; farikolo yɔrɔ wɛrɛw, i n’a fɔ sinw, wuluw, senw, farikolo tulumaw, ani kɔnɔnafɛnw cɛ ni muso fila bɛɛ la, ani kɔnɔbara basigilenw musow la.EcK2 bɛ sɔrɔ kosɛbɛ MAG ni Gambia atriw fila bɛɛ la, k’a sɔrɔ EPP bɛ sɔrɔ MAG dɔrɔn de la.c, Proteomic analysis of male ejaculate group translocation into female atria at 3, 12 and 24 hpm, min bɛ farikolojɔlifɛn 67 jira minnu ka ca kosɛbɛ.Musow lamɔna dumuni na min kɔnɔ 15N bɛ sɔrɔ walasa ka farikolojɔli dumuniw bɛɛ taamasiyɛn (ani ka u datugu).Cɛ minnu ma taamasiyɛn kɛ, olu farala ɲɔgɔn kan ni musow ye minnu taamasiyɛn kɛra, ani musow ka LRTw tigɛra ni 3, 12 ani 24 hpm ye walasa ka farikolojɔlifɛnw sɛgɛsɛgɛ (aw ye Tablo 1 lajɛ walasa ka cɛya furakisɛw lisɛli dafalen sɔrɔ).Inset, EPP, Eck1 ani EcK2 sɔrɔla cɛ npogotigininw ka MAG la ni o farikolo yɔrɔw sɛgɛsɛgɛli ye ni proteomiki ye.d, EPP sɔrɔla ni western blot ye muso furulenw ka MAG ni LRT la, nka a ma sɔrɔ muso npogotigininw na walima cɛw la walima muso farikolo tɔ la.Membranes tun bɛ sɛgɛsɛgɛli kɛ waati kelen na ni anti-actin (loading control) ani anti-EPP anticorps ye.Cɛ bɛɛ ye npogotigininw ye.See Jaa 1 faralen ɲɔgɔn kan ka ɲɛsin jeli sɔrɔyɔrɔ kunnafoniw ma.Western blots kɛra siɲɛ fila ni jaabi kelenw ye.
EPP ka ecdysteroid phosphatase baara sɛgɛsɛgɛra HPLC-MS/MS fɛ ni 3D20E22P ye min bɔra MAG la (Extended Data Fig. 2a).Ka fara o kan, n’an ye EPP da tugu ni ARN-mediated interference (RNAi) ye, an ye fosifatazi baara dɔgɔya kosɛbɛ nin cɛw (Ja 3a), ani musomanninw ka bangekɔlɔsi fɛɛrɛw la mated with EPP-silenced males showed significant A lower proportion of dephosphorylated 3D20E (Fig. 3b) despite partial gene silencing (Extended Data Fig. 2b,c).O ni ɲɔgɔn cɛ, an ma fɛn caman ye 20E22P/20E hakɛ la soso kelenw na, o bɛ se k’a jira ko anzimu in kɛrɛnkɛrɛnnen don 3D20E22P la (Ja 3b).
a, Fosfatazi baara dɔgɔyali MAG kɔnɔ min bɛ sɔrɔ EPP dabila fɛ ni EPP ARN fila-fila (dsEPP) walima GFP ARN fila-fila (dsGFP) kɔlɔsilikɛlaw ye.MAG pooli mugan kɛra baarakɛminɛn kelen-kelen bɛɛ la (P = 0,0046, t-test fila-fila, fan fila), o jiralen don ni dots danfaralenw ye.b, Muso minnu furula ni EPP-silenced cɛw ye, olu tun bɛ ni a 3D20E defosforilasi hakɛ dɔgɔyara kosɛbɛ 3 hpm la (P = 0,0043, t-test unpaired, two-sided), k’a sɔrɔ 20E hakɛw ma nɔ bila o la (P = 0,063, unpaired). t-test, two-sided).Data are presented as mean ± sem from three pools of 13, 16 and 19 women each.c, Muso minnu farala ɲɔgɔn kan ni EPP-silenced cɛw ye, olu ka segin-ka-bɔnye hakɛ tun ka ca kosɛbɛ (P = 0,0002, Fisher’s exact test, two-sided).Musow tun wajibiyalen don fɔlɔ ka furuɲɔgɔnya kɛ walasa k’u ka furusiri cogoya dɔn Tile 2 o kɔfɛ, u ye ɲɔgɔn sɔrɔ ni cɛ wɛrɛw ye minnu bɛ ni cɛya jiginni ye walasa ka furuɲɔgɔnya seginni hakɛ jateminɛ ni jɛnɛya caman sɔrɔli ye PCR fɛ.d, Muso minnu balola joli la, minnu ni cɛ minnu ye EPP makun, olu ka bange hakɛ dɔgɔyara kosɛbɛ (P < 0.0001; Mann-Whitney ka sɛgɛsɛgɛli, fan fila) ani kɔnɔ hakɛ dɔgɔyara dɔɔni (P = 0,088, Mann-Whitney ka sɛgɛsɛgɛli, fan fila) , k’a sɔrɔ den sɔrɔli hakɛ ma nɔ bila (P = 0,94, Fisher ka sɛgɛsɛgɛli tigitigi, fan fila).Panɛli bɛɛ kɔnɔ, n bɛ sosow tacogo hakɛ jira.NS, a tɛ nafa sɔrɔ.*P < 0,05, **P < 0,01, ***P < 0,001, ****P < 0,001 ye.
O kɔfɛ, an ye jateminɛ kɛ ni ɛkidizɔni defosforilasi nafa ka bon walasa ka furuɲɔgɔnya kunbɛnni bila musow la.Kɛrɛnkɛrɛnnenya la, muso minnu ye ɲɔgɔn sɔrɔ ni cɛ minnu ye EPP dɔgɔya, olu seginna ka ɲɔgɔn sɔrɔ siɲɛ caman na (44,9%) ka tɛmɛ muso kɔrɔlenw kan (10,4%) ni u kɛra cɛ wɛrɛw la (Ja 3c).An ye bangekɔlɔsi dɔgɔyaba fana kɔlɔsi (Ja 3c).An ye den sɔrɔli dɔgɔya kosɛbɛ fana (Ja 3c). 3d, numan fɛ) ani dɔ bɔ dɔɔnin o musow ka kɔnɔw da la (Ja 3d, cɛmancɛ), k’a sɔrɔ musow ye kɔnɔ minnu da kɛmɛsarada la (jaabi wɛrɛ min bɛ sɔrɔ musow la ni furuɲɔgɔnya ye )) ma nɔ bila o la (Ja 3d, kinin fɛ).Ni an y’a jateminɛ ko EPP ka kɛrɛnkɛrɛnnenya kɔlɔsira 3D20E22P kama, nin jaabi ninnu b’a jira ko 3D20E min bɛ kɛ EPP fɛ min bɛ wuli furuɲɔgɔnma waati la, o bɛ se ka jɔyɔrɔba ta muso ka sɔnni dabila la ka taa furuɲɔgɔnya wɛrɛw la, o kɛcogo min tun bɛ tali kɛ cɛnimusoya jiginni na fɔlɔ 20E .O la, nin cɛ kɛrɛnkɛrɛnnen in ɔrimɔni in fana bɛ nɔba bila muso ka den sɔrɔli la.
O kɔfɛ, an ye 20E ni 3D20E ka baarakɛcogo suma pikiri kɛcogo la npogotiginin minnu kɔgɔlen don cɛnimusoya siratigɛ la ni 3D20E ye min dilannen don ni kemikɛli ye (Ja 4a–c) ani 20E min bɛ sɔrɔ jago la.An y’a kɔlɔsi ko 3D20E tun bɛ se kosɛbɛ ka tɛmɛ 20E kan musow ka ɲɔgɔn sɔrɔli dabila la a hakɛ fila bɛɛ la (Ja 4d).A kɔlɔsira ko a tilancɛ 3D20E hakɛ farikolo ta fan fɛ LRT la (pg 1066 pikiri kɔfɛ ni pg 2022 ye furuɲɔgɔnma kɔfɛ) ye musomanninw hakɛ dɔ bila min tɛ se ka fari faga, o hakɛ tun ka ca ni 20E hakɛ ye siɲɛ 20 ka tɛmɛ 20E hakɛ kan (pg 361 pikiri kɔfɛ) sanga 24 pikiri kɛlen kɔfɛ ni a hakɛ cayalenba ye 18 pg furulen kɔfɛ Extended Data Table 1).Nin jaabi in bɛ bɛn hakilina ma ko 20E ka cɛnimusoya jiginni tɛ kɛ sababu ye ka furusiri waatiw kɛ minnu tɛ se ka furu, wa a b’a jira ka t’a fɛ ko 3D20E ye fɛnba ye min bɛ bangebagaw ni denw cɛsira sabati.3D20E fana tun bɛ baara kɛ kosɛbɛ ka tɛmɛ 20E kan kɔnɔsogonin sɛgɛsɛgɛliw la muso npogotigininw na (Ja 4e), o b’a jira ko kɔnɔw bɔli hakɛ bɛnnen don an ma min kɔlɔsira EPP damatɛmɛni yɔrɔ dɔ kɔfɛ, o sababu bɔra 3D20E baara tolen sɔrɔli la min bɛ sɔrɔ hali bi musow ka fɛnw fɛ minnu bɛ sɔrɔ Mating fɛ.
(a,b) 3D20E min dilannen don kemikɛli fɛ ka bɔ 20E la (a) ni fɛn caman tigɛli/nafa ka bon kosɛbɛ (kunnafoni minnu jirala i n’a fɔ cɛmancɛ ± sem ka bɔ sintezi yɛrɛmahɔrɔnya wale saba la) (b).c, Mass spectrum (jukɔrɔ tilancɛ) bɛ bɛn tigitigi ecdysone ma min bɛ sɔrɔ LRT muso furulen na (sanfɛ tilancɛ).d, Ni i ye a suma ni 20E ye (0,63 μg, P = 0,02;0,21 μg, P < 0,0001; (0,63 μg, P = 0,0002; 0,21 μg, P = 0,54; Fisher ka sɛgɛsɛgɛli tigitigi, fan 2).e, 3D20E pikiri ye den sɔrɔli hakɛ caya kosɛbɛ muso npogotigininw na ka tɛmɛ 10% etanɔli kunbɛnbagaw kan (0,21 μg, P < 0,0001; 0,13 μg, P = 0,0003 ; a tata hakɛ ka ca (0,21 μg, P = 0,0019; 0,13 μg, P = 0,075; Fisher ka sɛgɛsɛgɛli tigitigi, fan fila).Panɛli bɛɛ kɔnɔ, n bɛ sosow tacogo hakɛ jira.NS, o tɛ nafama ye.*P < 0,05, **P < 0,01, ***P < 0,001, ****P < 0.001.Dɔnniyaw bɛ bɔ ɲɔgɔndan saba la.
Kalan tɛmɛnenw na, an y’a jira ko cɛnimusoya hormones stéroïdes cili bɛ MISO (Mating-Induced Stimulator of Oogenesis 11) jirali bila mɔgɔ la, o ye muso ka bangekɔlɔsi jamu ye min bɛ A. gambiae musow tanga P. falciparum banakisɛ ma Kɛnɛya musaka minnu bɛ sɔrɔ 13 fɛ, o ye hadamadenw ka sumayabana banakisɛ ye min bɛ mɔgɔ faga kosɛbɛ.Ni MISO nafa ka bon Anopheles ka bangeko seko la sumayabana bɛ yɔrɔ minnu na, an y’a latigɛ k’a dɔn ɔrimɔni 3D20E walima 20E jumɛn bɛ nin jamu in jirali daminɛ.An y’a ye ko hali ni 20E pikiri ye kɛrɛnkɛrɛnnenya la walima ni fanga ye ka tɛmɛ o kan, o ye nukilila ɔrimɔni minɛbaga (HR) dɔw lawuli, i n’a fɔ HR3 ani HR4, ani duguma stɛroyi laɲinitaw, i n’a fɔ jirisunw Vg14, 15, 16, MISO tun ka ca strongly was induced by 3D20E (Extended Data Fig. 3).O cogo la, a bɛ i n’a fɔ nin andorojɛni stɛroyi ɔrimɔni in ka cɛnimusoya jiginni bɛ fɛɛrɛw bila minnu bɛ musow tanga musakaw ma minnu bɛ sɔrɔ paraziti banakisɛ fɛ.Ka fara o kan, 3D20E bɛ nɔ bila E minɛbaga EcR ka isofɔmu fila bɛɛ la cogo danfara la, ka EcR-A bila sen kan ani ka a damatɛmɛ EcR-B, ani ka fanga sɔrɔ ka furuɲɔgɔnya-minɛn wɛrɛw lawuli, HPX15 fana sen bɛ o la, o min bɛ nɔ bila muso ka bangeko la.O bɛ se ka bangebaliya nafama ɲɛfɔ min kɔlɔsira musow la minnu furula ni cɛw ye minnu ma kuma EPP fɛ (Dɔnniya janyalenw Jaa 3).O kunnafoniw b’a jira ko jiginɛ siraw bɛ yen minnu ka fisa ka baara kɛ ni ecdysone hormones fila ye minnu bɛ se ka kɛ cɛnimusoya kɛrɛnkɛrɛnnen baara sababu ye.
O kɔfɛ, an ye EcK jamu fila minnu jirala an ka biyoinfɔrmatiki pibiliki kɔnɔ, olu baarakɛcogo kɔrɔbɔ.EcK1 walima EcK2 dabilali kɛra sababu ye ka mɔgɔ caman faga cɛw la (Extended Data Fig. 4a), o b’a jira ko ecdysone phosphorylation, ani o cogo la, baarakɛbaliya, nafa ka bon ɲɛnamaya la.Sabula EcK2 jirala hakɛ caman na ka tɛmɛ EcK1 kan, wa a sɔrɔla MAGw kɔnɔ proteomics (Ja 2b,c ani Tablo Supplémentaire 2), an y’a ka ecdysteroid kinase baara sinsin ni a bilali ye ni 20E ye, o kɛra sababu ye ka fosforilasi 20E22P sɔrɔ (Extended Data Figure 2).4b).Ni an tun bɛ baara kɛ ni 3D20E ye i n’a fɔ substrat, an ma se ka fosforilasi sɔrɔlen 3D20E22P sɔrɔ (Extended Data Fig. 4c), o b’a jira ko 20E sanni ka kɛ 3D20E ye, o bɛ se ka kɛ EcK2 ka laɲini ɲuman ye.
Ka kɛɲɛ n’an ka ARN-seq sɛgɛsɛgɛli ye, EcK2 fana tun bɛ jira kosɛbɛ muso npogotigininw ka LRT la, a tun bɛ faga yɔrɔ min na furuɲɔgɔnma kɔfɛ (Ja 2b).An ye nin kunnafoniw sinsin ani k’a jira ko EcK2 jirali ma nɔ bila joli baloli la (Dɔnniya janyalen Jaa 5a).An ye an ka MS kɛlɛli fɔlɔw janya, an y’a ye ko 20E22P kuncɛyɔrɔ ni a kuncɛyɔrɔ bɛ tali kɛ ɲɔgɔn na kosɛbɛ ka 20E (lɛrɛ 22-26 joli dumuni kɔfɛ; Kunnafoni janyalenw Jaa 5b).EcK2 makunni muso npogotigininw na, o kɛra sababu ye ka 20E ni 20E22P hakɛ danfaralen caya siɲɛ 3 lɛrɛ 26 joli dumuni kɔfɛ (Dɔnniya janyalenw ja 2c ani 5c), o b’a jira ko EcK2 fana bɛ fosforilasi 20E in musow.Kɛrɛnkɛrɛnnenya la, npogotiginin minnu dɔgɔyara EcK2 la, olu ye cɛnimusoya jaabi dafalen mara (Extended Data Fig. 5d,e), o b’a jira ka t’a fɛ ko musow ka 20E sɔrɔli tɛ furusiri waatiw bila u la.Nka, o muso ninnu tun ye kɔnɔtiɲɛ hakɛ caya kosɛbɛ ni u ni kɔrɔsilikɛlaw ta kɛra ɲɔgɔn fɛ, ni npogotigininw ye kɔnɔw da ka tɛmɛ 30% kan (Extended Data Fig. 5f).Ni Eck2 ARN fila-fila (dsEcK2) pikiriw kɛra joli dilen kɔfɛ, den sɔrɔli ma kɛ, o waati la, 20E hakɛ min bɛ sɔrɔ joli dunni fɛ, o tun dɔgɔyara.Kuma bɛɛ la, nin jaabi ninnu bɛ misali dɔ sinsin min bɛ se ka kɛ sababu ye ka 20E sɔrɔ joli minni kɔfɛ, o bɛ se ka den sɔrɔli bila mɔgɔ la, nka ni den sɔrɔli bloki (EcK2 ani a bɛ se ka kɛ ko fɛn wɛrɛw) jiginna dɔrɔn off by mating.20E ni 3D20E pikiriw ma EcK2 jirali bali npogotigininw na (Extended Data Fig. 5g), o b’a jira ko fɛn wɛrɛw bɛ kɛ sababu ye ka nin kinase in balili kɛ.Nka, 20E hakɛ joli dili kɔfɛ, o tun tɛ se ka furuɲɔgɔnya dusukasi bila mɔgɔ la, nka a daminɛna ka ɲɛ ni 3D20E minnu jiginna cɛnimusoya siratigɛ la, olu hakɛ cayalenba fɛ.
An ka jaabiw bɛ hakilina nafamaw di A. gambiae ka bangeko ɲɛtaa ɲɛnabɔcogo fɛɛrɛw kan.Modèle dɔ bɔra kɛnɛ kan, cɛw ye 3D20E hakɛ caman dilan yɔrɔ min na, o ye ɛkdizɔni camanba ye min bɛ se ka kɛ cɛ kɛrɛnkɛrɛnnenw ye, n’o bɛ bangebagaw hakili sigi ni musomanninw tɛ u ka ɲɔgɔn sɔrɔ ka taa a fɛ.O waati kelen na, o sumayabana fɛnɲɛnamafagalan ninnu fana ye fɛɛrɛ ɲuman dɔ labɛn walasa ka se ka baara kɛ 3D20E musow la ka jaabi di cɛw ka EPP kɛrɛnkɛrɛnnen ka cɛnimusoya jiginni ma.An ka dɔnniya la, nin ye misali fɔlɔ ye cɛ ni muso ka stɛroyi ɔrimɔni sigida la min bɛ baara kɛrɛnkɛrɛnnen ni kɔrɔlen kɛ fɛnɲɛnamaw la.Cɛ kɛrɛnkɛrɛnnen ecdysone baarakɛcogo jateminɛna nka a ma jira k’a jɛya.Misali la, hakilina 18 min tɛ tiɲɛ kosɛbɛ, o ye ko nin baara ninnu bɛ se ka kɛ ni 20E precursor E1.A dɔnna kosɛbɛ ko Drosophila la, monandry bɛ daminɛ ni cɛnimusoya pepitiri misɛnninw jiginni ye19,20 minnu bɛ ɲɔgɔn sɔrɔ ni senfagabana ye minnu bɛ muso ka bangekɔlɔsi fɛɛrɛw sɔgɔ cɛnimusoya pepitiri sɔrɔyɔrɔ kɛrɛnkɛrɛnnenw fɛ21,22.Baara wɛrɛw ka kan ka kɛ walasa ka jiginɛ taamasiɲɛw ka cakɛdaw dɔn minnu bɛ kɔlɔsi 3D20E fɛ A. gambiae musomanninw na ani k’a dɔn ni o jirisunw bɛ se ka mara sosow ni Drosophila cɛ.
Ni an y’a jateminɛ ko 3D20E jɔyɔrɔ ka bon musow ka bangeko ni u ka kɛwalew la minnu jirala an ka kalan kɔnɔ, sira minnu bɛ taa ni 3D20E labɛnni ni a baarakɛcogo ye, olu bɛ cogo kura di sosow kɛlɛli fɛɛrɛ nataw ma, i n’a fɔ cɛ sterili minnu bɛ se ka ɲɔgɔn sɔrɔ fɛnɲɛnamafagalanw ka fɛɛrɛbɔ fɛɛrɛw la minnu bɛ se ka ɲɔgɔn sɔrɔ Baara kɛ ni kungo kɔnɔ bɔli ye walima ka 3D20E ladege npogotiginin tulonkɛ la.The 3D20E ka baara kɛrɛnkɛrɛnnen bɛ se ka kɛ cɛya la, n’a sɔrɔla A. gambiae ni Cellia suguya wɛrɛw ye se sɔrɔ k’u ka cɛya sɔgɔ ka kɛ furuɲɔgɔnmaw ye, bawo o bɛ kɛ sababu ye ka ɔrimɔni ni ɔrimɔni-walekɛlan caman cili kɛ ka ɲɛ bangekɔlɔsi seko ni dɔnko sumayabana ka ca yɔrɔ minnu na, o bɛ kɛ sababu ye ka Plasmodium jɛnsɛnni kɛ cogo la min tɛ ɲɛ.Ka da a kan a jirala ko muso 20E bɛ nɔba bila P. falciparum ka ɲɛnamaya n’a bonya la Anopheles soso musomanw na,24 cɛ ni muso fila bɛɛ ka stɛroyi ɔrimɔni siraw ye sisan soso ni paraziti cɛsira jɔnjɔnw ye.
A. gambiae G3 suguyaw lamɔna fɛnɲɛnamaw cogoya jɔnjɔnw na (26-28 °C, 65-80% nɛnɛ hakɛ, 12:12 h yeelen/dibi fotota waati).Larvaw balola ni jɛgɛ dumuni mugulen ye (TetraMin Tropical Flakes, Koi Pellets ani Tetra Pond Sticks ni hakɛ ye 7:7:2 ye).Mɔgɔkɔrɔba sosow tun bɛ balo ad libitum 10% dextrose solution ani dɔgɔkun o dɔgɔkun hadamaden joli (kalan joli yɔrɔw).Sogo npogotigininw sɔrɔla ni cɛya ni musoya danfara ye pupal stage la, u labanw sɛgɛsɛgɛli kɔfɛ ni mikroskɔpi ye.Cɛ minnu bɛ DsRed transgene ta, olu ɲɛfɔra ka tɛmɛ.
Kɔrɔbɔli fangamaw kɛra ka kɛɲɛ ni sariyaw ye minnu ɲɛfɔra ka tɛmɛ.Jɛɲɔgɔnya dacogo la, muso npogotiginin minnu si bɛ tile 4 la, olu tun bɛ mara ni 1:3 ye ni npogotigi npogotigininw ye minnu kɔgɔlen don cɛnimusoya siratigɛ la su fila kɔnɔ.Kɔrɔbɔliw la, cɛw pikiri kɛra ni dsEPP ye, co-caging bɛ bɛn tile 3-4 pikiri kɔfɛ, waati min na fosifatazi baara tun bɛ dabila kosɛbɛ (A janyalen don Donanw Jaa 2b).
Sosow farikolo yɔrɔw, su tɔw (farikolo tɔ), walima farikolo bɛɛ tigɛra ka kɛ mɛtanɔli 100% ye ka u kɛ kelen ye ni biɲɛ ye (milimɛtɛrɛ 2 gilasi biɲɛw, 2400 rpm, sec 90).Fura hakɛw ni mɛtanɔli hakɛ tun ye nin ye: farikolo tɔ, 50 μl 1000 kɔnɔ MAG, 50–100 80 μl; muso LRT, 25–50 80 μl.Ja jiginɛ kɛra mɛtanɔli bɔli filanan na ni mɛtanɔli hakɛ kelen ye.Selilu nɔgɔw bɔra ni santirifugare ye.Metanɔli min bɔra o bɔlen fila bɛɛ la, o farala ɲɔgɔn kan ka ja azote jibɔ kɔnɔ, o kɔfɛ, a bɛ segin ka kɛ nin hakɛ ninnu na, mɛtanɔli 80% ji la: farikolo tɔ, μl ( μl) ; MAGw ni muso LRT, 30 μl.
Sɛgɛsɛgɛli kɛra fɛnɲɛnamafagalanw kan (ID-X, Thermo Fisher) kan min sirilen don LC minɛn dɔ la (Vanquish, Thermo Fisher).Sɛbɛnni 5 μl pikiri kɛra 3 μm, 100 × 4,6 mm koloma kan (Inspire C8, Dikma) min maralen bɛ 25 °C la.LC ka fasa mobili tun ye A (ji, 0,1%) ye (ji, 0,1%). asidi foromiki) ani B (asetonitirili, asidi foromiki 0,1%).LC gradient tun ye nin ye: 5% B miniti 1 kɔnɔ, o kɔfɛ, a cayara ka se 100% B ma miniti 11 kɔnɔ.Minuti 8 kɔfɛ 100% kɔnɔ, aw bɛ segin ka kolotuguda balansi 5% B la miniti 4 kɔnɔ.A jibɔ hakɛ tun ye mililitiri 0,3 ye min-1.Ionization in the MS source is o bɛ kɛ ni ionizasiyɔn ye min bɛ kɛ ni kuran fiyɛli ye ni funteni ye cogo ɲuman ni cogo jugu la.
Mas spectromètre bɛ kunnafoniw suman m/z hakɛ la k’a ta 350 na ka se 680 ma ni 60.000 ye MS cogo dafalen na.MS/MS kunnafoniw sɔrɔla [M + H]+ (laɲini bɛɛ), [M - H2O + H]+ (laɲini bɛɛ), ani [M - H]- (laɲini fosifɔrilen) kan.MS/MS kunnafoniw kɛra ka laɲiniw ka ecdysone cogoyaw dafa, sariya tɛ minnu na tun bɛ sɔrɔ.Walisa ka ɛkidisitɛriw dɔn minnu ma laɲini, MS/MS kunnafoniw sɛgɛsɛgɛra HPLC kuncɛw bɛɛ kan minnu ka caya danfaralen >15%.Jateminɛ kɛ ni kurufe jɔnjɔnw ye minnu dabɔra ka bɔ sariya saniyalenw na (20E, 3D20E) walasa ka hakɛ dafalenw walima dilutions jateminɛ sample kɛrɛnkɛrɛnnen kelen na (laɲini tɔw bɛɛ) walasa k’u bɛn hakɛw jateminɛ minnu sɔrɔla cɛ kelen kɔnɔ.For 3D20E, hakɛ jateminɛ kɛra ni nin adduct ninnu faralen ye ɲɔgɔn kan: [M + TFA]-, [M + COOH]-, [M + Na]+, [M + Cl]-, [M + NO3]-.Dɔnniyaw bɔra ka hakɛ jateminɛ ni Tracefinder ye (version 4.1).MS/MS kunnafoniw sɛgɛsɛgɛra ni Xcalibur ye (version 4.4).The MS spectra of E, 20E ani 3D20E sumalen don ni sariyaw ye.3D20E22P sɛgɛsɛgɛra ni derivatization ye ni Girard ka reagent ye.20E22P sɛgɛsɛgɛra ni m/z ratio ye.
3D20E22P saniyalen don ka bɔ MAG la.Saniya kɛra sɛgɛsɛgɛli hakɛ la ni ultra-performance liquid chromatograph (Acquity, Waters) ye ni quadrupole mass-based detector (QDa, Acquity, Waters) ye LC cogoya kelen na ni HPLC-MS/MS sɛgɛsɛgɛli ye.Fraction collection daminɛna ni m/z bɛ bɛn 3D20E22P ma tun bɛ sɔrɔ a mara waati kelen na i n’a fɔ a tun latigɛra cogo min na ka tɛmɛ.O kɔfɛ, furakisɛ minnu bɔra, olu saniyacogo lajɛra ni HPLC-MS/MS ye i n’a fɔ a ɲɛfɔlen don cogo min na sanfɛ.
ARN bɛɛ lajɛlen bɔra bangekɔlɔsi fɛɛrɛ 10-12 la walima farikolo yɔrɔ wɛrɛw la (kunkolo tɛ minnu na) ni TRI reagent (Thermo Fisher) ye ka kɛɲɛ ni a dilabaga ka laadilikanw ye.ARN furakɛra ni TURBO DNase ye (Thermo Fisher).cDNA kɛra ni Moloney murine leukemia virus reverse transcriptase (M-MLV RT; Thermo Fisher) ye o kɔfɛ manufacturer’s instructions.Primers for reverse transcription quantitative PCR (RT-qPCR; Extended Data Table 2) tun bɔra ka tɛmɛ24 walima u dabɔra ni Primer-BLAST26 ye, ni ɲɛnatɔmɔli ye fɛnw ye minnu bonya ye 70-150 bp ye ani minnu bɛ ɛkzɔn ni ɛkzɔn ɲɔgɔndanw cɛ walima Primer pair primers bɛ ɛkzɔnw danfara.cDNA misaliw ka bɔ biologique replica saba la ka se naani ma tun bɛ dilan siɲɛ naani ji la RT-qPCR kama.Jateminɛ kɛra 15 μl replicate reactions kɔnɔ minnu kɔnɔ 1× PowerUp SYBR Green Master Mix (Thermo Fisher), primers, ani 5 μl diluted cDNA.Reactions kɛra QuantStudio 6 Pro real-time PCR system (Thermo Fisher) kan ani kunnafoniw lajɛra ka sɛgɛsɛgɛli kɛ ni Design ani Sɛgɛsɛgɛli (version 2.4.3).I n’a fɔ a jiralen bɛ cogo min na nin kalan in kɔnɔ, hakɛ danfaralenw kɛra normal ye ka kɛɲɛ ni ribosomal gène RpL19 (AGAP004422) ye, min jiracogo ma Changé kosɛbɛ ni joli baloli ye 27 walima ni furuɲɔgɔnma
ARN jogo sɛgɛsɛgɛra ni Agilent Bioanalyzer 2100 Bioanalyzer (Agilent) ye.Illumina paired-end libraries labɛnna ka boli Broad Institute of MIT ani Harvard la.Sequencing kalanw tun bɛ bɛn A. gambiae genome (PEST strain, version 4.12) ma ni HISAT2 (version 2.0.5) ye ni paramètres defaults ye.Reads with mapping quality (MAPQ) jatedenw <30 bɔra ni Samtools ye (version 1.3.1).Kalan hakɛ min kartilen don jeninida la, o jate kɛra ni htseq-count (version 0.9.1) ye ni paramètres defaults ye.Kalan hakɛ normalizelenw jatera ani ka jeninida jiracogo danfaralenw sɛgɛsɛgɛ ni DESeq2 pake ye (version 1.28.1) in R (version 4.0.3).
Ecdysone-modifying gene kandidaw dɔnna ni A. gambiae genome ɲinini fɔlɔ ye ni PSI-BLAST algorithm ye (https://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/2.8.1/), ka baara kɛ ni default values ​​ye Parameters with the following query protein sequences: from Bombyx mori (Accession No. NP_001038956.1), Musca domestica (Jɛkulu No. XP_005182020.1, XP_005175332.1 ani XP_011294434.1) ani Microplitis demolitor (Don No. XP_0052646,1 ani XP_008552645.1) EcK min bɛ bɔ B. mori (A donna No. NP_001036900), Drosophila melanogaster (Accession No. NP_651202), Apis mellifera (Don No. XP_394838) ani Acyrthosiphon pisum (Don No. XP_001947166) ani EPP min bɛ bɔ B. mori la (Accession No. XP_001947166) NP_001177919.1 ani NP_001243996.1) ani EO ka bɔ D. melanogaster la (Accession No. NP_572986.1) (taamasenɲɛ 1).O kɔfɛ, filɛri bɛ gosi ka da ARNm jiracogo caman kan (>100 fragments/kilobase exons per million mapped reads (FPKM) walima >85%) bangekɔlɔsi fɛɛrɛw la (muso LRT walima MAG) Gambia (taamasenɲɛ 2nan ). Walasa ka kɛrɛnkɛrɛnnenya ɲɛ, an ye anzimu kandidaw sugandi minnu fana bɛ jira A. albimanus ka bangekɔlɔsi fɛɛrɛw la, anopheles suguya min tɛ ecdysone dilan walima k’a bila a la furuɲɔgɔnma waati.Kandidati ka jamu filɛra ka da a jiracogo dɔgɔya kan (<100 FPKM walima <85th percentile) A. albimanus bangeyɔrɔ la (taamasen 3).As a final filter (step 4), kandida jamu ka kan ka ninnu dɔ wasa a dɔgɔyalenba la : (1) u bɛ wuli kosɛbɛ furuɲɔgɔnma kɔfɛ (P < 0,05) ka kɛɲɛ ni jamu minnu jiralen don danfara la, olu sɛgɛsɛgɛli ye ani (2) farikolo yɔrɔw la minnu tɛ bangekɔlɔsi fɛɛrɛw ye (< 85 % walima <100 FPKM).
An ye fɛɛrɛ 28,29,30 minnu ɲɛfɔra ka tɛmɛ, olu sɛmɛntiya walasa ka fɛnɲɛnama bɛɛ ka isotopu taamasiɲɛw sɔrɔ.Kuma surun na, kungo kɔnɔ Saccharomyces cerevisiae suguya II (YSC2, Sigma) sɛgɛsɛgɛra yezu azote basi la (BD Difco, DF0335) min kɔnɔ (wt/vol) 2% glucose (G7528, Sigma), 1.7% bɛ sɔrɔ. asidi aminiyɔmu tɛ min na ani sulfate amoniyɔmu. culture medium) ani 5% 15N amoniyɔmu sulfate (NLM-713, >99%, Cambridge Isotope Laboratories) ye azote sɔrɔyɔrɔ kelenpe ye.Jɛkulu sɔrɔla santirifugare fɛ ani sosow balola u yɛrɛ sago la fo ka se u ka wuluwulu ma.A bɛ fara jɛgɛmugu kan (miligaramu 0,5 wulu 300 o la) walasa ka soso naaninan bali saya.O kɔfɛ, musow dɔrɔn de kɛra furusiri kɔlɔsiliw la ni cɛw ye minnu tɔgɔ ma sɛbɛn walasa ka cɛw ka protéome sɛgɛsɛgɛ min jiginna furuɲɔgɔnma waati.
Tile 4-6 npogotiginin minnu taamasiyɛn kɛra ni 15N ye, olu wajibiyara ka ɲɔgɔn sɔrɔ ni npogotiginin cɛmanw ye minnu si hakɛ bɛ bɛn ɲɔgɔn ma.Jɛɲɔgɔnya ɲɛtaa sɛgɛsɛgɛra ni jɛɲɔgɔnya fɛnɲɛnamafagalanw sɔrɔli ye epifluorescence microscopie fɛ.Ni 3, 12 ani 24 hpm la, muso furulen 45-55 ka ɲɛgɛnɛsira tigɛra ka kɛ amoniyɔmu 50 μl ye bikarbonati tampon (pH 7,8) ani ka a kɛ kelen ye ni fɛnɲɛnamafagalan ye.Homojeni kɛra santirifuge la ka a sanfɛla ɲagami ni 50 μl ye 0,1% RapiGest (186001860, Jiw) la 50 mM amoniyɔmu bikaribonati la.Supernatant ni pellet minnu bɛ bɔ sɛgɛsɛgɛli kelen-kelen bɛɛ la, olu bɛ sumaya joona jisuma jalen kan ka taa su fɛ ka taa a bila MacCoss laboratuwari min bɛ Washinton Iniwɛrisite la, o yɔrɔ la, LC-MS/MS ka misali labɛnni dafara.Aw bɛ furakisɛ in wuli kokura 0,1% RapiGest 50 μl kɔnɔ bikaribonati amoniyɔmu 50 mM kɔnɔ ka sonikɛti kɛ jisuma na.Amugu ni a sanfɛla furakisɛ hakɛ jateminɛna ni BCA sɛgɛsɛgɛli ye, sɛgɛsɛgɛli kɛlenw dɔgɔyara ni dithiothreitol 5 mM ye (DTT; Sigma), alkilila ni iyodoasetamidi 15 mM (Sigma) ye ka a bila 37 °C la (1:0 50) fo lɛri 1 ni tripsinization: tripsin:substrate ratio).RapiGest kɛra lyse ye ni 200 mM HCl farali ye a kan, o kɔfɛ ka a bila 37 °C la miniti 45 kɔnɔ ka a bila santirifugare la 14.000 rpm miniti 10 kɔnɔ 4 °C la walasa ka nɔgɔw bɔ.Sɛgɛsɛgɛli kɛra ni dual-mode solid-phase extraction ye (Oasis MCX cartridges, Waters) ka segin ka u lajɔ asidi formiki 0,1% la walasa ka farikolojɔli dumuni laban hakɛ 0,33 μg μl-1 sɔrɔ.MAG proteome minnu ma taamasiyɛn kɛ, olu fana sɛgɛsɛgɛra o cogo kelen na ka bɔ npogotiginin na cɛw.Sɛgɛsɛgɛli kɛcogo fila sɛgɛsɛgɛra sɛgɛsɛgɛli kelen-kelen bɛɛ la.O kɔfɛ, u kelen-kelen bɛɛ la, μg 1 sɛgɛsɛgɛra ni silika fused 75-μm koloma ye min janya ye santimɛtɛrɛ 25 ye, ni silika Kasil1 (PQ) frit trap ye min falen don santimɛtɛrɛ 4 la, min falen don Jupiter C12 reversed-phase resin (Phenomenex) la ani miniti 180 liquid chromatography. Sample digests – MS/MS kɛra Q-Exactive HF mass spectrometer (Thermo Fisher) kan ni nanoACQUITY UPLC System (Waters) ye.Dɔnniya sɔrɔcogo kunnafoniw minnu sɔrɔla boli kelen-kelen bɛɛ kama, olu jiginna ka kɛ mzML cogo ye ni Proteowizard (version 3.0.20287) ye ani ka baara kɛ ni Comet31 (version 3.2) ye ka kɛɲɛ ni FASTA kunnafonidilan ye min kɔnɔ poroteyini bɛ sɔrɔ sequences from Anopheles gambiae (VectorBase version 54), Anopheles coluzzi Ɲini kɛra Mali-NIH (VectorBase version 54), Saccharomyces cerevisiae (Uniprot, Marisikalo 2021), A. gambiae RNA-seq, ani hadamaden nɔgɔ dɔntaw bamanankan saba bamanankan na.Peptide map-matched FDRs, olu latigɛra ni Percolator32 (version 3.05) ni a dancɛ ye 0,01 ye, ani pepitiriw lajɛra ka kɛ farikolojɔlifɛnw dɔnni ye ni poroteyini parsimoni ye Limelight33 (version 2.2.0) kɔnɔ.Protéines relatifs abundance jateminɛna ni normalized spectral abundance factor (NSAF) ye min jatera farikolojɔlifɛn kelen-kelen bɛɛ la boli kelen-kelen bɛɛ la i n’a fɔ a ɲɛfɔra cogo min na ka tɛmɛ.NSAF ka kɛɲɛ ni poroteyini kelen-kelen bɛɛ ye, o kɛra moyenne ye samples kɔnɔ minnu bɔra biologique suguya fila la replicates.15N labeling successfully masked the female proteome, although a little labeled protein could be detected from the labeled virgins.An ye cɛw ka farikolojɔli dumuniw dɔgɔyali sɔrɔli sɛbɛn (1-5 spectra) musow ka sɛgɛsɛgɛli kɛlenw na dɔrɔn fɛɛrɛbɔ boliw la, yɔrɔ min na sɛgɛsɛgɛli kɛlenw kɛra cɛw/furuɲɔgɔnma sɛgɛsɛgɛliw kɔfɛ, o kɛra HPLC “carry over” sababu ye.Tuma dɔw la, poroteyini minnu sɔrɔla i n’a fɔ ‘nɔgɔlenw’ minnu bɛ bɔ npogotigininw na minnu tɔgɔ sɛbɛnnen don, olu bɛ sɔrɔ jatebɔsen 1 faralen ɲɔgɔn kan.
Pepitidi antijeni fila, QTTDRVAPAPDQQQ (isotype PA kɔnɔ) ani MESDGTTPSGDSEQ (isotype PA ni PB kɔnɔ) Genscript kɔnɔ.Pepitiri fila ninnu farala ɲɔgɔn kan, o kɔfɛ, u farala ɲɔgɔn kan ni carrier protein KLH ye ka pikiri kɛ Nuwɛlika laɲɔw la.Laɲɔw saraka pikiri naaninan kɔfɛ, ani IgG bɛɛ lajɛlen farala ɲɔgɔn kan ni affinité ye purification.IgG min bɔra laɲɔ la min ka EPP kɛrɛnkɛrɛnnen don kosɛbɛ, o kɛra ka western blotting wɛrɛw kɛ.
Tlebinyanfan blotiw ta fan fɛ, MAG (n = 10, n bɛ sosow tacogo jira minnu tɛ ɲɛnamaya la) ani muso LRT (n = 30) minnu bɔra cɛ npogotigininw na minnu si bɛ tile 4 la ani muso npogotigininw walima muso minnu ye ɲɔgɔn sɔrɔ fanga la (<10 furuɲɔgɔnma kɔfɛ), Protein extraction buffer (50 mM Tris, pH 8,0; 1% NP-40; 0,25%) sodium deoxycholate;150 mM NaCl;1 mM EDTA; °C.Protéines hakɛ jateminɛna ni Bradford assay (Bio-Rad) ye.O kɔfɛ, MAG proteine ​​20 μg, LRT proteine ​​40 μg, ani protéine bulk tolen 20 μg denatured ka faranfasi ni 10% Bis-Tris NuPAGE ye ni MOPS buffer ye.Protéines bilala polyvinylidene fluoride membranes kɔnɔ ni iBlot2 transfert ye system (Thermo Fisher).Membraniw ko siɲɛ fila 1× PBS-T kɔnɔ (0,1% Tween-20 PBS kɔnɔ) ka sɔrɔ ka u datugu Odyssey blocking buffer (Li-Cor) la lɛri 1 kɔnɔ 22°C la.Membranes lamaga su fɛ 4°C la ni custom rabbit anti-EPP polyclonal primary antibody (1:700 in blocking buffer) ye anti-actin monoclonal primary antibody MAC237 (Abeam; 1:4,000).Membraniw ko ni PBS-T ye ka sɔrɔ ka u bila ni anticorps filananw ye (fali anti-lapin 800CW ani bakɔrɔn anti-rat 680LT (Li-Cor), u fila bɛɛ ye 1:20.000 ye) tampon bloquant kɔnɔ min kɔnɔ SDS 0,01% ani 0,2% bɛ sɔrɔ. Tween -20 sanga 1 kɔnɔ 22 °C la.Membranes ko ni PBS-T ye ka ja kɛ ni Odyssey CLx scanner ye.Jaw lajɛra ka baara kɛ Image Studio kɔnɔ (version 5.2).Bandi kɛrɛnkɛrɛnnen min bɛ bɛn EPP-RA isoform (82 kDa) ma, o ma sɔrɔ.
EPP (i n’a fɔ isoform AGAP002463-RB min kɔnɔ histidine phosphatase domaine bɛ sɔrɔ, NCBI conserved domain search 34) ani EcK2 (AGAP002181) ka kodeli yɔrɔw kɛra clone ye ka kɛ pET-21a(+) plasmide (Novagen Millipore Sigma) ye primers are listed in Extended Data Table 2.GS4 linkers 8 (in tandem) donna ka kɔn pET-21a(+)-EcK2 construct ka C-terminal 6xHis tag ɲɛ.Protéines recombinantes dilanna ni NEBExpress cell-free E. coli protein synthesis reaction ye (New England BioLabs).Protéines recombinantes tun saniyalen don ni NEBExpress Ni spin columns ye (New England BioLabs).Dihydrofolate reductase (DHFR) kɔlɔsili furakisɛ dilannen don ni ADN misali ye min bɔra NEBExpress Cell-Free E. coli Protein Synthesis Kit la.Protéinew maralen don 50% glycérol la PBS kɔnɔ -20 °C la fo kalo 3.
EPP ni farikolo yɔrɔw bɔlenw ka fosifatazi baara jateminɛna ni 4-nitrophenyl phosphate (pNPP; Sigma-Aldrich) ye.Tris 25 mM, asidi asetiki 50 mM, Bis-Tris 25 mM, NaCl 150 mM, EDTA 0,1 mM, ani DTT 1 mM tun bɛ sɔrɔ farikolojidɛsɛ tampon na.Tissue tun bɛ homojeni kɛ tampon kɔnɔ ani selilu nɔgɔw bɔra ni santirifugare ye.Aw bɛ farikoloɲɛnajɛ daminɛ ni anzimu walima farikolojidɛsɛ furakisɛ farali ye farikoloɲɛnajɛ tampon na min kɔnɔ miligaramu 2,5 mililitiri-1 pNPP bɛ sɔrɔ.Fɛɛrɛ ɲagaminen in bilala so funteni na dibi la, ani pNP hakɛ min jiginna ka bɔ pNPP la, o hakɛ jateminɛna ni a minɛcogo sumani ye 405 nm waati caman na.
Walasa ka EcK baara kɛ in vitro, farikolojɔlifɛn in tun bɛ tobi ni miligaramu 0,2 ye 20E walima 3D20E la tampon 200 μl (pH 7,5) kɔnɔ min kɔnɔ HEPES–NaOH 10 mM, 0,1% BSA, 2 mM ATP ani 10 mM MgCl2 bɛ sɔrɔ fo lɛri 2 kɔnɔ 27 °C la.A kɛcogo jɔra ni 800 farali ye a kan μl mɛtanɔli, o kɔ aw bɛ nɛnɛ -20 °C la lɛri 1 kɔnɔ, o kɔ aw bɛ a santirifuge kɛ garamu 20.000 na miniti 10 kɔnɔ 4 °C la.O kɔfɛ, a sanfɛla bɛ sɛgɛsɛgɛ ni HPLC-MS/MS ye.Walisa ka farikolojɔli dumuni minnu kɛra kɔrɔsili kulu kɔnɔ, olu ka funteni-wale kɛ, farikolojɔlifɛnw bɛ tobi 50% glycérol la PBS kɔnɔ miniti 20 kɔnɔ 95°C la.
Walasa ka EPP baara kɛ in vitro, farikolojɔli dumuni in tun bɛ wuli ni 3D20E22P ye (o bɛ bɛn hakɛ ma min bɛ sɔrɔ MAG fila 18 la, min saniyalen don ni HPLC-MS/MS ye) tampon 100 μl kɔnɔ (pH 7,5) min kɔnɔ Tris 25 mM, asidi asetiki 50 mM, Bis-Tris 25 mM, NaCl 150 mM, 0,1 mM EDTA, ani 1 mM DTT sanga 3 kɔnɔ 27 °C la.A furakɛcogo jɔra ni mɛtanɔli 400 μl farali ye a kan ka nɛnɛ -20 °C la lɛri 1 kɔnɔ, o kɔfɛ ka a kɛ santirifugare la 20.000 g la miniti 10 kɔnɔ 4 °C la.A ji min bɛ sanfɛ, o sɛgɛsɛgɛra ni HPLC-MS/MS ye.
PCR yɔrɔ minnu bɛ EPP (362 bp), EcK1 (AGAP004574, 365 bp) ani EcK2 (556 bp) la, olu cayara ka bɔ ADNc la min labɛnna ka bɔ sosow suw la minnu kunkolo tɛ cɛ ni muso ɲagaminenw na.EGFP kunbɛnni PCR yɔrɔ (495 bp) cayara ka bɔ pCR2.1-eGFP la min ɲɛfɔra ka tɛmɛ PCR daminɛfɛnw bɛ sɔrɔ kunnafonidilan janyalenw na Tablo 2 kɔnɔ.PCR yɔrɔ in donna T7 promotɛri inversés cɛ pL4440 plasmide kan.Plasmide constructs sɔrɔla NEB 5-α competent E. coli fɛ (New England Biolabs) ani ka sɛgɛsɛgɛli kɛ ni ADN sequencing ye sani u ka baara kɛ (aw ye Supplementary Data 1 lajɛ ka insert sequence lajɛ).Primers matched to the T7 promoter (Extended Data Table 2) kɛra ka fɛn donta bonya ka bɔ pL4440-based plasmide la.PCR fɛn dilannen hakɛ dafara ni agarose gel electrophoresis ye.dsRNA sɛbɛnna ka bɔ PCR templates la ni Megascript T7 Transcription Kit (Thermo Fisher) ye ka saniya ka kɛɲɛ ni a dilabaga ka laadilikanw ye ni fɛn caman tigɛli ye minnu ɲɛfɔra ka tɛmɛ.
Ni dsRNA pikiri kɛra, dsRNA ng 1380 (dsGFP, dsEcK1, dsEcK2, dsEPP) pikiri kɛra ni nl-1 ng 10 ye cɛkɔrɔba walima muso balikuw barajuru la (Nanoject III, Drummond) tile 1 kɔnɔ eclosion kɔfɛ.Jɛnɛya knockdown hakɛw jateminɛna biologique replicates saba la dɔgɔmannin na ARN fɛ extraction, cDNA synthesis, and RT-qPCR.Ecdysone pikiri kama, tile 4 npogotigi walima tile 6 npogotigi joli balolen musow pikiri kɛra ni 20E walima 3D20E (Nanoject III, Drummond) μg 0,13, 0,21 walima 0,63 ye (Nanoject III, Drummond) ni a hakɛ ye 1,3, 2,1, ye, ni a hakɛ ye 1,3, 2,1, ye, o cogo kelen na, ka kɛɲɛ ni jateminɛ kɛcogo ye walima 6,3 ng nl-1.Aw bɛ etanɔli 10% (vol/vol) nl 100 pikiri ji la; 3D20E22P nl 100 etanɔli 10% la (o bɛ bɛn hakɛ 75% ma min bɛ sɔrɔ MAG fila la).Sosow bilala pikiri kulu la k’a sɔrɔ u ma kɛ cogo si la.
Walasa ka den sɔrɔli sɛgɛsɛgɛliw kɛ, tile 3 musow tun bɛ balo ad libitum hadamaden joli la.Aw bɛ soso minnu balolen don walima minnu ma balo, aw bɛ olu bɔ.Ka kɛɲɛ ni furakɛli ye, musomanninw bilala den sɔrɔli tasaw danfaralenw kɔnɔ su naani kɔnɔ, a dɔgɔyalenba la lɛri 48 joli dumuni kɔfɛ.Kɔnɔw jatera stɛriyɔmu fɛ (Stemi 508, Zeiss) muso furulenw fɛ, kɔnɔ minnu bɛ Bɔ ka Kɛ wuluw ye, olu tùn bɛ jate den ye.
Ka ɲɛsin furusiri sɛgɛsɛgɛliw ma, musomanninw bilala tile 2 la dɔgɔmannin ka kɛɲɛ ni furakɛli ye walasa u ka se ka furuɲɔgɔnya kunbɛn, o kɔfɛ, kungo kɔnɔ cɛ minnu si hakɛ bɛ bɛn ɲɔgɔn ma, olu donna o kaburu kelen kɔnɔ.Su fila o kɔfɛ, musomanninw ka fɛnɲɛnamafagalanw tigɛra ka ADN genomic bɔ ni freeze-thaw ni sonication ye tampon kɔnɔ min kɔnɔ Tris-HCl 10 mM bɛ sɔrɔ, EDTA mM 1, ani NaCl 25 mM (pH 8,2).Sɛgɛsɛgɛliw kɛra ni Proteinase K (0,86 μg μl-1) ye miniti 15 kɔnɔ 55 °C la, o kɔfɛ miniti 10 kɔnɔ 95 °C la.ADN jamu labɛncogo nɔgɔlenw bɛ dilan siɲɛ 10 ka sɔrɔ ka Y kolosinsinnanw sɔrɔ qPCR fɛ primers are listed in Extended Data Table 2.Y chromosome sequence tɛ yen, o b’a jira ko furuɲɔgɔnma tɛ.
Walasa ka segin-ka-bɔnye sɛgɛsɛgɛliw kɛ, muso minnu ye ɲɔgɔn sɔrɔ ni fanga ye, olu sɛgɛsɛgɛra walasa ka furuɲɔgɔnya-minɛnw sɔrɔ walasa ka furuɲɔgɔnya cogoya sɛmɛntiya ani ka tile 2 to u ka se ka furusiribaliya sɔrɔ cɛw tɛ yen, i n’a fɔ a ɲɛfɔra cogo min ka tɛmɛ 36 .Cɛ minnu bɛ ni DsRed transgenic sperm ye, olu donna musomanninw ka kaburuw kɔnɔ o kɔfɛ.Su fila o kɔfɛ, fertilising vesicles donna a tigɛra ka bɔ musow la, ka ADN jamu labɛn i n’a fɔ a ɲɛfɔlen don cogo min sanfɛ, ka kɛ qPCR fɛ ka DsRed fɛnɲɛnamafagalan sɛgɛsɛgɛli kɛ; primers are listed in Extended Data Table 2.DsRed transgene tɛ yen, o y’a jira ko furuɲɔgɔnma kura si ma kɛ.
3D20E dilannen don i n’a fɔ a ɲɛfɔra cogo min ka tɛmɛ 37 .Kuma surun na, 20E miligaramu 10 (Sigma-Aldrich) bɛ wuli ji mililitiri 10 na, o kɔfɛ, platini nɛrɛmuguma miligaramu 30 farala a kan (mugu cogo la, Sigma-Aldrich).O2 jikuru nɔgɔman dɔ bɛ to ka wuli ka don farikoloɲɛnajɛ ɲagaminen na, min bɛ wuli so kɔnɔ funteni.Lɛrɛ 6 kɔfɛ, mɛtanɔli mililitiri 30 farala a kan walasa ka farikoloɲɛnajɛ dabila.A ɲagaminen bɛ kɛ santirifugu la walasa ka katalisita fɛnɲɛnamafagalanw bɔ.A sanfɛla bɛ wuli fo ka ja fo ka a ja, a bɛ kɛ fɛn ye min tɛ fɛn ye, so funteni na.Fɛɛrɛ jalen bɛ kɛ etanɔli 10% ni mɛtanɔli la walasa ka pikiri kɛ walasa ka HPLC-MS/MS sɛgɛsɛgɛli kɛ.A jiginni hakɛ (ka bɔ 20E la ka se 3D20E) tun bɛ 97% ɲɔgɔn na (Ja 4b), wa 3D20E min dilannen don, o ka MS spektrum tun bɛ bɛn ni min bɛ sɔrɔ muso furulenw na (Ja 4c).
Jateminɛ sɛgɛsɛgɛli minnu kɛra, olu kunnafoni kɛrɛnkɛrɛnnenw bɛ o nsiirin kɔnɔ.GraphPad (version 9.0) kɛra ka Fisher ka sɛgɛsɛgɛli tigitigi kɛ, Mantel-Cox ka sɛgɛsɛgɛli, ani Student ka t-test.Cochran-Mantel-Haenszel sɛgɛsɛgɛliw kɛra ni R sɛbɛnnikɛlan ye min bɛ kɛ ka kɛɲɛ ni mɔgɔw sago ye (a bɛ sɔrɔ https://github.com/duopeng/mantelhaen.test).Dɔnniya tilatilali sɛgɛsɛgɛra normalité ni Shapiro-Wilk test ye ni significance threshold ye 0,05.Ni kunnafoniw ma se normalité test ma, Mann-Whitney test kɛra.Survival data analysed with the Mantel-Cox test.DESeq2 pake (version 1.28.1) kɛra ka RNA-seq gene-level differential expression analysis kɛ.Baro horizontal min bɛ graph kan, o bɛ median jira.A nafama nafa min ye P = 0,05 ye, o kɛra sɛgɛsɛgɛli bɛɛ dantigɛli ye.
Walasa ka kunnafoni wɛrɛw sɔrɔ kalan kɛcogo kan, aw ye Nature Research Report abstract lajɛ min ni nin barokun cɛsirilen don.
MS proteomic kunnafoniw bilala ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) kɔnɔ PRIDE Partner Repository (https://www.ebi.ac.uk/pride/) fɛ ni kunnafonidilan taamasiyɛn ye PXD032157.
ARN-seq kunnafonidilan bɛ bila Gene Expression Comprehensive Library (https://www.ncbi.nlm.nih.gov/geo/) kɔnɔ, seriyali sɛbɛn GSE198665 kɔnɔ.
Kunnafoni wɛrɛw minnu bɔra ani/walima minnu sɛgɛsɛgɛra nin kalan in senfɛ, olu bɛ se ka sɔrɔ sɛbɛnnikɛlaw fɛ minnu bɛ bɛn o ma, ni ɲinini kɛra cogo bɛnnen na.Nin barokun bɛ kunnafonidilanw bɔyɔrɔ di.
De Loof, A. Ecdysteroids: Fɛnɲɛnamaw ka cɛnimusoya stɛroyi minnu ma jate?Cɛ: Black Box.Insect Science.13, 325–338 (2006).
Redfern, CPF 20-hydroxyecdysone ani kɔnɔbara falenni Anopheles stephens la.J. Fɛnɲɛnɛmaw ka farikoloɲɛnajɛ.28, 97–109 (1982).