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Kwiindana abanyama bajisi musana, tuzunda tuyeeyelwa kuti tatujisi mahomoni aagwasya koonana. Mu Anopheles gambiae, ecdysone steroid 20-hydroxyecdysone (20E) ulibonya kuti wakasanduka kweendelezya kukomena kwamazai aawo naakali kuchitwa abanakazi2 mpawo akutalisya chiindi chakoonana aawo naakali kuchitwa abasimbi. alimwi akuzyalana nzintu ziyandika kapati mukuzyala, kumvwisisisya mbuli nkumba zya Anopheles zyabanakazi mboziswaanganya mahomoni aaya kulakonzya kugwasya kubamba nzila zipya zyakulwana malwazi aamalangazuba. Aawa, twayubununa kuti milimo eeyi yakuzyala yeendelezyegwa ama steroid aasiyene kwiinda mukubelesya nzila zikatazya zya ecdysteroid-activating/inactivating enzymes. 3-dehydro-20E (3D20E), eeyo ikwabilila bazyali kwiinda mukujala kutambula koonana kwabanakazi nikwakamana kuchincha koonana akuchita kuti kube kubeleka kwiinda mukugusya fosiforila. Chazibinkene, 3D20E kuchincha akwalo kwakapa kuti kube kuzwa kwama genes aakuzyala aayo aabamba kukomena kwamazai muchiindi cha Plasmodium infection, ikusinizya buumi bwabanakazi bachiswa F-20E. nsandulo yakoonana, pesi ilazumizya bantu bayandana kuti babike mazai nikwakamana kulesyegwa 20E-inhibiting kinases. Kuzyibwa kwa steroid hormone eeyi iijatikizya tuzunda twabaalumi amulimu wayo mukulanganya koonana kwabanakazi, kuzyala akuswaangana a Plasmodium kutondeezya kuti ilakonzya kucesya kuzwidilila kwakuzyala kwatuzunda tutola malwazi aamalangazuba.
Kuchiswa malwazi aamalangazuba alufu kuli mukuyaambele alimwi4 akaambo kakukazya misamu iijaya tuzunda mumakumba aa Anopheles, ngaayo alikke aatola malwazi aamalangazuba aabantu. Ikuzyalana kwatuzunda ootu nchintu chibotu kapati mukulesya malangazuba nkaambo banakazi bakwatana biyo chiindi chomwe5; kuchita kuti eechi chintu chomwe chakuzyalana chibe chakuzyala inga kwagwasya loko kuchesya mweelwe wa nkumba mumuunda.
Banakazi tabakonzyi koonana nibamana kutambula mahomoni aa steroid manji kuzwa kuli baalumi. Ziiyo zyakatondezya kuti chintu chipa kuti kube buyumuyumu bwakuzumanana kuzyalana ni 20-hydroxyecdysone (20E), mahomoni aa steroid aazyibidwe kuti nguulanganya kuzyalana kwamasusu muchiindi chakuzyala. Ikukonzya kwabanaalumi kupanga akutola 20 E kwasanduka kapati mumahomoni aa Anocifele chibeela cha Cellia7, eecho chijanika muAfulika mpo chibikkilizya zintu ziyoosya kapati zyamalangazuba, kubikkilizya a Anopheles gambiae. Eezi zilayandika kapati nkaambo mumisyobo eeyi banakazi balazyala 20E musule akulya bulowa, mpawo 20E njiyeendelezya nzila ya oogenesis (langa 8). ecdysone (kutola mwaalumi akulya bulowa) kakunyina kunyonganya nguzu zyabo zyakuzyalana. Mubunike, ikuti 20E iipangwa abanakazi yachita kuti kutabi koonana, eezi zilakonzya kuleta kutazyala kuli baabo balya bana batakwetwe, nchito iidumide kapati kuliba nkumba5.
Bupanduluzi bukonzya kuba mbwakuti baalumi ba A. gambiae balatola ecdysone yabaalumi, eeyo iipa kuti kube nzila yakuzyala yabanakazi, eezi zipa kuti kutabi kukkalikana. Nokuba boobo, nikuba kuti banyama bajisi musana bajisi mahomoni manji aa steroid, mbuli estrogen a androgen (aalangwa-langwa mu ref. 9), muluzyibo lwesu, ma sterogen-roid tanaazyibwa pe mutuzunda.
Twakabikka nzila yakujana mahomoni aa steroid mumubili wamwaalumi (MAG) wa A. gambiae uusimide mumakani aakoonana mukuyandaula ma steroid aakonzya kuchincha. Kubelesya nzila ibeleka kabotu ya liquid chromatography antoomwe a tandem mass spectrometry (HPLC-MS/MS) kutali nzila iitabelesyegwi kabotu, twakajana edy edy (EE2020) aawa nyama, kusinizya zyakachitika musule. Nokuba boobo, musamu wakazwide ma oxidized phosphorylated steroids, kweendelana a fomula 3-dehydro-20E22P)12 (Mufanikisyo 1). Amwi mafoomu abikkilizya 3-dehydro-20E (3D20E22P) a 20E-20E-phosphate (20E22P). I HPLC-MS/MS signal intensity ya 3D20E22P yakali ziindi zyobile kwiinda nzila yayo ya dephosphorylated, 3D20E, mpawo ziindi zyotatwe kwiinda ya E a 20E (Chitondeezyo 1). Nikuba kuti kuli zimwi zibeela zya mubili amuzibeela zyakuzyala zili munsi (Chitondeezyo 1). alimwi bakavwuntauzya ma ecdysteroids mubantu bapya bakajalwa (MAG antoomwe a LRT yabanakazi (kubikkilizya atria, seminal vesicles, antoomwe a parovarium) zyakakosolwa kuzwa kuli 4-day-old (4-day-old) basankwa batakwetwe abanakazi batakwetwe (0.5, 3, antoomwe a 12 hpm). Ecdysone muzibeela eezi yakavwuntauzyigwa a HPLC-MS (anzizyo zitakwe zizo, t-t-pame, t-pamered ±; mweelwe wakubeja (FDR) wakalulamika; *P < 0.05, **P < 0.01 .3D20E: mawoola otatwe vs. 0.5 mawoola, P = 0.035; 12 mawoola vs. 3, P = 0.0015; 3D20E22P: mawoola otatwe kweelanisigwa amawoola aali 0.5, P = 0.25; mawoola aali kkumi aabili kweelanisigwa amawoola otatwe, P = 0.0032; mawoola aali kkumi aabili kweelanisigwa amawoola aali 0.5, P = 0.015). nkumba. Ecdysone wiimininwa amubala mbuli mboutobela: E, sokwe; 20E, orenji; 20E22P, ntumbula; 3D20E, buluu; 3D20E22P, pinki. Ikubikkwa kwakuyungizya chipimyo a y-axis kutondezya zyeelelo zya ecdysone zili munsi.
Kuvwuntauzya kuti naa 3D20E22P a 3D20E zilatolwa muchiindi chakuzyalana, twakakosola ma LRTs aabamakaintu muchiindi chiingi nikwakamana kuzyalana. Nikuba kuti ecdysone tiiyakajanwa mubanakazi, twakabona mweelwe mupati wa 3D20E22P mu LRT mpawo-mpawo nikwakamana kuzyalana (05 hpm, kakuli kucheya kwachiindi chilamfu. 3D20E yakatanta loko (Mufanikisyo 1). Kubelesya 3D20E yakapangwa amakemikalu mbuli chitondezyo, twakajana kuti zyeelelo zya steroid hormone eeyi muma LRTs zyakali kuvwula ziindi zili 100 kwiinda 20E (Extended Data Table 1). Aboobo, 3D20E22P ngo ecson mupati wamwaalumi uuzwa ku LRT yamwanakazi muchiindi chaLRT kuzyalana, mpawo 3D20E, ilavwula loko nikwakainda buyo kuzyalana. Eezi zitondezya kuti ecdysone ilaa mulimo mupati mubuumi bwabanakazi bamana kuzyalana.
Nitwakamana kupanga RNA mpya (RNA-seq) dataset (Mufanikisyo 2a), kubelesya nzila ya bioinformatics yakabambwa, twakayandaula ecdysone kinase (EcK), ecdysone oxidase (EO), antoomwe a ecdysone iijisi 20E-modified phosphatase genes EPP) iitondezyegwa muzibeela zyakuzyala. bakajana EPP gene yomwe antoomwe a EcK gene yobile (EcK1 a EcK2), pesi tiibakakonzya kujana EO gene iili kabotu. Chazibinkene, EPP genes yomwe-yomwe yakatondeezyegwa kapati (98.9th percentile) muma Gambian MAGs pesi kutali muma LRTs aabanakazi (Fig. 2b), mukwiimpana ambotwakali kulangila kuzwa 2020 DP3 mumubili ooyu wamwanakazi. Aboobo, tusyoma kuti EPP yamwaalumi ilakonzya kuchinchwa muchiindi chakuzyalana. Masimpe, twakabelesya in vivo stable isotope labeling kuti tuvwumbe ma protein aamwanakazi nikwakamana kuzyalana, enzyme yakajanwa a MS mu atrium yamukaintu (Fig. 2c a Supplementary Table 1). Kubako kwa EPP mu MAG amukaintu wakakwatwa (LRT LRT) akwalo tiikwakasinizyigwa kubelesya ma antibodies amwi (Fig. 2d).
a, Nzila ya bioinformatics yakapangwa kuti ivwuntauzye muzibeela zyakuzyala zyamuntu umwi awumwi kuti ujane ma genes aa EcKs, EOs, antoomwe a EPPs. Myeelwe iili afwaafwi amivwi itondezya mweelwe wabaalumi abanakazi bakonzya kunjila muchibaka chilichose. Ooku kuvwuntauzya kwakajana kuti EPP gene yomwe (EPP) antoomwe a EcK gene yomwe (Ec1) itondezyedwe muli EcKs yomwe, EcKs (EcK2) eeyo iitondezyegwa mubantu boonse bobile pesi tiikonzyi kupa EO gene.b, Heatmap kweezyanisya gene iikonzya kutondezyegwa mumubili uutakwetwe (V) akuzyalana (M) Anopheles gambiae antoomwe a Anopheles albicans tissues.Spca, fertilization; MAGs, ma gland aagwasya mubaalumi; zimwi zibeela zya mubili, kubikkilizya anseke, mababa, mawulu, mibili yamafuta, azibeela zyamukati kuli baalumi abanakazi boonse, alimwi ama ovary kubanakazi. EcK2 ilatondezyegwa kapati muli MAG alimwi amu atria yaku Gambia, kakuli EPP ilajanwa buyo mu MAG.c, Proteomic analysis of male ejaculate group translocation into femaria at 14 h, 24, kutondezya ma protein ali 67 manji. Banakazi bakakomezyegwa azyakulya zijisi 15N kuti balembe (akuvwumba) ma protein woonse. Baalumi batakwe ma tag bakakwetene abanakazi bakabikkwa ma tag, mpawo banakazi LRTs bakakosolwa a 3, 12 a 24 hpm kuti bavwuntauzye ma proteomic (Langa Supplementary Table 1 kuti ujane mulongo wa ma protein, Inset, EPP). Eck1 a EcK2 zyakajanwa mu MAG yabaalumi batakwetwe kwiinda mukulanga-langa ma proteomic aama tissue aaya.d, EPP yakajanwa kwiinda mukubelesya western blot mu MAG a LRT yabanakazi bakakwetene, pesi kutali mubanakazi batakwetwe naa baalumi naa mubili woonse wamwanakazi. Ma membrane akavwuntauzyigwa aciindi comwe ama antibodies aalwana malwazi (EPP) alimwi aa anti-APP ma antibodies. banakalindu.Langa mufanikisyo wakuyungizya 1 kuti ujane data ya gel. Ma Western blots akachitwa tubili mpawo zyakajana mpindu zikozyania.
Imulimo wa ecdysteroid phosphophosphatase wa EPP wakasinizyigwa nikwakamana kubikkwa a HPLC-MS/MS a 3D20E22P yakaandaanizyigwa kuzwa ku MAG (Extended Data Fig. 2a). Kuyungizya waawo, nitwakaumuna EPP kwiinda mu RNA-mediated interference (RNAi), twakabona kucheya kupati kwa phosphotase activity yakuzyala muma tisuphase aaya baalumi (Mufanikisyo 3a), abanakazi bakakwetene abaalumi batakwe EPP bakatondezya kuti mweelwe musyoonto wa dephosphorylated 3D20E (Mufanikisyo 3b) nikuba kuti kwakali kugwisigwa kwamajini (Extended Data Mufanikisyo 2b,c). Mukwiimpana, tiitwakabona kucinca kupati mu 20E22P/20E ratio mu njuka, eezyo zinga zitondezya kuti enzy chaamba 3D20E22P (Fig. 3b).
a, Kucheya kwa phosphatase mu MAG kwakachitwa akulesyegwa kwa EPP kubelesya double-stranded EPP RNA (dsEPP) naa double-stranded GFP RNA (dsGFP) controls. Mapopwe aa MAG aali makumi obile akabelesegwa muli umwi awumwi (P = 0.0046, paired t-test, two-sided), aatondezyedwe a Feb domales zyaandaanizyidwe. Baalumi bakaumuna EPP bakalijisi mweelwe musyoonto kapati wa dephosphorylated 3D20E a 3 hpm (P = 0.0043, unpaired t-test, two-sided), kakuli 20E levels tiizyakali kujatikizyigwa pe (P = 0.063, unpaired). t-test, two-sided).Data itondezyedwe mbuli mweelwe ± sem kuzwa kuziinga zyotatwe zya 13, 16 a 19 zyabanakazi umwi awumwi.c, Banakazi bakakwatana abaalumi batakwe EPP bakalijisi mweelwe mupati wakukwatana alimwi (P = 0.0002, Fisher's exact test, two-sided).Banakazi bakasaanguna kusinikizyigwa kuti basinizye bukkale bwabo bwakukwatana; Nikwakainda mazuba obile, bakaswaanganizyigwa abamwi baalumi bakanyamwide nseke zya transgenic kuti bazyibe mweelwe wakuswaangana alimwi kwiinda mukubelesya nzila ya PCR detection ya transgene.d, Banakazi bakali kulya bulowa bakakwetene abaalumi bakaangidwe a EPP bakaceya kapati kuzyala (P < 0.0001; Mann-Whitney test, two-sided) alimwi mweelwe wa egg wakaceya asyoonto (P < 0.088, 088,000). Musunko wa Mann-Whitney, wamabazu obile) , kakuli mweelwe wakuzyala tiiwakajatikizyigwa pe (P = 0.94, musunko wa Fisher wini, wamabazu obile). Muzibeela zyoonse, n wiiminina mweelwe wamasampu aa nkumba aatalibonyi pe. NS, taali mapati pe.*P < 0.05, **P < 0.01, *** P < 0.01 * P < 0.00.
Kuzwa waawo, twakalanga-langa naa ecdysone dephosphorylation ilayandika kapati mukupa kuti banakazi kabatayandi kuzyalana. Chazibinkene, banakazi bakakwatana abaalumi batakwe EPP bakali kukwatana alimwi ziindi zyiingi (44.9%) kwiinda banakazi batakwe EPP (10.4%) aawo nibakaswaangana abasankwa bambi (bachincha) (Fig. Wc 3 awalo wakayungizya kucheya). kuzyala (Mufanikisyo 3d, kulumwensyi) akucheya kusyoonto kwamweelwe wamazai aabikkwa abanakazi aaba (Mufanikisyo. 3d, akati), mpawo mweelwe wamazai aabikkwa abanakazi (kwiingula kumwi kwakajanwa kubanakazi kwiinda mukukwatana)) tiikwakali kujatikizyigwa pe (Mufanikisyo. 3d, kululyo). Twaambwa kuti EPP 2020 yakazwa kubelesya 3D20E a EPP yakapegwa muchiindi chakuzyalana kulakonzya kuba amulimu mupati mukulesya kutambula kwabanakazi kuti bazumanane kuzyalana, nchito yakali kwaambwa kuti yakazwa kukutola 20E mumakani aakoonana. Aboobo, eeyi homoni iijatikizya mwaalumi ilajatikizya kapati kuzyala kwabanakazi.
Kuzwa waawo, twakakozyanisya milimu ya 20E a 3D20E mumasunko aakubelesya injection kubana batakwetwe bapati mumakani aakoonana kubelesya 3D20E yakapangwa amakemikalu (Fig. 4a–c) antoomwe a 20E iijanwa mumakwebo. Twakabona kuti 3D20E yakali kubeleka obotu kwiinda 20E mukujala zyintu zyoonse zyobile (Fig. 4a-c). 4d). Chazibinkene, chisela cha 3D20E mu LRT (1,066 pg musule akujaigwa kweelanisigwa a 2,022 pg musule aakuzyalana) chakapa kuti kube mweelwe wabasimbi batakonzyi kulwana wakali 20-fold kwiinda mweelwe wa 20E (361 pg musule akujaigwa) mumawoola aali 24 musule aakujaigwa 18 mapeeji nikwakamana kukwatana; Extended Data Table 1).Eechi chakazwa chileendelana amuzeezo wakuti kusiyana kwa 20E mumakani aakoonana takupi kuti kutabi chiindi chakuzyalana, mpawo chitondezya kuti 3D20E nchintu chipati mukusinizya kuti bazyali abana balakonzya kujatana. 3D20E wakali kubeleka loko kwiinda 20E mukulanga-langa kuzyala banakazi batakwetwe (Mufanikisyo 4), kutondeezya kuti kuzyala-mazai kwakali kabotu. chakabonwa nikwakamana kuumuna kwa EPP kwakali akaambo kakujanika kwa 3D20E yakachili kuchitwa a Mating-induced female factors.
(a,b) 3D20E yakapangwa amakemikalu kuzwa ku 20E (a) akusanduka kupati loko (data itondezyedwe mbuli mweelwe ± sem kuzwa kuzintu zyotatwe zyakupanga zitazulide) (b).c, Mass spectrum (cisela caansi) cileendelana ncobeni a ecdysone iijanwa mu LRT yabanakazi bakwetene (cisela caatala, kweezyanisyigwa a (E2063). µg, P = 0.02; 0.21 µg, P < 0.0001; Fisher's exact test, two-sided) antoomwe a 10% ethanol (0.63 µg, P < 0.0001; 0.21 µg, P < 0.0001; Fisher's exactly, 2-sided test), kakuli Fisher's test 2000 wakali mupati loko, kakuli Fisher's 0.0001 wakali mupati loko. kwiinda kweendelezya buyo muzibeela zipati (0.63 μg, P = 0.0002; 0.21 µg, P = 0.54; Fisher's exact test, 2-sided).e, 3D20E injection yakapa kuti kube kuzyala kupati kapati mubanakazi batakwetwe kwiinda 10% ethanol control (P = 0.000 μg, P = 0.54. 0.0001; 0.13 μg, P = 0.0003; Fisher's exact test, two-sided), mpawo 20E kweezyanisyigwa abasikulela Buyo muzibeela zipati (0.21 μg, P = 0.022; 0.13 μg, P = 0.0822 Fisher's test two-sided;D). mweelwe wakuzyala uusumpukide kwiinda 20E muzibeela zipati (0.21 μg, P = 0.0019; 0.13 μg, P = 0.075; Fisher's exact test, two-sided). Muzibeela zyoonse, n wiiminina mweelwe wamasampu aa nkumba aatalibonyi pe. NS, kutali kupati

Muziiyo zyakainda, twakajana kuti kutola mahomoni aa steroid mumakani aakoonana kupa kuti MISO (Mating-Induced Stimulator of Oogenesis 11) itondezyegwe, musamu uukwabilila banakazi ba A. gambiae kuzwa ku P. falciparum Infection. masena aajisi malwazi aamalangazuba, twakayeeya kuti tuzyibe kuti 3D20E naa 20E nzi iipa kuti gene eeyi itondezyegwe. Twakajana kuti nikuba kuti 20E injection yakapa kuti kube mahomoni amwi aatambula (HR), mbuli HR3 a HR4, antoomwe a steroid targets, mbuli yolgkogenic genes V14, MISO yakali anguzu kapati, MISO zyakaletwa a 3D20E (Extended Data Fig. 3). Aboobo, kusanduka kwa homoni eeyi ya androgenic steroid kulibonya kuti kuletela nzila zikwabilila banakazi kuzwa kuzintu zikonzya kuchitika akaambo kakuciswa. Kunze lyaboobo, 3D20E ilajatikizya munzila ziindene zyoonse zyobile EcR receptor EcR, akucesya EcR-B, alimwi akucesya EcR-B changuzu zipa kuti kube kuzyalana, kubikkilizya a HPX15, eezyo zijatikizya kuzyala kwabanakazi. Eezi zilakonzya kupandulula kutazyala kupati kwakabonwa kubanakazi bakwetene abaalumi batakwe EPP (Extended Data Fig. 3). Eeyi data itondezya kubako kwanzila zyakuselemuka zibelesyegwa aamahomoni obile aa ecdyson aakonzya kubeleka munsi-munsi aakoonana.
Kuzwa waawo, twakasunka mulimo wama EcK genes obile aakajanwa mumulongo wesu wa bioinformatics. Kulesya EcK1 naa EcK2 kwakapa kuti kube lufu lupati mubaalumi (Extended Data Fig. 4a), kutondeezya kuti ecdysone phosphorylation, aboobo inactivation, nchintu chipati mukupona. Nkaambo EcK2 yakali kutondezyegwa kapati kwiinda EcK1 zyakajanwa mu MAGs kwiinda mukubelesya proteomics (Fig. 2b,c antoomwe a Supplementary Table 2), twakasinizya mulimo wayo wa ecdysteroid kinase kwiinda mukuzibikka antoomwe a 20E, eezyo zyakapa kuti kube phosphorylation 20E22P (Extended Data Figure 2).4b). Notwakabelesya 3D20E mbuli substrate, twakajana maphophorylation 3D20E22P (Extended Data Fig. 4c), kutondeezya kuti 20E kutali 3D20E inga yaba nzila iiyandisi ya EcK2.
Kweelana akuvwuntauzya kwesu kwa RNA-seq, EcK2 yakatondezyegwa kapati mu LRT yabanakazi batakwetwe, ooko nkoyakazimwa nikwakamana kuzyalana (Fig. 2b). Twakasinizya data eeyi mpawo twakajana kuti EcK2 tiiyakajatikizyigwa akulya bulowa (Extended Data Fig. 5a). Mukuyungizya MS yesu yakusaanguna, twakajana kuti peak ya 20 EcK2 yakali afwaafwi kweendelana a 20E (mawoola aali 22-26 kuzwa kulya bulowa; Data Iyungizidwe Fig. 5b). Kuumuna kwa EcK2 kubanakazi batakwetwe kwakapa kuti mweelwe wa 20E kusikila ku 20E22P uyungizigwe ziindi zyotatwe mumawoola aali 26 kuzwa kulya bulowa (Data Iyungizidwe Zifwanikiso 2c alimwi a EcphophoK2 akwalo kusinizya kuti EcK2 20E mubanakazi. Chazibinkene, banakalindu batakwe EcK2 bakazumanana kutambula koonana koonse (Extended Data Fig. 5d,e), mpawo kutondeezya kuti kupanga 20E kwabanakazi takupi kuti kutabi kuzyalana. Nokuba boobo, aaba banakazi bakayungizya mweelwe wakuzyala kweelanisigwa abanakazi batakwe 30% (Extended Data Fig. 5f). Ikuti naa double-stranded Eck2 RNA (dsEcK2) injections zyakachitilwa musule aakusanzya bulowa, kuzyala tiikwakachitika pe, aawo 20E peak akaambo kakunywa bulowa niyakaselemuka. Zyoonse antoomwe, eezyi mpindu zigwasyilizya nzila yakuti 20E yakapangwa musule aakunyonka bulowa ilakonzya kuzyala, pesi buyo kuti kuzyala akusinkilila (EKK2 ambweni twaambo tumwi) tulazimwa kwiinda mukuswaangana. Kunyina 20E naa 3D20E injections zyakalesya EcK2 kutondezyegwa mubanakalindu (Extended Data Fig. 5g), kutondeezya kuti zimwi twaambo zilalesya kulesyegwa kwa kinase eeyi. Nokuba boobo, 20E levels nikwakamana kusanina bulowa tiizyakali kuzula pe kuleta kutamvwana pesi zyakali kuchitwa a titer 3D20E yakabikkwa mumakani aakoonana.
Zyakajanwa kulindiswe zipa mizeezo iiyandika kapati kujatikizya nzila zyeendelezya kuzwidilila kwakuzyala kwa A. gambiae. Kwakazwa nzila yakuti baalumi bakasanduka kuti bapange 3D20E, ecdysone yakacincwa yabaalumi iipa kuti kube bazyali kwiinda mukulesya banakazi kuti bazumanane kukwatana. chita kuti 3D20E ichitwe kubanakazi mukwiingula kusanduka kwa EPP kwabaalumi abanakazi. Kweelana aluzibo ndotujisi, eechi nchikozyano chakusanguna cha steroid hormone system yabaalumi abanakazi iichita mulimu uulibedelede alimwi uuyandika kapati mutuzunda. Mulimo wa ecdysone wabaalumi wakayeeyelwa pesi tiiwakatondezyegwa pe. Muchikozyano chipati, 188 mpuwo eeyi njakutondezya kuti milimo eeyi yakasinizyigwa. chilakonzyeka kuchitwa a 20E precursor E1. Chilizyibidwe kuti mu Drosophila, monandry ilatalisyigwa akutola koonana kwama peptide masyoonto aakoonana19,20 aayo aswaangana ama neurons aagwasya nzila yakuzyala yamwanakazi kwiinda muma peptide aakoonana amwi21,22. Mulimo uumbi uyandika kutegwa kuzyibwe nzila zyakutobelezya zitondezyo zyeendelezyegwa a A20D. banakazi akujana kuti naa ma cascade aaya alakonzya kukwabililwa aakati ka nkumba a Drosophila.
Twapegwa mulimo mupati wa 3D20E mukuzyala kwabanakazi akulilemeka kwakajanwa mukwiiya kwesu, nzila zitola ku 3D20E kupanga akuchita kuti zibeleke zipa zyoolwe zipya zyakubelesya nzila zyakumbele zyakukwabilila nkumba, mbuli kuzyala bana basankwa batakonzyi kuzyala munzila zyakupanga tuzunda tutakwe bana. 3D20E ilakonzya kuti yakasanduka aawo A. gambiae amisyobo imwi ya Cellia niyakajana nguzu zyakuvwumbilila bulo bwabo kuti bube mbuli mapulagi aakuzyalana, nkaambo eezi zipa kuti kube kweenda kabotu kwamahomoni miingi ama enzymes aagwasya mahomoni. Mpawo, kusanduka kwa 3D20E kubelesya monandry kupa nzila yakugwasya banakazi (MISO) kuti bakonzye kuzyala masena ajisi bulwazi bwamalangazuba, alo aagwasya mukumwaika kwa Plasmodium. Mbwaanga 20E yabanakazi yakatondezyegwa kuti ilaampindu mpati mukupona akukomena kwa P. falciparum mubasimbi ba Anopheles,24 zyoonse zyobile nzila zya steroid hormone zyabasankwa azyamukaintu lino nzintu zipati zyakuyanzana kwa njuka.
Misyobo ya A. gambiae G3 yakakomezyegwa mubukkale bwatuzunda (26-28 °C, 65-80% busi, 12:12 h light/dark photoperiod). Bana bakapegwa chakulya chanswi (TetraMin Tropical Flakes, Koi Pellets a Tetra Pond Sticks mumweelwe wa 7:2A). nkumba zyakali kupegwa 10% ya dextrose solution antoomwe abulowa bwamuntu amviki amviki (zibeela zyagazi zyakuvwuntauzya). Mankumba aatakwetwe akajanwa kwiinda mukwaandaanya baalumi abanakazi aawo mpaakali kuzyala nikwakamana kulanga-langa kumamanino kwiinda mukubelesya microscopy. Basankwa bajisi DsRed transgene bakapandululwa kale.
Kusunkwa kwakusinikizya kuzyalana kwakachitwa kweendelana anzila zyakaambwa musule. Kuzyalana kwachilengwaleza, banakazi batakwetwe bamazuba one bakabikkwa muchiimo cha 1:3 abaalumi batakwetwe bakakomena kwamazuba obile. Kumasunko oomo baalumi nibakabikkwa dsEPP, kubamba antoomwe kwakachitika antoomwe amazuba aali 3-4 nikwakainda kujaigwa, aawo phosetaction niyakabikkwa muchiindi chifwiifwi (Extended Data Fig. 2b).
Zibeela zya nkumba, zidumbu zyakasyeede (mubili woonse), naakuti mubili woonse zyakakosolwa kuba 100% ya methanol mpawo zyakabikkwa antoomwe a beader (2 mm glass beads, 2,400 rpm, 90 sec). Mweelwe wanyama a methanol zyakali boobu: mubili woonse, 500 mu 100 μl; MAG, 50–100 80 μl; female LRT, 25–50 80 µl. Ikuzwa kwakagwisigwa methanol lwabili a methanol mbweenya. Zisofweede zyamaseelo zyakagwisigwa kwiinda mukubelesya nzila ya centrifugation. I methanol kuzwa kuzibeela zyoonse zyobile yakaswaanganizyigwa akuyuminizyigwa munsi lya nitrogen flow, mpawo yakabikkwa alimwi muzibeela zitobela zya 80% methanol mumaanzi: μl, 50 μl; MAGs a LRT yabanakazi, 30 μl.
Zitondezyo zyakali kuvwuntauzyigwa a mass spectrometer (ID-X, Thermo Fisher) zyakaswaangene a LC instrument (Vanquish, Thermo Fisher).5 μl yasampulu yakabikkwa a 3 μm, 100 × 4.6 mm column (Inspire C8, Dikma) yakabamba a 25 °C. (maanzi, 0.1% asidi ya foomu) a B (acetonitrile, 0.1% asidi ya foomu). I LC gradient yakali boobu: 5% B kwaminiti yomwe, mpawo yakatanta kusika ku 100% B mumaminiti ali kkumi abumwi. Musule lyamaminiti aali 8 a 100%, bambulula kkolumini a 5% B kwamaminiti aali 44. Ikweenda kwakali 30 ml. min-1. Ionization mu MS source ilazuzikizigwa a electrospray ionization muchiimo chibotu achibi.
I mass spectrometer ipima data mu m/z kuzwa ku 350 kusika ku 680 a 60,000 resolution mu MS mode yoonse. MS/MS data yakajanwa a [M + H]+ (zyoonse zyakali kulangilwa), [M - H2O + H]+ (zyoonse zyakali kulangilwa), antoomwe a [M - H]- (zyoonse zyakali kulangilwa), antoomwe a [M - H]- (zibeela zya phosphorylated data zyakali kubelesegwa kusinizya zyintu zyakali kubelesyegwa kusinizya zyintu zyakali kuyandika). taakwe chiimo chakaliwo. Kuti tuzyibe ma ecdysteroids aatabikkilizyidwe, data ya MS/MS ya HPLC yoonse iijisi >15% yabunji yakavwuntauzyigwa. Bala kubelesya zipimyo zyakabambwa kuzwa kuziimo zisalala (20E, 3D20E) kuti ubale mweelwe wini naa kugwisigwa kwasampulu yomwe (zyoonse zimbi) kweelana amweelwe wabo wakajanwa mumweelwe omwe mwalumi.Ku 3D20E, mweelwe wakachitwa kubelesya mweelwe wazintu zitobela: [M + TFA]-, [M + COOH]-, [M + Na]+, [M + Cl]-, [M + NO3]-.Data yakagwisigwa mpawo yakapimwa kubelesya Tracefinder (version 4.1). Data ya MS/MS yakavwuntauzyigwa kubelesya XMS. spectra ya E, 20E antoomwe a 3D20E yakakozyanisigwa azyeelelo.3D20E22P yakavwuntauzyigwa kwiinda mukubelesya Girard's reagent.20E22P yakavwuntauzyigwa a m/z ratio.
3D20E22P yakasalazyigwa kuzwa ku MAG. Kusalazyigwa kwakachitwa aachipimyo chakuvwuntauzya kubelesya ultra-performance liquid chromatograph (Acquity, Waters) antoomwe a quadrupole mass-based detector (QDa, Acquity, Waters) muchiimo cheelene cha LC mbuli HPLC-MS/MS analysis. Kuyobolola kwakacitwa aawo m/z 3D20E22P yakajanwa muchiindi chomwe chakubamba mbuli mbukwakajanwa musule. Kusalala kwa misamu yakagwisigwa kwakalangwa a HPLC-MS/MS mbuli mbokwaambidwe atala aawa.
RNA yoonse yakagwisigwa kuzwa kuzibeela zyakuzyala zili 10-12 naakuti zimwi zibeela zyamubili (zitakwe mutwe) kubelesya musamu wa TRI (Thermo Fisher) kweendelana amalailile aabasikupanga. RNA yakasilikwa a TURBO DNase (Thermo Fisher). cDNA yakapangwa kubelesya Moloney murine leukemia reverse transcript (MLVRT; Fisher) kutobela malailile aabasikupanga. Ma Primers aa reverse transcription quantitative PCR (RT-qPCR; Extended Data Table 2) akamwaigwa kale24 naa kupangwa kubelesya Primer-BLAST26, akuyandisya zintu zya 70-150 bp mubupati alimwi zijatikizya exon-exon junctions naa pairs primers zyotatwe zyaandeene kuzwa ku exon-exon pairs kuzwa ku exons yotatwe yama exon. zintu zipona zyakasanzyigwa ziindi zyone mumaanzi kuti zibelesyegwe mu RT-qPCR. Kuvwuntauzya kwakachitwa mu 15 μl zyakusanduka zyakalijisi 1× PowerUp SYBR Green Master Mix (Thermo Fisher), ma primers, antoomwe a 5 μl ya cDNA yakasanzyigwa. Zisobano zyakachitilwa a QuantStudio real-time PCR system (Thermo Fisher zyakayobolwa akuvwuntauzyigwa kubelesya nzila ya Design and Analysis (version 2.4.3). Mbuli mbokutondezyedwe muchiiyo eechi, mweelwe uukozyenye wakabambululwa ku ribosomal gene RpL19 (AGAP004422), ooyo kutondezya kwawo tiiwakachincha pe akulya bulowa 27 naa kuzyalana 3 .
Bubotu bwa RNA bwakalangwa kubelesya Agilent Bioanalyzer 2100 Bioanalyzer (Agilent). Illumina paired-end malaibbulali yakabambilwa akubelesegwa ku Broad Institute of MIT a Harvard. Sequencing reads yakabikkwa antoomwe a A. gambiae genome (PEST strain, version 4.12) kubelesya HISAT (2020). zyeelelo.Zibalo zili aabubotu bwamaapu (MAPQ) zibalo <30 zyakagwisigwa kubelesya Samtools (version 1.3.1). Mweelwe wazibalo zyakabikkwa kuma genes wakabalilwa kubelesya htseq-count (version 0.9.1) ama parameters aatalikomeneni. Mweelwe wakubala uuzumizyidwe wakabalilwa mpawo kwakavwuntauzyigwa kwiindana kwa gene expression kubelesya package (Deq2 Severy 1.28.1) mu R (musyobo 4.0.3).
Ecdysone-modifying gene candidates yakajanwa kwiinda mukusaanguna kuyanduulisisya A. gambiae genome kubelesya PSI-BLAST algorithm (https://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/2.8.1/), kubelesya default values ​​Parameters with the following query sequence proteins: Momb NP_001038956.1), Musca domestica (Accession No. XP_005182020.1, XP_005175332.1 a XP_011294434.1) antoomwe a Microplitis demolitor (Accession No. XP_008552641646161515615151) kuzwa kuli B. mori (Accession No. NP_001036900), Drosophila melanogaster (Accession No. NP_651202), Apis mellifera (Accession No. XP_394838) antoomwe a Acyrthosiphon pisum (Accession No. XP_001947166); a EPP kuzwa kuli B. mori (Accession No. XP_001947166) NP_001177919.1 a NP_001243996.1) antoomwe a EO ya D. melanogaster (Accession No. NP_572986.1) (ntaamu 1). Kuzwa waawo, sefa mRNAs (00> zibeela/ma exons aa kilobase muli miliyoni yazibalo (FPKM) naa >85%) muzibeela zyakuzyala (LRT naa MAG yabanakazi) muGambia (ntaamu 2). Kuti tusumpule bupanduluzi, twakasala ma enzyme aakonzya kujanika munyama yakuzyala ya A. albimanus, musyobo wa anopheles uutapangi naa kutola ecdysone muchiindi chakuzyalana. Ma genes aakonzya kubelesegwa akasefwa kweendelanya akutondezya kusyoonto (<100 FPKM naa <85th percentile) munyama yakuzyala ya A. albimanus (Asp 3 yamamanino). kusefa (ntaamu 4), ma genes ayelede kuzuzikizya chimwi chazeezi: (1) ayungizigwa loko nikwakamana kuzyalana (P < 0.05) kweendelanya akuvwuntauzya kwama genes aatondezyedwe munzila yiindene (2) muzibeela zitazyali (< 85 % naa <100 FPKM).
Twakachincha nzila zyakaambwa musule 28,29,30 kuti tuzuzikizye kulemba zyoonse zipona. Mubufwiifwi, Saccharomyces cerevisiae type II (YSC2, Sigma) yakasunkwa mu yeast nitrogen base (BD Difco, DF0335) iijisi (wt/vol) 2% gluse (Ggluse), Sigma, 275%. amino acid-free antoomwe ammonium sulfate. busena bwakukomezya) antoomwe a 5% 15N ammonium sulfate (NLM-713, >99%, Cambridge Isotope Laboratories) mbuli nzila ilikke ya nitrogen. Yisiti yakajanwa kwiinda mukusanzya mpawo bana ba nkumba bakapegwa chakulya kusikila bakazyala. Kuyungizya a fulaulu wanswi (0.5 mg300) ku nyenyezi yone. kufwa. Banakazi balikke mbabakabelesegwa mukusola kuzyalana abaalumi batakwe zyina kuti bavwuntauzye ma proteome aamwaalumi aakagwisigwa muchiindi chakuzyalana.
4-6 mazuba aakuzyalwa 15N-tagged virgin females bakasinikizyigwa kukwatana a age-matched age-matched untagged virgin females. Kuzwidilila kwakuzyalana kwakasinizyigwa kwiinda mukujana mating plugs kwiinda mukubelesya epifluorescence microscopy. At 3, 12, a 24 hpm, atria ya 45-55 banakazi bakakwetene bakakosolwa kuba ammonium 50 μl bicarbonate buffer (pH 7.8) mpawo yakabambwa a pestle. I homogenate yakabikkwa mu centrifuged mpawo a supernatant yakasanganyigwa a 50 μl ya 0.1% RapiGest (186001860, Waters) mu 50 mM ammonium bicarbonate. I supernatant a pellet kuzwa ku sampulu yomwe-yomwe yaka dpp akutumwa atala aa ice ku laboratory ya MacCoss ku Yunivesiti yaku Washington, ooko kulibambila sampulu ya LC-MS/MS nkokwakamanizyigwa. Bikka alimwi musamu mu 50 μl ya 0.1% RapiGest mu 50 mM ammonium bicarbonate mpawo ubweze mumeenda. Bunji bwama protein aamubulowa a supernatant bwakapimwa a BCA assay, sampulu zyakacesyegwa a 50 mM ool (DTT; Sigma), yakabikkwa a 15 mM iodoacetamide (Sigma) mpawo yakabikkwa a 37 °C (1:0 50) kwawoola lyomwe antoomwe a trypsinization: trypsin:substrate ratio).RapiGest yakagwisigwa akuyungizya 200 mM HCl, mpawo yakabikkwa a 37 °C a 54 maminiti 14,000 rpm kwamaminiti aali kkumi a 4 °C kugwisya tombe. Zitondezyo zyakasanzigwa anzila zyobile zyakugwisya zintu ziyumu (Oasis MCX cartridges, Waters) mpawo zyakabikkwa mu 0.1% ya asidi ya foomu kusikila kumamanino aa 0.33 μg µl-1. Zitondezyo zyakabikkwa zina lyamapulotini kuzwa ku MAG aalembedwe munzila iikozyenye kuzwa ku MAG. baalumi. Zibeela zyobile zyakuvwuntauzya zyakavwuntauzyigwa kuli musamu umwi aumwi. Kuzwa waawo, 1 μg yachimwi achimwi yakavwuntauzyigwa kubelesya 25-cm fused silica 75-μm column antoomwe a 4-cm fused silica Kasil1 (PQ) frit trap yakazwide Jupiter C12 reversed-phase resin (PQ) alimwi a liquid exute-min 18 chromatography. Zitondezyo zyakugaya - MS/MS zyakachitilwa a Q-Exactive HF mass spectrometer (Thermo Fisher) antoomwe a nanoACQUITY UPLC System (Waters). Data-related acquisition data yakabambwa kuli umwi awumwi yakasandulwa kuba mzML format kubelesya Proteowizard (version 3.0.2027) antoomwe a Comet (version 3.33) kulwana . FASTA database iijisi zibeela zya ma protein kuzwa ku Anopheles gambiae (VectorBase version 54), Anopheles coluzzi Kuvwuntauzya kwakachitwa a Mali-NIH (VectorBase version 54), Saccharomyces cerevisiae (Uniprot, March 2021), A. gambiae RNA-seq, antoomwe amapulani otatwe aakusandululwa kwa Peptinants zizyibidwe. FDRs zyeendelana amaapu zyakajanwa kubelesya Percolator32 (version 3.05) a threshold ya 0.01, mpawo ma peptides akabungikwa mukuziba ma protein kubelesya protein parsimony mu Limelight33 (version 2.2.0). Bunji bwama protein bwakayeeyelwa kubelesya normalized spectral abundance factor (NSAF) yakabalilwa kuli umwi awumwi protein mbuli mbokwakapandululwa musyule. kweendelanya a protein imwi aimwi kwakali mweelwe waakati-kati mumasampu kuzwa kuzintu zyobile zyaandeene. 15N labeling yakazwidilila kuvwumbilila proteome yabanakazi, nikuba kuti ma protein masyoonto aatakwe mabala akajanwa kuzwa kuli basimbi bakabikkwa mabala. Twakalemba kujana kwakucheya kwama protein aabaalumi (1-5 spectra) mumasampu aabanakazi buyo mumasampu aatakwe mulimo, ooko kwakacitwa masampu aabanakazi aatabelesyegwi pe zitondezyo, akaambo ka HPLC "kutola". Ma protein amwi aajanika kuti 'masofwaazya' kuzwa kuli batakwetwe balembedwe alilembedwe mu Supplementary Table 1.
Ma peptide obile, QTTDRVAPAPDQQQ (mukati ka isotype PA) antoomwe a MESDGTTPSGDSEQ (mukati ka isotype PA a PB) mu Genscript. Ma peptide obile akaswaanganyigwa, mpawo akaswaanganizyigwa a carrier protein KLH mpawo akabikwa mu New Zealand rabbits. Rabbits zyakajayigwa nikwakamana kujaigwa kwane, mpawo IG G yakagwisigwa kusalazya. IgG kuzwa ku EPP-specific rabbit yakabelesegwa kunembo kwa western blotting.
Ku western blots, MAG (n = 10, aawo n wiiminina mweelwe wamasampu aa nkumba zitakwe buumi) antoomwe a LRT yabamakaintu (n = 30) kuzwa kuli basankwa batakwetwe bamazuba one abanakazi batakwetwe naakuti bakakwatwa kusinikizyigwa (<10 nikwakainda kukwatana), Protein extraction buffer (50 mM Tris, 80; pH-10; sodium deoxycholate; 150 mM NaCl; 1 mM EDTA; 1× protease inhibitor cocktail (Roche)) yakayungizigwa alikke. Zitondezyo zyakabikkwa antoomwe mpawo-mpawo nikwakamana kugonkwa a beader (2 mm glass beads, 2,400 rpm, 90 sec Insoluble debrived was derived at 200 g). a 4 °C. Ma protein akabalilwa a Bradford assay (Bio-Rad). Mpoonya, 20 μg ya MAG protein, 40 μg ya LRT protein, antoomwe a 20 μg ya residual bulk protein zyakasandulwa mpawo zyaandaanizyigwa a 10% Bis-Tris NuPAGE kubelesya MOPS buffer. Ma protein akatolwa ku polyvinydene fluoridene membranes kubelesya fluorideni nzila yakutola (Thermo Fisher). Ma membrane akasanzyigwa tubili mu 1× PBS-T (0.1% Tween-20 mu PBS) mpawo akajalilidwe mu Odyssey blocking buffer (Li-Cor) kwawoola lyomwe a 22°C. Ma membrane akazungaanisigwa masiku woonse a 4 °C a custom rabbit anti-EPP polyclonal polyclonal antibody (10:10 buffer) antoomwe a rat anti-actin monoclonal primary antibody MAC237 (Abeam; 1:4,000). Zibeela zyakasanzigwa a PBS-T mpawo zyakabikkwa antoomwe a secondary antibodies (donkey anti-rabbit 800CW antoomwe a goat anti-rat 680LT (Li-Cor), zyoonse zyobile 1:2000, zili a 100% ya buffer. SDS a 0.2% Tween -20 kwawoola lyomwe a 22 °C. Zibeela zyakasanzyigwa a PBS-T mpawo zyakatolwa a Odyssey CLx scanner. Zifwanikiso zyakayobolwa akubelesegwa mu Image Studio (version 5.2). Bbandi limwi lyeendelana a EPP-RA isoform (82 kDa) tiilyakajanwa pe.
Masena aa EPP (mbuli isoform AGAP002463-RB iijisi histidine phosphatase domain, NCBI conserved domain search 34) antoomwe a EcK2 (AGAP002181) akabikkwa mu pET-21a(+) plasmid (Novagen Millipore Sigma) ; ma pulayima alilembedwe mu Extended Data Table 2. Zilongwe zili musanu atutatu zya GS4 (mu tandem) zyakabikkwa kakutanaba C-terminal 6xHis tag ya pET-21a(+)-EcK2 construct. Ma protein aasanganizidwe akapangwa kubelesya NEBExpress cell-free E. coli protein synthesis reaction (New England BioRecombinant proteins zyakali kubelesya NEBExpress ma protein aasalala). Ni spin columns (New England BioLabs).Dihydrofolate reductase (DHFR) control protein yakapangwa kubelesya DNA template kuzwa ku NEBExpress Cell-Free E. coli Protein Synthesis Kit. Ma protein akabikkwa mu 50% glycerol mu PBS a -20 °C kwamyezi yotatwe.
Ikubeleka kwa phosphatase kwa EPP antoomwe azibeela zyanyama kwakapimwa kubelesya 4-nitrophenyl phosphate (pNPP; Sigma-Aldrich). Chikwabilizyo chakusanduka chakajisi 25 mM Tris, 50 mM acetic acid, 25 mM Bis-Tris, 150 mM NaCl, 10 mM MTA, antoomwe a mM MTA. DTT. Nyama yakabikkwa antoomwe mu reaction buffer mpawo ma cell debris akagwisigwa kwiinda mukubelesya centrifugation. Amutalisye reaction kwiinda mukuyungizya enzyme naakuti tissue extract mu reaction buffer iijisi 2.5 mg ml-1 pNPP. Musamu wa reaction wakabikkwa mumudima mumudima, mpawo mweelwe wa pNP wakapimwa kuzwa ku pPP kuyoya a 405 nm muziindi zisiyene.
Muchiimo cha EcK, ma protein akabikkwa a 0.2 mg 20E naa 3D20E mu 200 μl buffer (pH 7.5) iijisi 10 mM HEPES–NaOH, 0.1% BSA, 2 mM ATP antoomwe a 10 mM MgCl2 ku 27 °C akuyungizya. 800 μl methanol, mpawo yakatontozyegwa a -20 °C kwawoola lyomwe, mpawo yakatontozyegwa a 20,000 g kwamaminiti aali kkumi a 4 °C. Maanzi aakasyaala mpawo akavwuntauzyigwa a HPLC-MS/MS. Kutegwa ma protein akabelesegwa mukabunga kakweendelezya aleke kubeleka, ma protein akabikkwa mu 50% mu PBS a 20% 95°C.
Muchiimo cha EPP, ma protein akabikkwa antoomwe a 3D20E22P (kweelana amweelwe uujanika mu 18 pairs zya MAG, zyakasalala a HPLC-MS/MS) mu 100 μl buffer (pH 7.5) iijisi 25 mM Tris, 50 mM acetic acid, 25 mMs, 50 mM Bi NaCl, 0.1 mM EDTA, antoomwe a 1 mM DTT kwamahola otatwe a 27 °C. Kuchincha ooku kwakaimikwa akuyungizya 400 μl methanol mpawo kwakatontozyegwa a -20 °C kwawoola lyomwe, mpawo kwakatontozyegwa a 20,000 g kwamaminiti aali kkumi a 4 °C. Kuvwuntauzya kwakacitwa a MSLC/MSLC.
Zibeela zya PCR zya EPP (362 bp), EcK1 (AGAP004574, 365 bp) antoomwe a EcK2 (556 bp) zyakayungizigwa kuzwa ku cDNA yakapangwa kuzwa kumitunta ya nkumba zitakwe mutwe zyabantu bamisyobo yiindene. Cibeela ca PCR ca eGFP (495 bp) chakayungizigwa kuzwa ku pCR-FPG yakapandululwa musyule; Ma PCR primers alilembedwe mu Extended Data Table 2. Chibeela cha PCR chakabikkwa aakati ka inverted T7 promoters a pL4440 plasmid. Plasmid constructs zyakajanwa kuzwa ku NEB 5-α competent E. coli (New England Biolabs) mpawo zyakasinizyigwa a DNA sequencing kaitanabelesegwa (Langa Supplement Data sequery 1 PL4440 sequencing fortersers). T7 promoter (Extended Data Table 2) yakabelesegwa kuyungizya insert kuzwa ku pL4440-based plasmid. Bupati bwachintu cha PCR bwakasinizyigwa a agarose gel electrophoresis. dsRNA yaka lembwa kuzwa ku PCR templates kubelesya Megascript T7 Transcription Kit (Thermo Fisher) mpawo yakasalazyigwa kweendelanya amalailile aabasikupanga aakapandululwa musule.
Mukujaigwa kwa dsRNA, 1,380 ng ya dsRNA (dsGFP, dsEcK1, dsEcK2, dsEPP) yakabikkwa mubunji bwa 10 ng nl-1 muchifuwa chabaalumi naa banakazi bapati (Nanoject III, Drummond) mukati kabuzuba bomwe kuzwa nikwakagwisigwa. Kugonkwa kwamajini kwakajanwa kwiinda mukuchita ziindi zyotatwe Kugwisigwa kwa RNA, kupanga cDNA, antoomwe a RT-qPCR. Mukujaigwa kwa ecdysone, banakazi batakwetwe bamazuba one naa batakwetwe bamazuba aali cisambomwe bakapegwa bulowa bwa 0.13, 0.21, naa 0.63 μg ya 20E naa 3D20E (Norum III, Dmond, 213. kweendelana, kweendelanya anzila yakusunka naa 6.3 ng nl-1. Kobikka 100 nl ya 10% (vol/vol) ethanol mumaanzi; 100 nl ya 3D20E22P mu 10% ethanol (kweelene a 75% yamweelwe uujanika mu MAGs). Inswi zyakabikkwa mukabunga kakubelesya injection.
Mukulanga-langa kuzyala, banakazi bamazuba otatwe bakapegwa bulowa bwamuntu. Gwisya nkumba zyakali kulya mbichani naakuti zitapedwe pe. Kweelana abusilisi, banakazi bakabikkwa munkomeki zyaandeene zyakuzyala kwamasiku one aatasiki ku 48 mahola kuzwa nikwakamana kulya bulowa. Mazai akabalilwa a stereoscope (Steo Zeimi, 508); kubanakazi bakakwatana, mazai aakazyala kuba nseke akali kubonwa kuti alakonzya kuzyala.
Mukusola kuzyalana, banakazi bakazumizigwa mazuba aatasiki kuli obile kweendelanya abusilisi kuti babe aanguzu zyakukazya kuzyalana, mpawo baalumi bamusyobo wamusyokwe bakabikkwa muchizumbo chomwe. Nikwakainda masiku obile, ma vesicles aabanakazi akazyalwa akakosolwa mpawo DNA ya genomic yakagwisigwa kwiinda mukubelesya freeze-thaw a sonication buffer iijisi m 10 M Tris-HCl, 1 mM EDTA, antoomwe a 25 mM NaCl (pH 8.2). Zitondezyo zyakabikkwa antoomwe a Proteinase K (0.86 μg μl-1) kwamaminiti ali kkumi amusanu a 55 °C, mpawo kwakatobela maminiti aali kkumi a 95 °C. Ma DNA aatakwe busani akasanzyigwa a Y 10 zibeela zya chromosome; ma pulayima alilembedwe mu Extended Data Table 2. Kubula Y chromosome sequence kutondeezya kuti taakwe kujatana.
Mukulanga-langa kuzyalana alimwi, banakazi bakasinikizyigwa kuzyalana bakalingulwa kuti naa kuli zijazyo zyakuzyalana kutegwa basinizye bukkale bwakuzyalana alimwi akuzumizyigwa mazuba obilo kuti batalike kuzyalana kakunyina baalumi, mbuli mbokwakapandululwa musyule 36 .Baalumi bakanyamwide DsRed transgenic sperm mpoonya bakabikkwa muzibbeeke zyabanakazi masiku masiku, zisyango zyakali kuzyala. yakakosolwa kuzwa kubanakazi, mpawo DNA ya genomic yakapangwa mbuli mbokwaambidwe atala aawa mpawo yakabikkwa mu qPCR detection ya DsRed transgene; ma pulayima alilembedwe mu Extended Data Table 2. Kubula kwa DsRed transgene kwakatondeezya kuti taakwe kuswaangana kwakachitika.
3D20E yakapangwa mbuli mbukwakapandululwa musule 37 . Mubufwiifwi, 10 mg ya 20E (Sigma-Aldrich) yakanyonyoonwa mu 10 ml yamaanzi, mpawo yakabikkwa 30 mg ya platinum black (muchikozyano cha Sigma-Aldrich). Mulonga uuteteete wa O2 wakazumanana kusanganizigwa mumusanganisyo aawo mpokutontola. Nikwakainda mawoola aali 6, 30 mL ya methanol yakabikkwa kuchitila kuti kutabi kuchincha. Musanganisyo wakabikkwa mu centrifuge kuchitila kuti kugwisye zibeela zya catalyst. Maanzi aakasyaala akayuminizyigwa kusikila akayume mu vacuum atempelecha yamukati. Ikuzwa kwakayuminina kwakanyonyoonwa mu 10% ya ethanol mpawo methanol yakabikkwa mu LCMS kuti ibelesyegwe kulanga-langa mweelwe wa HPMS. (kuzwa ku 20E kusikila ku 3D20E) yakali afwaafwi kusika ku 97% (Fig. 4b), mpawo MS spectrum ya 3D20E yakabambwa yakakozyanya ayeeyo yakajanika mubasimbi bakakwetene (Fig. 4c).
Makani aaya ajisi twaambo tujatikizya masunko aakachitwa. GraphPad (version 9.0) yakabelesegwa kuchita Fisher's exact test, Mantel-Cox test, antoomwe a Student's t-test. Cochran-Mantel-Haenszel tests yakachitwa kubelesya R script (ilajanika a https://github.com/haenszel. Data yakasunkwa). kuti ube kabotu kubelesya Shapiro-Wilk test a significance threshold ya 0.05. Aawo data niyakaalilwa kubelesya nzila ya Mann-Whitney yakachitwa. Data yakupona yakavwuntauzyigwa kubelesya nzila ya Mantel-Cox. I DESeq2 package (version 1.28.1) yakabelesegwa kuchita RNA-seq-level genetal expression a the horizontal expression. wiiminina mweelwe waakati-kati. Muulo uuyandika wa P = 0.05 wakabelesegwa mbuli mweelwe wamasunko woonse.
Kuti ujane makani manji aatala akubamba chiiyo, langa bbuku lya Nature Research Report lijatikizyidwe aachibalo eechi.
Data ya MS proteomic yakabikkwa mu ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) kwiinda mu PRIDE Partner Repository (https://www.ebi.ac.uk/pride/) antoomwe a dataset identifier PXD032157.
I RNA-seq dataset ilibikkidwe mu Gene Expression Comprehensive Library (https://www.ncbi.nlm.nih.gov/geo/) munsi lya serial record GSE198665.
Ma data aambi aakapangwa akuvwuntauzyigwa muchiindi chakwiiya alakonzya kujanwa kuzwa kuli balembi kuti kwalomba. Eechi chibalo chilapa data yakusaanguna.
De Loof, A. Ecdysteroids: Ma steroids aakoonana aatuzunda aatabikkilwi maanu? Mwaalumi: Black Box.Sayensi yatuzunda.13, 325–338 (2006).
Redfern, CPF 20-hydroxyecdysone akukomena kwamatumbu mu Anopheles stephens.J. Insect Physiology.28, 97–109 (1982).