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Kusiyana nemhuka dzine mapfupa emusana, zvipembenene zvinofungidzirwa kuti hazvina mahormone ebonde anotsigira varume. MuAnopheles gambiae, ecdysone steroid 20-hydroxyecdysone (20E) inoita kunge yakashanduka kudzora kukura kwemazai kana akagadzirwa nevakadzi2 uye kukonzera nguva yekusangana isingashande kana yatamisirwa pabonde nevarume3. Sezvo kukura kwemazai nekusangana kuri hunhu hwakakosha hwekubereka, kunzwisisa kuti umhutu hweAnopheles hwechikadzi hunobatanidza sei zviratidzo izvi zvemahomoni kunogona kubatsira kugadzirwa kwezvirongwa zvitsva zvekudzora malaria. Pano, tinozivisa kuti mabasa aya ekubereka anodzorwa nemasteroid akasiyana ebonde kuburikidza ne network yakaoma ye ecdysteroid-activating/inactivating enzymes. Takaona ecdysone yakasanganiswa nevarume, 3-dehydro-20E (3D20E), inodzivirira mubereki nekudzima kugamuchira pabonde kwevakadzi mushure mekutamisirwa pabonde uye kuvhurwa ne dephosphorylation. Zvinonyanya kukosha, kutamisirwa kwe3D20E kwakakonzerawo kuratidzwa kwemajini ekubereka anochengetedza kukura kwemazai panguva yehutachiona hwePlasmodium, zvichiita kuti hutano hwevakadzi vane hutachiona huvepo. 20E inobva kuvakadzi haiburitse maitiro epabonde, asi inobvumira vanhu vanoberekesana kuti vaise mazai mushure mekunge 20E-inhibiting kinases yadziviswa. Kuzivikanwa kweiyi steroid hormone yezvipembenene zvevarume uye basa rayo mukutonga kugamuchira kwevakadzi pabonde, kubereka uye kusangana nePlasmodium zvinoratidza mukana wekuderedza kubudirira kwekubereka kweumhutu hunotapurirana malaria.
Nyaya dzemarariya nerufu zviri kuwedzera zvakare4 nekuda kwekusakwanisa kuuraya zvipembenene muumhutu hweAnopheles, uhwo chete hunokonzera tupukanana twemarariya kuvanhu. Kusangana kweumhutu uhwu kunokwezva zvikuru pakurwisa malaria itsva nekuti hukadzi hunongosangana kamwe chete5; kuita kuti kusangana uku kuve kusina utachiona kungave nemukana mukuru wekuderedza kuwanda kweumhutu mumunda.
Vakadzi vanotadza kuita zvepabonde mushure mekugamuchira mahormone e steroid ane titer kubva kuvarume. Zvidzidzo zvakaratidza kuti chinokonzera kuoma kwekubatana kwepabonde i20-hydroxyecdysone (20E), hormone ye steroid inozivikanwa semutongi we molting cycle muchikamu chemakonzo. Kugona kwevarume kugadzira nekufambisa 20E kwashanduka zvakanyanya mumarudzi eAnopheles ari chikamu cheCellia7, iyo inowanikwa muAfrica uye inosanganisira hutachiona hune njodzi huru hwemarariya, kusanganisira Anopheles gambiae. Izvi zvinonyanya kukosha nekuti mumarudzi aya vakadzi vanogadzirawo 20E mushure mekudya kwega kwega kweropa, uye 20E inotungamidza oogenesis cycle (ona ref. 8). Zvisinei, zvishoma zvinozivikanwa nezvenzira iyo vakadzi vanobatanidza nayo zviratidzo kubva kumarudzi maviri akasiyana eecdysone (kutapurirana kwevarume uye kupihwa ropa) pasina kukanganisa kugona kwavo kufambidzana. Kutaura zvazviri, kana 20E inogadzirwa nevakadzi ikakonzera kusashivirirana pabonde, izvi zvinotungamira mukushaya mbereko muvanhu vanoyamwisa mhandara, maitiro akajairika muumhutu uhwu5.
Tsananguro ingangodaro ndeyekuti varume veA. gambiae vanotamisa ecdysone yakagadziriswa yemurume, iyo inomutsa kuyerera kwezviratidzo munzira yekubereka yemukadzi, zvichikonzera kusagadzikana kwekusangana. Zvisinei, kunyange zvazvo mhuka dzine vertebrate dzine mahormone akawanda esteroid, akadai seestrogen neandrogen (akaongororwa mu ref. 9), maererano neruzivo rwedu, masteroid ane androgenic-biased haana kuwanikwa muzvipembenene.
Takaronga kuona huwandu hwemahormone esteroid mugland remurume (MAG) reA. gambiae yakura pabonde tichitsvaga masteroid anogona kugadziriswa. Tichishandisa high performance liquid chromatography pamwe chete ne tandem mass spectrometry (HPLC-MS/MS) pane nzira isina kunyanya kushandiswa, takaona ecdysone (E) ne20E munyama iyi, tichisimbisa mhedzisiro yapfuura. Zvisinei, sampuro yacho yaive yakawanda nemasteroids e oxidized phosphorylated, zvinoenderana nefomula 3-dehydro-20E-22-phosphate (3D20E22P)12 (Mufananidzo 1). Mamwe mafomu anosanganisira 3-dehydro-20E (3D20E) uye 20E-22-phosphate (20E22P). HPLC-MS/MS signal intensity ye3D20E22P yaive miviri mikuru kupfuura dephosphorylated form yayo, 3D20E, uye mitatu mikuru kupfuura iyo yeE ne20E (Mufananidzo 1). Kunyangwe mune dzimwe nzvimbo dzemuviri. uye nzira yekubereka yepazasi (LRT; Extended Data Fig. 1a). Takaongororawo ma ecdysteroids muvarume nevakadzi vachangobva kuvharwa (pasi pezuva rimwe chete) uye takaona 3D20E ne3D20E22P chete muMAG; E, 20E ne20E22P zvaivepo muvarume nevakadzi vese (Extended Data Fig. 1b). Data iri rinoratidza kuti varume vakuru veA. gambiae vanogadzira ma titers akawanda ema modifying hormones muMAG dzavo dzisingagadzirwe nevakadzi.
MAG neLRT yechikadzi (kusanganisira atria, seminal vesicles, uye parovarium) zvakabviswa kubva kuvarume vane mazuva mana (4 ekuberekwa) nevasikana vakaroorwa (0.5, 3, uye 12 hpm). Ecdysone mumatishu aya yakaongororwa neHPLC-MS/MS (avhareji ± sem; unpaired t-test, two-sided, false discovery rate (FDR) yakagadziriswa; NS, haina kukosha; *P < 0.05, **P < 0.01 . 3D20E: maawa matatu vs. maawa 0.5, P = 0.035; maawa gumi nemaviri vs. maawa matatu, P = 0.0015; maawa gumi nemaviri vs. maawa 0.5, P = 0.030. 3D20E22P: maawa matatu vs. maawa 0.5, P = 0.25; maawa gumi nemaviri vs. maawa matatu, P = 0.0032; Maawa gumi nemaviri vs. maawa 0.5, P = 0.015). Data rinobva muzvikamu zvitatu zvehupenyu. Nzvimbo yepamusoro ye ecdysone yega yega yaifarirwa yakaverengerwa uye yakajairika nehuwandu hweumhutu. Ecdysone inomiririrwa neruvara seizvi: E, girini; 20E, orenji; 20E22P, yepepuru; 3D20E, bhuruu; 3D20E22P, pingi. Chinyorwa chinowedzera chiyero pa y-axis kuratidza mazinga akaderera e ecdysone.
Kuti tiongorore kana 3D20E22P ne3D20E zvichitapurirwa panguva yekusangana, takaongorora maLRT echikadzi panguva dzakasiyana-siyana mushure mekusangana. Kunyangwe ecdysone isina kuwanikwa mumhandara, takaona huwandu hwakawanda hwe3D20E22P muLRT mushure mekusangana (0.5 h mushure mekusangana, hpm), huchidzikira nekufamba kwenguva, nepo mazinga e3D20E akawedzera zvakanyanya (Mufananidzo 1). Tichishandisa 3D20E yakagadzirwa nemakemikari sechiyero, takaona kuti mazinga ehomoni iyi yesteroid muLRTs dzekusangana aive akakwira kanenge ka100 pane 20E (Extended Data Table 1). Saka, 3D20E22P ndiyo ecdysone huru yechirume inotapurirwa kuLRT yechikadzi panguva yekusangana, uye chimiro chayo che dephosphorylated, 3D20E, chinova chakawanda nguva pfupi mushure mekusangana. Izvi zvinoratidza basa rakakosha re ecdysone yekupedzisira mubiology yevakadzi mushure mekusangana.
Mushure mekugadzira data idzva reRNA sequencing (RNA-seq) (Mufananidzo 2a), tichishandisa pombi ye bioinformatics yakagadzirwa, takatsvaga ecdysone kinase (EcK), ecdysone oxidase (EO), uye ecdysone encoding 20E-modified phosphatase gene.EPP) inoratidzwa mumatishu ekubereka.Takawana gene rimwe chete reEPP uye majini maviri eEcK (EcK1 neEcK2), asi hatina kukwanisa kuwana gene rakanaka reEO. Zvinonyanya kukosha, majini ega ega eEPP akaratidzwa pamazinga akakwira (98.9th percentile) muGambian MAGs asi kwete muLRTs dzechikadzi (Mufananidzo 2b), zvinopesana nezvataitarisira kubvira pakaitika dephosphorylation ye3D20E22P mumatishu aya echikadzi.Naizvozvo, tinotenda kuti EPP yechirume inogona kutamiswa panguva yekusangana.Zvechokwadi, takashandisa in vivo stable isotope labeling kufukidza protein yechikadzi mushure mekusangana, iyo enzyme yakaonekwa neMS mu atrium yechikadzi (Mufananidzo 2c na Tafura Yekuwedzera 1). Kuvapo kweEPP muMAG uye LRT yevakadzi yakabatana (asi kwete mhandara) kwakasimbiswawo uchishandisa ma antibodies chaiwo (Mufananidzo 2d).
a, Nzira yekuongorora magene emhando ye bioinformatics yakagadzirwa nemaoko kuti iongorore magene emhando yega yega yebonde kuti ione magene anoumba maEcK, maEO, uye maEPP. Nhamba dziri padivi pemiseve dzinoratidza huwandu hwevanhurume nevakadzi padanho rega rega. Ongororo iyi yakaratidza gene rimwe chete reEPP (EPP) uye gene rimwe reEcK (EcK1) rinoratidzwa muvanhurume, uye gene rimwe reEcK (EcK2) rinoratidzwa muvanhurume vese asi risingaburitse gene reEO rinowanikwa.b, Heatmap inoenzanisa expression yemagene emhando ye candidate mu virgin (V) uye mating (M) Anopheles gambiae uye matishu eAnopheles albicans.Spca, fertilization; MAGs, accessory glands muvanhurume; Zvimwe zvikamu zvemuviri, kusanganisira mazamu, mapapiro, makumbo, miviri ine mafuta, uye nhengo dzemukati muvanhurume nevanhukadzi, uye maovari muvanhukadzi. EcK2 inoratidzwa zvakanyanya muMAG neatria yeGambia, nepo EPP inowanikwa chete muMAG.c, Proteomic analysis yekuchinja kweboka remarume ejaculate kuita atria yevakadzi pa3, 12 ne24 hpm, zvichiratidza mapuroteni 67 akawandisa. Vanhukadzi vakarerwa nekudya kune 15N kuti vanyore (uye vafukidze) mapuroteni ese. Vanhurume vasina tag vakasanganiswa nevakadzi vane tag, uye maLRT evakadzi akachekwa pa3, 12 ne24 hpm kuti vaongorore proteomic (ona Supplementary Table 1 kuti uwane runyorwa rwakakwana rwemapuroteni ejaculatory). Inset, EPP, Eck1 neEcK2 zvakaonekwa muMAG yevanhurume vasina kuroorana neproteomic analysis yematishu aya.d, EPP yakaonekwa newestern blot muMAG neLRT yevanhukadzi vasina kuroorana, asi kwete muvanhukadzi vasina kuroorana kana varume kana vamwe vakadzi. Muviri. Matumbu akaongororwa panguva imwe chete ne anti-actin (loading control) uye anti-EPP antibodies. Varume vese havana mhandara. Ona Mufananidzo Wekuwedzera 1 kuti uwane data remanyuko ejeli. Western blots yakaitwa kaviri nemhedzisiro yakafanana.
Basa reEPP reEcdysteroid phosphophosphatase rakaongororwa mushure mekuiswa mudumbu neHPLC-MS/MS ne3D20E22P yakabviswa kubva kuMAG (Extended Data Fig. 2a). Uyezve, patakanyaradza EPP neRNA-mediated interference (RNAi), takaona kudzikira kwakasimba kwekuita kwephosphatase mumatishu ekubereka evarume ava (Fig. 3a), uye vakadzi vakasangana nevarume vakanyaradzwa neEPP vakaratidza kukosha. Chikamu chidiki chedephosphorylated 3D20E (Fig. 3b) pasinei nekunyaradzwa kwemajini zvishoma (Extended Data Fig. 2b,c). Kusiyana neizvi, hatina kuona shanduko dzakakosha mu20E22P/20E ratio muumhutu humwe chete, izvo zvinogona kuratidza kuti enzyme iyi yakanangana ne3D20E22P (Fig. 3b).
a, Kudzikira kwekushanda kwephosphatase muMAG kunokonzerwa nekunyaradzwa kweEPP uchishandisa EPP RNA (dsEPP) ine tambo mbiri kana GFP RNA (dsGFP) ine tambo mbiri. Mapombi eMAG makumi maviri akashandiswa mune imwe neimwe kopi (P = 0.0046, paired t-test, two-sided), inomiririrwa nemadots akasiyana.b, Vakadzi vakasangana nevanhurume vakanyaradzwa neEPP vaiva nehuwandu hwakaderera hwe dephosphorylated 3D20E pa3 hpm (P = 0.0043, unpaired t-test, two-sided), nepo mazinga e20E asina kukanganiswa (P = 0.063, unpaired). bvunzo yet, ine mativi maviri). Data rinoratidzwa seavhareji ± sem kubva mumadziva matatu evakadzi 13, 16 ne19 mumwe nemumwe.c, Vakadzi vakasangana nevarume vakanyaradzwa neEPP vaiva nehuwandu hwakanyanya hwekusanganazve (P = 0.0002, bvunzo chaiyo yaFisher, ine mativi maviri). Vakadzi vakatanga kumanikidzwa kusangana kuti vave nechokwadi chekuti vanosangana sei; Mushure memazuva maviri, vakasangana nevamwe varume vaiva nembeu ye transgenic kuti vaongorore huwandu hwekusanganazve kwembeu kuburikidza nekuona huwandu hwePCR hwe transgene.d, vakadzi vakadyiswa ropa vakasangana nevarume vakanyaradzwa neEPP vakanga vaderedza kubereka (P < 0.0001; Mann-Whitney test, two-sided) uye nhamba yemazai yakaderera zvishoma (P = 0.088, Mann-Whitney test, two-sided), nepo huwandu hwekubereka husina kukanganiswa (P = 0.94, Fisher's exact test, two-sided).Mumapaneru ese, n inomiririra huwandu hwesamples dzeumhutu hwakazvimiririra.NS, hazvina kukosha.*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.001.
Tevere, takaongorora kana ecdysone dephosphorylation yakakosha pakukurudzira kusawirirana kwepabonde mumadzimai. Zvinonyanya kukosha kuti, vakadzi vakasangana nevarume vakapera simba reEPP vakasanganazve kakawanda (44.9%) kupfuura vakadzi vane simba rekudzivirira (10.4%) pavakasangana nevarume vekuwedzera (transgenic) (Mufananidzo 3c). Takaonawo kudzikira kukuru kwekubereka (Mufananidzo 3d, kuruboshwe) uye kudzikira kudiki kwehuwandu hwemazai akatarwa nemadzimai aya (Mufananidzo 3d, pakati), nepo huwandu hwemazai akatarwa nemadzimai (imwe mhinduro yakaunzwa nemadzimai nekusangana )) husina kukanganiswa (Mufananidzo 3d, kurudyi). Tichifunga nezvekunyatsoonekwa kweEPP ye3D20E22P, mhinduro idzi dzinoratidza kuti kushanda kwe3D20E neEPP yakatamiswa panguva yekusangana kunogona kuva nebasa rakakosha mukudzimisa kugamuchira kwevakadzi kune kumwe kusangana, maitiro akambotaurwa kare nekuda kwekufambiswa kwepabonde kwe20E. Nokudaro, iyi hormone yevarume inokanganisawo zvakanyanya kubereka kwevakadzi.
Tevere, takaenzanisa mabasa e20E ne3D20E mukuyedza majekiseni mumhandara dzakakura pabonde tichishandisa 3D20E yakagadzirwa nemakemikari (Mufananidzo 4a–c) uye 20E inowanikwa mumusika. Takaona kuti 3D20E yaishanda zvakanyanya kupfuura 20E mukudzivirira kunzwa kwevakadzi kujoinana pahuwandu hwese (Mufananidzo 4d). Zvinonyanya kukosha, hafu yehuwandu hwe3D20E muLRT (1,066 pg mushure mekujoina vs. 2,022 pg mushure mekujoina) yakakonzera chikamu chevakadzi vasingadzoreki chaive chakakwirira ka20 kupfuura huwandu hwe20E (361 pg mushure mekujoina) maawa makumi maviri nemana mushure mekujoina pahuwandu hwepamusoro hwe18 pg mushure mekujoina; Tafura yeData Yakawedzerwa 1). Mhedzisiro iyi inoenderana nepfungwa yekuti kutapurirana pabonde kwe20E hakukonzeri nguva yekusangana kwemwana, uye zvakare inoratidza 3D20E sechinhu chikuru mukuona hukama hwemubereki nemwana. 3D20E yaivewo yakashanda zvakanyanya kupfuura 20E mukuyedza kuisa mazai mumadzimai asina mhandara (Mufananidzo 4e), zvichiratidza kuti mwero wekuisa mazai watakaona mushure mekunyarara kwechikamu cheEPP wakakonzerwa nekuvapo kwemabasa e3D20E achiri kugadzirwa nezvinhu zvevakadzi zvinokonzereswa nekubatana.
(a,b) 3D20E yakagadzirwa nemakemikari kubva ku20E (a) ine shanduko/kushanda kwakanyanya (data rinoratidzwa seavhareji ± sem kubva kumaitiro matatu akazvimiririra ekugadzira) (b).c, Mass spectrum (hafu yepasi) inonyatsoenderana ne ecdysone inowanikwa muLRT yemukadzi yakasanganiswa (hafu yepamusoro).d, Zvichienzaniswa ne20E (0.63 µg, P = 0.02; 0.21 µg, P < 0.0001; Fisher's exact test, two-sided) uye 10% ethanol (0.63 µg, P < 0.0001; 0.21 µg, P < 0.0001; Fisher's exact test, 2-sided), nepo 20E yaive yakakwira zvakanyanya kupfuura control chete pamadhora akakwira (0.63 µg, P = 0.0002; 0.21 µg, P = 0.54; Fisher's exact test, 2-sided).e, jekiseni re3D20E rakakonzera mwero wekukura kwembeu muvakadzi vasina mhandara kupfuura 10% ethanol controls (0.21 µg, P < 0.0001; 0.13 µg, P = 0.0003; Fisher's exact test, two-sided), ukuwo 20E ichienzaniswa ne controls chete pa doses dzakakwirira (0.21 µg, P = 0.022; 0.13 µg, P = 0.0823; Fisher's exact test, two-sided).3D20E yakakonzera mwero wekukura kwembeu kupfuura 20E pa doses dzakakwirira (0.21 µg, P = 0.0019; 0.13 µg, P = 0.075; Fisher's exact test, two-sided).Mumapaneru ese, n inomiririra huwandu hwemasampuli eumhutu akazvimirira.NS, hazvina kukosha.*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.001. Data rinobva mumakopi matatu.
Muzvidzidzo zvekare, takaona kuti kutapurirana kwemahormone esteroid pabonde kunokonzera kuburitswa kweMISO (Mating-Induced Stimulator of Oogenesis 11), jini remukadzi rekubereka rinodzivirira vakadzi veA. gambiae kubva kuhutachiona hweP. falciparum. Mari yehutano inokonzerwa ne13, chipembenene chemarariya chinouraya vanhu. Tichifunga kukosha kweMISO pakubereka kweAnopheles munzvimbo dzine malaria, takasarudza kuona kuti ndeipi hormone 3D20E kana 20E inokonzera kuburitswa kwejini iri. Takaona kuti nepo jekiseni re20E rakanyanya kana kuti zvine simba rakanyanya kukonzera mamwe ma hormone receptors (HR), akadai seHR3 neHR4, uye zvinangwa zve steroid zvinouya pasi, zvakaita semajini eyolkogenic Vg14, 15, 16, MISO yakakurudzirwa zvakanyanya ne3D20E (Extended Data Fig. 3). Saka, kutapurirana pabonde kweiyi androgenic steroid hormone kunoita sekukonzera nzira dzinodzivirira vakadzi kubva kumari inokonzerwa nehutachiona hweparasite. Uyezve, 3D20E inokanganisa zvakasiyana ma isoform ese ari maviri e E receptor EcR, ichikonzera EcR-A uye ichidzvinyirira EcR-B, uye ichinyanya kukonzera mamwe majini anokonzera kusangana, kusanganisira HPX15, iyo inokanganisa kubereka kwevakadzi. Izvi zvinogona kutsanangura kusabereka kwakakosha kunoonekwa muvakadzi vanobatanidzwa nevarume vakanyaradzwa neEPP (Extended Data Fig. 3). Aya data anoratidza kuvapo kwenzira dzekudzika dzinoshandiswa zvakanyanya nemahormone maviri ecdysone anogona kukonzera basa repabonde.
Tevere, takaedza mashandiro emajini maviri eEcK akaonekwa mupaipi yedu ye bioinformatics. Kunyararidza EcK1 kana EcK2 kwakakonzera kufa kukuru muvarume (Extended Data Fig. 4a), zvichiratidza kuti ecdysone phosphorylation, uye nekudaro kusashanda, zvakakosha kuti munhu ararame. Nekuti EcK2 yakaratidzwa pamazinga akakwira kupfuura EcK1 uye yakaonekwa muMAGs neproteomics (Mufananidzo 2b,c uye Supplementary Table 2), takasimbisa mashandiro ayo ecdysteroid kinase nekuiisa mu20E, izvo zvakakonzera phosphorylation 20E22P (Extended Data Figure 2).4b). Patinoshandisa 3D20E se substrate, hatina kukwanisa kuona chigadzirwa che phosphorylated 3D20E22P (Extended Data Fig. 4c), zvichiratidza kuti 20E pane 3D20E ingave ndiyo chinangwa chakasarudzwa cheEcK2.
Sekureva kweongororo yedu yeRNA-seq, EcK2 yakaratidzwa zvakanyanya muLRT yevakadzi mhandara, uko yakadzimwa mushure mekusangana (Mufananidzo 2b). Takasimbisa data iri uye takaona kuti kutaura kweEcK2 hakuna kukanganiswa nekudyiswa ropa (Extended Data Fig. 5a). Tichiwedzera kuedza kwedu kwekutanga kweMS, takaona kuti huwandu hwe20E22P hwaive hwakabatana zvakanyanya nehukuru hwe20E (maawa 22-26 mushure mekudya ropa; Extended Data Fig. 5b). Kunyarara kweEcK2 muvakadzi mhandara kwakaguma nekuwedzera katatu kwehuwandu hwe20E kusvika 20E22P pamaawa makumi maviri nematanhatu mushure mekudya ropa (Extended Data Figures 2c na 5c), zvichiratidza kuti EcK2 inoisawo phosphorylates 20E muvakadzi. Zvinonyanya kukosha, vasikana vakapera simba reEcK2 vakaramba vachigamuchira pabonde zvizere (Extended Data Fig. 5d,e), zvichiratidzawo kuti kugadzirwa kwevakadzi kwe20E hakukonzere kuti kusangana kusagadzikane. Kusvika panguva dzepabonde. Zvisinei, vakadzi ava vaive nehuwandu hwakakura hwekubereka mazai zvichienzaniswa nevanochengetedza, nevanodarika 30% vevasikana vanoisa mazai (Extended Data Fig. 5f). Kana majekiseni eEck2 RNA (dsEcK2) ane tambo mbiri akaitwa mushure mekudyiswa ropa, kubereka hakuna kuitika, panguva iyo 20E peak nekuda kwekunwa ropa yakanga yadzikira. Kazhinji, mhedzisiro iyi inotsigira modhi iyo 20E yakagadzirwa mushure mekusvuta ropa inogona kukonzera kubereka, asi chete kana block yekubereka (EcK2 uye pamwe zvimwe zvinhu) yadzimwa nekusangana. Hapana majekiseni e20E kana 3D20E akadzivirira kutaura kweEcK2 muvasikana (Extended Data Fig. 5g), zvichiratidza kuti zvimwe zvinhu zvinodzivirira kudziviswa kwechinase ichi. Zvisinei, mazinga e20E mushure mekudyiswa ropa akanga asina kukwana kukonzera kusagadzikana kwekusangana, asi akakonzerwa nemazinga akakwira e3D20E inotapurirwa pabonde.
Zvakabuda muongororo yedu zvinopa ruzivo rwakakosha pamusoro penzira dzinodzora kubudirira kwekubereka kweA. gambiae. Muenzaniso wakabuda apo vanhurume vakashanduka kugadzira matiter akakwira e3D20E, ecdysone yakagadziriswa yevarume inovimbisa kubereka nekudzima tsinga dzevakadzi kuti varambe vachisangana. Panguva imwe chete, aya mavectors emalaria akagadzirawo sisitimu inoshanda yekuisa 3D20E muvanhukadzi mukupindura kutamiswa pabonde kweEPP yevarume. Sekuziva kwedu, uyu ndiwo muenzaniso wekutanga wesystem yesteroid hormone inodzorwa nevarume nevakadzi ichiita basa rakasiyana uye rakakosha muzvipembenene. Basa re ecdysone yevarume rakafungidzirwa asi harina kuratidzwa zvakajeka. Semuenzaniso, fungidziro isingabvumidzwe zvakanyanya 18 ndeyekuti mabasa aya anogona kuitwa ne20E precursor E1. Zvinozivikanwa kuti muDrosophila, monandry inokonzerwa nekutamiswa pabonde kwepeptides diki dzepabonde19,20 dzinosangana nemasero etsinga anochengetedza nzira yekubereka yemukadzi kuburikidza nemareceptors epeptide epabonde21,22. Basa rakawanda rinodiwa kuti uone pasi. ma cascade ekuratidza zviratidzo anodzorwa ne3D20E muvakadzi veA. gambiae uye kuona kana ma cascade aya achigona kuchengetedzwa pakati peumhutu neDrosophila.
Zvichienderana nebasa rakakosha re3D20E pakubereka kwevakadzi nemaitiro avo akaonekwa muchidzidzo chedu, nzira dzinotungamira mukugadzirwa kwe3D20E uye kushanda kwayo dzinopa mikana mitsva yemaitiro emangwana ekudzora umhutu, akadai sekugadzira varume vanokwikwidzana vasina utachiona mune tekinoroji yezvipembenene zvisina utachiona. Shandisa pakuburitsa mhuka kana kutevedzera 3D20E mumutambo wemhandara. Basa re3D20E revarume rinogona kunge rakashanduka apo A. gambiae nedzimwe mhando dzeCellia dzakawana kugona kuvharira mbeu yavo mumapulagi ekubatana, sezvo izvi zvichibvumira kutamiswa kwakanaka kwemahormone akawanda nema enzymes anogadzira mahomoni. Nekudaro, 3D20E evolution implementing monandry inopa nzira yekuti vakadzi (kuburikidza neMISO yakawanda) vabatsire kusimba kwavo kwekubereka munzvimbo dzine huwandu hwakawanda hwemarariya, izvo zvinopa mukana wekutapurirana kwePlasmodium. Zvichienderana nekuti 20E yevakadzi yakaratidzwa kuva nemigumisiro yakakura pakurarama nekukura kweP. falciparum muumhutu hwechikadzi hweAnopheles,24 nzira dzemahormone echirume neechikadzi ikozvino dzave zvinhu zvakakosha pakudyidzana kweumhutu nezvipembenene.
Marudzi eA. gambiae G3 akarerwa pasi pemamiriro ezvinhu akajairwa ezvipembenene (26-28 °C, hunyoro hwe65-80%, nguva yechiedza/rima kwemaawa 12:12). Makonye akadyiswa chikafu chehove cheupfu (TetraMin Tropical Flakes, Koi Pellets neTetra Pond Sticks muchiyero che7:7:2). Umhutu hwakura hwakadyiswa 10% dextrose solution uye ropa revanhu revhiki nevhiki (zvikamu zveropa rekudzidza). Umhutu hwemhandara hwakawanikwa nekupatsanura bonde padanho revana mushure mekuongorora magumo ne microscopy. Hono dzakatakura DsRed transgene dzakatsanangurwa kare.
Kuedza kwekumanikidza kusangana kwakaitwa zvichienderana nemitemo yakatsanangurwa kare. Pakusangana kwepabonde, vakadzi vane mazuva mana vakachengetwa muchikamu che1:3 nevarume vakakura pabonde kwehusiku huviri. Pakuedzwa uko varume vakabayiwa jekiseni redsEPP, co-caging yakabatana nemazuva matatu kusvika mana mushure mekubayiwa jekiseni, apo basa rephosphatase rakamiswa zvakanyanya (Extended Data Fig. 2b).
Matishu eumhutu, zvitunha zvasara (muviri wese), kana muviri wese zvakachekwa kuita 100% methanol uye zvakasanganiswa ne beader (2 mm glass beads, 2,400 rpm, 90 sec). Huwandu hwematishu uye methanol vhoriyamu zvaive seizvi: zororo remuviri, 50 mu 1,000 µl; MAG, 50–100 80 µl; LRT yevakadzi, 25–50 80 µl. Precipitate yakabviswa kechipiri methanol nevhoriyamu imwechete ye methanol. Marara emasero akabviswa ne centrifugation. Methanol kubva mukubvisa kwese kwakasanganiswa ndokuomeswa pasi pekuyerera kwenitrogen, ndokuzodzorerwa mumavhoriyamu anotevera e80% methanol mumvura: zororo remuviri, 50 µl; MAG neLRT yevakadzi, 30 µl.
Mienzaniso yakaongororwa pa mass spectrometer (ID-X, Thermo Fisher) yakabatana neLC instrument (Vanquish, Thermo Fisher). 5 µl yesample yakapinzwa pa3 µm, 100 × 4.6 mm column (Inspire C8, Dikma) yakachengetwa pa25 °C. Mafambiro emobile eLC aive A (mvura, 0.1% formic acid) uye B (acetonitrile, 0.1% formic acid). LC gradient yaive seinotevera: 5% B kweminiti imwe, ndokuzowedzerwa kusvika 100% B kwemaminetsi gumi nerimwe. Mushure memaminitsi masere pa100%, enzanisazve column pa5% B kwemaminetsi mana. flow rate yaive 0.3 ml min-1. Ionization muMS source inoitwa ne heated electrospray ionization mune positive uye negative modes.
Iyo mass spectrometer inoyera data mu m/z range kubva pa 350 kusvika 680 pa 60,000 resolution mu MS mode yakazara. Data reMS/MS rakatorwa pa [M + H]+ (all targets), [M - H2O + H]+ (all targets), uye [M - H]- (phosphorylated targets). Data reMS/MS rakashandiswa kusimbisa hunhu hwe ecdysone hwetargets dzisina standard yaiwanikwa. Kuti uone non-targeted ecdysteroids, data reMS/MS rema HPLC peaks ese ane >15% relative abundance rakaongororwa. Quantify uchishandisa standard curves dzakagadzirwa kubva ku pure standards (20E, 3D20E) kuverenga absolute amounts kana dilutions yeimwe chaiyo sampuro (all targets) kuverenga equivalence yavo nehuwandu hwakawanikwa mumurume mumwe chete. Kune 3D20E, quantification yakaitwa uchishandisa huwandu hwe adducts dzinotevera: [M + TFA]-, [M + COOH]-, [M + Na]+, [M + Cl]-, [M + NO3]-.Data rakatorwa uye rakayerwa uchishandisa Tracefinder (vhezheni 4.1).Data reMS/MS rakaongororwa uchishandisa Xcalibur (vhezheni 4.4). MS spectra yeE, 20E uye 3D20E yakaenzaniswa nemazinga akakodzera. 3D20E22P yakaongororwa nekubvisa neGirard's reagent. 20E22P yakaongororwa ne m/z ratio.
3D20E22P yakacheneswa kubva kuMAG. Kucheneswa kwakaitwa pachikero chekuongorora uchishandisa ultra-performance liquid chromatograph (Acquity, Waters) ne quadrupole mass-based detector (QDa, Acquity, Waters) pasi pemamiriro akafanana eLC seHPLC-MS/MS analysis. Kuunganidzwa kwezvikamu kwakatanga apo m/z inoenderana ne3D20E22P yakaonekwa panguva imwechete yekuchengetedza sezvakatsanangurwa kare. Kuchena kwemakomponi akabviswa kwakazoongororwa neHPLC-MS/MS sezvakatsanangurwa pamusoro apa.
RNA yese yakatorwa kubva mumatishu ekubereka 10-12 kana zvimwe zvikamu zvemuviri (isina musoro) uchishandisa TRI reagent (Thermo Fisher) zvichitevera mirairo yemugadziri. RNA yakarapwa neTURBO DNase (Thermo Fisher). cDNA yakagadzirwa uchishandisa Moloney murine leukemia virus reverse transcriptase (M-MLV RT; Thermo Fisher) zvichitevera mirairo yemugadziri. MaPrimers e reverse transcription quantitative PCR (RT-qPCR; Extended Data Table 2) akamboburitswa 24 kana kugadzirwa uchishandisa Primer-BLAST26, nesarudzo yakapihwa kune zvigadzirwa zve 70-150 bp muhukuru uye zvinopararira exon-exon junctions kana Primer pair primers dzakasiyana exons. cDNA samples kubva ku3 kusvika ku4 biological replicates dzakasanganiswa kane mumvura yeRT-qPCR. Quantification yakaitwa mu15 µl replica reactions ine 1 × PowerUp SYBR Green Master Mix (Thermo Fisher), primers, uye 5 µl ye diluted cDNA. Reactions dzakaitwa pa QuantStudio 6 Pro real-time PCR system (Thermo Fisher) nedata zvakaunganidzwa uye zvakaongororwa uchishandisa Design and Analysis (vhezheni 2.4.3). Sezvakaratidzwa muchidzidzo ichi, huwandu hwakaenzana hwakagadziriswa kune ribosomal gene RpL19 (AGAP004422), iyo kutaura kwayo kusina kuchinja zvakanyanya nekupa ropa 27 kana kusangana 3.
Hunhu hweRNA hwakaongororwa uchishandisa Agilent Bioanalyzer 2100 Bioanalyzer (Agilent). Maraibhurari eIllumina paired-end akagadzirwa uye akaendeswa kuBroad Institute of MIT neHarvard. Kuverengwa kwesequencing kwakanyatsoenderana neA. gambiae genome (PEST strain, version 4.12) uchishandisa HISAT2 (version 2.0.5) nema default parameters. Kuverengwa ne mapping quality (MAPQ) scores <30 kwakabviswa uchishandisa Samtools (version 1.3.1). Huwandu hwekuverenga hwakaiswa mumajini hwakaverengwa uchishandisa htseq-count (version 0.9.1) nema default parameters. Kuverengwa kwekuverenga kwakajairwa kwakaverengwa uye differential gene expression yakaongororwa uchishandisa DESeq2 package (version 1.28.1) muR (version 4.0.3).
Vanhu vane majini anochinja Ecdysone vakaonekwa nekutsvaga genome reA. gambiae vachishandisa PSI-BLAST algorithm (https://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/2.8.1/), vachishandisa default values. MaParameter ane zvinotevera query protein sequences: kubva kuBombyx mori (Accession No. NP_001038956.1), Musca domestica (Accession No. XP_005182020.1, XP_005175332.1 uye XP_011294434.1) uye Microplitis demolitor (Accession No. XP_008552646.1 uye XP_008552645.1) EcK kubva kuB. mori (Accession No. NP_001036900), Drosophila melanogaster (Accession No. NP_651202), Apis mellifera (Accession No. XP_394838) uye Acyrthosiphon pisum (Accession No. XP_001947166); uye EPP kubva kuB. mori (Accession No. XP_001947166) NP_001177919.1 uye NP_001243996.1) uye EO yeD. melanogaster (Accession No. NP_572986.1) (danho 1).Tevere, firita inorova zvichibva pakuratidzwa kwemRNA yakakwira (>100 fragments/kilobase exons per million mapped reads (FPKM) kana >85%) muzvinyama zvekubereka (female LRT kana MAG) muGambia (danho 2). Kuti tivandudze hunhu hwemuviri, takasarudza ma enzymes anowanikwawo munyama yekubereka yeA. albimanus, rudzi rweanopheles rusingagadzire kana kutamisa ecdysone panguva yekusangana. Majini akawanikwa akaseferwa zvichibva pahuwandu hwakaderera hwemasero (<100 FPKM kana <85th percentile) munyama yekubereka yeA. albimanus (nhanho 3). Sesefa yekupedzisira (nhanho 4), majini akawanikwa anofanira kugutsa chimwe chezvinotevera: (1) akawedzerwa zvakanyanya mushure mekusangana (P < 0.05) zvichienderana nekuongororwa kwemajini akaratidzwa zvakasiyana uye (2) munyama dzisina kubereka (< 85% kana <100 FPKM).
Takachinja nzira dzakambotsanangurwa kare 28,29,30 kuti tiwane labeling yemuviri wese. Muchidimbu, Saccharomyces cerevisiae type II (YSC2, Sigma) yakaedzwa muyeast nitrogen base (BD Difco, DF0335) ine (wt/vol) 2% glucose (G7528, Sigma), 1.7% amino acid-free uye ammonium sulfate. culture medium) uye 5% 15N ammonium sulfate (NLM-713, >99%, Cambridge Isotope Laboratories) senzvimbo yega yenitrogen. Mbiriso yakatorwa ne centrifugation uye mosquito larvae dzakapihwa ad libitum kusvika dzakura. Kuwedzera nehove (0.5 mg pa300 larvae) kudzivirira kufa kwevana. Hadzi chete ndidzo dzakashandiswa mukuyedza kusangana nevarume vasina label kuti vaongorore proteome yechirume yakaendeswa panguva yekusangana.
Mapoka echikadzi ane mazuva mana kusvika matanhatu ane 15N-tagged akamanikidzwa kusangana nevarume vasina tambo dzezera ravo. Kusangana kwakabudirira kwakaonekwa nekuona maplugs ekubatana achishandisa epifluorescence microscopy. Pa3, 12, uye 24 hpm, atria yevakadzi vane 45-55 vakasangana yakachekwa kuita 50 µl yeammonium bicarbonate buffer (pH 7.8) uye yakasanganiswa nepestle. Homogenate yakatenderedzwa uye supernatant yakasanganiswa ne50 µl ye0.1% RapiGest (186001860, Waters) mu50 mM ammonium bicarbonate. Supernatant nepellet kubva musampuli yega yega zvakatorwa nechando pachando chakaoma ndokutumirwa husiku hwese kuMacCoss laboratory paYunivhesiti yeWashington, uko kugadzirira sampuro yeLC-MS/MS kwakaitwa. Resume pellet mu50 µl ye0.1% RapiGest mu50 mM. Ammonium bicarbonate uye sonicate mumvura yekugezera. Kuwanda kwemapuroteni mupellet uye supernatant kwakayerwa neBCA assay, sampuro dzakaderedzwa ne5 mM dithiothreitol (DTT; Sigma), dzakasanganiswa ne15 mM iodoacetamide (Sigma) uye dzakaiswa pa37 °C (1:0 50) kweawa imwe chete netrypsinization: trypsin: substrate ratio). RapiGest yakaongororwa nekuwedzera 200 mM HCl, ichiteverwa nekudzikiswa pa37 °C kwemaminitsi makumi mana nemashanu uye centrifugation pa14,000 rpm kwemaminitsi gumi pa4 °C kubvisa marara. Sampuro dzakagezwa ne dual-mode solid-phase extraction (Oasis MCX cartridges, Waters) uye dzakaiswazve mu 0.1% formic acid kuti pave ne final protein concentration ye 0.33 µg µl-1. MaMAG proteomes asina kunyorwa akaongororwawo zvakafanana kubva ku virgin. Vanhurume. Makopi maviri ekuongorora akaongororwa pamuenzaniso wega wega. Tevere, 1 µg yemumwe nemumwe yakaongororwa uchishandisa 25-cm fused silica 75-μm column ine 4-cm fused silica Kasil1 (PQ) frit trap yakazadzwa neJupiter C12 reversed-phase resin (Phenomenex) uye 180-minute liquid chromatography. Kuongororwa kwemuenzaniso - MS/MS yakafambiswa paQ-Exactive HF mass spectrometer (Thermo Fisher) ine nanoACQUITY UPLC System (Waters). Data rekuwana data rakagadzirwa pakushandisa kwega kwega rakachinjwa kuita mzML format uchishandisa Proteowizard (vhezheni 3.0.20287) uye uchishandisa Comet31 (vhezheni 3.2) uchipesana neFASTA database ine mapuroteni sequences kubva kuAnopheles gambiae (VectorBase vhezheni 54), Anopheles coluzzi Kutsvaga kwakaitwa paMali-NIH (VectorBase vhezheni 54), Saccharomyces cerevisiae (Uniprot, Kurume 2021), A. gambiae RNA-seq, uye shanduro dzemafuremu matatu dzezvinosvibisa vanhu zvinozivikanwa. Mapeptide map-matched FDRs akawanikwa achishandisa Percolator32 (vhezheni 3.05) ane threshold ye0.01, uye mapeptides akaunganidzwa kuita mapuroteni anoshandiswa kushandisa protein parsimony muLimelight33 (vhezheni 2.2.0). Kuwanda kwemapuroteni kwakafungidzirwa uchishandisa normalized spectral abundance factor (NSAF) yakaverengerwa puroteni yega yega mukumhanya kwega kwega sezvakatsanangurwa kare. NSAF maererano nepuroteni yega yega yakaverengerwa paavhareji kubva kumasampuli maviri akasiyana ezvehupenyu. 15N labeling yakavhara proteome yechikadzi zvinobudirira, kunyangwe huwandu hushoma hweprotein isina label hwaigona kuonekwa kubva kumhandara dzakanyorwa label. Takanyora kuwanikwa kwekuderedza mapuroteni echirume (1-5 spectra) musampuli dzisina kunyorwa dzechikadzi chete mukumhanya kwehunyanzvi, uko sampuro dzisina kunyorwa dzakaitwa mushure mesampuli dzechirume/dzekusangana, nekuda kweHPLC "kutakura". Dzimwe nguva mapuroteni anowanikwa se'tsvina' kubva kumhandara dzakanyorwa label akanyorwa muSupplementary Table 1.
Mapeptide maviri eantigenic, QTTDRVAPAPDQQQ (mukati me isotype PA) uye MESGTTPSGDSEQ (mukati me isotype PA nePB) muGenscript. Mapeptide maviri aya akabatanidzwa, ndokuzobatanidzwa ne carrier protein KLH ndokupinzwa mutsuro dzeNew Zealand. Tsuro dzakabayirwa mushure me jekiseni rechina, uye total IgG yakaparadzaniswa ne affinity cleansing. IgG kubva kutsuro yakanyanya EPP yakashandiswa kune mamwe ma Western blotting.
Kune mablots ekumadokero, MAG (n = 10, apo n inomiririra huwandu hwesamples dzeumhutu hwakazvimiririra) uye LRT yechikadzi (n = 30) kubva kuvarume vane mazuva mana ekuberekwa mhandara uye vakadzi vakamanikidzwa (<10 mushure mekusangana), 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)) yakawedzerwa yoga. Mienzaniso yakasanganiswa pakarepo mushure mekupatsanurwa nebeader (2 mm glass beads, 2,400 rpm, 90 sec). Marara asinganyunguduke akabviswa ne centrifugation pa 20,000 g pa 4 °C. Mapuroteni akayerwa neBradford assay (Bio-Rad). Zvadaro, 20 µg yeMAG protein, 40 µg yeLRT protein, uye 20 µg yeresidual bulk protein. Zvakabviswa ganda ndokupatsanurwa ne10% Bis-Tris NuPAGE uchishandisa MOPS buffer. Mapuroteni akaendeswa ku polyvinylidene fluoride membranes uchishandisa iBlot2 transfer system (Thermo Fisher). Mamembrane akagezwa kaviri mu 1× PBS-T (0.1% Tween-20 muPBS) ndokuzovharwa muOdyssey blocking buffer (Li-Cor) kweawa imwe pa22°C. Mamembrane akazunguzwa husiku hwese pa4°C ne custom rabbit anti-EPP polyclonal primary antibody (1:700 mu blocking buffer) uye rat anti-actin monoclonal primary antibody MAC237 (Abeam; 1:4,000). Mamembrane akagezwa nePBS-T ndokuzoiswa ma secondary antibodies (dhonkey anti-rabbit 800CW uye goat anti-rat 680LT (Li-Cor), ese ari 1:20,000) mu blocking buffer ine 0.01% SDS uye 0.2% Tween -20 kweawa imwe chete pa22 °C. Matanda akagezwa nePBS-T ndokutorwa mifananidzo neOdyssey CLx scanner. Mifananidzo yakaunganidzwa uye yakagadziriswa muImage Studio (vhezheni 5.2). Bhandi rakati rinoenderana neEPP-RA isoform (82 kDa) harina kuonekwa.
Nzvimbo dzeEPP (se isoform AGAP002463-RB dzine histidine phosphatase domain, NCBI conserved domain search 34) uye EcK2 (AGAP002181) dzakaunganidzwa mu pET-21a(+) plasmid (Novagen Millipore Sigma); Ma primer akanyorwa muExtended Data Table 2. Ma GS4 linkers masere (ari mu tandem) akaiswa C-terminal 6xHis tag ye pET-21a(+)-EcK2 construct isati yagadzirwa. Mapuroteni e recombinant akagadzirwa achishandisa NEBEExpress cell-free E. coli protein synthesis reaction (New England BioLabs). Mapuroteni e recombinant akacheneswa achishandisa NEBEExpress Ni spin columns (New England BioLabs). Dihydrofolate reductase (DHFR) control protein yakagadzirwa uchishandisa DNA template kubva kuNEBEExpress Cell-Free E. coli Protein Synthesis Kit. Mapuroteni akachengetwa mu50% glycerol muPBS pa -20 °C kwemwedzi inosvika mitatu.
Basa rePhosphatase reEPP netissue extracts rakayerwa uchishandisa 4-nitrophenyl phosphate (pNPP; Sigma-Aldrich). Reaction buffer yaive ne25 mM Tris, 50 mM acetic acid, 25 mM Bis-Tris, 150 mM NaCl, 0.1 mM EDTA, uye 1 mM DTT. Tishu dzakasanganiswa mu reaction buffer uye tsvina yemasero yakabviswa ne centrifugation. Tanga reaction nekuwedzera enzyme kana tissue extract ku reaction buffer ine 2.5 mg ml-1 pNPP. Musanganiswa we reaction wakaiswa patembiricha yekamuri murima, uye huwandu hwepNP hwakachinjwa kubva ku pNPP hwakayerwa nekuyera absorbance pa405 nm panguva dzakasiyana.
Pakuita kweEcK mu in vitro, protein iyi yakaiswa mu 0.2 mg 20E kana 3D20E mu 200 µl buffer (pH 7.5) ine 10 mM HEPES–NaOH, 0.1% BSA, 2 mM ATP uye 10 mM MgCl2 kwemaawa maviri pa 27 °C. Kuita kwacho kwakamiswa nekuwedzera 800 µl methanol, yozotonhodzwa pa -20 °C kweawa imwe, yozoiswa mu centrifuge pa 20,000 g kwemaminetsi gumi pa 4 °C. Supernatant yakazoongororwa neHPLC-MS/MS. Kuti mapuroteni anoshandiswa muboka rekudzora apiswe, mapuroteni akaiswa mu 50% glycerol muPBS kwemaminetsi makumi maviri pa 95 °C.
Pakuita kweEPP mu vitro, protein iyi yakaiswa mu 3D20E22P (yakaenzana nehuwandu hwakawanikwa mumapaya gumi nemasere eMAG, akacheneswa neHPLC-MS/MS) mu 100 µl buffer (pH 7.5) ine 25 mM Tris, 50 mM acetic acid, 25 mM Bis-Tris, 150 mM NaCl, 0.1 mM EDTA, uye 1 mM DTT kwemaawa matatu pa27 °C. Kuita kwacho kwakamiswa nekuwedzera 400 µl methanol ndokutonhodzwa pa -20 °C kweawa imwe, ndokuzoiswa mu centrifuge pa20,000 g kwemaminetsi gumi pa4 °C. Supernatant yakaongororwa neHPLC-MS/MS.
Zvidimbu zvePCR zveEPP (362 bp), EcK1 (AGAP004574, 365 bp) uye EcK2 (556 bp) zvakawedzerwa kubva ku cDNA yakagadzirwa kubva muzvitunha zveumhutu zvisina musoro zvebonde rakasanganiswa. Chidimbu chePCR che eGFP control (495 bp) chakawedzerwa kubva pa pCR2.1-eGFP yakatsanangurwa kare; MaPCR primers akanyorwa muExtended Data Table 2. Chidimbu chePCR chakaiswa pakati pema inverted T7 promoters pa pL4440 plasmid. Plasmid constructs dzakatorwa kubva ku NEB 5-α competent E. coli (New England Biolabs) uye dzakasimbiswa neDNA sequencing vasati vashandisa (ona Supplementary Data 1 ye insert sequence). MaPrimers akabatana neT7 promoter (Extended Data Table 2) akashandiswa kuwedzera insert kubva ku pL4440-based plasmid. Saizi yechigadzirwa chePCR yakasimbiswa ne agarose gel electrophoresis.dsRNA yakatorwa kubva kumatemplate ePCR uchishandisa Megascript T7 Transcription Kit (Thermo Fisher) uye yakacheneswa zvichienderana nemirairo yemugadziri nekuchinja kwakatsanangurwa kare.
Pakubaiwa jekiseni re dsRNA, 1,380 ng ye dsRNA (dsGFP, dsEcK1, dsEcK2, dsEPP) yakabayiwa ne 10 ng nl-1 muchipfuva chevarume kana vakadzi vakuru (Nanoject III, Drummond) mukati mezuva rimwe chete mushure mekubuda kweropa. Kuwanda kwe gene knockdown kwakaonekwa muma genetic replicates matatu e biology nekubvisa RNA, cDNA synthesis, uye RT-qPCR. Pakubaiwa jekiseni re ecdysone, vakadzi vane mazuva mana ekuberekwa mhandara kana vane mazuva matanhatu ekuberekwa mhandara vakadyiswa ropa vakabayiwa jekiseni re 0.13, 0.21, kana 0.63 µg ye 20E kana 3D20E (Nanoject III, Drummond) pa 1.3, 2.1, zvichiteerana, zvichienderana nekugadzirwa kwekuyedza kana 6.3 ng nl-1. Baiwa jekiseni re 100 nl ye 10% (vol/vol) ethanol. mumvura; 100 nl ye3D20E22P mu10% ethanol (yakaenzana ne75% yehuwandu hwakawanikwa mumaMAG maviri). Umhutu hwakaiswa muboka remajekiseni zvisina tsarukano.
Pakuyedzwa kwekubereka, hadzi dzine mazuva matatu dzakapihwa ropa revanhu pasina muripo. Bvisai umhutu hwakadyiswa zvishoma kana husina kudyiswa. Zvichienderana nekurapwa, hadzi dzakaiswa mumakapu akasiyana ekubereka kwehusiku huna maawa makumi mana nemasere mushure mekudya ropa. Mazai akaverengwa pasi pe stereoscope (Stemi 508, Zeiss); kune hadzi dzakaberekeswa, mazai aibuda muhonye aionekwa seane gonye.
Pakuyedzwa kwekusangana kwemhuka, mhuka dzechikadzi dzakabvumirwa kwemazuva maviri zvichienderana nekurapwa kwadzo kuti dzisarambe kusangana, uye mhuka dzechikadzi dzakafanana nezera remhuka dzakazopinzwa mukeji imwe chete. Mushure memazuva maviri, maburi echikadzi akachekwa uye genomic DNA yakaburitswa nekunyungudika kwechando uye sonication mubuffer ine 10 mM Tris-HCl, 1 mM EDTA, uye 25 mM NaCl (pH 8.2). Mienzaniso yakaiswa muProteinase K (0.86 µg µl-1) kwemaminitsi gumi nemashanu pa55 °C, ichiteverwa nemaminitsi gumi pa95 °C. Kugadzirira kweDNA yegenomic isina kusimba kwakaderedzwa ka10 uye kwakaongororwa qPCR yeY chromosome sequences; primers dzakanyorwa muExtended Data Table 2. Kusavapo kweY chromosome sequence kunoratidza kuti hapana kusangana.
Pakuyedzwa kwekusanganazve, vakadzi vakamanikidzwa vakaongororwa kuti vaone kana paine maplug ekusangana kuti vaone kana vari varume, uye vakapa mazuva maviri kuti vasakwanise kusangana kana varume vasipo, sezvakatsanangurwa kare. 36. Varume vakatakura mbeu yeDsRed transgenic vakabva vaiswa mumakeji evakadzi. Mushure memazuva maviri, ma vesicles ekuberekesa akabviswa kubva kuvakadzi, uye genomic DNA yakagadzirwa sezvakatsanangurwa pamusoro uye yakaongororwa kuti ionekwe neQPCR transgene yeDsRed; ma primer akanyorwa muExtended Data Table 2. Kusavapo kweDsRed transgene kwakaratidza kuti hapana kusanganazve kwakaitika.
3D20E yakagadzirwa sezvakatsanangurwa kare 37. Muchidimbu, 10 mg ye20E (Sigma-Aldrich) yakanyungudutswa mu10 ml yemvura, zvichiteverwa nekuwedzera 30 mg yeplatinum black (muchimiro cheupfu, Sigma-Aldrich). Rukova runyoro rweO2 rwakaramba ruchiburitswa mumusanganiswa wereaction, uyo wakakurunga patembiricha yemumba. Mushure memaawa matanhatu, 30 mL yemethanol yakawedzerwa kumisa reaction. Musanganiswa wacho wakaiswa pakati kuti ubvise catalyst particles. Supernatant yakabviswa kuita yakaoma mu vacuo patembiricha yemumba. Chigadzirwa chereaction chakaomeswa chakanyungudutswa mu10% ethanol ne methanol kuti ibayiwe HPLC-MS/MS analysis. Chiyero chekushandura (kubva pa20E kuenda ku3D20E) chaive chinenge 97% (Mufananidzo 4b), uye MS spectrum ye3D20E yakagadzirwa yakaenzana neyakawanikwa muvakadzi vakaroorwa (Mufananidzo 4c).
Ngano iyi ine ruzivo rwakadzama rwebvunzo dzehuwandu dzakaitwa. GraphPad (vhezheni 9.0) yakashandiswa kuita bvunzo chaiyo yaFisher, bvunzo yeMantel-Cox, uye bvunzo yet-test yeMudzidzi. Miedzo yeCochran-Mantel-Haenszel yakaitwa uchishandisa script yeR yakasarudzika (inowanikwa pa https://github.com/duopeng/mantelhaen.test). Kugoverwa kwedata kwakaedzwa kuti kuonekwe kuti kwakajairika sei uchishandisa bvunzo yeShapiro-Wilk ine muganhu wekukosha kwe0.05. Data parakakundikana bvunzo yekukosha, bvunzo yeMann-Whitney yakaitwa. Data rekupona rakaongororwa uchishandisa bvunzo yeMantel-Cox. Pakeji yeDESeq2 (vhezheni 1.28.1) yakashandiswa kuita ongororo yeRNA-seq gene-level differential expression. Bhara rakatwasuka pagirafu rinomiririra median. Kukosha kwekukosha kweP = 0.05 kwakashandiswa semuganhu webvunzo dzese.
Kuti uwane rumwe ruzivo nezve dhizaini yekudzidza, ona pfupiso yeNature Research Report yakabatana nechinyorwa chino.
Ruzivo rweMS proteomic rwakaiswa muProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) kuburikidza nePRIDE Partner Repository (https://www.ebi.ac.uk/pride/) nedataset identifier PXD032157.
Dataset yeRNA-seq inoiswa muGene Expression Comprehensive Library (https://www.ncbi.nlm.nih.gov/geo/) pasi pegwaro reGSE198665.
Mamwe madata akagadzirwa uye/kana kuongororwa panguva yekudzidza kuri kuitwa anogona kuwanikwa kubva kuvanyori vanoenderana kana zvikakumbirwa zvine musoro. Chinyorwa chino chinopa ruzivo rwekwakabva.
De Loof, A. Ecdysteroids: Masteroid epabonde ezvipembenene asina hanya? Murume: Black Box. Sainzi yezvipembenene.13, 325–338 (2006).
Redfern, CPF 20-hydroxyecdysone uye kukura kweovarian muAnopheles stephens.J. Insect Physiology.28, 97–109 (1982).