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Ngokungafani nezilwane ezinomgogodla, izinambuzane kucatshangwa kabanzi ukuthi azinawo ama-hormone e-sex steroid ahambisana nabesilisa. Ku-Anopheles gambiae, i-ecdysone steroid 20-hydroxyecdysone (20E) ibonakala sengathi ishintshe ukulawula ukukhula kwamaqanda lapho kwenziwa ngabesifazane2 kanye nokukhuthaza isikhathi sokungazali lapho kudluliswa ngocansi ngabesilisa3. Njengoba ukukhula kwamaqanda kanye nokuzalisana kuyizimpawu ezibalulekile zokuzala, ukuqonda ukuthi omiyane besifazane be-Anopheles bahlanganisa kanjani lezi zimpawu zama-hormone kungasiza ekwakhiweni kwezinhlelo ezintsha zokulawula umalaleveva. Lapha, sembula ukuthi le misebenzi yokuzala ilawulwa yi-sex steroid ehlukile ngenethiwekhi eyinkimbinkimbi yama-enzyme avuselela/aqeda ukusebenza kwe-ecdysteroid. Sithole i-ecdysone e-oxidized ethize yabesilisa, i-3-dehydro-20E (3D20E), evikela ubuzali ngokuvala ukwamukelwa kocansi kwabesifazane ngemuva kokudluliselwa ngokocansi kanye nokusebenza nge-dephosphorylation. Okuphawulekayo ukuthi, ukudluliselwa kwe-3D20E nakho kwabangela ukuvezwa kwezakhi zofuzo zokuzala ezigcina ukukhula kwamaqanda ngesikhathi sokutheleleka kwe-Plasmodium, ukuqinisekisa impilo yabesifazane abathelelekile. I-20E ethathwe kwabesifazane ayivezi impendulo yocansi, kodwa ivumela abantu abahlanganayo ukuba babeke amaqanda ngemva kokuvinjelwa kwama-kinase angu-20E. Ukuhlonza le hormone ye-steroid yezinambuzane ethize yabesilisa kanye nendima yayo ekulawuleni ukwamukelwa kocansi kwabesifazane, ukuzala kanye nokusebenzisana nePlasmodium kusikisela ukuthi kungenzeka ukunciphisa impumelelo yokuzala komiyane abadlulisela umalaleveva.
Amacala nokufa kukamalaleveva kuyanda futhi4 ngenxa yokumelana okubanzi kwezibulala-zinambuzane komiyane be-Anopheles, okuwukuphela kwembangela yezinambuzane ezibangelwa umaleveva kubantu. Ibhayoloji yokuzalana kwalawa mamiyane iyisisulu esikhangayo kakhulu sokungenelela okusha kokulawula umaleveva ngoba abesifazane bazala kanye kuphela5; ukwenza lesi senzakalo sokuzalana okukodwa kube yinyumba kungaba namandla amakhulu okunciphisa inani lomiyane ensimini.
Abesifazane bayakhubazeka ngokocansi ngemva kokuthola ama-hormone e-steroid ane-titer ephezulu emadodeni. Izifundo zikhombisile ukuthi imbangela yobunzima ekuzaleni okuqhubekayo yi-20-hydroxyecdysone (20E), i-hormone ye-steroid eyaziwa kangcono njengomlawuli womjikelezo wokubola esigabeni sama-larval. Amandla abesilisa okukhiqiza nokudlulisa i-20E avele ikakhulukazi ezinhlotsheni ze-Anopheles eziyingxenye ye-subgenus Cellia7, esakazeke e-Afrika futhi ifaka phakathi izimbangela eziyingozi kakhulu ze-malaria, kufaka phakathi i-Anopheles gambiae. Lokhu kuyaphawuleka kakhulu ngoba kulezi zinhlobo abesifazane nabo bakhiqiza i-20E ngemva kokudla kwegazi ngakunye, kanti i-20E iqhuba umjikelezo we-oogenesis (bheka ireferensi 8). Kodwa-ke, kuncane okwaziwayo ngendlela abesifazane abahlanganisa ngayo izimpawu ezivela emithonjeni emibili ehlukene ye-ecdysone (ukudluliselwa kwabesilisa kanye nokungeniswa kokuphakelwa kwegazi) ngaphandle kokubeka engcupheni ikhono labo lokuzala. Eqinisweni, uma i-20E ekhiqizwa abesifazane ibangela ukungabekezelelani ngokocansi, lokhu kuzoholela ekungazalini kubantu abancelisa intombazane, ukuziphatha okuvamile kakhulu kulawa ma-moth5.
Incazelo engaba khona ukuthi abesilisa be-A. gambiae badlulisela i-ecdysone eguquliwe eqondene nabesilisa, evuselela i-cascade yesiginali endleleni yokuzala yabesifazane, okuholela ekungazinzini kokuhlangana. Kodwa-ke, yize izilwane ezinomgogodla zinama-hormone amaningi e-steroid, njenge-estrogen ne-androgen (kubuyekezwe ku-ref. 9), ngokwazi kwethu, ama-steroid ahambisana ne-androgenic awazange atholakale ezinambuzaneni.
Siqale ukuthola uhlu lwama-hormone e-steroid ku-gland yesilisa evuthwe ngokocansi (MAG) ye-A. gambiae sifuna ama-steroid angashintshayo angaba khona. Sisebenzisa i-chromatography yoketshezi esebenza kahle kakhulu ehlanganiswe ne-tandem mass spectrometry (HPLC-MS/MS) kunendlela engacacile kangako esetshenziswe ngaphambilini, sithole i-ecdysone (E) kanye ne-20E kulesi zicubu, siqinisekisa umphumela wangaphambilini. Kodwa-ke, isampula yayibuswa ama-steroids e-phosphorylated axidized, ahambisana nefomula 3-dehydro-20E-22-phosphate (3D20E22P)12 (Isithombe 1). Ezinye izinhlobo zifaka phakathi i-3-dehydro-20E (3D20E) kanye ne-20E-22-phosphate (20E22P). Ukuqina kwesignali ye-HPLC-MS/MS ye-3D20E22P kwakungama-oda amabili amakhulu kunefomu layo le-dephosphorylated, i-3D20E, kanye nama-oda amathathu amakhulu aphezulu kune-E kanye ne-20E (Isithombe 1). Nakuba kwezinye izingxenye zomzimba kanye nomgudu wokuzala ophansi (LRT; Idatha Enwetshiwe Umfanekiso 1a). Siphinde sahlaziya ama-ecdysteroids kwabesilisa nabesifazane abasanda kuvalwa (abangaphansi kosuku olulodwa ubudala) futhi sathola i-3D20E kanye ne-3D20E22P kuphela ku-MAG; i-E, 20E kanye ne-20E22P zazikhona kubo bobabili ubulili (Idatha Enwetshiwe Umfanekiso 1b). Le datha isikisela ukuthi abesilisa abadala be-A. gambiae bakhiqiza amazinga aphezulu ama-hormone aguqulayo kuma-MAG abo angakhiqizwa ngabesifazane.
I-MAG kanye ne-LRT yesifazane (kufaka phakathi i-atria, ama-seminal vesicles, kanye ne-parovarium) kukhishwe kwabesilisa abaneminyaka engu-4 ubudala (abaneminyaka engu-4 ubudala) kanye nabesifazane abaneminyaka engu-0.5, 3, kanye ne-12 hpm). I-Ecdysone kulezi zicubu ihlaziywe yi-HPLC-MS/MS (isilinganiso ± sem; ukuhlolwa kwe-t okungabhangqiwe, izinga lokutholakala kwamanga elizinhlangothi zombili (i-FDR) elilungisiwe; i-NS, ayibalulekile; *P < 0.05, **P < 0.01 . 3D20E: amahora ama-3 vs. amahora angu-0.5, P = 0.035; amahora ayi-12 vs. amahora ama-3, P = 0.0015; amahora ayi-12 vs. amahora angu-0.5, P = 0.030. 3D20E22P: amahora ama-3 vs. amahora angu-0.5, P = 0.25; amahora ayi-12 vs. amahora ama-3, P = 0.0032; Amahora ayi-12 vs. amahora angu-0.5, P = 0.015).Idatha ivela eziphindaphindweni ezintathu zebhayoloji. Indawo ephezulu ye-ecdysone ngayinye ethakazelisayo yabalwa futhi yalungiswa ngenani lomiyane. I-Ecdysone imelelwa ngombala kanje: E, oluhlaza okotshani; 20E, o-orenji; 20E22P, onsomi; 3D20E, oluhlaza okwesibhakabhaka; 3D20E22P, opinki.Isithombe esingaphakathi sandisa isikali ku-y-axis ukuze sibonise amazinga aphansi e-ecdysone.
Ukuze sihlole ukuthi i-3D20E22P kanye ne-3D20E ziyadluliselwa yini ngesikhathi sokuhlangana, sihlukanise ama-LRT abesifazane ngezikhathi ezahlukene ngemva kokuhlangana. Nakuba i-ecdysone ingatholakalanga ezintombini, sibone inani elikhulu le-3D20E22P ku-LRT ngokushesha ngemva kokuhlangana (amahora angu-0.5 ngemva kokuhlangana, hpm), lehla ngokuhamba kwesikhathi, kuyilapho amazinga e-3D20E enyuka kakhulu (Isithombe 1). Sisebenzisa i-3D20E eyenziwe ngamakhemikhali njengendinganiso, sithole ukuthi amazinga ale hormone ye-steroid kuma-LRT okuhlangana ayephakeme okungenani ngokuphindwe kayi-100 kune-20E (Ithebula Ledatha Elandisiwe 1). Ngakho-ke, i-3D20E22P iyi-ecdysone enkulu yesilisa edluliselwa ku-LRT yesifazane ngesikhathi sokuhlangana, futhi isimo sayo se-dephosphorylated, i-3D20E, siba siningi kakhulu ngemva nje kokuhlangana. Lokhu kusikisela indima ebalulekile ye-ecdysone yokugcina kubhayoloji yabesifazane ngemva kokuhlangana.
Ngemva kokudala isethi entsha yedatha yokulandelelana kwe-RNA (i-RNA-seq) (Umfanekiso 2a), sisebenzisa ipayipi le-bioinformatics elakhelwe ngokwezifiso, sifune i-ecdysone kinase (EcK), i-ecdysone oxidase (EO), kanye ne-ecdysone ebhala i-20E-modified phosphatase gene. I-EPP) ivezwa ezicutshini zokuzala. Sithole i-gene eyodwa ye-EPP efanelekile kanye nama-gene amabili e-EcK angenzeka (i-EcK1 kanye ne-EcK2), kodwa asikwazanga ukuthola i-gene enhle ye-EO efanelekile. Okuphawulekayo ukuthi, ama-gene e-EPP ngamanye avezwa emazingeni aphezulu (i-percentile engama-98.9th) kuma-MAG aseGambia kodwa hhayi kuma-LRT abesifazane (Umfanekiso 2b), ngokuphambene nokulindela kwethu selokhu i-dephosphorylation ye-3D20E22P yenzeka kulesi zicubu sowesifazane. Ngakho-ke, sikholelwa ukuthi i-EPP yesilisa ingadluliselwa ngesikhathi sokukhulelwa. Ngempela, sisebenzise ilebula ye-isotope eqinile ye-in vivo ukufihla iphrotheni yesifazane ngemva kokukhulelwa, i-enzyme ekhonjwe yi-MS ku-atrium yesifazane (Umfanekiso 2c kanye Ithebula Elingeziwe 1). Ukuba khona kwe-EPP ku-MAG kanye ne-LRT yesifazane ekhulelwe (kodwa hhayi emsulwa) kwaqinisekiswa futhi kusetshenziswa ama-antibodies athile (Isithombe 2d).
a, Ipayipi le-bioinformatics elakhelwe ngokwezifiso lokusesha izicubu zokuzala zobulili ngabunye ukuthola izakhi zofuzo ezifaka ama-EcK, ama-EO, nama-EPP. Izinombolo eziseduze nemicibisholo zibonisa inani labesifazane nabesilisa abazongenela isinyathelo ngasinye. Lokhu kuhlaziya kuthole isakhi sofuzo esisodwa se-EPP (EPP) kanye nesakhi sofuzo esisodwa se-EcK (EcK1) esivezwa kwabesilisa, kanye nesakhi sofuzo esisodwa se-EcK (EcK2) esivezwa kubo bobabili ubulili kodwa esingavezi isakhi sofuzo se-EO esizongenela.b, Imephu yokushisa eqhathanisa ukubonakaliswa kwesakhi sofuzo esizongenela ku-virgin (V) kanye nokuzala (M) Izicubu ze-Anopheles gambiae kanye nezicubu ze-Anopheles albicans. I-Spca, ukukhulelwa; Ama-MAG, izindlala ezingeziwe kwabesilisa; ezinye izingxenye zomzimba, okuhlanganisa amabele, amaphiko, imilenze, imizimba enamafutha, kanye nezitho zangaphakathi kubo bobabili ubulili, kanye nama-ovari kwabesifazane. I-EcK2 ibonakaliswa kakhulu ku-MAG kanye ne-atria yaseGambia, kanti i-EPP itholakala kuphela ku-MAG.c, ukuhlaziywa kwe-Proteomic kokudluliselwa kweqembu le-ejaculate labesilisa libe yi-atria yabesifazane ku-3, 12 kanye no-24 hpm, okubonisa amaprotheni angu-67 amaningi kakhulu. Abesifazane bakhuliswa ngokudla okuqukethe i-15N ukuze babhale (futhi bafake imaski) wonke amaprotheni. Abesilisa abanganamathegi bahlanganiswa nabesifazane abanamathegi, kanti ama-LRT abesifazane ahlukaniswa ku-3, 12 kanye no-24 hpm ukuze kuhlaziywe i-proteomic (bheka Ithebula Elingeziwe 1 ukuze uthole uhlu oluphelele lwamaprotheni okukhipha i-ejaculatory). I-Inset, i-EPP, i-Eck1 kanye ne-EcK2 kutholakale ku-MAG yabesilisa abamsulwa ngokuhlaziywa kwe-proteomic kwalezi zicubu.d, i-EPP itholakale yi-western blot ku-MAG kanye ne-LRT yabesifazane abakhulelwe, kodwa hhayi kwabesifazane abamsulwa noma kwabesilisa noma abanye besifazane. umzimba. Ama-membrane ahlolwe ngasikhathi sinye nge-anti-actin (ukulawula ukulayisha) kanye nama-anti-EPP antibodies. Bonke abesilisa bayizintombi. Bheka uMfanekiso Ongeziwe 1 ukuthola idatha yomthombo wejeli. Ama-Western blots enziwe kabili ngemiphumela efanayo.
Umsebenzi we-ecdysteroid phosphophosphatase we-EPP uqinisekiswe ngemuva kokukhuliswa yi-HPLC-MS/MS nge-3D20E22P ehlukaniswe ne-MAG (Idatha Enwetshiwe Umfanekiso 2a). Ngaphezu kwalokho, lapho sithulisa i-EPP ngokuphazamiseka okubangelwa yi-RNA (i-RNAi), sithole ukwehla okukhulu komsebenzi we-phosphatase ezicutshini zokuzala zala madoda (Isithombe 3a), kanti abesifazane abahlanganyelwe nabesilisa abathuliswe yi-EPP babonise okuphawulekayo. Ingxenye ephansi ye-3D20E e-dephosphorylated (Isithombe 3b) naphezu kokuthuliswa kwezakhi zofuzo okungaphelele (Idatha Enwetshiwe Umfanekiso 2b,c). Ngokuphambene nalokho, asizange sithole izinguquko ezibalulekile ku-20E22P/20E ratio komiyane abafanayo, okungase kusikisele ukuthi i-enzyme iqondene ne-3D20E22P (Isithombe 3b).
a, Ukwehla komsebenzi we-phosphatase ku-MAG okubangelwa ukuthulisa i-EPP kusetshenziswa izilawuli ze-EPP RNA (dsEPP) ezinezintambo ezimbili noma i-GFP RNA (dsGFP) ezinezintambo ezimbili. Kusetshenziswe amachibi e-MAG angamashumi amabili ku-replica ngayinye (P = 0.0046, i-t-test ebhangqiwe, enezinhlangothi ezimbili), emelelwe amachashazi ahlukene.b, Abesifazane abahlanganyelwe nabesilisa abane-EPP-silenced babenesilinganiso esiphansi kakhulu se-3D20E e-dephosphorylated ku-3 hpm (P = 0.0043, i-t-test engabhangqiwe, enezinhlangothi ezimbili), kanti amazinga angu-20E awazange athinteke (P = 0.063, engabhangqiwe). ukuhlolwa kwe-t, okunezinhlangothi ezimbili). Idatha yethulwa njengesilinganiso ± sem esivela emachibini amathathu abesifazane abangu-13, 16 kanye no-19 ngamunye. c, Abesifazane abakhwelwe ngabesilisa abathuliswe yi-EPP babenezinga eliphezulu kakhulu lokuphinda bakhwelwe (P = 0.0002, ukuhlolwa okuqondile kukaFisher, okunezinhlangothi ezimbili). Abesifazane baphoqeleka okokuqala ukuthi bakhwelwe ukuqinisekisa isimo sabo sokukhwelana; Ngemva kwezinsuku ezimbili, baxhumana namanye amadoda aphethe isidoda esiguqulwe ufuzo ukuze bahlole amazinga okuhlangana kabusha ngokuthola i-PCR yobuningi be-transgene.d, abesifazane abanceliswa ngegazi abahlanganiswe nabesilisa abathuliswe yi-EPP babenokuzala okunciphile kakhulu (P < 0.0001; ukuhlolwa kwe-Mann-Whitney, okunezinhlangothi ezimbili) kanye nenani lamaqanda elinciphile kancane (P = 0.088, ukuhlolwa kwe-Mann-Whitney, okunezinhlangothi ezimbili), kuyilapho izinga lokuzala lingathintekile (P = 0.94, ukuhlolwa okuqondile kukaFisher, okunezinhlangothi ezimbili).Kuwo wonke amaphaneli, u-n umele inani lamasampula omiyane azimele ngokwebhayoloji.NS, akubalulekile.*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.001.
Okulandelayo, sihlole ukuthi i-ecdysone dephosphorylation ibalulekile yini ekubangeleni ukumelana nokuhlangana kwabesifazane. Okuphawulekayo ukuthi abesifazane abahlangana nabesilisa abaphelelwe yi-EPP baphinde bahlangana kaningi kakhulu (44.9%) kunabesifazane abalawulayo (10.4%) lapho bevezwa kwabesilisa abengeziwe (abashintshe ufuzo) (Isithombe 3c). Siphinde sabona ukwehla okukhulu kokuzala (Isithombe 3d, kwesobunxele) kanye nokwehla okuncane kwenani lamaqanda abekwa yilaba besifazane (Isithombe 3d, maphakathi), kuyilapho iphesenti lamaqanda abekwa yilaba besifazane (elinye impendulo elivezwe kwabesifazane ngokuhlangana )) lingathintekile (Isithombe 3d, kwesokudla). Njengoba kunikezwe ukucaciswa okubonwe kwe-EPP kwe-3D20E22P, le miphumela iphakamisa ukuthi ukusebenza kwe-3D20E yi-EPP okudluliselwe ngesikhathi sokuhlangana kungaba nendima ebalulekile ekuvaleni ukwamukelwa kwabesifazane ekuhlanganeni okuqhubekayo, ukuziphatha okwakubangelwa ukudluliselwa ngokocansi kwe-20E ngaphambilini. Ngakho-ke, le hormone ethile yabesilisa nayo ithinta kakhulu ukuzala kwabesifazane.
Okulandelayo, siqhathanise imisebenzi ye-20E kanye ne-3D20E ekuhlolweni kokujova ezintombini ezivuthiwe ngokocansi sisebenzisa i-3D20E eyenziwe ngamakhemikhali (Isithombe 4a–c) kanye ne-20E etholakala kwezentengiselwano. Sibone ukuthi i-3D20E yayisebenza kahle kakhulu kune-20E ekuvaleni ukuzwela kwabesifazane ekuzalweni kokubili kokugxila (Isithombe 4d). Okuphawulekayo ukuthi ingxenye yezinga lomzimba le-3D20E ku-LRT (1,066 pg ngemuva kokujova vs. 2,022 pg ngemuva kokujova) ibangele ingxenye yabesifazane abangaphili kahle eyayiphakeme ngokuphindwe ka-20 kunezinga lomzimba le-20E (361 pg ngemuva kokujova) emahoreni angu-24 ngemuva kokujova ekugxilweni okuphezulu kakhulu kwe-18 pg ngemuva kokujova; Ithebula Ledatha Elandisiwe 1). Lo mphumela uhambisana nomqondo wokuthi ukudluliselwa ngokocansi kwe-20E akubangeli izikhathi zokuhlangana ezingavumi ukuhlangana, futhi ukhomba ku-3D20E njengesici esiyinhloko ekuqinisekiseni ubudlelwano bomzali nengane. I-3D20E nayo yayisebenza kakhulu kune-20E ekuhlolweni kokubekwa kwamaqanda kwabesifazane abamsulwa (Isithombe 4e), okuphakamisa ukuthi izinga elijwayelekile lokubekwa kwamaqanda esilibonile ngemuva kokuthulisa ingxenye ye-EPP lalibangelwa ukuba khona komsebenzi we-3D20E osele osakhiqizwa yizici zabesifazane ezibangelwa ukuhlangana.
(a,b) I-3D20E eyenziwe ngamakhemikhali kusukela ku-20E (a) ngokuguqulwa/ukusebenza kahle okuphezulu kakhulu (idatha ethulwe njengesilinganiso ± sem kusuka ekuphenduleni okuthathu okuzimele kokwenziwa) (b).c, i-Mass spectrum (ingxenye engezansi) ifana ncamashi ne-ecdysone etholakala ku-LRT yesifazane ehlanganisiwe (ingxenye ephezulu).d, Uma kuqhathaniswa ne-20E (0.63 µg, P = 0.02; 0.21 µg, P < 0.0001; Ukuhlolwa okuqondile kukaFisher, okunezinhlangothi ezimbili) kanye ne-ethanol engu-10% (0.63 µg, P < 0.0001; 0.21 µg, P < 0.0001; Ukuhlolwa okuqondile kukaFisher, okunezinhlangothi ezimbili), kuyilapho i-20E yayiphakeme kakhulu kunokulawula kuphela kumthamo ophezulu (0.63 µg, P = 0.0002; 0.21 µg, P = 0.54; Ukuhlolwa okuqondile kukaFisher, 2-side).e, umjovo we-3D20E ubangele amazinga aphezulu kakhulu okuzalela kwabesifazane abamsulwa kunokulawula kwe-ethanol okungu-10% (0.21 µg, P < 0.0001; 0.13 µg, P = 0.0003; Ukuhlolwa okuqondile kukaFisher, okunezinhlangothi ezimbili), kuyilapho u-20E uqhathaniswa nokulawulwa kuphela kumanani aphezulu (0.21 µg, P = 0.022; 0.13 µg, P = 0.0823; Ukuhlolwa okuqondile kukaFisher, okunezinhlangothi ezimbili).I-3D20E ibangele amazinga aphezulu kakhulu okuzalela kunama-20E kumanani aphezulu (0.21 µg, P = 0.0019; 0.13 µg, P = 0.075; Ukuhlolwa okuqondile kukaFisher, okunezinhlangothi ezimbili).Kuwo wonke amaphaneli, u-n umele inani lamasampula omiyane azimele ngokwebhayoloji.NS, akubalulekile.*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.001. Idatha ivela kumakhophi amathathu.
Ezifundweni zangaphambilini, sithole ukuthi ukudluliselwa ngokocansi kwama-hormone e-steroid kubangela ukuvezwa kwe-MISO (Ukuvuselela Okubangelwa Ukuhlangana kwe-Oogenesis 11), isakhi sofuzo sowesifazane esivikela abesifazane be-A. gambiae ekuthelelekeni kwe-P. falciparum. Izindleko zempilo ezibangelwa yi-13, i-parasite ebulalayo kakhulu yomuntu ebulawa yi-malaria. Njengoba sibheka ukubaluleka kwe-MISO empilweni yokuzala ye-Anopheles ezindaweni ezivame ukuba khona yi-malaria, sinqume ukunquma ukuthi iyiphi i-hormone i-3D20E noma i-20E ebangela ukuvezwa kwalesi sakhi sofuzo. Sithole ukuthi ngenkathi umjovo we-20E ubangela ngokuqondile noma ngamandla ama-receptor e-nuclear hormone (HR), njenge-HR3 ne-HR4, kanye nezinhloso ezijwayelekile ze-steroid, njengezakhi zofuzo ze-yolkogenic Vg14, 15, 16, i-MISO yabangelwa kakhulu yi-3D20E (Idatha Enwetshiwe Umfanekiso 3). Ngakho-ke, ukudluliselwa ngokocansi kwale hormone ye-androgenic steroid kubonakala sengathi kubangela izindlela ezivikela abesifazane ezindlekweni ezibangelwa ukutheleleka yi-parasite. Ngaphezu kwalokho, i-3D20E ithinta ngokuhlukile zombili ama-isoforms e- i-E receptor EcR, ebangela i-EcR-A futhi icindezela i-EcR-B, futhi ibangela kakhulu ezinye izakhi zofuzo ezibangela ukuhlangana, okuhlanganisa ne-HPX15, ethinta ukuzala kwabesifazane. Lokhu kungachaza ukungabi nabantwana okuphawulekayo okubonwa kwabesifazane abakhulelwe nabesilisa abathuliswe yi-EPP (Isithombe Sedatha Esandisiwe 3). Le datha iphakamisa ukuthi kukhona izindlela ezingezansi ezivuselelwa ngokukhethekile ngama-hormone amabili e-ecdysone angase abe ngaphansi komsebenzi othize wobulili.
Okulandelayo, sihlole umsebenzi wezakhi zofuzo ezimbili ze-EcK ezitholakale kuphayiphi yethu ye-bioinformatics. Ukuthulisa i-EcK1 noma i-EcK2 kuholele ekufeni okukhulu kwabesilisa (I-Extended Data Fig. 4a), okuphakamisa ukuthi i-ecdysone phosphorylation, ngaleyo ndlela ukungasebenzi, kubalulekile ekusindeni. Ngenxa yokuthi i-EcK2 ivezwe emazingeni aphezulu kune-EcK1 futhi yatholakala kuma-MAG yi-proteomics (Isithombe 2b,c kanye neThebula Elingeziwe 2), siqinisekisile umsebenzi wayo we-ecdysteroid kinase ngokuyifushanisa ne-20E, okwaholela ku-phosphorylation 20E22P (Isithombe Sedatha Esandisiwe 2).4b). Lapho sisebenzisa i-3D20E njenge-substrate, asikwazanga ukuthola umkhiqizo we-phosphorylated 3D20E22P (Isithombe Sedatha Esandisiwe 4c), okuphakamisa ukuthi i-20E kune-3D20E ingaba yinhloso ekhethwayo ye-EcK2.
Ngokusho kokuhlaziywa kwethu kwe-RNA-seq, i-EcK2 nayo yavezwa kakhulu ku-LRT yabesifazane abamsulwa, lapho yayivalwa khona ngemva kokuhlangana (Isithombe 2b). Siqinisekisile le datha futhi sathola ukuthi ukuvezwa kwe-EcK2 akuzange kuthinteke ngokudla igazi (Isithombe Esinwetshiwe Sedatha 5a). Sandisa izivivinyo zethu zokuqala ze-MS, sathola ukuthi inani eliphakeme le-20E22P lalihlobene eduze nenani eliphakeme le-20E (amahora angu-22-26 ngemva kokudla igazi; Isithombe Esinwetshiwe Sedatha 5b). Ukuthuliswa kwe-EcK2 kwabesifazane abamsulwa kwaholela ekukhuleni okuphindwe kathathu kwesilinganiso esilinganiselwe se-20E kuya ku-20E22P emahoreni angu-26 ngemva kokudla kwegazi (Izibalo Zedatha Ezinwetshiwe 2c no-5c), okuqinisekisa ukuthi i-EcK2 iphinde ifake i-phosphorylates engu-20E kwabesifazane. Okuphawulekayo ukuthi, abesifazane abamsulwa be-EcK2 bagcina ukwamukelwa okugcwele ngokocansi (Isithombe Esinwetshiwe Sedatha 5d,e), okuphakamisa ukuthi ukukhiqizwa kwabesifazane kwe-20E akukukhuthazi ukuhlangana kowesifazane. Izikhathi. Kodwa-ke, laba besifazane babenezinga eliphezulu kakhulu lokubeka amaqanda uma kuqhathaniswa nezilawuli, kanye nama-30% ezintombi ezibeka amaqanda (Extended Data Fig. 5f). Uma imijovo ye-Eck2 RNA (dsEcK2) enezintambo ezimbili yenziwe ngemva kokuncelisa igazi, ukuzala akuzange kwenzeke, lapho i-peak ye-20E ngenxa yokuncelisa igazi yehlile. Sekukonke, le miphumela isekela imodeli eyenziwe yi-20E ngemuva kokuncelisa igazi engabangela ukuzala, kodwa kuphela lapho i-block yokuzala (EcK2 kanye nezinye izici) ivaliwe ngokuncelisa. Imijovo ye-20E noma ye-3D20E ayivimbelanga ukubonakaliswa kwe-EcK2 ezintombini (Extended Data Fig. 5g), okuphakamisa ukuthi ezinye izici zilawula ukuvinjelwa kwalesi kinase. Kodwa-ke, amazinga e-20E ngemva kokuncelisa igazi ayenganele ukudala ukungakhululeki kokuzala, kodwa abangelwa ngempumelelo yi-titers ephezulu ye-3D20E edluliselwa ngocansi.
Imiphumela yethu inikeza ukuqonda okubalulekile ngezindlela ezilawula impumelelo yokuzala ye-A. gambiae. Kuvele imodeli lapho abesilisa beguqukele khona ekwakheni amazinga aphezulu e-3D20E, i-ecdysone eguquliwe eqondene nabesilisa eqinisekisa ukuzala ngokususa ukuzwela kwabesifazane ukuze baqhubeke nokuzala. Ngesikhathi esifanayo, lawa ma-vectors e-malaria athuthukise nohlelo olusebenzayo lokusebenzisa i-3D20E kwabesifazane ukuphendula ekudlulisweni ngokocansi kwe-EPP eqondene nabesilisa. Ngokwazi kwethu, lesi yisibonelo sokuqala sesistimu ye-steroid hormone ephethwe ngabesilisa nabesifazane eyenza umsebenzi oyingqayizivele nobalulekile ezinambuzaneni. Umsebenzi we-ecdysone eqondene nabesilisa ucatshangelwe kodwa awuzange uboniswe ngokusobala. Isibonelo, umbono ongaphikiswa kakhulu 18 ukuthi le misebenzi ingenziwa yi-20E precursor E1. Kwaziwa kahle ukuthi ku-Drosophila, i-monandry ibangelwa ukudluliselwa ngokocansi kwama-peptide amancane ocansi19,20 asebenzisana nama-neurons agcina indlela yokuzala yabesifazane ngokusebenzisa ama-receptor athile e-peptide ocansi21,22. Kudingeka umsebenzi owengeziwe ukuthola ukuthi yini engezansi. ama-cascade okubonisa izimpawu alawulwa yi-3D20E kwabesifazane base-A. gambiae kanye nokunquma ukuthi la ma-cascade angalondolozwa yini phakathi komiyane ne-Drosophila.
Njengoba kunikezwe indima ebalulekile ye-3D20E ekuzaleni nasekuziphatheni kwabesifazane okubonwe ocwaningweni lwethu, izindlela eziholela ekuhlanganisweni nasekusebenzeni kwe-3D20E zinikeza amathuba amasha amasu okulawula omiyane esikhathi esizayo, njengokukhiqiza abesilisa abancintisanayo abangabodwa kumasu obuchwepheshe bezinambuzane abangabodwa. Sebenzisa ukukhululwa kwasendle noma ukulingisa i-3D20E ekudlaleni okumsulwa. Umsebenzi we-3D20E oqondene nabesilisa kungenzeka ukuthi washintsha lapho i-A. gambiae kanye nezinye izinhlobo ze-Cellia zithola ikhono lokuhlanganisa isidoda sazo kuma-plugs okuhlangana, njengoba lokhu kuvumela ukudluliselwa okuphumelelayo kwama-hormone amaningi nama-enzyme avuselela ama-hormone. Ngakolunye uhlangothi, ukusebenzisa i-monandry ye-3D20E yokuziphendukela kwemvelo kunikeza indlela yabesifazane (ngokuveza okuphezulu kwe-MISO) ukuthi bathande ukufaneleka kwabo kokuzala ezindaweni ezivame kakhulu umalaleveva, okufaka isandla ngokungaqondile ekudlulisweni kwe-Plasmodium. Njengoba kuboniswe ukuthi i-20E yabesifazane i-20E inemiphumela ejulile ekusindeni nasekukhuleni kwe-P. falciparum komiyane besifazane be-Anopheles, izindlela ze-steroid zabesilisa nabesifazane manje seziyizici ezibalulekile zokusebenzisana komiyane nezinambuzane.
Izinhlobo ze-A. gambiae G3 zakhuliswa ngaphansi kwezimo ezijwayelekile zezinambuzane (26-28 °C, umswakama ohlobene ongu-65-80%, isikhathi sokukhanya/sobumnyama samahora angu-12:12). Izibungu zondliwa ngokudla kwezinhlanzi okuphuzwayo (i-TetraMin Tropical Flakes, ama-Koi Pellets kanye ne-Tetra Pond Sticks ngesilinganiso esingu-7:7:2). Omiyane abadala bondliwa ngesisombululo se-dextrose esingu-10% kanye negazi lomuntu lamasonto onke (izingxenye zegazi ezifundwayo). Omiyane abangowesifazane batholakala ngokuhlukanisa ubulili esigabeni se-pupal ngemuva kokuhlola iziphetho nge-microscopy. Omiyane abangamadoda abaphethe i-DsRed transgene bachazwe ngaphambilini.
Ukuhlolwa kokuhlangana okuphoqelelwe kwenziwa ngokwemigomo echazwe ngaphambilini. Ekuhlanganeni kwemvelo, abesifazane abangowesifazane abaneminyaka engu-4 ubudala bagcinwa ngesilinganiso esingu-1:3 nabesilisa abangowesifazane abavuthiwe ngokocansi ubusuku obubili. Ekuhlolweni lapho abesilisa bajovwa khona nge-dsEPP, ukuhlangana ndawonye kwahambisana nezinsuku ezingu-3-4 ngemuva kokujova, lapho umsebenzi we-phosphatase uthuliswa kakhulu (Idatha Enwetshiwe Umfanekiso 2b).
Izicubu zomiyane, izidumbu ezisele (umzimba wonke), noma umzimba wonke zahlukaniswa zaba yi-100% methanol futhi zahlanganiswa nge-beader (2 mm glass beads, 2,400 rpm, 90 sec). Amanani ezicubu kanye nomthamo we-methanol kwakungokulandelayo: ingxenye esele yomzimba, 50 ku-1,000 µl; I-MAG, 50–100 80 µl; i-LRT yesifazane, 25–50 80 µl. I-precipitate yafakwa ekukhishweni kwesibili kwe-methanol ngenani elifanayo le-methanol. Udoti weseli wasuswa nge-centrifugation. I-methanol evela kokubili okukhishwe yahlanganiswa futhi yomiswa ngaphansi kokugeleza kwe-nitrogen, yabe isiphinde yaxhunywa kumanani alandelayo e-80% methanol emanzini: ingxenye esele yomzimba, 50 µl; i-MAG kanye ne-LRT yesifazane, 30 µl.
Amasampula ahlaziywe ku-mass spectrometer (ID-X, Thermo Fisher) exhunywe nethuluzi le-LC (Vanquish, Thermo Fisher). I-5 µl yesampula ifakwe kukholomu engu-3 µm, engu-100 × 4.6 mm (Inspire C8, Dikma) egcinwe ku-25 °C. Izigaba ezihambayo ze-LC kwakuyi-A (amanzi, i-0.1% formic acid) kanye ne-B (acetonitrile, i-0.1% formic acid). I-gradient ye-LC yayinje: 5% B umzuzu owodwa, bese ikhushulwa ibe yi-100% B umzuzu owodwa. Ngemuva kwemizuzu engu-8 ku-100%, phinda ulinganise ikholomu ku-5% B imizuzu engu-4. Izinga lokugeleza lalingu-0.3 ml min-1. I-Ionization emthonjeni we-MS ifezwa yi-heated electrospray ionization ngezindlela ezinhle nezimbi.
I-mass spectrometer ilinganisa idatha ebangeni le-m/z kusukela ku-350 kuya ku-680 ku-60,000 resolution kumodi ye-MS egcwele. Idatha ye-MS/MS itholakale ku-[M + H]+ (zonke izinhloso), [M - H2O + H]+ (zonke izinhloso), kanye no-[M - H]- (izinhloso ze-phosphorylated). Idatha ye-MS/MS isetshenziswe ukuqinisekisa izakhiwo ze-ecdysone zama-targets okwakungatholakali standard yazo. Ukuze kutholakale ama-ecdysteroids angahlosiwe, idatha ye-MS/MS yawo wonke ama-peak e-HPLC anobuningi obungaphezulu kuka-15% yahlaziywa. Bala usebenzisa ama-curve ajwayelekile adalwe kusuka kumazinga amsulwa (20E, 3D20E) ukuze kubalwe amanani aphelele noma ukuncishiswa kwesampula eyodwa ethile (zonke ezinye izinhloso) ukuze kubalwe ukulingana kwazo namanani atholakala kumuntu oyedwa wesilisa. Ku-3D20E, ukulinganisa kwenziwa kusetshenziswa isamba sezinto ezilandelayo: [M + TFA]-, [M + COOH]-, [M + Na]+, [M + Cl]-, [M + NO3]-.Idatha ikhishwe futhi yalinganiswa kusetshenziswa i-Tracefinder (inguqulo 4.1).Idatha ye-MS/MS ihlaziywe kusetshenziswa i-Xcalibur (inguqulo 4.4).I-MS spectra ye-E, 20E kanye ne-3D20E iqhathaniswa nezindinganiso ezifanele.I-3D20E22P ihlaziywe ngokususelwa ku-reagent kaGirard.I-20E22P ihlaziywe ngesilinganiso se-m/z.
I-3D20E22P ihlanzwe ku-MAG. Ukuhlanzwa kwenziwe ngesikali sokuhlaziya kusetshenziswa i-chromatograph yoketshezi esebenza kahle kakhulu (i-Acquity, Amanzi) nge-quadrupole mass-based detector (i-QDa, i-Acquity, Amanzi) ngaphansi kwezimo ezifanayo ze-LC njengokuhlaziywa kwe-HPLC-MS/MS. Ukuqoqwa kwezingxenyana kwaqala lapho i-m/z ehambisana ne-3D20E22P itholakala ngesikhathi esifanayo sokugcinwa njengoba kunqunywe ngaphambilini. Ubumsulwa bamakhemikhali akhishwe babuye bahlolwa yi-HPLC-MS/MS njengoba kuchaziwe ngenhla.
I-RNA iyonke ikhishwe ezicutshini zokuzala eziyi-10-12 noma kwezinye izingxenye zomzimba (ezingenakhanda) kusetshenziswa i-TRI reagent (Thermo Fisher) kulandela imiyalelo yomenzi. I-RNA yelashwa nge-TURBO DNase (Thermo Fisher). I-cDNA yakhiwe kusetshenziswa i-Moloney murine leukemia virus reverse transcriptase (M-MLV RT; Thermo Fisher) kulandela imiyalelo yomenzi. Ama-primer e-reverse transcription quantitative PCR (RT-qPCR; Extended Data Table 2) ashicilelwe ngaphambilini24 noma aklanywa kusetshenziswa i-Primer-BLAST26, kwakhethwa imikhiqizo enobukhulu obungu-70-150 bp futhi ihlanganisa ama-exon-exon junctions noma ama-Primer pair primer ama-exons ahlukene. Amasampula e-cDNA avela kuma-replica amathathu kuya kwamane e-biological axutshwe kane emanzini e-RT-qPCR. Ukuhlolwa kwenziwa kuma-replica reaction angu-15 µl aqukethe i-1 × PowerUp SYBR Green Master Mix (Thermo Fisher), ama-primer, kanye ne-5 µl ye-cDNA exutshwe. Ukusabela kuqhutshwe ku- Uhlelo lwe-QuantStudio 6 Pro lwe-PCR lwesikhathi sangempela (i-Thermo Fisher) kanye nedatha kwaqoqwa futhi kwahlaziywa kusetshenziswa i-Design and Analysis (inguqulo 2.4.3). Njengoba kuboniswe kulolu cwaningo, amanani ahlobene ajwayelekile kwi-ribosomal gene RpL19 (AGAP004422), okuvezwa kwayo kungazange kushintshe kakhulu ngokudla igazi 27 noma ukuhlangana 3.
Ikhwalithi ye-RNA ihlolwe kusetshenziswa i-Agilent Bioanalyzer 2100 Bioanalyzer (Agilent). Amalabhulali e-Illumina paired-end alungiselelwe futhi aqhutshwa e-Broad Institute of MIT naseHarvard. Ukufundwa kokulandelana kwaqondaniswa ne-genome ye-A. gambiae (uhlobo lwe-PEST, inguqulo 4.12) kusetshenziswa i-HISAT2 (inguqulo 2.0.5) ngamapharamitha azenzakalelayo. Ukufundwa okunezilinganiso zekhwalithi yokumapha (MAPQ) <30 kususiwe kusetshenziswa i-Samtools (inguqulo 1.3.1). Inani lokufundwa okumakwe kuma-gene labalwa kusetshenziswa i-htseq-count (inguqulo 0.9.1) ngamapharamitha azenzakalelayo. Ukufundwa okujwayelekile kwabalwa futhi ukuvezwa kwezakhi zofuzo okuhlukile kwahlaziywa kusetshenziswa iphakheji ye-DESeq2 (inguqulo 1.28.1) ku-R (inguqulo 4.0.3).
Abantu abazongenela i-Ecdysone-modifying gene batholwe ngokusesha okokuqala i-genome ye-A. gambiae besebenzisa i-algorithm ye-PSI-BLAST (https://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/2.8.1/), besebenzisa amanani azenzakalelayo Amapharamitha anezinhlu ezilandelayo zeprotheyini yombuzo: kusuka ku-Bombyx mori (Inombolo Yokufinyelela. NP_001038956.1), i-Musca domestica (Inombolo Yokufinyelela. XP_005182020.1, XP_005175332.1 kanye ne-XP_011294434.1) kanye ne-Microplitis demolitor (Inombolo Yokufinyelela. XP_008552646.1 kanye ne-XP_008552645.1) i-EcK evela ku-B. mori (Inombolo Yokufinyelela. NP_001036900), i-Drosophila melanogaster (Inombolo Yokufinyelela. NP_651202), i-Apis i-mellifera (Inombolo Yokufinyelela XP_394838) kanye ne-Acyrthosiphon pisum (Inombolo Yokufinyelela XP_001947166); kanye ne-EPP evela ku-B. mori (Inombolo Yokufinyelela XP_001947166) NP_001177919.1 kanye ne-NP_001243996.1) kanye ne-EO ye-D. melanogaster (Inombolo Yokufinyelela NP_572986.1) (isinyathelo 1). Okulandelayo, isihlungi sithinta ngokusekelwe ekubonakalisweni okuphezulu kwe-mRNA (izingcezu eziyi->100/ama-kilobase exons ngezigidi ze-mapped reads (FPKM) noma >85%) ezicutshini zokuzala (i-LRT yabesifazane noma i-MAG) eGambia (isinyathelo 2). Ukuze sithuthukise ukucaciswa, sikhethe ama-enzyme afanele aphinde avele ezicutshini zokuzala ze-A. albimanus, uhlobo lwe-anopheles olungakhiqizi noma ludlulisele i-ecdysone ngesikhathi sokuhlangana. Izakhi zofuzo ezifanelekile zihlungiwe ngokusekelwe ekubonakalisweni okuphansi (<100 FPKM noma <85th percentile) ezicutshini zokuzala ze-A. albimanus (isinyathelo 3). Njengesihlungi sokugcina (isinyathelo 4), izakhi zofuzo ezifanelekile zidinga ukwanelisa okungenani okukodwa kwalokhu okulandelayo: (1) zikhuliswe kakhulu ngemva kokuhlangana (P < 0.05) ngokusho kokuhlaziywa kwezakhi zofuzo ezivezwe ngokuhlukile kanye (2) ezicutshini ezingezona ezokuzala (< 85 % noma <100 FPKM).
Sishintshe izindlela ezichazwe ngaphambilini ezingu-28,29,30 ukuze sifinyelele ukulebula kwe-isotopic kwento yonke. Kafushane nje, uhlobo lwe-Saccharomyces cerevisiae lwe-wild-type II (YSC2, Sigma) luhlolwe ku-yeast nitrogen base (BD Difco, DF0335) equkethe (wt/vol) i-2% glucose (G7528, Sigma), i-1.7% amino acid-free kanye ne-ammonium sulfate. culture medium) kanye ne-5% 15N ammonium sulfate (NLM-713, >99%, Cambridge Isotope Laboratories) njengomthombo we-nitrogen kuphela. Imvubelo yatholakala nge-centrifugation futhi izibungu zomiyane zondliwa ngokushesha kuze kube yilapho zikhula. Isengezo ngenhlanzi (0.5 mg ngezibungu ezingu-300) ukuvimbela ukufa kwesine kwenkanyezi. Abesifazane kuphela ababe sebesetshenziswa ekuhlolweni kokuhlangana nezinduna ezingenamalebula ukuhlaziya i-proteome yesilisa edluliselwe ngesikhathi sokuhlangana.
Izinsikazi ezineminyaka engu-4-6 ubudala ezine-tag engu-15N zaphoqeleka ukuhlangana nezinsikazi ezineminyaka engafani. Ukuhlangana okuphumelelayo kwaqinisekiswa ngokuthola ama-plug okuhlangana ngaphansi kwe-epifluorescence microscopy. Ku-3, 12, kanye no-24 hpm, i-atria yezinsikazi ezineminyaka engu-45-55 ezihlangene yahlukaniswa yaba yi-50 µl ye-ammonium bicarbonate buffer (pH 7.8) futhi yahlanganiswa nge-pestle. I-homogenate yafakwa phakathi kwe-centrifuged kwathi i-supernatant yaxutshwa ne-50 µl ye-0.1% RapiGest (186001860, Waters) ku-50 mM ammonium bicarbonate. I-supernatant kanye ne-pellet evela kusampula ngayinye kwaqotshwa eqhweni elomile kwathunyelwa ubusuku bonke elabhorethri ye-MacCoss e-University of Washington, lapho kwaqedwa khona ukulungiselela isampula ye-LC-MS/MS. Phinda umise i-pellet ku-50 µl ye-0.1% RapiGest ku-50 mM. I-ammonium bicarbonate kanye ne-sonicate ebhavini lamanzi. Ukuhlushwa kwamaprotheni e-pellet kanye ne-supernatant kwalinganiswa nge-BCA assay, amasampula ancishisiwe nge-5 mM dithiothreitol (DTT; Sigma), axutshwe ne-15 mM iodoacetamide (Sigma) futhi afakwa ku-37 °C (1:0 50) ihora eli-1 nge-trypsinization: trypsin: substrate ratio). I-RapiGest yahlungwa ngokungezwa kwe-200 mM HCl, kwalandela i-incubation ku-37 °C imizuzu engama-45 kanye ne-centrifugation ku-14,000 rpm imizuzu eyi-10 ku-4 °C ukususa udoti. Amasampula ahlanzwa nge-dual-mode solid-phase extraction (Oasis MCX cartridges, Waters) futhi aphinde axutshwe ku-0.1% formic acid ukuze kuhlushwe amaprotheni okugcina angu-0.33 µg µl-1. Ama-proteome e-MAG angabhalwanga ahlaziywa ngokufanayo kusukela ku-virgin. Kwahlaziywa amakhophi amabili okuhlaziya esampula ngayinye. Okulandelayo, i-1 µg ngayinye yahlaziywa kusetshenziswa ikholomu ye-silica ehlanganisiwe engu-25-cm engu-75-μm ene-frit trap engu-4-cm ehlanganisiwe ye-silica Kasil1 (PQ) egcwele i-resin ye-Jupiter C12 reversed-phase (Phenomenex) kanye ne-chromatography yoketshezi yemizuzu engu-180. Ukuhlolwa kwesampula – I-MS/MS yaqhutshwa ku-Q-Exactive HF mass spectrometer (Thermo Fisher) ene-nanoACQUITY UPLC System (Waters). Idatha yokuthola idatha ehlobene nedatha ekhiqizwe ekuqhutshweni ngakunye yaguqulwa yaba yifomethi ye-mzML kusetshenziswa i-Proteowizard (inguqulo 3.0.20287) kanye nokusebenzisa i-Comet31 (inguqulo 3.2) ngokumelene nesizindalwazi se-FASTA esiqukethe ukulandelana kwamaprotheni kusuka ku-Anopheles gambiae (inguqulo ye-VectorBase 54), i-Anopheles coluzzi Kwenziwe usesho ku-Mali-NIH (inguqulo ye-VectorBase 54), i-Saccharomyces cerevisiae (Uniprot, Mashi 2021), i-A. gambiae RNA-seq, kanye nokuhumusha okunezinhlaka ezintathu kwezinto ezingcolisayo zabantu ezaziwayo. Ama-FDR ahambisana nemephu ye-Peptide anqunywa kusetshenziswa i-Percolator32 (inguqulo 3.05) enomkhawulo ongu-0.01, kanti ama-peptide ahlanganiswa abe ukuhlonza amaprotheni kusetshenziswa i-protein parsimony ku-Limelight33 (inguqulo 2.2.0). Ubuningi bamaprotheni ahlobene bulinganiswe kusetshenziswa i-normalized spectral abundance factor (NSAF) ebalwe ngephrotheni ngayinye ekugijimeni ngakunye njengoba kuchaziwe ngaphambilini. I-NSAF maqondana nephrotheni ngayinye ilinganiswe kumasampula avela kuma-replica amabili ahlukene ebhayoloji. Ukufakwa kwelebula kwe-15N kufihlwe ngempumelelo i-proteome yesifazane, yize inani elincane lephrotheni elingenalebula lingatholakala kuma-virgin anelebula. Siqophe ukutholakala kokuncipha kwamaprotheni esilisa (ama-spectra angu-1-5) kumasampula angaphekiwe esifazane kuphela ekugijimeni kobuchwepheshe, lapho amasampula angaphekiwe aqhutshwa khona ngemva kwamasampula esilisa/okuhlangana, ngenxa yokuthi i-HPLC "iyaqhubeka". Amaprotheni ngezikhathi ezithile atholakala 'njengokungcola' avela kuma-virgin anelebula abhalwe kuThebula Elingeziwe 1.
Ama-peptide amabili e-antigenic, i-QTTDRVAPAPDQQQ (ngaphakathi kwe-isotype PA) kanye ne-MESGTTTPSGDSEQ (ngaphakathi kwe-isotype PA kanye ne-PB) ku-Genscript. Ama-peptide amabili ahlanganiswa, abese ehlanganiswa ne-carrier protein KLH futhi afakwa konogwaja baseNew Zealand. Onogwaja bahlatshwa ngemuva komjovo wesine, kanti i-IgG iyonke yahlukaniswa ngokuhlanzwa kwe-affinity. I-IgG kunogwaja okhethekile kakhulu we-EPP yasetshenziselwa ukubhula okwengeziwe kwe-western.
Kuma-western blots, i-MAG (n = 10, lapho i-n imelela inani lamasampula omiyane abazimele ngokwebhayoloji) kanye ne-LRT yabesifazane (n = 30) evela kwabesilisa abaneminyaka engu-4 ubudala abamsulwa kanye nabesifazane abamsulwa noma abaphoqelelwe (<10 ngemuva kokuhlangana), i-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)) yanezelwa ngokwehlukana. Amasampula ahlanganiswa ngendlela efanayo ngokushesha ngemva kokuhlukanisa nge-beader (2 mm glass beads, 2,400 rpm, 90 sec). Imfucumfucu enganyibiliki isuswe nge-centrifugation ku-20,000 g ku-4 °C. Amaprotheni alinganiswa nge-Bradford assay (Bio-Rad). Ngemuva kwalokho, i-20 µg ye-MAG protein, i-40 µg ye-LRT protein, kanye ne-20 µg ye-residual bulk protein. zahlukaniswa nge-10% Bis-Tris NuPAGE kusetshenziswa i-MOPS buffer. Amaprotheni adluliselwe kuma-membrane e-polyvinylidene fluoride kusetshenziswa uhlelo lokudlulisa i-iBlot2 (Thermo Fisher). Ama-membrane agezwa kabili ku-1× PBS-T (0.1% Tween-20 ku-PBS) bese evalwa ku-Odyssey blocking buffer (Li-Cor) ihora eli-1 ku-22°C. Ama-membrane anyakaziswa ubusuku bonke ku-4°C nge-custom rabbit anti-EPP polyclonal primary antibody (1:700 ku-blocking buffer) kanye ne-rat anti-actin monoclonal primary antibody MAC237 (Abeam; 1:4,000). Ama-membrane agezwa nge-PBS-T bese efakwa kuma-antibodies esibili (idonkey anti-rabbit 800CW kanye ne-goat anti-rat 680LT (Li-Cor), womabili angu-1:20,000) ku-blocking buffer equkethe i-0.01% SDS kanye ne-0.2% Tween. -20 ihora eli-1 ku-22 °C. Ama-membrane ahlanzwe nge-PBS-T futhi athathwa izithombe ngesikena se-Odyssey CLx. Izithombe zaqoqwa futhi zacutshungulwa ku-Image Studio (inguqulo 5.2). Ibhendi ethile ehambisana ne-isoform ye-EPP-RA (82 kDa) ayizange itholakale.
Izindawo zokubhala ikhodi ze-EPP (njenge-isoform AGAP002463-RB equkethe isizinda se-histidine phosphatase, isizinda esigciniwe se-NCBI 34) kanye ne-EcK2 (AGAP002181) zahlanganiswa ku-pET-21a(+) plasmid (Novagen Millipore Sigma); Ama-primer abhalwe ku-Extended Data Table 2. Ama-linkers ayisishiyagalombili e-GS4 (ahambisanayo) afakwa ngaphambi kwethegi ye-C-terminal 6xHis yesakhiwo se-pET-21a(+)-EcK2. Amaphrotheni ahlanganisiwe akhiqizwa kusetshenziswa i-NEBEExpress cell-free E. coli protein synthesis reaction (New England BioLabs). Amaphrotheni ahlanganisiwe ahlanzwa kusetshenziswa amakholomu e-NEBEExpress Ni spin (New England BioLabs). Iphrotheni yokulawula i-Dihydrofolate reductase (DHFR) yakhiqizwa kusetshenziswa ithempulethi ye-DNA evela ku-NEBEExpress Cell-Free E. coli Protein Synthesis Kit. Amaphrotheni agcinwa ku-50% glycerol ku-PBS ku--20 °C kuze kube yizinyanga ezi-3.
Umsebenzi we-phosphatase we-EPP kanye neziqeshana zezicubu ulinganiswe kusetshenziswa i-4-nitrophenyl phosphate (pNPP; Sigma-Aldrich). I-reaction buffer yayiqukethe i-25 mM Tris, i-50 mM acetic acid, i-25 mM Bis-Tris, i-150 mM NaCl, i-0.1 mM EDTA, kanye ne-1 mM DTT. Izicubu zahlanganiswa ku-reaction buffer futhi udoti weseli wasuswa nge-centrifugation. Qala ukusabela ngokungeza i-enzyme noma i-tissue extract ku-reaction buffer equkethe i-2.5 mg ml-1 pNPP. Ingxube yokusabela yafakwa ekushiseni kwegumbi ebumnyameni, futhi inani le-pNP eliguqulwe kusuka ku-pNPP lalinganiswa ngokulinganisa ukumuncwa ku-405 nm ngezikhathi ezahlukene.
Ngomsebenzi we-EcK we-in vitro, iphrotheni yafakwa ku-0.2 mg 20E noma i-3D20E ku-200 µl buffer (pH 7.5) equkethe i-10 mM HEPES–NaOH, i-0.1% BSA, i-2 mM ATP kanye ne-10 mM MgCl2 amahora ama-2 ku-27 °C. Ukusabela kwamiswa ngokungeza i-800 µl methanol, yabe isipholiswa ku--20 °C ihora eli-1, yabe isifakwa ku-centrifuge ku-20,000 g imizuzu eyi-10 ku-4 °C. I-supernatant yabe isihlaziywa yi-HPLC-MS/MS. Ukuze kuvinjwe ukushisa kwamaprotheni asetshenziswa eqenjini lokulawula, amaprotheni afakwa ku-50% glycerol ku-PBS imizuzu engama-20 ku-95°C.
Ngomsebenzi we-EPP we-in vitro, iphrotheni yafakwa i-3D20E22P (elingana nenani elitholakale kuma-pair angu-18 e-MAG, ahlanzwe yi-HPLC-MS/MS) ku-buffer engu-100 µl (pH 7.5) equkethe i-25 mM Tris, i-50 mM acetic acid, i-25 mM Bis-Tris, i-150 mM NaCl, i-0.1 mM EDTA, kanye ne-1 mM DTT amahora ama-3 ku-27 °C. Ukusabela kwamiswa ngokungeza i-400 µl methanol futhi kwapholiswa ku--20 °C ihora eli-1, kwabe sekufakwa i-centrifuge ku-20,000 g imizuzu eyi-10 ku-4 °C. I-supernatant yahlaziywa yi-HPLC-MS/MS.
Izingcezu ze-PCR ze-EPP (362 bp), i-EcK1 (AGAP004574, 365 bp) kanye ne-EcK2 (556 bp) zandiswa kusukela ku-cDNA elungiselelwe kusuka ezidunjini zomiyane ezingenamakhanda zobulili obuxubile. Isingcezu se-PCR sokulawula i-eGFP (495 bp) sandiswa kusukela ku-pCR2.1-eGFP echazwe ngaphambilini; Ama-primer e-PCR abhalwe kuThebula Ledatha Elandisiwe 2. Isiqephu se-PCR sifakwe phakathi kwama-promoter e-T7 aguquliwe ku-plasmid ye-pL4440. Ama-Plasmid constructs atholwe ku-NEB 5-α competent E. coli (New England Biolabs) futhi aqinisekiswa nge-DNA sequencing ngaphambi kokusetshenziswa (bheka Idatha Engeziwe 1 ukuthola ukulandelana kokufaka). Ama-Primer afaniswe ne-promoter ye-T7 (Ithebula Ledatha Elandisiwe 2) asetshenziswa ukukhulisa i-insert evela ku-plasmid esekelwe ku-pL4440. Usayizi womkhiqizo we-PCR uqinisekiswe yi-agarose gel electrophoresis.dsI-RNA iqoshwe kusuka kumathempulethi e-PCR kusetshenziswa i-Megascript T7 Transcription Kit (Thermo Fisher) futhi yahlanzwa ngokwemiyalelo yomenzi ngezinguquko ezichazwe ngaphambilini.
Ngokujova kwe-dsRNA, i-1,380 ng ye-dsRNA (dsGFP, dsEcK1, dsEcK2, dsEPP) yajovwa nge-concentration ye-10 ng nl-1 esifubeni sabesilisa noma abesifazane abadala (i-Nanoject III, i-Drummond) zingakapheli usuku olulodwa ngemuva kokuvaleka. Amazinga okudonswa kwezakhi zofuzo anqunywa okungenani kuma-replica amathathu ebhayoloji ngokukhishwa kwe-RNA, ukwakheka kwe-cDNA, kanye ne-RT-qPCR. Ngokujova kwe-ecdysone, abesifazane abaneminyaka engu-4 ubudala noma abaneminyaka engu-6 ubudala abanceliswe ngegazi bajovwa nge-0.13, 0.21, noma i-0.63 µg ye-20E noma i-3D20E (i-Nanoject III, i-Drummond) ngama-concentration angu-1.3, 2.1, ngokulandelana, kuye ngomklamo wokuhlola noma i-6.3 ng nl-1. Jova i-100 nl ye-10% (vol/vol) ethanol emanzini; 100 nl ye-3D20E22P ku-10% ethanol (okulingana no-75% wenani elitholakale kuma-MAG amabili). Omiyane babelwa ngokungahleliwe eqenjini elijovwe.
Ekuhlolweni kokuzalela, izinsikazi ezinezinsuku ezintathu ubudala zaziphiwa igazi lomuntu. Susa omiyane abanceliswe kancane noma abanganceliswanga. Kuye ngokwelashwa, izinsikazi zafakwa ezinkomishini zokuzalela ezihlukene ubusuku obune okungenani amahora angu-48 ngemva kokudla igazi. Amaqanda abalwa ngaphansi kwe-stereoscope (Stemi 508, Zeiss); kwezinsikazi ezizalelwe, amaqanda aqanduselwa abe yizibungu ayebhekwa njenganothe.
Ezivivinyweni zokuhlangana, abesifazane bavunyelwa okungenani izinsuku ezi-2 kuye ngokuthi ukwelashwa kunjani ukuze bamelane nokuhlangana, futhi abesilisa abalingana nobudala bezinhlobo zasendle kamuva bangeniswa ekhejini elifanayo. Ebusuku obubili kamuva, ama-vesicles avundisiwe ayisikiwe futhi i-DNA ye-genomic yakhishwa ngokuqandisa kanye nokukhipha i-sonication ku-buffer equkethe i-10 mM Tris-HCl, i-1 mM EDTA, kanye ne-25 mM NaCl (pH 8.2). Amasampula afakwa i-Proteinase K (0.86 µg µl-1) imizuzu eyi-15 ku-55 °C, kulandelwe yimizuzu eyi-10 ku-95 °C. Amalungiselelo e-genomic DNA aluhlaza ancishisiwe izikhathi eziyi-10 futhi atholwa yi-qPCR ye-chromosome sequences; ama-primer abhalwe ku-Extended Data Table 2. Ukungabikho kwe-chromosome sequence ye-Y kubonisa ukuthi akukho ukuhlangana.
Ekuhlolweni kokuphinda kuqhathaniswe, abesifazane abaphoqelelwe bahlolwe ukuthi bakhona yini ama-plug okuhlangana ukuze kuqinisekiswe isimo sokuhlangana futhi bavumela izinsuku ezi-2 ukuthi bakhule bengakwazi ukuhlangana uma kungekho abesilisa, njengoba kuchaziwe ngaphambilini. Abesilisa abaphethe isidoda se-DsRed transgenic babe sebefakwa ezindlini zabesifazane. Ngemva kwezinsuku ezimbili, ama-vesicles avundisayo akhishwa kwabesifazane, kwathi i-DNA ye-genomic yalungiswa njengoba kuchaziwe ngenhla futhi yahlolwa nge-qPCR ye-DsRed transgene; ama-primer abhalwe kuThebula Ledatha Elandisiwe 2. Ukungabikho kwe-DsRed transgene kubonise ukuthi akukho ukuhlangana kabusha okwenzekile.
I-3D20E yakhiwe njengoba kuchaziwe ngaphambilini 37. Ngamafuphi, i-10 mg ye-20E (i-Sigma-Aldrich) yancibilikiswa ku-10 ml wamanzi, kwalandela ukwengeza i-30 mg ye-platinum black (ngesimo sempuphu, i-Sigma-Aldrich). Umfudlana omnene we-O2 waqhubeka nokufakwa engxubeni yokusabela, eyavuselelwa ekushiseni kwegumbi. Ngemva kwamahora ayi-6, kwanezelwa i-30 mL ye-methanol ukuze kumiswe ukusabela. Ingxube yafakwa phakathi ukuze kususwe izinhlayiya ze-catalyst. I-supernatant yashibilikiswa yaba yomile ku-vacuo ekushiseni kwegumbi. Umkhiqizo wokusabela owomile wancibilikiswa ku-10% ethanol kanye ne-methanol ukuze kujovwe ukuhlaziywa kwe-HPLC-MS/MS. Izinga lokuguqulwa (kusuka ku-20E kuya ku-3D20E) lalicishe libe ngu-97% (Isithombe 4b), kanye ne-MS spectrum ye-3D20E eyenziwe yafana neyatholakala kwabesifazane abakhulelwe (Isithombe 4c).
Le nganekwane iqukethe imininingwane ethile yokuhlolwa kwezibalo okwenziwe. I-GraphPad (inguqulo 9.0) yasetshenziswa ukwenza ukuhlolwa okuqondile kukaFisher, ukuhlolwa kweMantel-Cox, kanye nokuhlolwa kwe-t komfundi. Ukuhlolwa kwe-Cochran-Mantel-Haenszel kwenziwa kusetshenziswa iskripthi se-R esenziwe ngokwezifiso (esitholakala ku-https://github.com/duopeng/mantelhaen.test). Ukusatshalaliswa kwedatha kwahlolwa ukuthi kujwayelekile kusetshenziswa ukuhlolwa kwe-Shapiro-Wilk okunomkhawulo wokubaluleka ongu-0.05. Lapho idatha yehluleka ukuhlolwa kokujwayelekile, kwenziwa ukuhlolwa kwe-Mann-Whitney. Idatha yokusinda yahlaziywa kusetshenziswa ukuhlolwa kwe-Mantel-Cox. Iphakheji ye-DESeq2 (inguqulo 1.28.1) yasetshenziswa ukwenza ukuhlaziywa kokubonakaliswa komehluko wezinga lezakhi zofuzo le-RNA-seq. Ibha evundlile kugrafu imelela i-median. Inani lokubaluleka elingu-P = 0.05 lasetshenziswa njengomkhawulo wazo zonke izivivinyo.
Ukuze uthole ulwazi olwengeziwe mayelana nomklamo wocwaningo, bheka isifinyezo se-Nature Research Report esixhunywe kulesi sihloko.
Idatha ye-MS proteomic ifakwe ku-ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) nge-PRIDE Partner Repository (https://www.ebi.ac.uk/pride/) nge-dataset identifier PXD032157.
Isethi yedatha ye-RNA-seq ibekwe ku-Gene Expression Comprehensive Library (https://www.ncbi.nlm.nih.gov/geo/) ngaphansi kwerekhodi le-serial GSE198665.
Amasethi edatha engeziwe akhiqizwe kanye/noma ahlaziywe ngesikhathi socwaningo lwamanje angatholakala kubabhali abahambisanayo uma kucelwa ngokufanele. Lesi sihloko sinikeza idatha yomthombo.
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I-Redfern, i-CPF 20-hydroxyecdysone kanye nokuthuthukiswa kwama-ovari ku-Anopheles stephens.J. I-Insect Physiology.28, 97–109 (1982).