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Ba kamar ƙasusuwan baya ba, ana kyautata zaton kwari ba su da hormones na steroid na jima'i na maza. A cikin Anopheles gambiae, da alama ecdysone steroid 20-hydroxyecdysone (20E) ya samo asali ne don sarrafa ci gaban ƙwai lokacin da mata suka haɗa shi2 da kuma haifar da lokacin da maza suka yi jima'i. Tunda ci gaban ƙwai da haɗuwa sune muhimman halaye na haihuwa, fahimtar yadda sauro na mata na Anopheles ke haɗa waɗannan siginar hormones zai iya sauƙaƙe ƙirƙirar sabbin shirye-shiryen kula da zazzabin cizon sauro. A nan, mun bayyana cewa waɗannan ayyukan haihuwa ana sarrafa su ta hanyar steroids na jima'i daban-daban ta hanyar hanyar sadarwa mai rikitarwa ta enzymes masu kunna ecdysteroid/wanda ke hana aiki. Mun gano ecdysone na musamman na maza, 3-dehydro-20E (3D20E), wanda ke kare iyaye ta hanyar rufe karɓar jima'i na mata bayan canja wurin jima'i da kunnawa ta hanyar dephosphorylation. Abin lura shi ne, canja wurin 3D20E shi ma ya haifar da bayyanar kwayoyin halittar haihuwa waɗanda ke kula da ci gaban ƙwai yayin kamuwa da cutar Plasmodium, yana tabbatar da lafiyar mata masu kamuwa da cutar. 20E da aka samo daga mata ba ya haifar da jima'i. martani, amma yana bawa mutane masu haɗuwa damar yin ƙwai bayan an hana 20E-inhibiting kinases. Gano wannan hormone na steroid na kwari na musamman ga namiji da kuma rawar da yake takawa wajen daidaita karɓar jima'i ga mace, haihuwa da hulɗa da Plasmodium yana nuna yuwuwar rage nasarar haihuwa na sauro masu yaɗa cutar malaria.
Yawan mace-mace da kuma mace-mace na karuwa saboda yawan shan magungunan kashe kwari a cikin sauro na Anopheles, wanda shi ne kawai ya haifar da ƙwayoyin cuta na maleriya a cikin ɗan adam. Ilimin halittar waɗannan sauro wani abu ne mai jan hankali musamman ga sabbin hanyoyin magance maleriya domin mata suna saduwa sau ɗaya kawai; sanya wannan haɗuwar ta zama marar lafiya zai sami babban damar rage yawan sauro a cikin filin.
Mata suna samun matsalar rashin ƙarfin jima'i bayan sun sami hormones na steroid masu yawan gaske daga maza. Bincike ya nuna cewa abin da ke haifar da matsala wajen ƙarin haɗuwa shine 20-hydroxyecdysone (20E), wani hormone na steroid wanda aka fi sani da mai kula da zagayowar molting a matakin tsutsa. Ikon maza don haɗawa da canja wurin 20E ya samo asali musamman a cikin nau'ikan Anopheles waɗanda ke cikin ƙananan ƙwayoyin halitta Cellia7, wanda aka rarraba a Afirka kuma ya haɗa da ƙwayoyin cuta mafi haɗari na zazzabin cizon sauro, gami da Anopheles gambiae. Wannan abin lura ne musamman saboda a cikin waɗannan nau'ikan mata kuma suna samar da 20E bayan kowace cin abinci na jini, kuma 20E yana jagorantar zagayowar oogenesis (duba ref. 8). Duk da haka, ba a san komai ba game da yadda mata ke haɗa sigina daga tushe biyu daban-daban na ecdysone (canja wurin maza da kuma shigar da jini) ba tare da lalata ikon su na haɗuwa ba. A gaskiya ma, idan 20E da mata ke samarwa yana haifar da rashin haƙuri ga jima'i, wannan zai haifar da rashin haihuwa a cikin mutanen da ke ciyar da budurwa, wani hali da ya zama ruwan dare a cikin waɗannan sauro5.
Wata ma'ana mai yiwuwa ita ce mazan A. gambiae suna canja wurin ecdysone na musamman ga maza, wanda ke kunna siginar sigina a cikin hanyar haihuwa ta mace, wanda ke haifar da rashin kwanciyar hankali na haɗuwa. Duk da haka, kodayake ƙasusuwan baya suna da hormones na steroid da yawa, kamar estrogen da androgen (an sake duba su a cikin ref. 9), gwargwadon iliminmu, ba a gano steroids masu son androgen a cikin kwari ba.
Mun yi niyyar tantance yawan hormones na steroid a cikin glandar haɗin maza na maza (MAG) na A. gambiae na jima'i don neman yiwuwar canza steroids. Ta amfani da ingantaccen chromatography na ruwa tare da tandem mass spectrometry (HPLC-MS/MS) maimakon hanyar da ba ta da takamaiman tsari da aka yi amfani da ita a baya, mun gano ecdysone (E) da 20E a cikin wannan kyallen, wanda ya tabbatar da sakamakon da ya gabata. Duk da haka, samfurin ya mamaye steroids na oxidized phosphorylated, daidai da dabarar 3-dehydro-20E-22-phosphate (3D20E22P)12 (Hoto na 1). Sauran siffofi sun haɗa da 3-dehydro-20E (3D20E) da 20E-22-phosphate (20E22P). Ƙarfin siginar HPLC-MS/MS na 3D20E22P ya kasance matakai biyu na girma fiye da siffar dephosphorylated, 3D20E, da kuma matakai uku na girma sama da na E da 20E (Hoto na 1). Kodayake a wasu sassan jiki da kuma ƙananan hanyoyin haihuwa (LRT; Bayanan da aka faɗaɗa Hoto na 1a). Mun kuma yi nazarin ecdysteroids a cikin maza da mata da aka rufe (ƙasa da kwana ɗaya) kuma mun gano 3D20E da 3D20E22P kawai a cikin MAG; E, 20E da 20E22P sun kasance a cikin jinsi biyu (Bayanan da aka faɗaɗa Hoto na 1b). Waɗannan bayanan sun nuna cewa maza manya na A. gambiae suna samar da manyan adadin hormones masu gyara a cikin MAGs ɗinsu waɗanda mata ba su haɗa su ba.
An cire MAG da mace LRT (gami da atria, vesicles na maniyyi, da parovarium) daga mazan da suka yi kwana 4 (na kwana 4) budurwa da mata masu juna biyu (0.5, 3, da 12 hpm). An yi nazarin Ecdysone a cikin waɗannan kyallen ta hanyar HPLC-MS/MS (matsakaicin ± sem; gwajin t-test mara haɗin kai, mai gefe biyu, ƙimar gano ƙarya (FDR) da aka gyara; NS, ba mai mahimmanci ba; *P < 0.05, **P < 0.01 . 3D20E: awanni 3 vs. awanni 0.5, P = 0.035; awanni 12 vs. awanni 3, P = 0.0015; awanni 12 vs. awanni 0.5, P = 0.030. 3D20E22P: awanni 3 vs. awanni 0.5, P = 0.25; awanni 12 vs. awanni 3, P = 0.0032; awanni 12 idan aka kwatanta da awanni 0.5, P = 0.015). Bayanan sun fito ne daga kwafi uku na halittu. An ƙididdige yankin kololuwar kowane ecdysone mai sha'awa kuma an daidaita shi da adadin sauro. Ana wakiltar Ecdysone ta launi kamar haka: E, kore; 20E, lemu; 20E22P, shunayya; 3D20E, shuɗi; 3D20E22P, ruwan hoda. Inset ɗin yana ƙara sikelin akan axis y don nuna ƙananan matakan ecdysone.
Domin bincika ko an canza 3D20E22P da 3D20E yayin haɗuwa, mun cire mata LRTs a lokutan daban-daban bayan haɗuwa. Duk da cewa ba a sami ecdysone a cikin budurwai ba, mun lura da adadi mai yawa na 3D20E22P a cikin LRT nan da nan bayan haɗuwa (awanni 0.5 bayan haɗuwa, hpm), yana raguwa akan lokaci, yayin da matakan 3D20E suka ƙaru sosai (Hoto na 1). Ta amfani da 3D20E da aka haɗa ta hanyar sinadarai a matsayin mizani, mun ƙaddara cewa matakan wannan hormone na steroid a cikin haɗuwar LRTs sun fi 20E ninki 100 girma (Teburin Bayanai Mai Tsawo na 1). Don haka, 3D20E22P shine babban ecdysone na namiji wanda aka canza zuwa LRT na mace yayin haɗuwa, kuma sifar sa ta dephosphorylated, 3D20E, ta zama mai yawa jim kaɗan bayan haɗuwa. Wannan yana nuna muhimmiyar rawa ga ecdysone na ƙarshe a cikin ilimin halittar mace bayan haɗuwa.
Bayan samar da sabon tsarin tattara bayanai na RNA (RNA-seq) (Hoto na 2a), ta amfani da bututun bioinformatics da aka gina musamman, mun nemi ecdysone kinase (EcK), ecdysone oxidase (EO), da kuma ecdysone wanda ke ƙunshe da kwayar halittar phosphatase mai 20E. An bayyana EPP) a cikin kyallen haihuwa. Mun gano kwayar halittar EPP guda ɗaya da kuma kwayoyin halittar ECK guda biyu masu yuwuwa (EcK1 da Eck2), amma ba mu sami ingantaccen kwayar halittar EO ba. Abin lura, an bayyana kwayoyin halittar EPP guda ɗaya a manyan matakai (kashi 98.9 cikin ɗari) a cikin MAGs na Gambia amma ba a cikin LRTs na mata ba (Hoto na 2b), sabanin tsammaninmu tun lokacin da aka sami dephosphorylation na 3D20E22P a cikin wannan kyallen mace. Saboda haka, mun yi imanin cewa ana iya canja wurin EPP na maza yayin haɗuwa. Hakika, mun yi amfani da lakabin isotope mai karko a cikin jiki don ɓoye furotin na mata bayan haɗuwa, wani enzyme da MS ya gano a cikin atrium na mace (Hoto na 2c da Tebur na Ƙarin 1). An kuma tabbatar da kasancewar EPP a cikin MAG da kuma LRT na mace mai haɗuwa (amma ba budurwa ba) ta amfani da takamaiman ƙwayoyin rigakafi (Hoto na 2d).
a, Tsarin bioinformatics da aka gina musamman don bincika kyallen haihuwa na kowace jinsi don gano kwayoyin halittar da ke ƙunshe da EcKs, EOs, da EPPs. Lambobin da ke kusa da kibiyoyi suna nuna adadin 'yan takara maza da mata a kowane mataki. Wannan bincike ya gano kwayar halittar EPP guda ɗaya (EPP) da kwayar halittar Eck guda ɗaya (EcK1) da aka bayyana a cikin maza, da kuma kwayar halittar Eck guda ɗaya (EcK2) wacce aka bayyana a cikin jinsi biyu amma ba ta samar da kwayar halittar EO guda ɗaya ba.b, Taswirar zafi tana kwatanta bayyanar kwayar halittar ɗan takara a cikin budurwa (V) da haɗuwa (M) Anopheles gambiae da Anopheles albicans kyallen.Spca, hadi; MAGs, glands masu haɗawa a cikin maza; Sauran sassan jiki, ciki har da nono, fikafikai, ƙafafu, jikin mai, da gabobin ciki a cikin jinsi biyu, da kuma kwai a cikin mata. EcK2 yana da matuƙar bayyana a cikin MAG da atria na Gambia, yayin da EPP ke samuwa ne kawai a cikin MAG.c, nazarin Proteomic na canjin ƙungiyar maniyyi na maza zuwa atria na mace a 3, 12 da 24 hpm, yana nuna furotin 67 mafi yawan gaske. An renon mata a kan abinci mai ɗauke da 15N don yiwa (da kuma rufe) duk furotin. An haɗa mazan da ba a yiwa alama da mata masu alama ba, kuma an rarraba LRT na mata a 3, 12 da 24 hpm don nazarin proteomic (duba Ƙarin Tebur 1 don cikakken jerin furotin maniyyi). An gano inset, EPP, Eck1 da Eck2 a cikin MAG na mazan budurwa ta hanyar nazarin proteomic na waɗannan kyallen.d, an gano EPP ta hanyar western blot a cikin MAG da LRT na mata masu aure, amma ba a cikin mata budurwa ko maza ko sauran jikin mace ba. An yi bincike a lokaci guda tare da maganin hana ɗaukar kaya (mai sarrafa lodi) da kuma maganin hana EPP. Duk mazan budurwa ne. Duba Ƙarin Hoto na 1 don bayanan tushen gel. An yi wa Western blots sau biyu tare da sakamako iri ɗaya.
An tabbatar da aikin ecdysteroid phosphophosphatase na EPP bayan shigar da shi ta hanyar HPLC-MS/MS tare da 3D20E22P da aka ware daga MAG (Bayanan da aka faɗaɗa Hoto na 2a). Bugu da ƙari, lokacin da muka rufe EPP ta hanyar tsangwama ta hanyar RNA (RNAi), mun gano raguwa mai ƙarfi a cikin ayyukan phosphatase a cikin kyallen haihuwa na waɗannan maza (Hoto na 3a), kuma mata da suka haɗu da mazan da aka ɗaure da EPP sun nuna mahimmanci Ƙananan rabo na dephosphorylated 3D20E (Hoto na 3b) duk da rufewar kwayoyin halitta (Hoto na 2b,c). Sabanin haka, ba mu gano manyan canje-canje a cikin rabo na 20E22P/20E a cikin sauro iri ɗaya ba, wanda zai iya nuna cewa enzyme ɗin ya keɓance ga 3D20E22P (Hoto na 3b).
a, Raguwar aikin phosphatase a cikin MAG wanda ya faru sakamakon rufewar EPP ta amfani da EPP RNA mai nau'i biyu (dsEPP) ko kuma GFP RNA mai nau'i biyu (dsGFP). An yi amfani da wuraren MAG guda ashirin a cikin kowane kwafi (P = 0.0046, gwajin t-gwaji mai nau'i biyu, mai gefe biyu), wanda aka wakilta ta hanyar ɗigo daban-daban.b, Matan da suka haɗu da mazan da suka yi shiru na EPP suna da ƙarancin kaso na dephosphorylated 3D20E a 3 hpm (P = 0.0043, gwajin t-gwaji mara nau'i biyu, mai gefe biyu), yayin da matakan 20E ba su shafi ba (P = 0.063, ba a haɗa su ba). gwajin t, mai gefe biyu). An gabatar da bayanai a matsayin matsakaicin ± sem daga tafkuna uku na mata 13, 16 da 19 kowannensu.c, Matan da suka haɗu da mazan da aka yi shiru da EPP suna da yawan sake haɗuwa sosai (P = 0.0002, gwajin Fisher na ainihi, mai gefe biyu). An fara tilasta wa mata su haɗu don tabbatar da matsayin haɗuwarsu; Bayan kwana 2, an yi hulɗa da su da wasu mazan da ke ɗauke da maniyyi mai canza jinsi don tantance yawan sake haɗuwa ta hanyar gano PCR na adadi na transgene.d. Matan da aka shayar da jini waɗanda suka haɗu da mazan da aka yi wa shiru na EPP sun rage yawan haihuwa sosai (P < 0.0001; gwajin Mann-Whitney, mai gefe biyu) da kuma ɗan raguwar adadin ƙwai (P = 0.088, gwajin Mann-Whitney, mai gefe biyu), yayin da ƙimar haihuwa ba ta shafi ba (P = 0.94, gwajin Fisher daidai, mai gefe biyu). A cikin dukkan bangarorin, n yana wakiltar adadin samfuran sauro masu zaman kansu na halitta.NS, ba mai mahimmanci ba.*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.001.
Na gaba, mun tantance ko ecdysone dephosphorylation yana da mahimmanci don haifar da juriya ga haɗuwa a cikin mata. Abin lura shi ne, mata da suka haɗu da mazan da suka ƙare da EPP sun sake haɗuwa a mafi yawan lokuta (44.9%) fiye da mata masu iko (10.4%) lokacin da aka fallasa su ga ƙarin maza (transgenic) (Hoto na 3c). Mun kuma lura da raguwar haihuwa mai yawa (Hoto na 3d, hagu) da ɗan raguwar adadin ƙwai da waɗannan mata suka saka (Hoto na 3d, tsakiya), yayin da kashi na ƙwai da mata suka saka (wani martani da aka samu ga mata ta hanyar haɗuwa)) bai shafi ba (Hoto na 3d, dama). Ganin takamaiman EPP da aka lura don 3D20E22P, waɗannan sakamakon sun nuna cewa kunna 3D20E ta EPP da aka canjawa wuri yayin haɗuwa na iya samun muhimmiyar rawa wajen kashe karɓar mata zuwa ƙarin haɗuwa, wani hali da aka danganta da canja wurin jima'i na 20E. Saboda haka, wannan hormone na musamman ga maza kuma yana da tasiri sosai ga haihuwa ta mata.
Na gaba, mun kwatanta ayyukan 20E da 3D20E a gwaje-gwajen allura a cikin budurwoyi masu shekaru ta hanyar amfani da 3D20E da aka haɗa ta hanyar sinadarai (Hoto na 4a–c) kuma ana samun su a kasuwa 20E. Mun lura cewa 3D20E ya fi tasiri fiye da 20E wajen kashe hankalin mata ga haɗuwa a duka yawan haɗuwa (Hoto na 4d). Abin lura shi ne, rabin matakin ilimin halittar jiki na 3D20E a cikin LRT (1,066 pg bayan allura da 2,022 pg bayan haɗuwa) ya haifar da rabon mata masu tsaurin ra'ayi wanda ya ninka matakin ilimin halittar jiki na 20E (361 pg bayan allura) sau 24 bayan allura a mafi girman yawan haɗuwa 18 pg bayan haɗuwa; Teburin Bayanai Mai Tsawaita na 1). Wannan sakamakon ya yi daidai da ra'ayin cewa canja wurin jima'i na 20E ba ya haifar da lokutan da ba sa haɗuwa da juna, kuma ya nuna cewa 3D20E a matsayin babban abin da ke tabbatar da dangantakar iyaye da yara. 3D20E shi ma ya fi aiki fiye da 20E a cikin gwaje-gwajen kwanciya ƙwai a cikin mata budurwa (Hoto na 4e), yana nuna cewa yawan kwanciya ƙwai na yau da kullun da muka lura bayan rufe EPP na ɗan lokaci ya kasance saboda kasancewar sauran ayyukan 3D20E da har yanzu ke samarwa ta hanyar abubuwan mata da ke haifar da haɗuwa da juna.
(a,b) 3D20E wanda aka haɗa ta hanyar sinadarai daga 20E (a) tare da babban canji/inganci sosai (bayanan da aka gabatar a matsayin matsakaicin ± sem daga halayen haɗa guda uku masu zaman kansu) (b).c, Tsarin taro (rabin ƙasa) yayi daidai da ecdysone da aka samu a cikin LRT na mace da aka haɗu (rabin sama).d, Idan aka kwatanta da 20E (0.63 µg, P = 0.02; 0.21 µg, P < 0.0001; Gwajin Fisher na daidai, mai gefe biyu) da 10% ethanol (0.63 µg, P < 0.0001; 0.21 µg, P < 0.0001; Gwajin Fisher na daidai, mai gefe biyu), yayin da 20E ya fi iko sosai fiye da sarrafawa kawai a manyan allurai (0.63 µg, P = 0.0002; 0.21 µg, P = 0.54; Daidaitaccen Fisher gwaji, mai gefe biyu).e, allurar 3D20E ta haifar da ƙaruwar yawan haihuwa a cikin mata budurwa fiye da kashi 10% na sarrafa ethanol (0.21 µg, P < 0.0001; 0.13 µg, P = 0.0003; gwajin Fisher na daidai, mai gefe biyu), yayin da 20E idan aka kwatanta da sarrafawa. Kawai a cikin allurai mafi girma (0.21 µg, P = 0.022; 0.13 µg, P = 0.0823; gwajin Fisher na daidai, mai gefe biyu). 3D20E ta haifar da ƙaruwar haihuwa fiye da 20E a manyan allurai (0.21 µg, P = 0.0019; 0.13 µg, P = 0.075; gwajin Fisher na daidai, mai gefe biyu). A cikin dukkan bangarori, n yana wakiltar adadin samfuran sauro masu zaman kansu na halitta.NS, ba mai mahimmanci ba.*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.001. Bayanai sun fito ne daga kwafi uku.
A cikin binciken da muka yi a baya, mun gano cewa canja wurin hormones na steroid ta hanyar jima'i yana haifar da bayyanar MISO (Mating-Induced Stimulator of Oogenesis 11), wata kwayar halitta ta haihuwa ta mace wadda ke kare mata A. gambiae daga kamuwa da cutar P. falciparum Kudaden kiwon lafiya da 13 ke haifarwa, mafi munin kwayar cutar malaria ta ɗan adam. Ganin muhimmancin MISO ga lafiyar haihuwa na Anopheles a yankunan da cutar malaria ta fi kamari, mun yanke shawarar tantance wane hormone 3D20E ko 20E ke haifar da bayyanar wannan kwayar halitta. Mun gano cewa yayin da allurar 20E musamman ko mafi ƙarfi ta haifar da wasu masu karɓar hormone na nukiliya (HR), kamar HR3 da HR4, da kuma abubuwan da ake so a ƙasa na steroid, kamar kwayoyin halittar yolkogenic Vg14, 15, 16, MISO ya fi ƙarfin ta hanyar 3D20E (Bayanan da aka faɗaɗa Hoto na 3). Don haka, canja wurin wannan hormone na steroid na androgenic yana haifar da hanyoyin da ke kare mata daga farashin da kamuwa da cuta ke haifarwa. Bugu da ƙari, 3D20E yana shafar nau'ikan isoforms guda biyu na EcR mai karɓar E, yana haifar da EcR-A da kuma danne EcR-B, kuma yana haifar da wasu kwayoyin halitta masu haifar da haɗuwa, gami da HPX15, wanda ke shafar haihuwa ta mata. Wannan zai iya bayyana mahimmancin rashin haihuwa da aka gani a cikin mata da suka haɗu da mazan da EPP ya rufe (Bayanan da aka faɗaɗa Hoto na 3). Waɗannan bayanan suna nuna wanzuwar hanyoyin da ke ƙasa waɗanda aka fi amfani da su ta hanyar hormones guda biyu na ecdysone waɗanda zasu iya zama tushen aikin jima'i.
Na gaba, mun gwada aikin kwayoyin halittar Eck guda biyu da aka gano a cikin bututun bioinformatics ɗinmu. Yin shiru EcK1 ko EcK2 ya haifar da mace-mace mai yawa a cikin maza (Fitowar Bayanan da aka Tsara 4a), yana nuna cewa ecdysone phosphorylation, don haka rashin kunnawa, yana da mahimmanci don rayuwa. Saboda an bayyana EcK2 a matakai mafi girma fiye da EcK1 kuma an gano shi a cikin MAGs ta hanyar proteomics (Figogi na 2b,c da Tebur na Ƙarin 2), mun tabbatar da aikin ecdysteroid kinase ta hanyar saka shi cikin 20E, wanda ya haifar da phosphorylation 20E22P (Fitowar Bayanan da aka Tsara 2).4b). Lokacin amfani da 3D20E azaman substrate, ba mu iya gano samfurin phosphorylated 3D20E22P ba (Fitowar Bayanan da aka Tsara 4c), yana nuna cewa 20E maimakon 3D20E na iya zama abin da Eck2 ya fi so.
A bisa ga bincikenmu na RNA-seq, EcK2 ya kuma bayyana sosai a cikin LRT na mata budurwa, inda aka kashe shi bayan haɗuwa (Hoto na 2b). Mun tabbatar da waɗannan bayanan kuma mun tabbatar da cewa ba a shafar yanayin EcK2 ta hanyar ciyar da jini ba (Hoto na 5a na Bayanan da aka Tsara). Bayan faɗaɗa gwaje-gwajen MS na farko, mun gano cewa kololuwar 20E22P tana da alaƙa da kololuwar 20E (awanni 22-26 bayan cin abinci; Bayanan da aka Tsara Hoto na 5b). Rufe Eck2 a cikin mata budurwa ya haifar da ƙaruwa sau 3 a cikin rabon 20E zuwa 20E22P a awanni 26 bayan cin abinci (Hoto na Bayanan da aka Tsara 2c da 5c), yana tabbatar da cewa EcK2 kuma yana fitar da 20E a cikin mata. Abin lura shi ne, budurwai da suka ƙare da Eck2 sun ci gaba da karɓar cikakkiyar jima'i (Hoto na Bayanan da aka Tsara 5d,e), yana ƙara nuna cewa samar da mata na 20E ba ya haifar da lokacin rashin jituwa tsakanin jima'i. Koyaya, waɗannan Mata sun sami ƙaruwa sosai a yawan kwanciya da ƙwai idan aka kwatanta da na sarrafawa, inda sama da kashi 30% na budurwai ke yin ƙwai (Fitowar Bayanan da aka Tsara 5f). Idan an yi allurar Eck2 RNA (dsEcK2) mai nau'i biyu bayan ciyar da jini, haihuwa ba ta faru ba, a lokacin ne kololuwar 20E saboda shan jini ya ragu. Gabaɗaya, waɗannan sakamakon suna goyan bayan samfurin da 20E da aka samar bayan tsotsar jini zai iya haifar da haihuwa, amma sai lokacin da aka kashe toshewar haihuwa (EcK2 da wataƙila wasu abubuwa) ta hanyar haɗuwa. Babu allurar 20E ko 3D20E da ta hana bayyanar Eck2 a cikin budurwai (Fitowar Bayanan da aka Tsara 5g), tana nuna cewa wasu dalilai suna haifar da hana wannan kinase. Duk da haka, matakan 20E bayan ciyar da jini bai isa ba don haifar da rashin jin daɗin saduwa, amma an haifar da su ta hanyar yawan adadin 3D20E da aka ɗauka ta hanyar jima'i.
Sakamakonmu ya ba da muhimman bayanai game da hanyoyin da ke daidaita nasarar haihuwa na A. gambiae. Wani samfuri ya bayyana inda maza suka haɓaka yawan adadin 3D20E, wani ecdysone da aka gyara musamman ga maza wanda ke tabbatar da iyaye ta hanyar rage jin daɗin mata don ƙarin haɗuwa. A lokaci guda, waɗannan ƙwayoyin cuta na malaria sun kuma haɓaka ingantaccen tsarin don kunna 3D20E a cikin mata don mayar da martani ga canja wurin jima'i na EPP na musamman ga maza. A saninmu, wannan shine misali na farko na tsarin hormone steroid wanda maza da mata suka mamaye yana yin aiki na musamman da mahimmanci a cikin kwari. An yi hasashen aikin ecdysone na musamman ga maza amma ba a nuna shi ba tukuna. Misali, wata zato da aka musanta 18 ita ce waɗannan ayyukan za a iya yin su ta hanyar precursor na 20E E1. An san cewa a cikin Drosophila, monandry yana haifar da canja wurin jima'i na ƙananan peptides na jima'i19,20 waɗanda ke hulɗa da neurons waɗanda ke shiga cikin hanyar haihuwa ta mace ta hanyar masu karɓar peptide na jima'i21,22. Ana buƙatar ƙarin aiki don tantance siginar ƙasa cascades da 3D20E ke sarrafawa a cikin mata A. gambiae da kuma don tantance ko za a iya kiyaye waɗannan cascades tsakanin sauro da Drosophila.
Ganin muhimmancin rawar da 3D20E ke takawa wajen haihuwa da halayyar mata da aka gano a cikin bincikenmu, hanyoyin da ke haifar da hadawa da kunnawar 3D20E suna ba da sabbin damammaki ga dabarun sarrafa sauro na gaba, kamar samar da maza masu fafatawa a cikin dabarun fasahar kwari marasa tsafta. Amfani da shi don sakin daji ko kuma yin kwaikwayon 3D20E a cikin wasan budurwa. Aikin musamman na maza na 3D20E na iya canzawa lokacin da A. gambiae da sauran nau'ikan Cellia suka sami ikon haɗa maniyyinsu zuwa matosai masu haɗuwa, saboda wannan yana ba da damar canja wurin adadi mai yawa na hormones da enzymes masu kunna hormone. A gefe guda kuma, aiwatar da juyin halittar 3D20E yana ba da hanya ga mata (ta hanyar yawan bayyanar MISO) don fifita lafiyarsu ta haihuwa a yankunan da ke da yawan zazzabin cizon sauro, wanda ke ba da gudummawa kai tsaye ga yaduwar Plasmodium. Ganin cewa an nuna cewa mata 20E suna da tasiri mai zurfi akan rayuwa da ci gaban P. falciparum a cikin sauro na mata na Anopheles, 24 duka hanyoyin hormone na steroid na maza da mata yanzu manyan fannoni ne na hulɗar sauro da ƙwayoyin cuta.
An haifi nau'in G3 na gambiae a ƙarƙashin yanayin kwari na yau da kullun (26-28 °C, zafi mai kyau 65-80%, haske/duhu na awanni 12:12). An ciyar da tsutsotsi da abincin kifi mai foda (TetraMin Tropical Flakes, Koi Pellets da Tetra Pond Sticks a cikin rabo na 7:7:2). An ciyar da sauro manya ad libitum 10% maganin dextrose da jinin ɗan adam na mako-mako (nazarin abubuwan da ke cikin jinin). An samo sauro budurwa ta hanyar raba jinsi a matakin pupal bayan an duba ƙarshen ta hanyar na'urar microscopy. An bayyana mazan da ke ɗauke da transgene na DsRed a baya.
An yi gwaje-gwajen da aka tilasta wa ma'aurata bisa ga ka'idojin da aka bayyana a baya. Don ma'aurata na halitta, an ajiye mata 'yan kwanaki 4 a cikin rabo 1:3 tare da mazan da suka manyanta ta hanyar jima'i na tsawon dare biyu. Don gwaje-gwajen da aka yi wa maza allurar dsEPP, haɗin gwiwa ya yi daidai da kwanaki 3-4 bayan allurar, lokacin da aka dakatar da aikin phosphatase gaba ɗaya (Bayanan da aka faɗaɗa Hoto na 2b).
An rarraba kyallen sauro, sauran gawarwakin da suka rage (sauran jikin), ko dukkan jiki zuwa kashi 100% na methanol kuma an haɗa su da beader (ƙwallon gilashi 2 mm, 2,400 rpm, 90 sec). Adadin nama da girman methanol sune kamar haka: sauran jiki, 50 a cikin 1,000 µl; MAG, 50–100 80 µl; LRT na mace, 25–50 80 µl. An cire ruwan da ya fashe a karo na biyu na fitar da methanol tare da wannan adadin methanol. An cire tarkacen ƙwayoyin halitta ta hanyar centrifugation. An haɗa methanol daga duka cirewar kuma an busar da shi a ƙarƙashin kwararar nitrogen, sannan aka sake dakatar da shi a cikin waɗannan adadin methanol 80% a cikin ruwa: sauran jiki, 50 µl; MAGs da LRT na mace, 30 µl.
An yi nazarin samfura a kan na'urar auna mass spectrometer (ID-X, Thermo Fisher) da aka haɗa da kayan aikin LC (Vanquish, Thermo Fisher). An allurar µl na samfurin a kan ginshiƙi mai girman 3 µm, 100 × 4.6 mm (Inspire C8, Dikma) wanda aka kiyaye a 25 °C. Matakan motsi na LC sune A (ruwa, 0.1% formic acid) da B (acetonitrile, 0.1% formic acid). Matsakaicin LC kamar haka: 5% B na minti 1, sannan aka ƙara shi zuwa 100% B a cikin mintuna 11. Bayan mintuna 8 a 100%, sake daidaita ginshiƙin a 5% B na mintuna 4. Yawan kwararar ya kasance 0.3 ml min-1. Ana samun ionization a cikin tushen MS ta hanyar ionization mai zafi na electrospray a cikin yanayi masu kyau da marasa kyau.
Mita mai auna ma'aunin nauyi yana auna bayanai a cikin kewayon m/z daga 350 zuwa 680 a ƙuduri 60,000 a cikin cikakken yanayin MS. An samo bayanan MS/MS akan [M + H]+ (duk abubuwan da aka nufa), [M - H2O + H]+ (duk abubuwan da aka nufa), da [M - H]- (manufa ta phosphorylated). An yi amfani da bayanan MS/MS don tabbatar da kaddarorin ecdysone na abubuwan da aka nufa waɗanda babu wani mizani da aka nufa. Don gano ecdysteroids marasa niyya, an yi nazarin bayanan MS/MS don duk kololuwar HPLC tare da yawan dangi sama da 15%. A ƙididdige ta amfani da lanƙwasa na yau da kullun da aka ƙirƙira daga tsarkakkun ƙa'idodi (20E, 3D20E) don ƙididdige adadi cikakke ko narkewar samfuri ɗaya (duk sauran abubuwan da aka nufa) don ƙididdige daidaiton su da adadin da aka samu a cikin namiji ɗaya. Ga 3D20E, an yi ƙididdigewa ta amfani da jimlar abubuwan da aka nufa: [M + TFA]-, [M + COOH]-, [M + Na]+, [M + Cl]-, [M + NO3]-.An cire bayanai kuma an ƙididdige su ta amfani da Tracefinder (sigar 4.1).An yi nazarin bayanan MS/MS ta amfani da Xcalibur (sigar 4.4).An kwatanta siginar MS na E, 20E da 3D20E da mizanan da suka dace.An yi nazarin 3D20E22P ta hanyar cirewa tare da reagent na Girard.An yi nazarin 20E22P ta hanyar rabon m/z.
An tsarkake 3D20E22P daga MAG. An yi tsarkakewa akan sikelin nazari ta amfani da chromatograph na ruwa mai aiki sosai (Acquity, Waters) tare da na'urar gano mass-based quadrupole (QDa, Acquity, Waters) a ƙarƙashin yanayin LC iri ɗaya kamar yadda aka yi nazarin HPLC-MS/MS. An fara tattara fraction lokacin da aka gano m/z daidai da 3D20E22P a daidai lokacin riƙewa kamar yadda aka ƙayyade a baya. Sannan an duba tsarkin mahaɗan da aka cire ta HPLC-MS/MS kamar yadda aka bayyana a sama.
An cire jimillar RNA daga kyallen haihuwa guda 10-12 ko wasu sassan jiki (mara kai) ta amfani da TRI reagent (Thermo Fisher) bisa ga umarnin masana'anta. An yi wa RNA magani da TURBO DNase (Thermo Fisher). An haɗa cDNA ta amfani da Moloney murine leukemia virus reverse transcriptase (M-MLV RT; Thermo Fisher) bisa ga umarnin masana'anta. An buga firam ɗin PCR na reverse transcription quantitative PCR (RT-qPCR; Extended Data Table 2) a baya 24 ko an tsara su ta amfani da Primer-BLAST26, tare da fifiko ga samfuran girman 70-150 bp da kuma haɗin exon-exon ko Primer pair primers daban-daban exons. An narkar da samfuran cDNA daga kwafi uku zuwa huɗu na halitta sau huɗu a cikin ruwa don RT-qPCR. An yi ƙima a cikin halayen kwafi 15 µl waɗanda suka ƙunshi 1 × PowerUp SYBR Green Master Mix (Thermo Fisher), firam, da 5 µl na cDNA mai narkewa. An gudanar da halayen akan An tattara kuma an yi nazarin tsarin PCR na QuantStudio 6 Pro na ainihin lokaci (Thermo Fisher) da bayanai ta amfani da Tsarin Zane da Nazari (sigar 2.4.3). Kamar yadda aka nuna a cikin wannan binciken, an daidaita adadin dangi zuwa ga kwayar halittar ribosomal RpL19 (AGAP004422), wacce yanayinta bai canza sosai ba lokacin ciyar da jini 27 ko haɗuwa 3.
An duba ingancin RNA ta amfani da Agilent Bioanalyzer 2100 Bioanalyzer (Agilent). An shirya ɗakunan karatu na Illumina kuma an gudanar da su a Broad Institute of MIT da Harvard. An daidaita karatun jerin gwano zuwa ga A. gambiae genome (PEST iri, sigar 4.12) ta amfani da HISAT2 (sigar 2.0.5) tare da sigogi na tsoho. An cire karatun da ke da ingancin taswira (MAPQ) maki <30 ta amfani da Samtools (sigar 1.3.1). An ƙidaya adadin karantawa da aka tsara zuwa kwayoyin halitta ta amfani da htseq-count (sigar 0.9.1) tare da sigogi na tsoho. An ƙididdige ƙididdigar karantawa na yau da kullun kuma an bincika bayyanar kwayar halitta ta daban ta amfani da fakitin DESeq2 (sigar 1.28.1) a cikin R (sigar 4.0.3).
An gano waɗanda ke canza kwayoyin halittar Ecdysone ta hanyar fara binciken kwayar halittar A. gambiae ta amfani da tsarin PSI-BLAST (https://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/2.8.1/), ta amfani da ƙimar tsoho. Sigogi tare da jerin sunadaran tambaya masu zuwa: daga Bombyx mori (Accesion No. NP_001038956.1), Musca domestica (Accesion No. XP_005182020.1, XP_005175332.1 da XP_011294434.1) da kuma Microplitus demolitor (Accesion No. XP_008552646.1 da XP_008552645.1) Eck daga B. mori (Accesion No. NP_001036900), Drosophila melanogaster (Accesion No. NP_651202), Apis mellifera (Lambar Samun dama. XP_394838) da Acyrthosiphon pisum (Lambar Samun dama. XP_001947166); da EPP daga B. mori (Lambar Samun dama. XP_001947166) NP_001177919.1 da NP_001243996.1) da EO na D. melanogaster (Lambar Samun dama. NP_572986.1) (mataki na 1). Na gaba, ana samun matattara bisa ga yawan bayyanar mRNA (> gutsuttsura 100/kilobase exons a kowace miliyan da aka zana taswirar (FPKM) ko >85%) a cikin kyallen haihuwa (LRT ko MAG na mata) a Gambia (mataki na 2). Domin inganta takamaiman bayanai, mun zaɓi enzymes masu cancanta waɗanda kuma ake bayyana su a cikin ƙwayar haihuwa ta A. albimanus, wani nau'in anopheles wanda ba ya haɗa ko canja wurin ecdysone yayin haɗuwa. An tace kwayoyin halitta na 'yan takara bisa ga ƙarancin bayyanawa (<100 FPKM ko <85th percentile) a cikin ƙwayar haihuwa ta A. albimanus (mataki na 3). A matsayin matattara ta ƙarshe (mataki na 4), kwayoyin halitta masu cancanta suna buƙatar gamsar da aƙalla ɗaya daga cikin waɗannan: (1) an daidaita su sosai bayan haɗuwa (P < 0.05) bisa ga nazarin kwayoyin halitta da aka bayyana daban-daban da kuma (2) a cikin kyallen da ba sa haihuwa (< 85% ko <100 FPKM).
Mun gyara hanyoyin da aka bayyana a baya 28,29,30 don cimma lakabin isotopic na dukkan kwayoyin halitta. A takaice, an gwada nau'in Saccharomyces cerevisiae na nau'in II (YSC2, Sigma) a cikin tushen nitrogen na yisti (BD Difco, DF0335) wanda ke ɗauke da (wt/vol) 2% glucose (G7528, Sigma), 1.7% marasa amino acid da ammonium sulfate. matsakaici na al'ada) da 5% 15N ammonium sulfate (NLM-713, >99%, Cambridge Isotope Laboratories) a matsayin tushen nitrogen kawai. An dawo da yisti ta hanyar centrifugation kuma an ciyar da tsutsotsi sauro ad libitum har sai sun girma. Ƙari da abincin kifi (0.5 mg a kowace tsutsotsi 300) don hana mace-macen haihuwa na huɗu. Sannan aka yi amfani da mata kawai a gwaje-gwajen haɗuwa da maza marasa lakabi don nazarin furotin na namiji da aka canjawa wuri yayin haɗuwa.
An tilasta wa mata 'yan kwanaki 4-6 masu alamar 15N masu shekaru 4-6 su haɗu da mazan da ba su da alamar shekaru. An tabbatar da nasarar haɗuwa ta hanyar gano matosai na haɗuwa a ƙarƙashin na'urar hangen nesa ta epifluorescence. A cikin 3, 12, da 24 hpm, an rarraba atria na mata masu haɗuwa 45-55 zuwa 50 µl na ammonium bicarbonate buffer (pH 7.8) kuma an haɗa su da pestle. An sanya homogenate a cikin centrifuge kuma an haɗa supernatant da 50 µl na 0.1% RapiGest (186001860, Waters) a cikin 50 mM ammonium bicarbonate. An daskare supernatant da pellet daga kowane samfurin a kan busasshen kankara kuma an aika su cikin dare zuwa dakin gwaje-gwaje na MacCoss a Jami'ar Washington, inda aka kammala shirya samfurin LC-MS/MS. Sake dawo da pellet ɗin a cikin 50 µl na 0.1% RapiGest a cikin 50 mM ammonium bicarbonate da sonicate a cikin wanka mai ruwa. An auna yawan furotin na pellet da supernatant ta hanyar gwajin BCA, an rage samfuran da dithiothreitol 5 mM (DTT; Sigma), an haɗa su da alkylated da iodoacetamide 15 mM (Sigma) sannan aka saka su a zafin jiki na 37 °C (1:0 50) na tsawon awa 1 tare da trypsinization: trypsin:substrate ratio). An yi amfani da RapiGest ta hanyar ƙara 200 mM HCl, sannan aka saka su a zafin jiki na 37 °C na tsawon mintuna 45 da kuma centrifugation a 14,000 rpm na tsawon mintuna 10 a zafin jiki na 4 °C don cire tarkace. An wanke samfuran ta hanyar cirewa mai ƙarfi na yanayin dual-mode (katunan Oasis MCX, Ruwa) sannan aka sake dakatar da su a cikin 0.1% formic acid don samun yawan furotin na ƙarshe na 0.33 µg µl-1. An yi nazarin furotin na MAG marasa lakabi iri ɗaya daga mazan budurwa. Biyu An yi nazarin kwafi na nazari ga kowane samfurin. Na gaba, an yi nazarin 1 µg na kowanne ta amfani da ginshiƙin silica mai girman 25-cm mai siffar silica 75-μm tare da tarko mai siffar silica mai girman 4-cm mai siffar silica Kasil1 (PQ) wanda aka cika da resin Jupiter C12 mai juyawa (Phenomenex) da kuma chromatography na ruwa na minti 180. Samfurin nassoshi - An gudanar da MS/MS akan na'urar auna ma'aunin HF mai suna Q-Exactive (Thermo Fisher) tare da nanoACQUITY UPLC System (Waters). An canza bayanan da suka shafi bayanai don kowane gudu zuwa tsarin mzML ta amfani da Proteowizard (sigar 3.0.20287) da kuma amfani da Comet31 (sigar 3.2) akan bayanan FASTA da ke ɗauke da jerin furotin daga Anopheles gambiae (sigar VectorBase 54), Anopheles coluzzi An gudanar da bincike akan Mali-NIH (sigar VectorBase 54), Saccharomyces cerevisiae (Uniprot, Maris 2021), A. gambiae RNA-seq, da fassarar firam uku na gurɓatattun mutane da aka sani. An tantance FDRs da aka daidaita da taswirar Peptide ta amfani da Percolator32 (sigar 3.05) tare da matakin 0.01, kuma an haɗa peptides cikin gano furotin ta amfani da parsimony furotin a cikin Limelight33 (sigar 2.2.0). An kiyasta yawan furotin mai alaƙa ta amfani da ma'aunin yawan spectral (NSAF) wanda aka ƙididdige ga kowane furotin a kowace gudu kamar yadda aka bayyana a baya. An auna matsakaicin NSAF dangane da kowace furotin a cikin samfuran daga kwafi biyu daban-daban na halittu. Lakabin 15N ya yi nasarar ɓoye furotin na mace, kodayake an iya gano ƙaramin adadin furotin mara lakabi daga budurwai masu lakabi. Mun rubuta gano raguwar furotin na maza (1-5 spectra) a cikin samfuran da ba a saka ba na mata kawai a cikin gwaje-gwajen fasaha, inda aka gudanar da samfuran da ba a saka ba bayan samfuran maza/masoya, sakamakon "kwashewar HPLC". Sunadaran da ake samu a matsayin 'gurɓatattu' daga budurwai masu lakabi an jera su a cikin Tebur na Ƙarin 1.
Peptides guda biyu na antigenic, QTTDRVAPAPDQQQ (cikin isotype PA) da MESDGTTPSGDSEQ (cikin isotype PA da PB) a cikin Genscript. An haɗa peptides guda biyu, sannan aka haɗa su da furotin mai ɗauke da KLH aka kuma yi musu allurar a cikin zomaye na New Zealand. An yanka zomaye bayan allura ta huɗu, kuma an ware IgG gaba ɗaya ta hanyar tsarkakewar affiliation. An yi amfani da IgG daga mafi yawan zomo na musamman na EPP don ƙarin blotting na yamma.
Ga ƙwayoyin cuta na yamma, MAG (n = 10, inda n ke wakiltar adadin samfuran sauro masu zaman kansu ta hanyar halitta) da kuma LRT na mace (n = 30) daga mazan budurwa 'yan kwanaki 4 da mata masu budurwa ko waɗanda aka tilasta musu haɗuwa (<10 bayan haɗuwa), ma'aunin cire furotin (50 mM Tris, pH 8.0; 1% NP-40; 0.25% sodium deoxycholate; 150 mM NaCl; 1 mM EDTA; 1 × protease inhibitor cocktail (Roche)) an ƙara su daban. An haɗa samfuran nan da nan bayan an cire su da beader (ƙwallon gilashi 2 mm, 2,400 rpm, 90 sec). An cire tarkace marasa narkewa ta hanyar centrifugation a 20,000 g a 4 °C. An auna sunadaran ta hanyar gwajin Bradford (Bio-Rad). Sannan, 20 µg na furotin MAG, 40 µg na furotin LRT, da 20 µg na An cire ragowar furotin mai yawa kuma an raba shi da kashi 10% na Bis-Tris NuPAGE ta amfani da MOPS buffer. An canza sunadaran zuwa membranes na polyvinylidene fluoride ta amfani da tsarin canja wurin iBlot2 (Thermo Fisher). An wanke membranes sau biyu a cikin 1 × PBS-T (0.1% Tween-20 a cikin PBS) sannan aka toshe su a cikin Odyssey blocking buffer (Li-Cor) na tsawon awa 1 a 22°C. An girgiza membranes cikin dare a 4°C tare da rabbit anti-EPP polyclonal primary antibody (1:700 a cikin block buffer) da beraye anti-actin monoclonal primary antibody MAC237 (Abeam; 1:4,000). An wanke membranes da PBS-T sannan aka saka su da secondary antibodies (jaki anti-rabbit 800CW da goat anti-rat 680LT (Li-Cor), duka 1:20,000) a cikin block buffer wanda ke ɗauke da 0.01% SDS da 0.2% Tween -20 na tsawon awa 1 a zafin jiki na 22 °C. An wanke membranes da PBS-T kuma an yi musu hotonsu da na'urar daukar hoto ta Odyssey CLx. An tattara hotuna kuma an sarrafa su a cikin Image Studio (sigar 5.2). Ba a gano wani takamaiman madauri da ya dace da isoform na EPP-RA (82 kDa) ba.
An haɗa yankunan lambar EPP (kamar isoform AGAP002463-RB wanda ke ɗauke da yankin histidine phosphatase, NCBI conserved domain search 34) da Eck2 (AGAP002181) cikin plasmid pET-21a(+) (Novagen Millipore Sigma); An jera firam ɗin a cikin Teburin Bayanai Mai Faɗi 2. An saka mahaɗin GS4 guda takwas (a lokaci guda) kafin alamar C-terminal 6xHis na ginin pET-21a(+)-EcK2. An samar da sunadaran sake haɗawa ta amfani da amsawar sunadaran E. coli na ƙwayoyin halitta (New England BioLabs). An tsarkake sunadaran sake haɗawa ta amfani da ginshiƙan juyawa na NEBExpress Ni (New England BioLabs). An samar da sunadaran sarrafawa na Dihydrofolate reductase (DHFR) ta amfani da samfurin DNA daga Kit ɗin Haɗa Protein na NEBExpress Cell-Free E. coli. An adana sunadaran a cikin glycerol 50% a cikin PBS a -20 °C har zuwa watanni 3.
An auna aikin phosphatase na EPP da cirewar nama ta amfani da 4-nitrophenyl phosphate (pNPP; Sigma-Aldrich). Ma'ajiyar amsawar ta ƙunshi 25 mM Tris, 50 mM acetic acid, 25 mM Bis-Tris, 150 mM NaCl, 0.1 mM EDTA, da 1 mM DTT. An haɗa nama a cikin ma'ajiyar amsawar kuma an cire tarkacen tantanin halitta ta hanyar centrifugation. Fara amsawar ta hanyar ƙara enzyme ko cirewar nama zuwa ma'ajiyar amsawar da ke ɗauke da 2.5 mg ml-1 pNPP. An saka cakudawar amsawar a zafin ɗaki a cikin duhu, kuma an auna adadin pNP da aka canza daga pNPP ta hanyar auna shan ruwa a 405 nm a lokuta daban-daban.
Don aikin Eck a cikin vitro, an saka furotin da 0.2 mg 20E ko 3D20E a cikin buffer 200 µl (pH 7.5) wanda ke ɗauke da 10 mM HEPES–NaOH, 0.1% BSA, 2 mM ATP da 10 mM MgCl2 na tsawon awanni 2 a zafin jiki na 27 °C. An dakatar da amsawar ta hanyar ƙara 800 µl methanol, sannan aka sanyaya a -20 °C na tsawon awa 1, sannan aka sanyaya a 20,000 g na tsawon mintuna 10 a zafin jiki na 4 °C. Sannan HPLC-MS/MS ta yi nazarin saman sinadarin. Don dumama-da kashe sunadaran da ake amfani da su a cikin rukunin sarrafawa, an saka sunadaran a cikin glycerol 50% a cikin PBS na tsawon mintuna 20 a zafin jiki na 95 °C.
Don aikin EPP na in vitro, an haɗa furotin da 3D20E22P (daidai da adadin da aka samu a cikin nau'i-nau'i 18 na MAG, waɗanda aka tsarkake ta HPLC-MS/MS) a cikin buffer 100 µl (pH 7.5) wanda ke ɗauke da 25 mM Tris, 50 mM acetic acid, 25 mM Bis-Tris, 150 mM NaCl, 0.1 mM EDTA, da 1 mM DTT na tsawon awanni 3 a zafin jiki na 27 °C. An dakatar da amsawar ta hanyar ƙara 400 µl methanol kuma aka sanyaya a -20 °C na tsawon awa 1, sannan a sanyaya a 20,000 g na mintuna 10 a zafin jiki na 4 °C. An yi nazarin saman sinadarin ta hanyar HPLC-MS/MS.
An ƙara girman gutsuttsuran PCR na EPP (362 bp), EcK1 (AGAP004574, 365 bp) da EcK2 (556 bp) daga cDNA da aka shirya daga gawar sauro marasa kai gauraye. An ƙara girman guntun PCR na sarrafa eGFP (495 bp) daga pCR2.1-eGFP da aka bayyana a baya; An jera firam ɗin PCR a cikin Teburin Bayanai Mai Tsawo na 2. An saka ɓangaren PCR tsakanin masu haɓaka T7 da aka juya akan plasmid na pL4440. An samo gine-ginen Plasmid daga NEB 5-α E. coli (New England Biolabs) kuma an tabbatar da su ta hanyar jerin DNA kafin amfani (duba Ƙarin Bayanan 1 don jerin sakawa). An yi amfani da firam ɗin da suka dace da mai haɓaka T7 (Teburin Bayanan da aka Tsawo na 2) don haɓaka shigarwar daga plasmid na tushen pL4440. An tabbatar da girman samfurin PCR ta hanyar agarose gel electrophoresis.dsRNA an rubuta shi daga samfuran PCR ta amfani da Megascript T7 Transcription Kit (Thermo Fisher) kuma an tsarkake shi bisa ga umarnin masana'anta tare da gyare-gyaren da aka bayyana a baya.
Don allurar dsRNA, an yi allurar 1,380 ng na dsRNA (dsGFP, dsEcK1, dsEcK2, dsEPP) a cikin yawan 10 ng nl-1 a ​​cikin ƙirjin manya maza ko mata (Nanoject III, Drummond) cikin kwana 1 bayan eclosion. An ƙayyade matakan bugun kwayar halitta a cikin aƙalla kwafi uku na halitta ta hanyar cire RNA, haɗa cDNA, da RT-qPCR. Don allurar ecdysone, an yi wa mata 'yan kwanaki 4 ko 'yan kwanaki 6 allurar 0.13, 0.21, ko 0.63 µg na 20E ko 3D20E (Nanoject III, Drummond) a yawan 1.3, 2.1, bi da bi, ya danganta da ƙirar gwaji ko 6.3 ng nl-1. Yi allurar 100 nl na 10% (vol/vol) ethanol a cikin ruwa; 100 nl na 3D20E22P a cikin ethanol 10% (daidai yake da kashi 75% na adadin da aka samu a cikin MAG guda biyu). An rarraba sauro bazuwar zuwa rukunin allura.
Don gwajin haihuwa, an ciyar da mata 'yan kwana 3 ad libitum akan jinin ɗan adam. Cire sauro da aka ciyar da su kaɗan ko kuma waɗanda ba a ciyar da su ba. Dangane da maganin, an sanya mata a cikin kofuna daban-daban na haihuwa na tsawon dare huɗu aƙalla awanni 48 bayan cin jinin. An ƙirga ƙwai a ƙarƙashin na'urar auna sitiriyo (Stemi 508, Zeiss); ga mata da suka haɗu, ƙwai da suka ƙyanƙyashe cikin tsutsotsi ana ɗaukar su masu haihuwa.
Don gwaje-gwajen haɗuwa, an ba wa mata aƙalla kwana 2 dangane da maganin don samun juriya ga haɗuwa, kuma daga baya aka shigar da mazan da suka yi daidai da shekaru iri na daji a cikin keji ɗaya. Bayan kwana biyu, an rarraba ƙwayoyin halittar mace da aka haɗa kuma an saki DNA na genomic ta hanyar daskarewa-narkewa da sonication a cikin wani ma'ajiyar da ke ɗauke da 10 mM Tris-HCl, 1 mM EDTA, da 25 mM NaCl (pH 8.2). An saka samfuran da aka haɗa da Proteinase K (0.86 µg µl-1) na tsawon mintuna 15 a zafin jiki na 55 °C, sannan aka biyo baya na mintuna 10 a zafin jiki na 95 °C. An narkar da shirye-shiryen DNA na genomic sau 10 kuma an yi musu gwaji na qPCR na jerin ƙwayoyin halittar Y; an jera firam ɗin a cikin Tebur Bayani Mai Tsawo na 2. Rashin jerin ƙwayoyin halittar Y yana nuna babu haɗuwa.
Don sake yin gwaje-gwajen haɗuwa, an binciki mata da aka tilasta wa haɗuwa don ganin akwai toshewar haɗuwa don tabbatar da yanayin haɗuwa kuma an ba su damar kwana 2 don haɓaka rashin jituwa ga haɗuwa idan babu maza, kamar yadda aka bayyana a baya 36 . Sannan aka shigar da mazan da ke ɗauke da maniyyi na DsRed a cikin kejin mata. Bayan kwana biyu, an cire ƙwayoyin halittar da ke tarawa daga mata, kuma an shirya DNA na genomic kamar yadda aka bayyana a sama kuma an yi wa qPCR gano transgene na DsRed gwaji; an jera firam ɗin a cikin Tebur na Bayanai Mai Tsawo na 2. Rashin transgene na DsRed ya nuna cewa babu sake haɗuwa da ya faru.
An haɗa 3D20E kamar yadda aka bayyana a baya 37. A takaice, an narkar da 10 MG na 20E (Sigma-Aldrich) a cikin ruwa 10 ml, sannan aka ƙara 30 MG na platinum black (a cikin foda, Sigma-Aldrich). An ci gaba da zuba ruwa mai laushi na O2 a cikin cakudawar amsawar, wanda aka juya a zafin ɗaki. Bayan awanni 6, an ƙara 30 mL na methanol don dakatar da amsawar. An sanya cakuda a cikin centrifuge don cire ƙwayoyin catalyst. An fitar da ruwan sama zuwa busasshe a cikin vacuo a zafin ɗaki. An narkar da busasshen samfurin amsawar a cikin 10% ethanol da methanol don allura don nazarin HPLC-MS/MS. Yawan juyawa (daga 20E zuwa 3D20E) ya kasance kusan 97% (Hoto na 4b), kuma bakan MS na 3D20E da aka haɗa ya yi daidai da wanda aka samu a cikin mata masu haɗuwa (Hoto na 4c).
Tatsuniyar ta ƙunshi takamaiman bayanai game da gwaje-gwajen ƙididdiga da aka yi. An yi amfani da GraphPad (sigar 9.0) don yin gwajin Fisher daidai, gwajin Mantel-Cox, da gwajin t na ɗalibi. An yi gwaje-gwajen Cochran-Mantel-Haenszel ta amfani da rubutun R na musamman (wanda ake samu a https://github.com/duopeng/mantelhaen.test). An gwada rarraba bayanai don daidaito ta amfani da gwajin Shapiro-Wilk tare da ma'aunin mahimmanci na 0.05. Lokacin da bayanan suka gaza gwajin daidaito, an yi gwajin Mann-Whitney. An yi nazarin bayanan tsira ta amfani da gwajin Mantel-Cox. An yi amfani da fakitin DESeq2 (sigar 1.28.1) don yin nazarin bambancin matakin kwayar halitta na RNA-seq. Sandar kwance akan jadawalin tana wakiltar matsakaici. An yi amfani da ƙimar mahimmanci na P = 0.05 a matsayin ma'aunin duk gwaje-gwaje.
Don ƙarin bayani game da tsarin binciken, duba taƙaitaccen Rahoton Bincike na Yanayi wanda aka haɗa da wannan labarin.
An saka bayanan MS proteomic a cikin ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) ta hanyar PRIDE Partner Repository (https://www.ebi.ac.uk/pride/) tare da mai gano bayanai PXD032157.
An ajiye bayanan RNA-seq a cikin Laburare Mai Cikakken Bayani na Gene Expression (https://www.ncbi.nlm.nih.gov/geo/) a ƙarƙashin rikodin jerin GSE198665.
Ana iya samun ƙarin bayanan da aka samar da/ko aka yi nazari a kansu a lokacin binciken na yanzu daga marubutan da suka dace bisa ga buƙata mai ma'ana. Wannan labarin yana ba da bayanan tushe.
De Loof, A. Ecdysteroids: Maganin steroid na jima'i na kwari da aka yi watsi da su? Namiji: Akwatin Baƙi. Kimiyyar Kwari.13, 325–338 (2006).
Redfern, CPF 20-hydroxyecdysone da ci gaban ovarian a cikin Anopheles stephens.J. Ilimin Halittar Kwari.28, 97–109 (1982).