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Tsis zoo li cov tsiaj txhu, cov kab feem ntau xav tias tsis muaj cov tshuaj hormones poj niam txiv neej uas cuam tshuam rau txiv neej. Hauv Anopheles gambiae, cov tshuaj ecdysone steroid 20-hydroxyecdysone (20E) zoo li tau hloov zuj zus los tswj kev loj hlob ntawm qe thaum cov poj niam tsim los2 thiab ua rau lub sijhawm tsis sib haum xeeb thaum cov txiv neej hloov kev sib deev3. Vim tias kev loj hlob ntawm qe thiab kev sib deev yog cov yam ntxwv tseem ceeb ntawm kev yug me nyuam, kev nkag siab txog seb cov yoov tshaj cum Anopheles poj niam koom ua ke cov cim hormonal no li cas tuaj yeem pab tsim cov kev pab cuam tswj kab mob malaria tshiab. Ntawm no, peb qhia tias cov haujlwm yug me nyuam no raug tswj hwm los ntawm cov tshuaj steroids sib txawv los ntawm lub network ntawm cov enzymes ecdysteroid-activating/inactivating. Peb tau txheeb xyuas cov tshuaj ecdysone oxidized tshwj xeeb rau txiv neej, 3-dehydro-20E (3D20E), uas tiv thaiv kev ua niam txiv los ntawm kev kaw cov poj niam kev sib deev tom qab kev hloov kev sib deev thiab kev ua haujlwm los ntawm dephosphorylation. Qhov tseem ceeb, kev hloov 3D20E kuj tau ua rau muaj kev qhia tawm ntawm cov noob caj noob ces yug me nyuam uas tswj kev loj hlob ntawm qe thaum muaj kab mob Plasmodium, ua kom ntseeg tau tias cov poj niam kis kab mob muaj kev noj qab haus huv. Cov poj niam los ntawm 20E tsis ua rau muaj kev teb rau kev sib deev, tab sis tso cai rau cov tib neeg sib yuav tso qe tom qab 20E-inhibiting kinases raug txwv. Kev txheeb xyuas cov tshuaj hormones steroid tshwj xeeb rau cov kab no thiab nws lub luag haujlwm hauv kev tswj hwm kev sib deev ntawm cov poj niam, kev muaj menyuam thiab kev sib cuam tshuam nrog Plasmodium qhia txog lub peev xwm los txo qhov kev vam meej ntawm cov yoov tshaj cum uas kis tus kab mob malaria.
Cov kab mob malaria thiab cov neeg tuag rov qab nce ntxiv4 vim yog cov kab Anopheles tsis kam tshuaj tua kab, uas yog tib hom kab mob malaria uas kis tau rau tib neeg. Cov kab no yog ib qho chaw zoo rau kev tswj kab mob malaria vim tias cov poj niam tsuas yog sib yuav ib zaug xwb5; kev ua kom qhov kev sib yuav no tsis muaj kab mob yuav muaj peev xwm txo cov kab hauv thaj teb.
Cov poj niam ua rau tsis muaj peev xwm sib deev tom qab tau txais cov tshuaj hormones steroid uas muaj titer siab los ntawm cov txiv neej. Cov kev tshawb fawb tau qhia tias qhov ua rau muaj teeb meem hauv kev sib deev ntxiv yog 20-hydroxyecdysone (20E), ib yam tshuaj hormones steroid uas paub zoo tias yog tus tswj hwm lub voj voog molting hauv theem larval. Lub peev xwm ntawm cov txiv neej los tsim thiab hloov 20E tau hloov pauv tshwj xeeb hauv cov hom Anopheles uas yog ib feem ntawm subgenus Cellia7, uas tau faib tawm hauv Africa thiab suav nrog cov kab mob malaria txaus ntshai tshaj plaws, suav nrog Anopheles gambiae. Qhov no yog qhov tseem ceeb tshaj plaws vim tias hauv cov hom no cov poj niam kuj tsim 20E tom qab txhua pluas noj ntshav, thiab 20E tsav lub voj voog oogenesis (saib ref. 8). Txawm li cas los xij, me ntsis paub txog txoj kev uas cov poj niam sib koom ua ke cov cim los ntawm ob qhov chaw sib txawv ntawm ecdysone (kev hloov txiv neej thiab kev pub ntshav) yam tsis cuam tshuam lawv tus kheej lub peev xwm los sib deev. Qhov tseeb, yog tias 20E tsim los ntawm cov poj niam ua rau tsis kam sib deev, qhov no yuav ua rau tsis muaj menyuam hauv cov neeg pub niam txiv, ib qho kev coj cwj pwm ntau heev hauv cov yoov tshaj cum5.
Ib qho kev piav qhia tau yog tias A. gambiae cov txiv neej hloov pauv ecdysone tshwj xeeb rau txiv neej, uas ua rau muaj kev sib txuas lus hauv txoj hlab ntaws poj niam, ua rau kev sib yuav tsis ruaj khov. Txawm li cas los xij, txawm hais tias cov vertebrates muaj ntau yam tshuaj hormones steroid, xws li estrogen thiab androgen (saib hauv ref. 9), raws li peb paub, cov tshuaj steroids uas muaj androgenic tsis tau pom hauv cov kab.
Peb tau teem sijhawm los txiav txim siab cov npe ntawm cov tshuaj hormones steroid hauv cov txiv neej cov khoom siv caj pas (MAG) ntawm cov poj niam A. gambiae uas loj hlob hauv kev tshawb nrhiav cov tshuaj steroids uas hloov pauv tau. Siv cov kua chromatography ua haujlwm siab ua ke nrog tandem mass spectrometry (HPLC-MS/MS) es tsis yog txoj kev tshwj xeeb uas siv yav dhau los, peb tau pom ecdysone (E) thiab 20E hauv cov ntaub so ntswg no, lees paub qhov tshwm sim yav dhau los. Txawm li cas los xij, cov qauv tau tswj hwm los ntawm cov tshuaj steroids oxidized phosphorylated, sib xws nrog cov mis 3-dehydro-20E-22-phosphate (3D20E22P)12 (Daim Duab 1). Lwm daim ntawv suav nrog 3-dehydro-20E (3D20E) thiab 20E-22-phosphate (20E22P). Lub zog ntawm HPLC-MS/MS ntawm 3D20E22P yog ob qho kev txiav txim siab ntawm qhov loj dua li nws daim ntawv dephosphorylated, 3D20E, thiab peb qhov kev txiav txim siab ntawm qhov loj dua li ntawm E thiab 20E (Daim Duab 1). Txawm hais tias nyob rau lwm qhov chaw ntawm lub cev thiab txoj hlab qe menyuam qis dua (LRT; Daim Duab Ntxiv 1a). Peb kuj tau tshuaj xyuas cov tshuaj ecdysteroids hauv cov txiv neej thiab poj niam uas nyuam qhuav kaw (<1 hnub) thiab pom 3D20E thiab 3D20E22P tsuas yog hauv MAG; E, 20E thiab 20E22P tau muaj nyob rau hauv ob qho tib si poj niam txiv neej (Daim Duab Ntxiv 1b). Cov ntaub ntawv no qhia tias A. gambiae cov txiv neej laus tsim cov tshuaj hormones hloov kho siab hauv lawv cov MAGs uas tsis tau tsim los ntawm cov poj niam.
MAG thiab poj niam LRT (suav nrog atria, seminal vesicles, thiab parovarium) tau raug txiav tawm ntawm cov txiv neej uas muaj 4 hnub (4 hnub) thiab cov poj niam uas muaj virgin thiab mated (0.5, 3, thiab 12 hpm). Ecdysone hauv cov ntaub so ntswg no tau raug soj ntsuam los ntawm HPLC-MS/MS (qhov nruab nrab ± sem; unpaired t-test, two-sided, false discovery rate (FDR) kho; NS, tsis tseem ceeb; *P < 0.05, **P < 0.01. 3D20E: 3 teev vs. 0.5 teev, P = 0.035; 12 teev vs. 3 teev, P = 0.0015; 12 teev vs. 0.5 teev, P = 0.030. 3D20E22P: 3 teev vs. 0.5 teev, P = 0.25; 12 teev vs. 3 teev, P = 0.0032; 12 teev vs. 0.5 teev, P = 0.015). Cov ntaub ntawv yog los ntawm peb qhov kev rov ua dua ntawm cov tsiaj txhu. Thaj chaw siab tshaj plaws rau txhua tus ecdysone uas xav tau raug suav thiab ua kom zoo los ntawm tus lej ntawm cov yoov tshaj cum. Ecdysone yog sawv cev los ntawm xim raws li nram no: E, ntsuab; 20E, txiv kab ntxwv; 20E22P, ntshav; 3D20E, xiav; 3D20E22P, liab dawb. Daim duab ntxig ua rau qhov ntsuas ntawm y-axis nce ntxiv los qhia cov qib ecdysone qis dua.
Yuav tshawb xyuas seb 3D20E22P thiab 3D20E puas raug hloov thaum lub sijhawm sib deev, peb tau txiav cov poj niam LRT ntawm ntau lub sijhawm tom qab sib deev. Txawm hais tias ecdysone tsis pom muaj nyob rau hauv cov poj niam hluas, peb tau pom ntau ntawm 3D20E22P hauv LRT tam sim ntawd tom qab sib deev (0.5 teev tom qab sib deev, hpm), txo qis dua lub sijhawm, thaum 3D20E qib nce ntxiv ntau (Daim Duab 1). Siv cov tshuaj synthesized 3D20E ua tus qauv, peb tau txiav txim siab tias cov theem ntawm cov tshuaj steroid no hauv kev sib deev LRT tsawg kawg yog 100 npaug siab dua 20E (Extended Data Table 1). Yog li, 3D20E22P yog tus txiv neej ecdysone tseem ceeb uas tau hloov mus rau poj niam LRT thaum lub sijhawm sib deev, thiab nws daim ntawv dephosphorylated, 3D20E, dhau los ua ntau heev tom qab sib deev. Qhov no qhia txog lub luag haujlwm tseem ceeb rau ecdysone tom kawg hauv poj niam tom qab sib deev biology.
Tom qab tsim cov ntaub ntawv RNA sequencing tshiab (RNA-seq) (Daim Duab 2a), siv cov kav dej bioinformatics uas tsim tshwj xeeb, peb tau tshawb nrhiav ecdysone kinase (EcK), ecdysone oxidase (EO), thiab ecdysone encoding 20E-modified phosphatase gene.EPP) tau qhia tawm hauv cov ntaub so ntswg reproductive. Peb tau txheeb xyuas ib tus neeg sib tw EPP gene thiab ob tus neeg sib tw EcK gene (EcK1 thiab EcK2), tab sis tsis tuaj yeem nrhiav tau tus neeg sib tw EO gene zoo. Qhov tseem ceeb, cov noob EPP ib leeg tau qhia tawm ntawm cov theem siab (98.9th percentile) hauv Gambian MAGs tab sis tsis yog hauv poj niam LRTs (Daim Duab 2b), tsis zoo li peb qhov kev cia siab txij li thaum dephosphorylation ntawm 3D20E22P tshwm sim hauv cov ntaub so ntswg poj niam no. Yog li ntawd, peb ntseeg tias txiv neej EPP yuav raug hloov pauv thaum lub sijhawm sib deev. Tseeb tiag, peb tau siv hauv vivo ruaj khov isotope labeling los npog cov protein poj niam tom qab sib deev, ib qho enzyme txheeb xyuas los ntawm MS hauv poj niam atrium (Daim Duab 2c thiab Cov Lus Qhia Ntxiv 1). Qhov muaj EPP hauv MAG thiab tus poj niam LRT uas tau sib yuav (tab sis tsis yog tus poj niam tsis tau sib yuav) kuj tau lees paub siv cov tshuaj tiv thaiv tshwj xeeb (Daim Duab 2d).
a, Ib txoj kab ke bioinformatics uas tsim tshwj xeeb los tshawb nrhiav cov ntaub so ntswg ntawm txhua tus poj niam txiv neej rau cov noob caj noob ces uas encoded EcKs, EOs, thiab EPPs. Cov lej nyob ib sab ntawm cov xub qhia txog tus lej ntawm cov txiv neej thiab poj niam sib tw ntawm txhua kauj ruam. Qhov kev tshuaj xyuas no tau txheeb xyuas ib qho EPP gene (EPP) thiab ib qho EcK gene (EcK1) uas tau qhia tawm hauv cov txiv neej, thiab ib qho EcK gene (EcK2) uas tau qhia tawm hauv ob qho tib si poj niam txiv neej tab sis tsis muab cov noob caj noob ces EO sib tw.b, Heatmap piv rau cov noob caj noob ces sib tw hauv cov ntaub so ntswg virgin (V) thiab mating (M) Anopheles gambiae thiab Anopheles albicans.Spca, kev xeeb tub; MAGs, cov qog ntxiv hauv cov txiv neej; lwm qhov ntawm lub cev, suav nrog lub mis, tis, ceg, lub cev rog, thiab cov kab mob sab hauv hauv ob qho tib si poj niam txiv neej, thiab zes qe menyuam hauv cov poj niam. EcK2 tau qhia ntau heev hauv ob qho tib si MAG thiab atria ntawm Gambia, thaum EPP tsuas pom hauv MAG.c, Proteomic kev tshuaj xyuas ntawm cov txiv neej ejaculate pawg translocation rau hauv poj niam atria ntawm 3, 12 thiab 24 hpm, qhia 67 cov protein ntau tshaj plaws. Cov poj niam tau loj hlob ntawm cov zaub mov uas muaj 15N los cim (thiab npog) tag nrho cov protein. Cov txiv neej tsis muaj cim tau sib yuav nrog cov poj niam cim, thiab poj niam LRTs tau txiav ntawm 3, 12 thiab 24 hpm rau kev tshuaj xyuas proteomic (saib Supplementary Table 1 rau daim ntawv teev tag nrho ntawm cov protein ejaculatory). Inset, EPP, Eck1 thiab EcK2 tau pom hauv MAG ntawm cov txiv neej nkauj xwb los ntawm kev tshuaj xyuas proteomic ntawm cov ntaub so ntswg no.d, EPP tau pom los ntawm western blot hauv MAG thiab LRT ntawm cov poj niam sib yuav, tab sis tsis yog hauv cov poj niam nkauj xwb lossis txiv neej lossis lwm qhov ntawm lub cev poj niam. Cov membranes tau tib lub sijhawm kuaj nrog cov tshuaj anti-actin (loading control) thiab anti-EPP antibodies. Tag nrho cov txiv neej yog nkauj xwb. Saib Daim Duab Ntxiv 1 rau cov ntaub ntawv gel qhov chaw. Western blots tau ua ob zaug nrog cov txiaj ntsig zoo sib xws.
Qhov kev ua haujlwm ntawm ecdysteroid phosphophosphatase ntawm EPP tau raug txheeb xyuas tom qab kev sib xyaw los ntawm HPLC-MS / MS nrog 3D20E22P cais los ntawm MAG (Extended Data Fig. 2a). Ntxiv mus, thaum peb ua kom EPP ntsiag to los ntawm RNA-mediated interference (RNAi), peb tau pom tias muaj kev txo qis ntawm kev ua haujlwm phosphatase hauv cov ntaub so ntswg ntawm cov txiv neej no (Daim duab 3a), thiab cov poj niam uas tau sib yuav nrog EPP-silenced txiv neej tau pom tias muaj qhov sib piv qis dua ntawm dephosphorylated 3D20E (Daim duab 3b) txawm tias muaj ib feem ntawm gene silencing (Extended Data Fig. 2b, c). Hauv kev sib piv, peb tsis tau pom qhov kev hloov pauv tseem ceeb hauv qhov sib piv 20E22P / 20E hauv tib cov yoov tshaj cum, uas yuav qhia tias cov enzyme yog tshwj xeeb rau 3D20E22P (Daim duab 3b).
a, Txo qis phosphatase kev ua haujlwm hauv MAG los ntawm EPP silencing siv ob-stranded EPP RNA (dsEPP) lossis ob-stranded GFP RNA (dsGFP) tswj. Nees nkaum MAG pas dej tau siv hauv txhua qhov rov ua dua (P = 0.0046, paired t-test, ob-sided), sawv cev los ntawm cov dots sib cais.b, Cov poj niam mated nrog EPP-silenced txiv neej muaj qhov sib piv qis dua ntawm dephosphorylated 3D20E ntawm 3 hpm (P = 0.0043, unpaired t-test, ob-sided), thaum 20E theem tsis cuam tshuam (P = 0.063, unpaired). t-xeem, ob sab). Cov ntaub ntawv raug nthuav tawm ua qhov nruab nrab ± sem los ntawm peb lub pas dej ntawm 13, 16 thiab 19 tus poj niam txhua tus.c, Cov poj niam sib yuav nrog EPP-silenced txiv neej muaj tus nqi siab dua ntawm kev rov sib yuav (P = 0.0002, Fisher qhov kev xeem tseeb, ob sab). Cov poj niam raug yuam kom sib yuav ua ntej kom ntseeg tau tias lawv txoj kev sib yuav; 2 hnub tom qab, lawv tau raug tiv tauj nrog lwm tus txiv neej uas nqa cov phev transgenic los ntsuas qhov rov sib yuav los ntawm kev kuaj pom PCR ntawm cov transgene.d, Cov poj niam pub ntshav uas tau sib yuav nrog cov txiv neej EPP uas tau ntsiag to muaj kev xeeb tub tsawg dua (P < 0.0001; Mann-Whitney xeem, ob sab) thiab txo qis me ntsis ntawm cov qe (P = 0.088, Mann-Whitney xeem, ob sab), thaum qhov kev tso qe tsis cuam tshuam (P = 0.94, Fisher's exact test, ob sab). Hauv txhua lub vaj huam sib luag, n sawv cev rau tus lej ntawm cov qauv yoov tshaj cum ywj pheej.NS, tsis tseem ceeb.*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.001.
Tom ntej no, peb tau soj ntsuam seb ecdysone dephosphorylation puas tseem ceeb rau kev ua kom cov poj niam tsis kam sib yuav. Qhov tseem ceeb, cov poj niam uas sib yuav nrog cov txiv neej uas tsis muaj EPP tau sib yuav dua ntau zaus dua (44.9%) dua li cov poj niam tswj (10.4%) thaum raug rau lwm cov txiv neej (transgenic) (Daim Duab 3c). Peb kuj tau pom tias muaj kev txo qis ntawm kev muaj menyuam (Daim Duab 3d, sab laug) thiab txo qis me ntsis ntawm cov qe uas cov poj niam no tso (Daim Duab 3d, nruab nrab), thaum feem pua ​​ntawm cov qe uas cov poj niam tso (lwm qhov kev teb tau los ntawm cov poj niam los ntawm kev sib yuav)) tsis raug cuam tshuam (Daim Duab 3d, sab xis). Muab qhov tshwj xeeb ntawm EPP rau 3D20E22P, cov txiaj ntsig no qhia tias kev ua haujlwm ntawm 3D20E los ntawm EPP hloov pauv thaum lub sijhawm sib yuav yuav muaj lub luag haujlwm tseem ceeb hauv kev tua cov poj niam txais tos rau kev sib yuav ntxiv, tus cwj pwm yav dhau los tau muab rau kev hloov pauv kev sib deev ntawm 20E. Yog li ntawd, cov tshuaj hormones tshwj xeeb rau txiv neej no kuj cuam tshuam rau kev muaj menyuam ntawm poj niam.
Tom ntej no, peb piv cov haujlwm ntawm 20E thiab 3D20E hauv kev sim txhaj tshuaj rau cov ntxhais hluas uas laus lawm siv cov tshuaj 3D20E uas tau tsim los ntawm tshuaj (Daim Duab 4a-c) thiab 20E uas muaj nyob hauv khw. Peb pom tias 3D20E muaj txiaj ntsig zoo dua li 20E hauv kev kaw cov poj niam tsis xis nyob rau kev sib yuav ntawm ob qho kev sib xyaw (Daim Duab 4d). Qhov tseem ceeb, ib nrab ntawm qib kev ua haujlwm ntawm 3D20E hauv LRT (1,066 pg tom qab txhaj tshuaj vs. 2,022 pg tom qab sib yuav) ua rau muaj feem pua ​​ntawm cov poj niam tsis kam txhaj tshuaj uas siab dua 20 npaug ntawm qib kev ua haujlwm ntawm 20E (361 pg tom qab txhaj tshuaj) 24 teev tom qab txhaj tshuaj ntawm qhov siab tshaj plaws 18 pg tom qab sib yuav; Cov Ntaub Ntawv Ntxiv 1). Qhov tshwm sim no yog sib xws nrog lub tswv yim tias kev hloov pauv ntawm kev sib deev ntawm 20E tsis ua rau lub sijhawm tsis sib haum xeeb, thiab ntxiv rau qhov taw qhia rau 3D20E ua qhov tseem ceeb hauv kev ua kom muaj kev sib raug zoo ntawm niam txiv thiab menyuam. 3D20E kuj tseem ua haujlwm ntau dua li 20E hauv kev ntsuam xyuas qe hauv cov poj niam tsis tau muaj poj niam (Daim Duab 4e), qhia tias tus nqi tso qe ib txwm uas peb tau pom tom qab ib feem ntawm EPP silencing yog vim muaj cov haujlwm 3D20E seem uas tseem tsim los ntawm Mating-induced poj niam yam.
(a,b) 3D20E tau tsim los ntawm 20E (a) nrog kev hloov pauv / ua haujlwm siab heev (cov ntaub ntawv nthuav tawm ua qhov nruab nrab ± sem los ntawm peb qhov kev sib xyaw ua ke ywj pheej) (b).c, Mass spectrum (ib nrab hauv qab) phim ecdysone pom hauv mated poj niam LRT (ib nrab sab saud).d, Piv nrog 20E (0.63 µg, P = 0.02; 0.21 µg, P < 0.0001; Fisher qhov kev xeem tseeb, ob sab) thiab 10% ethanol (0.63 µg, P < 0.0001; 0.21 µg, P < 0.0001; Fisher qhov kev xeem tseeb, 2 sab), thaum 20E siab dua li kev tswj hwm tsuas yog ntawm cov koob tshuaj ntau dua (0.63 µg, P = 0.0002; 0.21 µg, P = 0.54; Fisher qhov kev xeem tseeb, 2-sab).e, kev txhaj tshuaj 3D20E ua rau muaj kev tso qe ntau dua hauv cov poj niam tsis muaj poj niam dua li 10% ethanol tswj (0.21 µg, P < 0.0001; 0.13 µg, P = 0.0003; Fisher qhov kev xeem tseeb, ob sab), thaum 20E piv rau cov kev tswj Tsuas yog ntawm cov koob tshuaj ntau dua (0.21 µg, P = 0.022; 0.13 µg, P = 0.0823; Fisher qhov kev xeem tseeb, ob sab).3D20E ua rau muaj kev tso qe ntau dua li 20E ntawm cov koob tshuaj ntau dua (0.21 µg, P = 0.0019; 0.13 µg, P = 0.075; Fisher qhov kev xeem tseeb, ob sab).Hauv txhua lub vaj huam sib luag, n sawv cev rau tus lej ntawm cov qauv yoov tshaj cum ywj pheej.NS, tsis tseem ceeb.*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.001.Cov ntaub ntawv yog los ntawm peb qhov rov ua dua.
Hauv kev tshawb fawb yav dhau los, peb tau txiav txim siab tias kev hloov pauv ntawm cov tshuaj hormones steroid los ntawm kev sib deev ua rau muaj kev qhia txog MISO (Mating-Induced Stimulator of Oogenesis 11), uas yog cov noob caj noob ces poj niam uas tiv thaiv A. gambiae cov poj niam los ntawm kev kis tus kab mob P. falciparum. Cov nqi kho mob tshwm sim los ntawm 13, tus kab mob malaria uas ua rau tib neeg tuag taus. Muab qhov tseem ceeb ntawm MISO rau kev ua kom muaj zog ntawm Anopheles hauv thaj chaw muaj kab mob malaria, peb tau txiav txim siab txiav txim siab seb cov tshuaj hormones twg 3D20E lossis 20E ua rau muaj kev qhia txog cov noob caj noob ces no. Peb pom tias thaum kev txhaj tshuaj 20E tshwj xeeb lossis muaj zog dua ua rau qee cov tshuaj hormones nuclear (HR), xws li HR3 thiab HR4, thiab cov hom phiaj steroid downstream, xws li cov noob caj noob ces yolkogenic Vg14, 15, 16, MISO tau raug tshoov siab ntau dua los ntawm 3D20E (Extended Data Fig. 3). Yog li, kev hloov pauv ntawm cov tshuaj hormones steroid androgenic no zoo li ua rau muaj cov txheej txheem uas tiv thaiv cov poj niam los ntawm cov nqi uas ua los ntawm kev kis tus kab mob parasitic. Ntxiv mus, 3D20E cuam tshuam rau ob qho tib si. cov isoforms ntawm E receptor EcR, ua rau EcR-A thiab txo EcR-B, thiab ua rau lwm cov noob sib deev muaj zog dua, suav nrog HPX15, uas cuam tshuam rau poj niam txoj kev muaj menyuam. Qhov no tuaj yeem piav qhia txog qhov tsis muaj menyuam tseem ceeb uas pom hauv cov poj niam sib deev nrog cov txiv neej EPP uas tsis muaj zog (Extended Data Fig. 3). Cov ntaub ntawv no qhia txog qhov muaj cov txoj hauv kev nqes mus uas ua haujlwm los ntawm ob lub tshuaj hormones ecdysone uas yuav ua rau muaj kev ua haujlwm tshwj xeeb ntawm poj niam txiv neej.
Tom ntej no, peb tau sim qhov kev ua haujlwm ntawm ob lub noob EcK uas tau txheeb xyuas hauv peb cov kab ke bioinformatics. Kev ntsiag to EcK1 lossis EcK2 ua rau muaj kev tuag ntau hauv cov txiv neej (Extended Data Fig. 4a), qhia tias ecdysone phosphorylation, thiab yog li ntawd inactivation, yog qhov tseem ceeb rau kev ciaj sia. Vim tias EcK2 tau qhia tawm ntawm cov qib siab dua EcK1 thiab tau kuaj pom hauv MAGs los ntawm proteomics (Daim duab 2b, c thiab Supplementary Table 2), peb tau lees paub nws cov haujlwm ecdysteroid kinase los ntawm kev sib xyaw nrog 20E, uas ua rau phosphorylation 20E22P (Extended Data Fig. 2). 4b). Thaum siv 3D20E ua lub substrate, peb tsis tuaj yeem pom cov khoom phosphorylated 3D20E22P (Extended Data Fig. 4c), qhia tias 20E es tsis yog 3D20E tej zaum yuav yog lub hom phiaj nyiam ntawm EcK2.
Raws li peb qhov kev tshuaj xyuas RNA-seq, EcK2 kuj tau qhia tawm ntau heev hauv LRT ntawm cov poj niam uas tsis tau muaj menyuam, qhov chaw uas nws tau kaw tom qab sib deev (Daim Duab 2b). Peb tau lees paub cov ntaub ntawv no thiab txiav txim siab tias EcK2 kev qhia tawm tsis raug cuam tshuam los ntawm kev noj ntshav (Extended Data Fig. 5a). Txuas ntxiv peb qhov kev sim MS thawj zaug, peb tau txiav txim siab tias qhov siab tshaj plaws ntawm 20E22P muaj feem cuam tshuam nrog qhov siab tshaj plaws ntawm 20E (22-26 teev tom qab noj ntshav; Extended Data Fig. 5b). Kev ntsiag to ntawm EcK2 hauv cov poj niam uas tsis tau muaj menyuam ua rau muaj kev nce 3 npaug ntawm qhov sib piv ntawm 20E rau 20E22P ntawm 26 teev tom qab noj ntshav (Extended Data Figures 2c thiab 5c), lees paub tias EcK2 kuj phosphorylates 20E hauv cov poj niam. Qhov tseem ceeb, cov poj niam uas tsis muaj EcK2 tau tswj hwm kev sib deev tag nrho (Extended Data Fig. 5d, e), ntxiv qhia tias cov poj niam tsim 20E tsis ua rau lub sijhawm sib deev tsis zoo. Txawm li cas los xij, cov poj niam no tau muaj Cov qe tso ntau dua piv rau cov neeg tswj, nrog ntau dua 30% ntawm cov ntxhais nkauj xwb tso qe (Extended Data Fig. 5f). Yog tias muaj ob txoj hlua Eck2 RNA (dsEcK2) txhaj tshuaj tom qab pub ntshav, kev tso qe tsis tshwm sim, thaum ntawd qhov siab tshaj plaws 20E vim kev noj ntshav tau poob qis. Zuag qhia tag nrho, cov txiaj ntsig no txhawb nqa tus qauv uas 20E tsim tawm tom qab haus ntshav tuaj yeem ua rau muaj kev tso qe, tab sis tsuas yog thaum lub thaiv kev tso qe (EcK2 thiab tej zaum lwm yam) raug tua los ntawm kev sib deev. Tsis yog 20E lossis 3D20E txhaj tshuaj tiv thaiv EcK2 kev qhia tawm hauv cov ntxhais nkauj xwb (Extended Data Fig. 5g), qhia tias lwm yam tseem ceeb ua rau muaj kev txwv ntawm kinase no. Txawm li cas los xij, cov qib 20E tom qab pub ntshav tsis txaus los ua rau muaj kev tsis xis nyob ntawm kev sib deev, tab sis tau ua rau muaj kev cuam tshuam los ntawm cov titers siab ntawm kev sib deev 3D20E.
Peb cov txiaj ntsig muab cov kev nkag siab tseem ceeb rau cov txheej txheem tswj kev vam meej ntawm A. gambiae. Ib qho qauv tau tshwm sim qhov twg cov txiv neej tau hloov zuj zus los tsim cov titers siab ntawm 3D20E, ib qho kev hloov kho ecdysone tshwj xeeb rau txiv neej uas lav qhov ua niam txiv los ntawm kev tsis hnov ​​​​​​mob rau cov poj niam kom sib deev ntxiv. Tib lub sijhawm, cov kab mob malaria no kuj tau hloov zuj zus ib lub kaw lus zoo los ua kom 3D20E hauv cov poj niam teb rau kev hloov pauv kev sib deev ntawm EPP tshwj xeeb rau txiv neej. Raws li peb paub, qhov no yog thawj qhov piv txwv ntawm cov tshuaj hormones steroid txiv neej thiab poj niam ua lub luag haujlwm tshwj xeeb thiab tseem ceeb hauv cov kab. Kev ua haujlwm tshwj xeeb ecdysone rau txiv neej tau raug tshaj tawm tab sis tsis tau ua pov thawj meej. Piv txwv li, ib qho kev xav tsis zoo 18 yog tias cov haujlwm no yuav raug ua los ntawm 20E precursor E1. Nws paub zoo tias hauv Drosophila, monandry yog tshwm sim los ntawm kev hloov pauv kev sib deev ntawm cov peptides me me 19,20 uas cuam tshuam nrog cov neurons innervating txoj kev sib deev poj niam los ntawm cov peptide receptors tshwj xeeb 21,22. Yuav tsum ua haujlwm ntxiv los txiav txim siab qhov downstream cov teeb liab qhia uas tswj los ntawm 3D20E hauv A. gambiae poj niam thiab txiav txim siab seb cov dej tsaws tsag no puas tuaj yeem khaws cia ntawm cov yoov tshaj cum thiab Drosophila.
Muab lub luag haujlwm tseem ceeb ntawm 3D20E rau kev muaj menyuam thiab tus cwj pwm ntawm poj niam uas tau txheeb xyuas hauv peb txoj kev tshawb fawb, cov kev coj ua rau 3D20E tsim thiab ua kom muaj kev ua haujlwm muab cov cib fim tshiab rau cov tswv yim tswj yoov tshaj cum yav tom ntej, xws li kev tsim cov txiv neej sib tw hauv cov tswv yim siv tshuaj tua kab mob siv rau kev tso tawm qus lossis ua raws li 3D20E hauv kev ua si nkauj xwb. Lub luag haujlwm tshwj xeeb ntawm txiv neej ntawm 3D20E tej zaum yuav tau hloov pauv thaum A. gambiae thiab lwm hom Cellia tau txais lub peev xwm los coagulate lawv cov phev rau hauv cov plugs mating, vim qhov no tso cai rau kev hloov pauv ntawm ntau cov tshuaj hormones thiab cov enzymes ua haujlwm hormone. Nyob rau hauv lem, 3D20E kev hloov pauv siv monandry muab lub tshuab rau cov poj niam (los ntawm kev qhia siab ntawm MISO) los txhawb lawv txoj kev muaj menyuam hauv thaj chaw muaj kev kis kab mob malaria ntau, uas tsis ncaj qha pab txhawb rau Plasmodium kis. Muab hais tias poj niam 20E tau pom tias muaj cov teebmeem loj heev rau kev ciaj sia thiab kev loj hlob ntawm P. falciparum hauv poj niam Anopheles yoov tshaj cum,24 ob qho tib si txiv neej thiab poj niam steroid hormone txoj kev tam sim no yog cov ntsiab lus tseem ceeb ntawm kev sib cuam tshuam ntawm yoov tshaj cum-parasite.
Cov kab A. gambiae G3 tau loj hlob hauv qab cov kab mob ib txwm muaj (26-28 °C, 65-80% av noo, 12:12 teev lub teeb/tsaus ntuj). Cov kab menyuam tau noj cov hmoov ntses (TetraMin Tropical Flakes, Koi Pellets thiab Tetra Pond Sticks hauv qhov sib piv ntawm 7:7:2). Cov yoov tshaj cum laus tau noj ad libitum 10% dextrose kua thiab cov ntshav tib neeg txhua lub lim tiam (kev kawm cov khoom xyaw ntshav). Cov yoov tshaj cum nkauj xwb tau los ntawm kev cais cov poj niam txiv neej thaum lub sijhawm menyuam tom qab kuaj xyuas cov kawg los ntawm kev siv lub tshuab microscopy. Cov txiv neej uas nqa DsRed transgene tau piav qhia ua ntej lawm.
Kev sim ua khub niam txiv uas yuam tau ua raws li cov txheej txheem tau piav qhia ua ntej. Rau kev sib deev ntuj, cov poj niam uas tsis tau muaj 4 hnub tau khaws cia rau hauv qhov sib piv 1: 3 nrog cov txiv neej uas tsis tau muaj hnub nyoog sib deev rau ob hmos. Rau kev sim uas cov txiv neej tau txhaj tshuaj nrog dsEPP, kev sib koom ua ke tau sib xws nrog hnub 3-4 tom qab txhaj tshuaj, thaum cov haujlwm phosphatase tau raug kaw kom siab tshaj plaws (Extended Data Fig. 2b).
Cov ntaub so ntswg ntawm yoov tshaj cum, cov neeg tuag uas tseem tshuav (qhov seem ntawm lub cev), lossis tag nrho lub cev tau raug txiav ua 100% methanol thiab homogenized nrog lub beader (2 hli iav hlaws dai, 2,400 rpm, 90 sec). Cov ntaub so ntswg thiab cov ntim methanol yog raws li nram no: qhov seem ntawm lub cev, 50 hauv 1,000 µl; MAG, 50–100 80 µl; poj niam LRT, 25–50 80 µl. Cov precipitate tau raug rau qhov thib ob methanol extraction nrog tib lub ntim ntawm methanol. Cov khib nyiab ntawm tes tau raug tshem tawm los ntawm centrifugation. Cov methanol los ntawm ob qho kev rho tawm tau sib xyaw thiab ziab hauv qab nitrogen ntws, tom qab ntawd resuspended hauv cov ntim hauv qab no ntawm 80% methanol hauv dej: qhov seem ntawm lub cev, 50 µl; MAGs thiab poj niam LRT, 30 µl.
Cov qauv kuaj tau raug tshuaj xyuas ntawm lub tshuab ntsuas huab hwm coj (ID-X, Thermo Fisher) txuas nrog lub cuab yeej LC (Vanquish, Thermo Fisher). 5 µl ntawm cov qauv tau txhaj rau ntawm 3 µm, 100 × 4.6 mm kem (Inspire C8, Dikma) khaws cia ntawm 25 ° C. Cov theem txav mus los rau LC yog A (dej, 0.1% formic acid) thiab B (acetonitrile, 0.1% formic acid). Qhov LC gradient yog raws li nram no: 5% B rau 1 feeb, tom qab ntawd nce mus rau 100% B dhau 11 feeb. Tom qab 8 feeb ntawm 100%, rov sib npaug kem ntawm 5% B rau 4 feeb. Tus nqi ntws yog 0.3 ml min-1. Ionization hauv qhov chaw MS yog ua tiav los ntawm kev ua kom sov electrospray ionization hauv hom zoo thiab tsis zoo.
Lub tshuab ntsuas qhov loj ntsuas cov ntaub ntawv hauv m/z ntau ntawm 350 txog 680 ntawm 60,000 qhov kev daws teeb meem hauv hom MS tag nrho. Cov ntaub ntawv MS/MS tau txais ntawm [M + H]+ (txhua lub hom phiaj), [M - H2O + H]+ (txhua lub hom phiaj), thiab [M - H]- (cov hom phiaj phosphorylated). Cov ntaub ntawv MS/MS tau siv los lees paub cov khoom ecdysone ntawm cov hom phiaj uas tsis muaj tus qauv muaj. Txhawm rau txheeb xyuas cov ecdysteroids tsis raug tsom, cov ntaub ntawv MS/MS rau txhua lub HPLC peaks nrog >15% qhov muaj ntau ntxiv tau raug soj ntsuam. Ntsuas siv cov qauv kab uas tsim los ntawm cov qauv ntshiab (20E, 3D20E) los xam cov nqi tag nrho lossis kev dilutions ntawm ib qho qauv tshwj xeeb (tag nrho lwm lub hom phiaj) los xam lawv qhov sib npaug rau cov nqi pom hauv ib tus txiv neej. Rau 3D20E, kev ntsuas tau ua tiav siv qhov sib sau ua ke ntawm cov khoom hauv qab no: [M + TFA]-, [M + COOH]-, [M + Na]+, [M + Cl]-, [M + NO3]-. Cov ntaub ntawv tau rho tawm thiab ntsuas siv Tracefinder (version 4.1). Cov ntaub ntawv MS/MS tau soj ntsuam siv Xcalibur (version 4.4). Cov spectra MS ntawm E, 20E thiab 3D20E tau piv rau cov qauv sib xws.3D20E22P tau soj ntsuam los ntawm kev derivatization nrog Girard's reagent.20E22P tau soj ntsuam los ntawm m/z piv.
3D20E22P tau raug lim los ntawm MAG. Kev lim dej tau ua tiav ntawm qhov ntsuas kev tshuaj xyuas siv cov kua chromatograph ua haujlwm zoo heev (Acquity, Waters) nrog lub tshuab ntes quadrupole mass-based (QDa, Acquity, Waters) nyob rau hauv tib lub LC mob li HPLC-MS/MS kev tshuaj xyuas. Kev sau ua feem tau tshwm sim thaum m/z sib raug rau 3D20E22P tau raug kuaj pom tib lub sijhawm khaws cia raws li tau txiav txim siab ua ntej. Qhov huv ntawm cov tshuaj rho tawm tau raug kuaj xyuas los ntawm HPLC-MS/MS raws li tau piav qhia saum toj no.
Tag nrho RNA tau rho tawm los ntawm 10-12 cov ntaub so ntswg yug me nyuam lossis lwm qhov ntawm lub cev (tsis muaj taub hau) siv TRI reagent (Thermo Fisher) ua raws li cov lus qhia ntawm cov chaw tsim khoom. RNA tau kho nrog TURBO DNase (Thermo Fisher). cDNA tau tsim los ntawm kev siv Moloney murine leukemia virus rov qab transcriptase (M-MLV RT; Thermo Fisher) ua raws li cov lus qhia ntawm cov chaw tsim khoom. Cov primers rau kev rov qab transcription quantitative PCR (RT-qPCR; Extended Data Table 2) tau luam tawm ua ntej24 lossis tsim los ntawm kev siv Primer-BLAST26, nrog rau kev nyiam rau cov khoom ntawm 70-150 bp hauv qhov loj me thiab spanning exon-exon junctions lossis Primer pair primers cais exons. cDNA cov qauv los ntawm peb mus rau plaub qhov kev rov ua dua tshiab tau diluted plaub npaug hauv dej rau RT-qPCR. Kev ntsuas tau ua tiav hauv 15 µl replicate reactions uas muaj 1 × PowerUp SYBR Green Master Mix (Thermo Fisher), primers, thiab 5 µl ntawm diluted cDNA. Cov tshuaj tiv thaiv tau khiav ntawm a QuantStudio 6 Pro real-time PCR system (Thermo Fisher) thiab cov ntaub ntawv tau sau thiab tshuaj xyuas siv Design and Analysis (version 2.4.3). Raws li tau pom hauv kev tshawb fawb no, cov nqi sib piv tau raug kho kom zoo rau ribosomal gene RpL19 (AGAP004422), uas nws qhov kev qhia tsis hloov pauv ntau nrog kev pub ntshav 27 lossis kev sib deev 3.
RNA zoo tau kuaj xyuas siv Agilent Bioanalyzer 2100 Bioanalyzer (Agilent). Illumina paired-end libraries tau npaj thiab khiav ntawm Broad Institute ntawm MIT thiab Harvard. Sequencing nyeem tau sib phim rau A. gambiae genome (PEST strain, version 4.12) siv HISAT2 (version 2.0.5) nrog cov kev teeb tsa tsis siv neeg. Nyeem nrog mapping zoo (MAPQ) qhab nia <30 tau raug tshem tawm siv Samtools (version 1.3.1). Tus naj npawb ntawm kev nyeem mapped rau cov noob tau suav siv htseq-count (version 0.9.1) nrog cov kev teeb tsa tsis siv neeg. Cov lej nyeem normalized tau suav thiab kev qhia tawm noob sib txawv tau soj ntsuam siv DESeq2 pob (version 1.28.1) hauv R (version 4.0.3).
Cov neeg sib tw hloov kho Ecdysone tau txheeb xyuas los ntawm kev tshawb nrhiav thawj zaug ntawm A. gambiae genome siv PSI-BLAST algorithm (https://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/2.8.1/), siv cov nqi tsis siv neeg nrog cov lus nug protein sequences hauv qab no: los ntawm Bombyx mori (Accession No. NP_001038956.1), Musca domestica (Accession No. XP_005182020.1, XP_005175332.1 thiab XP_011294434.1) thiab Microplitis demolitor (Accession No. XP_008552646.1 thiab XP_008552645.1) EcK los ntawm B. mori (Accession No. NP_001036900), Drosophila melanogaster (Accession No. NP_651202), Apis mellifera (Action No. XP_394838) thiab Acyrthosiphon pisum (Action No. XP_001947166); thiab EPP los ntawm B. mori (Action No. XP_001947166) NP_001177919.1 thiab NP_001243996.1) thiab EO ntawm D. melanogaster (Action No. NP_572986.1) (kauj ruam 1). Tom ntej no, lim cov hits raws li kev qhia mRNA siab (>100 fragments/kilobase exons ib lab mapped nyeem (FPKM) lossis >85%) hauv cov ntaub so ntswg reproductive (poj niam LRT lossis MAG) hauv Gambia (kauj ruam 2). Txhawm rau txhim kho qhov tshwj xeeb, peb tau xaiv cov enzymes sib tw uas kuj tau qhia tawm hauv cov ntaub so ntswg ntawm A. albimanus, ib hom anopheles uas tsis tsim lossis hloov ecdysone thaum lub sijhawm sib deev. Cov noob sib tw tau lim raws li kev qhia tawm qis (<100 FPKM lossis <85th feem pua) hauv A. albimanus cov ntaub so ntswg (kauj ruam 3). Raws li qhov lim zaum kawg (kauj ruam 4), cov noob sib tw yuav tsum txaus siab tsawg kawg ib qho ntawm cov hauv qab no: (1) nce siab heev tom qab sib deev (P < 0.05) raws li kev tshuaj xyuas ntawm cov noob sib txawv thiab (2) hauv cov ntaub so ntswg tsis muaj menyuam (< 85% lossis <100 FPKM).
Peb tau hloov kho cov txheej txheem piav qhia ua ntej 28, 29, 30 kom ua tiav tag nrho cov kab mob isotopic labeling. Luv luv, hom tsiaj qus Saccharomyces cerevisiae hom II (YSC2, Sigma) tau sim hauv cov poov xab nitrogen puag (BD Difco, DF0335) uas muaj (wt / vol) 2% glucose (G7528, Sigma), 1.7% amino acid-free thiab ammonium sulfate. kab lis kev cai nruab nrab) thiab 5% 15N ammonium sulfate (NLM-713, >99%, Cambridge Isotope Laboratories) ua qhov chaw tsuas yog ntawm nitrogen. Cov poov xab tau rov qab los ntawm centrifugation thiab cov kab laug sab yoov tshaj cum tau pub ad libitum kom txog thaum pupation. Ntxiv nrog ntses hmoov (0.5 mg ib 300 tus kab laug sab) kom tiv thaiv kev tuag thib plaub. Tsuas yog cov poj niam tau siv rau hauv kev sim mating nrog cov txiv neej tsis muaj daim ntawv lo los tshuaj xyuas cov txiv neej proteome hloov pauv thaum lub sijhawm mating.
Cov poj niam uas tsis tau muaj hnub nyoog 4-6 hnub uas muaj daim ntawv cim 15N raug yuam kom sib yuav nrog cov txiv neej uas tsis tau muaj daim ntawv cim uas muaj hnub nyoog sib xws. Kev sib yuav tau ua tiav los ntawm kev kuaj pom cov ntsaws sib yuav hauv qab lub tshuab kuaj mob epifluorescence. Thaum 3, 12, thiab 24 hpm, lub atria ntawm 45-55 tus poj niam uas tau sib yuav tau raug txiav ua 50 µl ntawm ammonium bicarbonate buffer (pH 7.8) thiab homogenized nrog lub pestle. Cov homogenate tau centrifuged thiab cov supernatant sib xyaw nrog 50 µl ntawm 0.1% RapiGest (186001860, Waters) hauv 50 mM ammonium bicarbonate. Cov supernatant thiab pellet ntawm txhua tus qauv tau snap khov rau ntawm cov dej khov qhuav thiab xa mus rau MacCoss laboratory ntawm University of Washington ib hmos, qhov chaw uas kev npaj qauv rau LC-MS/MS tau ua tiav. Rov ua dua cov pellet hauv 50 µl ntawm 0.1% RapiGest hauv 50 mM ammonium. bicarbonate thiab sonicate hauv dej da dej. Cov protein concentration ntawm cov pellet thiab supernatant tau ntsuas los ntawm BCA assay, cov qauv tau txo nrog 5 mM dithiothreitol (DTT; Sigma), alkylated nrog 15 mM iodoacetamide (Sigma) thiab incubated ntawm 37 ° C (1:0 50) rau 1 teev nrog trypsinization: trypsin:substrate ratio). RapiGest tau lysed los ntawm kev ntxiv ntawm 200 mM HCl, ua raws li kev incubation ntawm 37 ° C rau 45 feeb thiab centrifugation ntawm 14,000 rpm rau 10 feeb ntawm 4 ° C kom tshem tawm cov khib nyiab. Cov qauv tau ntxuav los ntawm dual-mode solid-phase extraction (Oasis MCX cartridges, Waters) thiab resuspended hauv 0.1% formic acid rau qhov kawg protein concentration ntawm 0.33 µg µl-1. Unlabeled MAG proteomes tau soj ntsuam zoo sib xws los ntawm cov txiv neej nkauj xwb. Ob qho kev tshuaj xyuas rov ua dua tau raug tshuaj xyuas rau txhua tus qauv. Tom ntej no, 1 µg ntawm txhua tus tau raug tshuaj xyuas siv 25-cm fused silica 75-μm kem nrog 4-cm fused silica Kasil1 (PQ) frit trap ntim nrog Jupiter C12 reversed-phase resin (Phenomenex) thiab 180-feeb kua chromatography. Cov qauv zom zaub mov - MS/MS tau khiav ntawm Q-Exactive HF mass spectrometer (Thermo Fisher) nrog nanoACQUITY UPLC System (Waters). Cov ntaub ntawv txog kev tau txais cov ntaub ntawv tsim rau txhua qhov kev khiav tau hloov mus rau hom ntawv mzML siv Proteowizard (version 3.0.20287) thiab siv Comet31 (version 3.2) tawm tsam FASTA database uas muaj cov protein sequences los ntawm Anopheles gambiae (VectorBase version 54), Anopheles coluzzi Ib qho kev tshawb nrhiav tau ua rau Mali-NIH (VectorBase version 54), Saccharomyces cerevisiae (Uniprot, Lub Peb Hlis 2021), A. gambiae RNA-seq, thiab peb-frame translations ntawm cov neeg paub cov pa phem. Peptide daim ntawv qhia-matched FDRs tau txiav txim siab siv Percolator32 (version 3.05) nrog qhov txwv ntawm 0.01, thiab peptides tau sib sau ua ke rau hauv cov protein identifications siv protein parsimony hauv Limelight33 (version 2.2.0). Qhov muaj protein ntau npaum li cas tau kwv yees siv qhov normalized spectral abundance factor (NSAF) xam rau txhua tus protein hauv txhua qhov kev khiav raws li tau piav qhia ua ntej. NSAF piv rau txhua tus protein tau nruab nrab ntawm cov qauv los ntawm ob qhov kev rov ua dua ntawm cov tsiaj txhu. 15N daim ntawv lo tau ua tiav zais cov proteome poj niam, txawm hais tias muaj me ntsis ntawm cov protein tsis muaj daim ntawv lo tuaj yeem pom los ntawm cov ntxhais nkauj xwb. Peb tau sau tseg qhov kev pom ntawm cov txiv neej txo cov protein (1-5 spectra) hauv cov poj niam cov qauv nyoos tsuas yog hauv kev khiav haujlwm, qhov twg cov qauv nyoos tau khiav tom qab cov qauv txiv neej / mating, vim yog HPLC "nqa mus". Qee zaum cov protein pom tias yog 'cov pa phem' los ntawm cov ntxhais nkauj xwb tau teev nyob rau hauv Supplementary Table 1.
Ob tug antigenic peptides, QTTDRVAPAPDQQQ (nyob rau hauv isotype PA) thiab MESDGTTPSGDSEQ (nyob rau hauv isotype PA thiab PB) nyob rau hauv Genscript. Ob tug peptides tau muab ua ke, ces conjugated rau tus carrier protein KLH thiab txhaj rau hauv New Zealand luav. Cov luav tau raug tua tom qab txhaj tshuaj zaum plaub, thiab tag nrho IgG tau raug rho tawm los ntawm affinity purification. IgG los ntawm feem ntau EPP-specific luav tau siv rau ntxiv western blotting.
Rau cov Western blots, MAG (n = 10, qhov twg n sawv cev rau tus lej ntawm cov qauv yoov tshaj cum ywj pheej) thiab poj niam LRT (n = 30) los ntawm cov txiv neej 4-hnub-laus thiab cov poj niam virgin lossis force-mated (<10 post-mating), Protein extraction buffer (50 mM Tris, pH 8.0; 1% NP-40; 0.25% sodium deoxycholate; 150 mM NaCl; 1 mM EDTA; 1 × protease inhibitor cocktail (Roche)) tau ntxiv cais. Cov qauv tau homogenized tam sim ntawd tom qab dissection nrog lub beader (2 hli iav hlaws dai, 2,400 rpm, 90 sec). Cov khib nyiab tsis yaj tau raug tshem tawm los ntawm centrifugation ntawm 20,000 g ntawm 4 ° C. Cov protein tau ntsuas los ntawm Bradford assay (Bio-Rad). Tom qab ntawd, 20 µg ntawm MAG protein, 40 µg ntawm LRT protein, thiab 20 µg ntawm cov protein seem tau denatured thiab sib cais los ntawm 10% Bis-Tris NuPAGE siv MOPS buffer. Cov protein tau hloov mus rau polyvinylidene fluoride daim nyias nyias siv lub iBlot2 hloov pauv system (Thermo Fisher). Daim nyias nyias tau ntxuav ob zaug hauv 1 × PBS-T (0.1% Tween-20 hauv PBS) thiab tom qab ntawd thaiv hauv Odyssey blocking buffer (Li-Cor) rau 1 teev ntawm 22 ° C. Daim nyias nyias tau co ib hmos ntawm 4 ° C nrog kev cai luav anti-EPP polyclonal thawj antibody (1: 700 hauv blocking buffer) thiab nas anti-actin monoclonal thawj antibody MAC237 (Abeam; 1: 4,000). Daim nyias nyias tau ntxuav nrog PBS-T thiab tom qab ntawd incubated nrog cov tshuaj tiv thaiv theem nrab (donkey anti-rabbit 800CW thiab tshis anti-rat 680LT (Li-Cor), ob qho tib si 1: 20,000) hauv blocking buffer uas muaj 0.01% SDS thiab 0.2% Tween -20. rau 1 teev ntawm 22 ° C. Cov ntaub so ntswg tau ntxuav nrog PBS-T thiab thaij duab nrog Odyssey CLx scanner. Cov duab tau sau thiab ua tiav hauv Image Studio (version 5.2). Ib qho band tshwj xeeb sib raug rau EPP-RA isoform (82 kDa) tsis tau pom.
Cov cheeb tsam coding ntawm EPP (raws li isoform AGAP002463-RB uas muaj histidine phosphatase domain, NCBI conserved domain search 34) thiab EcK2 (AGAP002181) tau cloned rau hauv pET-21a (+) plasmid (Novagen Millipore Sigma); Cov primers tau teev nyob rau hauv Cov Ntaub Ntawv Txuas Ntxiv 2. Yim GS4 txuas (ua ke) tau ntxig ua ntej C-terminal 6xHis tag ntawm pET-21a (+) -EcK2 construct. Cov protein recombinant tau tsim los ntawm kev siv NEBExpress cell-free E. coli protein synthesis reaction (New England BioLabs). Cov protein recombinant tau purified siv NEBExpress Ni spin columns (New England BioLabs). Dihydrofolate reductase (DHFR) tswj protein tau tsim los ntawm kev siv DNA template los ntawm NEBExpress Cell-Free E. coli Protein Synthesis Kit. Cov protein tau khaws cia rau hauv 50% glycerol hauv PBS ntawm -20 ° C rau txog 3 lub hlis.
Kev ua haujlwm phosphatase ntawm EPP thiab cov ntaub so ntswg tau ntsuas siv 4-nitrophenyl phosphate (pNPP; Sigma-Aldrich). Lub buffer tshuaj tiv thaiv muaj 25 mM Tris, 50 mM acetic acid, 25 mM Bis-Tris, 150 mM NaCl, 0.1 mM EDTA, thiab 1 mM DTT. Cov ntaub so ntswg tau homogenized hauv lub buffer tshuaj tiv thaiv thiab cov khib nyiab ntawm tes tau raug tshem tawm los ntawm centrifugation. Pib qhov tshuaj tiv thaiv los ntawm kev ntxiv enzyme lossis cov ntaub so ntswg extract rau lub buffer tshuaj tiv thaiv uas muaj 2.5 mg ml-1 pNPP. Cov tshuaj tiv thaiv sib tov tau incubated ntawm chav tsev kub hauv qhov tsaus ntuj, thiab qhov ntau ntawm pNP hloov pauv los ntawm pNPP tau ntsuas los ntawm kev ntsuas qhov absorbance ntawm 405 nm ntawm ntau lub sijhawm.
Rau kev ua haujlwm EcK hauv vitro, cov protein tau incubated nrog 0.2 mg 20E lossis 3D20E hauv 200 µl buffer (pH 7.5) uas muaj 10 mM HEPES-NaOH, 0.1% BSA, 2 mM ATP thiab 10 mM MgCl2 rau 2 teev ntawm 27 ° C. Qhov kev tshuaj tiv thaiv tau nres los ntawm kev ntxiv 800 µl methanol, tom qab ntawd txias ntawm -20 ° C rau 1 teev, tom qab ntawd centrifuged ntawm 20,000 g rau 10 feeb ntawm 4 ° C. Lub supernatant tom qab ntawd tau soj ntsuam los ntawm HPLC-MS / MS. Txhawm rau kom sov-inactivate cov protein siv hauv pawg tswj hwm, cov protein tau incubated hauv 50% glycerol hauv PBS rau 20 feeb ntawm 95 ° C.
Rau kev ua haujlwm EPP hauv vitro, cov protein tau incubated nrog 3D20E22P (sib npaug rau qhov nyiaj pom hauv 18 khub ntawm MAG, purified los ntawm HPLC-MS / MS) hauv 100 µl buffer (pH 7.5) uas muaj 25 mM Tris, 50 mM acetic acid, 25 mM Bis-Tris, 150 mM NaCl, 0.1 mM EDTA, thiab 1 mM DTT rau 3 teev ntawm 27 ° C. Qhov tshuaj tiv thaiv tau nres los ntawm kev ntxiv 400 µl methanol thiab txias ntawm -20 ° C rau 1 teev, tom qab ntawd centrifuged ntawm 20,000 g rau 10 feeb ntawm 4 ° C. Lub supernatant tau soj ntsuam los ntawm HPLC-MS / MS.
Cov khoom PCR rau EPP (362 bp), EcK1 (AGAP004574, 365 bp) thiab EcK2 (556 bp) tau raug nthuav dav los ntawm cDNA uas tau npaj los ntawm cov kab laug sab tsis muaj taub hau sib xyaw. Cov khoom PCR ntawm eGFP tswj (495 bp) tau raug nthuav dav los ntawm pCR2.1-eGFP uas tau piav qhia ua ntej; Cov PCR primers tau teev nyob rau hauv Extended Data Table 2. Cov PCR fragment tau ntxig rau ntawm cov inverted T7 promoters ntawm pL4440 plasmid. Cov plasmid constructs tau rov qab los ntawm NEB 5-α competent E. coli (New England Biolabs) thiab tau txheeb xyuas los ntawm DNA sequencing ua ntej siv (saib Supplementary Data 1 rau cov kab lus ntxig). Cov primers phim rau T7 promoter (Extended Data Table 2) tau siv los ua kom cov ntxig los ntawm pL4440-based plasmid loj dua. PCR khoom loj tau lees paub los ntawm agarose gel electrophoresis. dsRNA tau sau los ntawm PCR templates siv Megascript T7 Transcription Kit (Thermo Fisher) thiab purified raws li cov lus qhia ntawm cov chaw tsim khoom nrog cov kev hloov kho tau piav qhia ua ntej.
Rau kev txhaj tshuaj dsRNA, 1,380 ng ntawm dsRNA (dsGFP, dsEcK1, dsEcK2, dsEPP) tau txhaj rau ntawm qhov concentration ntawm 10 ng nl-1 rau hauv lub hauv siab ntawm cov txiv neej lossis poj niam laus (Nanoject III, Drummond) hauv 1 hnub tom qab eclosion. Cov theem ntawm kev knockdown tau txiav txim siab hauv tsawg kawg peb qhov kev rov ua dua los ntawm RNA extraction, cDNA synthesis, thiab RT-qPCR. Rau kev txhaj tshuaj ecdysone, cov poj niam uas muaj 4-hnub lossis 6-hnub uas muaj ntshav tau txhaj nrog 0.13, 0.21, lossis 0.63 µg ntawm 20E lossis 3D20E (Nanoject III, Drummond) ntawm qhov concentration ntawm 1.3, 2.1, raws li, nyob ntawm qhov kev tsim qauv sim lossis 6.3 ng nl-1. Txhaj 100 nl ntawm 10% (vol/vol) ethanol hauv dej; 100 nl ntawm 3D20E22P hauv 10% ethanol (sib npaug rau 75% ntawm qhov ntau uas pom muaj nyob rau hauv ib khub MAGs). Cov yoov tshaj cum tau raug xaiv rau pawg txhaj tshuaj.
Rau kev kuaj xyuas cov qe menyuam, cov poj niam uas muaj 3 hnub tau noj cov ntshav tib neeg tsis tu ncua. Tshem tawm cov yoov tshaj cum uas tau noj ib ntus lossis tsis tau noj. Nyob ntawm seb kev kho mob zoo li cas, cov poj niam tau muab tso rau hauv cov khob qe sib cais rau plaub hmos tsawg kawg 48 teev tom qab noj cov ntshav. Cov qe tau suav nrog hauv lub stereoscope (Stemi 508, Zeiss); rau cov poj niam uas tau sib yuav, cov qe uas tawm los ua cov kab menyuam tau suav tias yog cov menyuam muaj menyuam.
Rau kev sim sib deev, cov poj niam tau tso cai tsawg kawg 2 hnub nyob ntawm kev kho mob kom tsim kev tiv thaiv kev sib deev, thiab cov txiv neej qus-hom hnub nyoog sib phim tau raug coj mus rau hauv tib lub tawb. Ob hmos tom qab, cov poj niam fertilized vesicles tau txiav thiab genomic DNA tau tso tawm los ntawm freeze-thaw thiab sonication hauv lub buffer uas muaj 10 mM Tris-HCl, 1 mM EDTA, thiab 25 mM NaCl (pH 8.2). Cov qauv tau incubated nrog Proteinase K (0.86 µg µl-1) rau 15 feeb ntawm 55 ° C, ua raws li 10 feeb ntawm 95 ° C. Crude genomic DNA npaj tau diluted 10-npaug thiab raug qPCR nrhiav tau ntawm Y chromosome sequences; primers tau teev nyob rau hauv Extended Data Table 2. Tsis muaj Y chromosome sequence qhia tias tsis muaj kev sib deev.
Rau kev ntsuam xyuas rov sib yuav, cov poj niam uas raug yuam kom sib yuav raug kuaj xyuas seb puas muaj cov ntsaws sib yuav kom paub meej tias muaj kev sib yuav thiab tso cai 2 hnub los tsim kev tsis kam sib yuav thaum tsis muaj txiv neej, raws li tau piav qhia ua ntej 36. Cov txiv neej uas nqa DsRed transgenic phev ces raug coj mus rau hauv cov tawb poj niam. Ob hmos tom qab, cov vesicles fertilizing tau raug txiav tawm ntawm cov poj niam, thiab genomic DNA tau npaj raws li tau piav qhia saum toj no thiab raug qPCR kuaj pom ntawm DsRed transgene; cov primers tau teev nyob rau hauv Extended Data Table 2. Qhov tsis muaj DsRed transgene qhia tias tsis muaj kev sib yuav rov qab tshwm sim.
3D20E tau tsim ua raws li tau piav qhia ua ntej 37. Hauv luv luv, 10 mg ntawm 20E (Sigma-Aldrich) tau yaj rau hauv 10 ml dej, ua raws li ntxiv 30 mg ntawm platinum dub (hauv daim ntawv hmoov, Sigma-Aldrich). Ib qho dej ntws maj mam ntawm O2 tau txuas ntxiv mus rau hauv cov tshuaj sib tov, uas tau do ntawm chav sov. Tom qab 6 teev, 30 mL ntawm methanol tau ntxiv los nres qhov tshuaj sib tov. Cov tshuaj sib tov tau centrifuged kom tshem tawm cov khoom catalyst. Cov supernatant tau evaporated kom qhuav hauv vacuo ntawm chav sov. Cov khoom qhuav tshuaj sib tov tau yaj rau hauv 10% ethanol thiab methanol rau kev txhaj tshuaj rau HPLC-MS / MS kev tshuaj xyuas. Tus nqi hloov pauv (los ntawm 20E rau 3D20E) yog kwv yees li 97% (Daim duab 4b), thiab MS spectrum ntawm cov tshuaj sib tov 3D20E sib phim uas pom muaj nyob rau hauv cov poj niam sib yuav (Daim duab 4c).
Cov lus piav qhia muaj cov ntsiab lus tshwj xeeb ntawm cov kev xeem suav lej uas tau ua. GraphPad (version 9.0) tau siv los ua Fisher qhov kev xeem tseeb, Mantel-Cox kev xeem, thiab Student's t-test. Cochran-Mantel-Haenszel kev xeem tau ua tiav siv cov ntawv sau R (muaj nyob ntawm https://github.com/duopeng/mantelhaen.test). Kev faib cov ntaub ntawv tau sim rau qhov ib txwm siv kev xeem Shapiro-Wilk nrog qhov tseem ceeb ntawm 0.05. Thaum cov ntaub ntawv tsis ua tiav qhov kev xeem ib txwm, Mann-Whitney kev xeem tau ua tiav. Cov ntaub ntawv ciaj sia tau soj ntsuam siv kev xeem Mantel-Cox. Lub pob DESeq2 (version 1.28.1) tau siv los ua RNA-seq gene-level differential expression analysis. Lub kab kab rov tav ntawm daim duab sawv cev rau qhov nruab nrab. Tus nqi tseem ceeb ntawm P = 0.05 tau siv ua qhov txwv rau txhua qhov kev xeem.
Yog xav paub ntxiv txog kev tsim txoj kev tshawb fawb, mus saib daim ntawv qhia txog Kev Tshawb Fawb Txog Xwm Txheej uas txuas nrog tsab xov xwm no.
Cov ntaub ntawv MS proteomic tau muab tso rau hauv ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) los ntawm PRIDE Partner Repository (https://www.ebi.ac.uk/pride/) nrog cov ntaub ntawv qhia txog tus lej PXD032157.
Cov ntaub ntawv RNA-seq tau muab tso rau hauv Gene Expression Comprehensive Library (https://www.ncbi.nlm.nih.gov/geo/) nyob rau hauv cov ntaub ntawv serial GSE198665.
Cov ntaub ntawv ntxiv uas tau tsim thiab/lossis tshuaj xyuas thaum lub sijhawm kawm tam sim no yuav tau txais los ntawm cov kws sau ntawv sib xws thaum muaj kev thov tsim nyog. Tsab xov xwm no muab cov ntaub ntawv qhov chaw.
De Loof, A. Ecdysteroids: Cov tshuaj steroids sib deev uas tsis quav ntsej?Txiv neej: Black Box.Insect Science.13, 325–338 (2006).
Redfern, CPF 20-hydroxyecdysone thiab kev loj hlob ntawm zes qe menyuam hauv Anopheles stephens.J. Insect Physiology.28, 97–109 (1982).