Ndza khensa ku endzela Nature.com.Vuhundzuluxeri bya browser lebyi u byi tirhisaka byi na nseketelo lowu nga nyawuriki wa CSS.Ku kuma ntokoto lowunene, hi ringanyeta leswaku u tirhisa browser leyi pfuxetiweke (kumbe u tima movha wa ku fambisana eka Internet Explorer).Hi nkarhi wolowo, ku tiyisisa nseketelo lowu yaka emahlweni, hi ta kombisa sayiti handle ka switayele na JavaScript.
Kuhambana na swihadyani swa marhambu ya nhloko, switsotswana swi ehleketiwa ngopfu leswaku swi pfumala ti hormones ta steroid ta rimbewu leti nga na xihlawuhlawu xa xinuna.Eka Anopheles gambiae, ecdysone steroid 20-hydroxyecdysone (20E) yi vonaka yi hundzukile ku lawula ku kula ka tandza loko yi endliwa hi ta xisati2 na ku hlohlotela nkarhi wa mating refractory period loko yi hundziseriwa hi swa masangu hi ta xinuna3.Ku sukela eka ku kula ka tandza na ku hlangana i swihlawulekisi swa nkoka swa ku veleka, ku twisisa ndlela leyi switsotswana swa xisati swa Anopheles swi hlanganisaka swikombiso leswi swa tihomoni swi nga olovisa ku endliwa ka minongonoko leyintshwa yo lawula malariya.Laha, hi paluxa leswaku mintirho leyi ya ku veleka yi lawuriwa hi ti-steroid ta rimbewu leti hambaneke hi ku tirhisa netiweke leyi rharhanganeke ya ti-enzyme leti rharhanganeke ta ecdysteroid-activating/inactivating.We identified a male-specific oxidized ecdysone, 3-dehydro-20E (3D20E), leyi sirhelelaka vutswari hi ku pfala ku amukela ka rimbewu ka xisati endzhaku ka ku hundziseriwa ka rimbewu na ku tirhisiwa hi dephosphorylation.Lexi xiyekaka, ku hundziseriwa ka 3D20E ku tlhele ku hlohlotela ku paluxiwa ka tijini ta ku veleka leti hlayisaka ku kula ka tandza hi nkarhi wa ku tluleriwa hi Plasmodium, ku tiyisisa rihanyo ra ta xisati leti tluleriweke.20E leyi humaka eka xisati a yi humesi rimbewu nhlamulo, kambe yi pfumelela vanhu lava hlanganisaka ku veka tandza endzhaku ka loko ti kinases leti sivelaka 20E ti siveriwile.Ku tiviwa ka hormone leyi ya steroid ya switsotswana leswi kongomisiweke eka xinuna na xiave xa yona eka ku lawula ku amukela ka rimbewu ka xisati, ku veleka na ku tirhisana na Plasmodium swi ringanyeta vuswikoti byo hunguta ku humelela ka ku veleka ka switsotswana leswi hundziselaka malaria.
Timhangu ta malariya na mafu swi le ku tlakukeni nakambe4 hikwalaho ka ku lwisana loku hangalakeke ka swidlaya-switsotswana eka switsotswana swa Anopheles, leswi nga swona ntsena leswi rhwalelaka switsotswana swa malariya swa vanhu.Ntivo-vutomi bya ku hlangana ka switsotswana leswi i xikongomelo xo koka rinoko ngopfu eka ku nghenelela ka novhele ka vulawuri bya malariya hikuva ta xisati ti hlangana kan’we ntsena5; ku endla leswaku xiendlakalo lexi xa ku hlangana xin’we xi nga ri na switsongwatsongwana swi ta va na vuswikoti lebyikulu byo hunguta nhlayo ya switsotswana ensin’wini.
Vavasati va va va nga ha koti ku endla timhaka ta masangu endzhaku ko amukela tihomoni ta steroid ta xiyimo xa le henhla eka vavanuna.Mindzavisiso yi kombisile leswaku xihlohloteri xa ku tika eka ku ya emahlweni ka ku hlangana i 20-hydroxyecdysone (20E), hormone ya steroid leyi tivekaka ngopfu tanihi mulawuri wa xirhendzevutani xa molting eka xiteji xa larval.Vuswikoti bya ta xinuna ku hlanganisa na ku hundzisela 20E byi hundzukile hi ku kongoma eka Tinxaka ta Anopheles letinga xiphemu xa subgenus Cellia7, leyi hangalakeke e Afrika naswona yikatsa ti vectors letinga na khombo swinene ta malaria, kukatsa na Anopheles gambiae.Leswi swa xiyeka ngopfu hikuva eka tinxaka leti ta xisati titlhela ti humesa 20E endzhaku ka swakudya swinwana na swinwana swa ngati, naswona 20E yi fambisa xirhendzevutani xa oogenesis (vona ref. 8).Hambiswiritano, aswi tiveki ngopfu hi ndlela leyi ti females ti hlanganaka ha yona swikombiso leswi humaka eka swihlovo swimbirhi swo hambana swa ecdysone (ku hundziseriwa ka xinuna na ku nghenisiwa ka ku dyisa ngati) handle ko kavanyeta vuswikoti bya swona byo hlangana.Entiyisweni, loko 20E leyi humesiwaka hi ta xisati yi hlohlotela ku nga tiyiseleleki ka rimbewu, leswi swi ta endla leswaku ku va na ku pfumala n’wana eka vanhu lava dyisaka nhwana, ku nga mahanyelo lama tolovelekeke swinene eka switsotswana leswi5.
Nhlamuselo leyi nga kotekaka hileswaku ya xinuna ya A. gambiae yi hundzisela ecdysone leyi cinciweke leyi kongomisiweke eka xinuna, leyi endlaka leswaku signaling cascade yi tirha eka xirho xa xisati xa ku veleka, leswi endlaka leswaku ku va na ku nga tshamiseki ka ku hlangana.Hambiswiritano, hambi leswi swihadyani swa marhambu ya nhloko swi nga na tihomoni to tala ta steroid, to fana na estrogen na androgen (leswi kambisisiweke eka ref. 9), hi ku tiva ka hina, ti steroid leti nga na xihlawuhlawu xa androgenic a ti si tiviwa eka switsotswana.
Hi veke ku kumisisa repertoire ya tihomoni ta steroid eka ndzhuti wa xinuna wa xinuna wa xipfuneti (MAG) wa A. gambiae leyi vupfeke hi tlhelo ra rimbewu hi lava ti-steroid leti nga cincaka.Hi ku tirhisa chromatography ya mati ya matirhelo ya le henhla leyi hlanganisiweke na tandem mass spectrometry (HPLC-MS/MS) ku tlula ndlela leyi nga kongomangiki ngopfu leyi tirhisiweke khale, hi kumile ecdysone (E) na 20E eka tinyama leti, ku tiyisisa khale mbuyelo.Hambiswiritano, xikombiso a xi lawuriwa hi ti-steroid leti oxidized phosphorylated, leti fambelanaka na fomula ya 3-dehydro-20E-22-phosphate (3D20E22P)12 (Xifaniso 1).Swivumbeko swin’wana swi katsa 3-dehydro-20E (3D20E) na 20E-22-phosphate (20E22P).The HPLC-MS/MS signality of 3D20E22P a yi ri ehenhla ka tioda timbirhi ta vukulu ku tlula xivumbeko xa yona lexi nga dephosphorylated, 3D20E, na tioda tinharhu ta vukulu ehenhla ku tlula ya E na 20E (Xifaniso 1).Hambi leswi eka swiphemu swin’wana swa miri na xirho xa le hansi xa ku veleka (LRT; Extended Data Fig. 1a).Hi tlhele hi kambisisa ti-ecdy eka ti-ecdy eka males ya xinuna lama ha ku pfariweke (<1 day old) na ya xisati naswona yi kumiwile 3D20E na 3D20E22P ntsena eka MAG; E, 20E na 20E22P ayiri kona eka rimbewu hinkwaro (Extended Data Fig. 1b).Data leyi yi ringanyeta leswaku A. gambiae ya xinuna leyikulu yi humesa ti titers tale henhla ta ti hormones to cinca eka ti MAGs ta tona letinga endliwa hi ta xisati.
MAG na LRT ya xisati (kukatsa na atria, seminal vesicles, na parovarium) swi tsemiwile kusuka eka 4-day-old (4-day-old) virgin males na virgin na mated ya xisati (0.5, 3, na 12 hpm).Ecdysone eka ti tissues leti yi kamberiwile hi HPLC-MS/MS (mean ± sem; unpaired t-test, two-sided, two-sided, two-sided, two-sided, two-sided, two-sided, unpaired t-test, two-sided, two-sided. mpimo wa ku tshuburiwa ka mavunwa (FDR) wu lulamisiwile; 3 wa tiawara vs. 0.5 wa tiawara, P = 0.25;12 wa tiawara ku hambana na 3 wa tiawara, P = 0.0032;12 wa tiawara vs. 0.5 wa tiawara, P = 0.015).Data yi huma eka ti biological replicates tinharhu.Ndhawu ya nhlohlorhi ya ecdysone yin’wana na yin’wana ya ntsakelo yi hlayiwile no normaliseriwa hi nhlayo ya switsotswana.Ecdysone yi yimeriwile hi muhlovo hi ndlela leyi landzelaka: E, rihlaza; 20E, ya muhlovo wa orenji; 20E22P, ya xivunguvungu; 3D20E, ya wasi; 3D20E22P, pink.Inset yi engetela xikalo eka y-axis ku kombisa ti levele ta le hansi ta ecdysone.
Ku lavisisa loko 3D20E22P na 3D20E swi hundziseriwa hi nkarhi wa ku hlangana, hi tseme ti LRT ta xisati eka tindzhawu to hambana ta nkarhi endzhaku ka ku hlangana.Hambi leswi ecdysone yinga kumeka eka vanhwana, hi vonile nhlayo leyikulu ya 3D20E22P eka LRT hiku hatlisa endzhaku ka ku hlangana (0.5 h post-mating, hpm), yihunguteka hiku famba ka nkarhi, loko Ti levele ta 3D20E ti tlakukile swinene (Fig. 1).Hi ku tirhisa 3D20E leyi endliweke hi tikhemikhali tanihi mpimanyeto, hi kumile leswaku ti levele ta hormone leyi ya steroid eka ti LRT ta ku hlangana a ti ri kwalomu ka 100-fold ehenhla ku tlula 20E (Extended Data Table 1).Xisweswo, 3D20E22P i ecdysone leyikulu ya xinuna leyi hundziseriwaka eka LRT ya xisati hi nkarhi wa ku hlangana, naswona xivumbeko xa yona xa dephosphorylated, 3D20E, xiva xitala swinene endzhakunyana ka ku hlangana.Leswi swiringanyeta xiave xa nkoka eka ecdysone yo hetelela eka biology ya xisati endzhaku ka ku hlangana.
Endzhaku ko tumbuluxa dataset leyintshwa ya RNA sequencing (RNA-seq) (Fig. 2a), hi ku tirhisa phayiphi ya bioinformatics leyi akiweke hi ku landza mukhuva, hi lavisile ecdysone kinase (EcK), ecdysone oxidase (EO), na ecdysone leyi khodaka 20E-modified phosphatase gene.EPP) yi kombisiwa eka tinyama to veleka.Hi kumile xitekela xin’we xa EPP na timbirhi tijini ta EcK leti nga vaka kona (EcK1 na EcK2), kambe a va swi kotanga ku kuma jini ya kahle ya EO ya nkandziyiso.Lexi xiyekaka, tijini ta EPP ha yin’we yin’we ti kombisiwile eka swiyimo swa le henhla (98.9th percentile) eka ti-MAG ta Gambia kambe ku nga ri eka ti-LRT ta xisati (Fig. 2b), ku hambana na leswi hi swi languteleke ku sukela loko dephosphorylation ya 3D20E22P yi humelerile eka tissue leyi ya xisati.Hikokwalaho, ha pfumela leswaku EPP ya xinuna yi nga hundziseriwa hi nkarhi wa ku hlangana.Hakunene, hi tirhise in vivo stable isotope labeling ku fihla protein ya xisati endzhaku ka ku hlangana, enzyme leyi hlawuriweke hi MS eka atrium ya xisati (Fig. 2c na Supplementary Table 1).Vukona bya EPP eka MAG na LRT ya xisati leyi hlanganisiweke (kambe ku nga ri virgin) yi tlhele yi tiyisisiwa hi ku tirhisa ti antibodies to karhi (Fig. 2d).
a, Phayiphi ya bioinformatics leyi akiweke hi ku landza mukhuva ku lavisisa tinyama ta ku veleka ta rimbewu rin’wana na rin’wana ku kuma tijini leti khodaka EcKs, EOs, na EPPs.Tinomboro leti nga ekusuhi na miseve ti kombisa nhlayo ya vahlawuriwa va xinuna na va xisati eka goza rin’wana na rin’wana.Nxopaxopo lowu wu kumile jini yin’we ya EPP (EPP) na jini yin’we ya EcK (EcK1) leyi kombisiwaka eka ta xinuna, na jini yin’we ya EcK (EcK2) leyi kombisiwaka eka rimbewu hinkwaro kambe yi nga humesi candidate EO gene.b, Heatmap yi pimanisa candidate gene expression eka virgin (V) na ku hlangana (M) Anopheles gambiae na Anopheles albicans tissues.Spca, ku nonisa; MAGs, ti accessory glands eka va xinuna; swiphemu swin’wana swa miri, ku katsa na mavele, timpapa, milenge, miri wo nona, na swirho swa le ndzeni eka rimbewu hinkwaro, na ti ovary eka ta xisati.EcK2 yi kombisiwa ngopfu eka MAG na ti atria ta Gambia, kasi EPP yi kumeka ntsena eka MAG.c, Proteomic analysis of male ejaculate group translocation into female atria at 3, 12 and 24 hpm, ku kombisa ti protein ta 67 letinga tala swinene.Ti ta xisati ti fuyisiwile eka swakudya leswingana 15N kuva ti lebula (na ku fihla) ti protein hinkwato.Ti ta xinuna letinga tagged ti hlanganisiwile na ta xisati leti tagged, naswona ti LRTs ta xisati ti tsemiwile eka 3, 12 na 24 hpm kuva ti endliwa proteomic analysis (vona Supplementary Table 1 ku kuma nxaxamelo lowu heleleke wa ti ejaculatory proteins).Inset, EPP, Eck1 na EcK2 yi kumiwile eka MAG ya va xinuna lava nga virgin hi proteomic analysis ya ti tissues leti.d, EPP yi kumiwile hi western blot eka MAG na LRT ya ta xisati leti hlanganisiweke, kambe ku nga ri eka ta xisati leti nga virgin kumbe ta xinuna kumbe miri hinkwawo wa xisati.Membranes yi kamberiwile hi nkarhi wun’we hi anti-actin (loading control) na anti-EPP antibodies.Hinkwaswo swa xinuna i virgins.See Xifaniso xo Engetela xa 1 xa datha ya xihlovo xa gel.Western blots yi endliwile kambirhi hi mbuyelo lowu fanaka.
Ntirho wa ecdysteroid phosphophosphatase wa EPP wu tiyisisiwile endzhaku ko pfaleriwa hi HPLC-MS/MS na 3D20E22P leyi hambanisiweke kusuka eka MAG (Extended Data Fig. 2a).Ku tlula kwalaho, loko hi miyeta EPP hi RNA-mediated interference (RNAi), hi kumile ku hunguteka lokukulu ka ntirho wa phosphatase eka tinyama to veleka ta tinyama leti ta xinuna (Fig. 3a), na ta xisati mated with EPP-silenced males showed significant Xiphemu xa le hansi xa dephosphorylated 3D20E (Fig. 3b) hambi leswi ku nga na xiphemu xa gene silencing (Extended Data Fig. 2b,c).Ku hambana na sweswo, a hi kumanga ku cinca lokukulu eka 20E22P/20E ratio eka switsotswana leswi fanaka, leswi nga ha ringanyetaka leswaku enzyme yi kongomile eka 3D20E22P (Fig. 3b). 3b).
a, Kuhunguteka ka ntirho wa phosphatase eka MAG leswi vangiwaka hi EPP silencing hiku tirhisa double-stranded EPP RNA (dsEPP) kumbe double-stranded GFP RNA (dsGFP) controls.Makhume mbirhi wa swidziva swa MAG switirhisiwile eka replicate yin’wana na yin’wana (P = 0.0046, paired t-test, two-sided), leswi yimeriweke hi ti dots leti hambaneke.b, Ta xisati leti hlanganisiweke na EPP-silenced males a ti ri na a xiphemu xa le hansi swinene xa dephosphorylated 3D20E eka 3 hpm (P = 0.0043, unpaired t-test, two-sided), kasi ti levele ta 20E a ti khumbekanga (P = 0.063, unpaired). t-test, two-sided).Data yi nyikeriwile tani hi mean ± sem kusuka eka swidziva swinharhu swa 13, 16 na 19 wa xisati xin’wana na xin’wana.c, Ta xisati leti hlanganisiweke na ta xinuna leti miyeleriweke hi EPP a ti ri na tihakelo ta le henhla swinene ta ku hlangana nakambe (P = 0.0002, Fisher’s exact test, two-sided).Ti ta xisati ti sungule ku sindzisiwa ku hlangana ku tiyisisa xiyimo xa tona xa ku hlangana; Endzhaku ka masiku ya 2, va hlanganisiwile na van’wana va xinuna lava rhwaleke mbewu ya vununa ya transgenic ku kambela ti re-mating rates hi quantitative PCR detection ya transgene.d, Ta xisati leti dyiwaka hi ngati leti hlanganisiweke na ta xinuna leti pfaleriweke hi EPP a ti hungute ngopfu ku veleka (P < 0.0001; Mann-Whitney test, two-sided) na nhlayo ya tandza leyi hungutekeke nyana (P = 0.088, Mann-Whitney test, two-sided) , kasi mpimo wa ku tswala a wu khumbekanga (P = 0.94, Fisher’s exact test, two-sided).Eka tiphaneleni hinkwato, n yi yimela nhlayo ya swikombiso swa switsotswana leswi tiyimeleke hi tlhelo ra biology.NS, a swi na nkoka.*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.001.
Endzhaku ka sweswo, hi kambisisile loko ecdysone dephosphorylation yiri ya nkoka eka ku hlohletela ku lwisana na ku hlangana eka ta xisati.Lexi xiyekaka, ta xisati leti hlanganisiweke na ta xinuna letinga heleriwa hi EPP titlhele ti hlangana hi nhlayo yale henhla swinene (44.9%) kutlula ta xisati ta vulawuri (10.4%) loko ti hlangana na ta xinuna to engetela (transgenic) (Fig. 3c).Hi tlhele hi vona kuhunguteka lokukulu ka ku veleka (Fig. 3c). 3d, ximatsi) na kuhunguteka nyana ka nhlayo ya tandza leri vekiweke hi ta xisati leti (Fig. 3d, exikarhi), kasi tiphesente ta tandza leri vekiweke hi ta xisati (nhlamulo yin’wana leyi humesiweke eka ta xisati hiku hlangana )) aswi khumbekanga (Fig. 3d, right).Given the observed specificity of EPP for 3D20E22P, these results suggest that the activation of 3D20E hi EPP leyi hundziseriwaka hi nkarhi wa ku hlangana yingava na xiave xa nkoka eka ku tima ku amukela ka xisati eka ku hlangana kun’wana, mahanyelo lawa khale aya boxiwa eka ku hundziseriwa ka rimbewu ka 20E .Hikokwalaho, hormone leyi yo hlawuleka ya xinuna yitlhela yi khumba swinene ku veleka ka xisati.
Endzhaku ka sweswo, hi pimanisile migingiriko ya 20E na 3D20E eka swikambelo swa injection eka vanhwanyana lava vupfeke hi swa masangu hi ku tirhisa 3D20E leyi endliweke hi tikhemikhali (Fig. 4a–c) na 20E leyi xavisiwaka.Hi xiye leswaku 3D20E a yi tirha swinene ku tlula 20E eku pfala ku twisisa ka xisati eka ku hlangana eka nhlayo leyimbirhi (Fig. 4d).Notably, half the levele ya physiological ya 3D20E eka LRT (1,066 pg endzhaku ka injection vs. 2,022 pg endzhaku ka ku hlangana) yi hlohletele xiphemu xa ta xisati letinga tiyelangiki lexi axi tlakukile hi 20-fold kutlula levele ya physiological ya 20E (361 pg endzhaku ka injection) 24 wa tiawara endzhaku ka injection eka nhlayo yale henhla swinene 18 pg endzhaku ka ku hlanganisiwa; Extended Data Table 1).Mbuyelo lowu wu fambisana na miehleketo ya leswaku ku hundziseriwa ka rimbewu ka 20E a swi vangi minkarhi yo nga tiyiseki ku hlangana, naswona ku ya emahlweni yi kombisa 3D20E tanihi xivangelo lexikulu eku tiyisiseni ka vuxaka bya mutswari na n’wana.3D20E na yona a yi tirha swinene ku tlula 20E eka swikambelo swa ku veka tandza eka ta xisati leti nga riki na nhwana (Fig. 4e), leswi ringanyetaka leswaku mpimo wa ntolovelo wa ku veka tandza hi leswi voniweke endzhaku ka ku miyetiwa ka xiphemu xa EPP a swi vangiwa hi vukona bya ntirho wa 3D20E lowu saleke lowu ha humelerisiwaka hi swilo swa xisati leswi hlohloteriwaka hi Mating.
(a,b) 3D20E leyi endliweke hi tikhemikhali ku suka eka 20E (a) hi ku hundzuka/ku tirha kahle ka le henhla swinene (data leyi nyikeriweke tanihi xiringaniso ± sem ku suka eka swiendlo swinharhu swa ku hlanganisiwa leswi tiyimeleke) (b).c, Mass spectrum (hafu ya le hansi) yi fambelana kahle na ecdysone leyi kumekaka eka LRT ya xisati leyi hlanganisiweke (hafu ya le henhla).d, Loko yi pimanisiwa na 20E (0.63 μg, P = 0.02 ; (0.63 μg, P = 0.0002; 0.21 μg, P = 0.54; Fisher’s exact test, 2-sided).e, ku cheriwa ka 3D20E swi hlohletele nhlayo yale henhla swinene ya ku tswala eka ta xisati letinga virgin kutlula 10% wa vulawuri bya ethanol (0.21 μg, P < 0.0001; 0.13 μg, P = 0.0003; Fisher’s exact test, two-sided), kasi 20E loko yi pimanisiwa na controls Ntsena eka mimpimo yale henhla (0.21 μg, P = 0.022; 0.13 μg, P = 0.0823; Fisher’s exact test, two-sided).3D20E yi hlohletele ti spawning rates tale henhla swinene kutlula 20E eka mimpimo ya le henhla (0.21 μg, P = 0.0019; 0.13 μg, P = 0.075; Xikambelo xa Fisher lexi kongomeke, xa matlhelo mambirhi).Eka tiphaneleni hinkwato, n yi yimela nhlayo ya swikombiso swa switsotswana leswi tiyimeleke hi tlhelo ra biology.NS, a swi na nkoka.*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.001.Data yi huma eka ti-replicate tinharhu.
Eka tidyondzo ta khale, hi kumile leswaku ku hundziseriwa ka rimbewu ka tihomoni ta steroid swi hlohlotela ku paluxiwa ka MISO (Mating-Induced Stimulator of Oogenesis 11), jini ya xisati ya ku veleka leyi sirhelelaka ya xisati ya A. gambiae eka ku tluleriwa hi P. falciparum Tihakelo ta rihanyo leti vangiwaka hi 13, xitsongwatsongwana xa malariya xa vanhu lexi dlayaka swinene.Hi ku nyikiwa nkoka wa MISO eka ku ringanela ka ku veleka ka Anopheles eka tindhawu leti nga ni malariya, hi endle xiboho xo kumisisa leswaku hi yihi homoni ya 3D20E kumbe 20E leyi hlohlotelaka ku paluxiwa ka jini leyi.Hi kume leswaku hambileswi ku cheriwa ka 20E hi ku kongoma kumbe hi matimba swinene ku hlohloteleke ti- receptor tin’wana ta tihomoni ta nyutliya (HR), to tanihi HR3 na HR4, ni swikongomelo swa steroid swa le hansi leswi tolovelekeke, swo tanihi tijini ta yolkogenic Vg14, 15, 16, MISO a yi tele strongly was induced by 3D20E (Extended Data Fig. 3).Xisweswo, ku hundziseriwa ka rimbewu ka hormone leyi ya androgenic steroid swi vonaka swi hlohlotela tindlela leti sirhelelaka ta xisati eka ku durha loku vangiwaka hi ku tluleriwa hi switsotswana.Ku tlula kwalaho, 3D20E hi ku hambana yi khumba ti-isoform hi vumbirhi bya E receptor EcR, yi hlohlotela EcR-A na ku tshikilela EcR-B, naswona hi matimba swinene ku hlohlotela tijini tin’wana leti hlohlotelaka ku hlangana, kukatsa na HPX15, leswi khumbaka ku veleka ka xisati.Leswi swinga hlamusela ku pfumala n’wana lokukulu loku voniweke eka ta xisati leti hlanganisiweke na ta xinuna leti pfaleriweke hi EPP (Extended Data Fig. 3).Data leyi yi ringanyeta vukona bya tindlela ta le hansi leti rhandzekaka ti tirhisiwa hi tihomoni timbirhi ta ecdysone leti nga vaka ehansi ka ntirho wo karhi wa rimbewu.
Endzhaku ka sweswo, hi ringetile ntirho wa tijini timbirhi ta EcK leti hlawuriweke eka phayiphi ya hina ya bioinformatics.Ku miyeta EcK1 kumbe EcK2 swi endle leswaku ku va na ku fa lokukulu eka va xinuna (Extended Data Fig. 4a), leswi ringanyetaka leswaku ecdysone phosphorylation, naswona xisweswo ku nga tirhi, i swa nkoka eka ku hanya.Hikuva EcK2 yi kombisiwile eka swiyimo swa le henhla ku tlula EcK1 naswona yi kumiwile eka MAGs hi proteomics (Fig. 2b,c na Tafula ro Engetela 2), hi tiyisisile ntirho wa yona wa ecdysteroid kinase hi ku yi pfalela na 20E, leswi endleke leswaku ku va na phosphorylation 20E22P (Extended Data Figure 2).4b).Loko hi tirhisa 3D20E tanihi substrate, a hi swi kotanga ku kuma xiendliwa xa phosphorylated 3D20E22P (Extended Data Fig. 4c), leswi ringanyetaka leswaku 20E ku tlula 3D20E yingava xikongomelo lexi tsakeriwaka xa EcK2.
Hikuya hi nxopaxopo wa hina wa RNA-seq, EcK2 yitlhele yi kombisiwa swinene eka LRT ya ta xisati letinga virgin, laha yinga timiwa endzhaku ka ku hlangana (Fig. 2b).Hi tiyisisile data leyi naswona hi kumile leswaku EcK2 expression ayi khumbekanga hi ku phameriwa ka ngati (Extended Data Fig. 5a).Hi ku andlala swikambelo swa hina swosungula swa MS, hi kumile leswaku nhlohlorhi ya 20E22P ayi fambelana swinene na nhlohlorhi ya 20E (22-26 wa tiawara endzhaku ka swakudya swa ngati; Extended Data Fig. 5b).Ku miyetiwa ka EcK2 eka ta xisati ta nhwana swi endle leswaku ku va na ku tlakuka ka 3-fold eka relative ratio ya 20E ku ya eka 20E22P eka 26 h endzhaku ka swakudya swa ngati (Extended Data Figures 2c na 5c), leswi tiyisisaka leswaku EcK2 yi tlhela yi phosphorylate 20E in ta xisati.Lexi xiyekaka, vanhwanyana lava nga na EcK2-depleted va hlayise ku amukela ka rimbewu loku heleleke (Extended Data Fig. 5d,e), leswi yaka emahlweni swi ringanyeta leswaku vuhumelerisi bya xisati bya 20E a byi hlohloteli minkarhi yo ka yi nga tiyiseli ku hlangana.Hambiswiritano, ta xisati leti a ti tlakukile swinene hi mpimo wa ku veka tandza loko ku pimanisiwa na leti lawuriwaka, laha ku tlula 30% wa vanhwanyana lava vekaka tandza (Extended Data Fig. 5f).Loko ku endliwile ti double-stranded Eck2 RNA (dsEcK2) injections endzhaku ka ku dyisiwa ka ngati, ku tswala a ku humelelanga, laha nhlohlorhi ya 20E hikwalaho ka ku dyiwa ka ngati a yi hungutekile.Hi ku angarhela, mimbuyelo leyi yi seketela modele wa leswaku 20E leyi humesiweke endzhaku ka ku mama ngati yi nga hlohlotela ku tswala, kambe ntsena loko spawning block (EcK2 naswona swi nga endleka swin’wana) yi hundzukile off by mating.Ni 20E kumbe 3D20E injections a yi sivela EcK2 expression eka vanhwanyana (Extended Data Fig. 5g), leswi ringanyetaka leswaku swin’wana swi hlanganisa ku sivela kinase leyi.Hambiswiritano, ti levele ta 20E endzhaku ka ku dyisiwa ka ngati a ti nga enelanga ku hlohlotela ku nga tsaki ka ku hlangana, kambe hi ndlela leyinene ti hlohloteriwile hi titers ta le henhla ta 3D20E leyi hundziseriweke hi rimbewu.
Mimbuyelo ya hina yi nyika vutivi bya nkoka eka tindlela leti lawulaka ku humelela ka ku veleka ka A. gambiae.Ku humelerile modele laha ta xinuna ti hundzukeke ku hlanganisa titers ta le henhla ta 3D20E, ecdysone leyi cinciweke leyi kongomeke ya xinuna leyi tiyisisaka vutswari hi ku endla leswaku ta xisati ti nga twisisi ku ya emahlweni ti hlangana.Hi nkarhi lowu fanaka, ti vectors leti ta malaria ti tlhele ti tumbuluxa sisiteme leyi tirhaka kahle ku endla leswaku ti tirha 3D20E eka ta xisati ku hlamula eka ku hundziseriwa ka rimbewu ka EPP leyi kongomisiweke eka xinuna.Hi ku tiva ka hina, lexi i xikombiso xo sungula xa sisiteme ya tihomoni ta steroid leyi lawuriwaka hi xinuna na xisati leyi endlaka ntirho wo hlawuleka na wa nkoka eka switsotswana.Ntirho wa ecdysone lowu kongomeke wa xinuna wu postulated kambe a wu kombisiwanga hi ku hetiseka.Xikombiso, xiringanyeto lexi nga kanetiwiki ngopfu xa 18 hi leswaku mintirho leyi yi nga endliwa hi 20E precursor E1.Swa tiveka kahle leswaku eka Drosophila, monandry yi hlohleteriwa hi ku hundziseriwa ka rimbewu ka ti peptides letintsongo ta rimbewu19,20 leti tirhisanaka na ti neurons leti nghenisaka xirho xa xisati xa ku veleka hi ku tirhisa ti sex peptide receptors to karhi21,22.Ku laveka ntirho wun’wana ku kumisisa ti downstream signaling cascades leti lawuriwaka hi 3D20E eka A. gambiae females na ku kumisisa loko ti cascades leti tinga hlayisiwa exikarhi ka switsotswana na Drosophila.
Hi ku nyikiwa xiave xa nkoka xa 3D20E eka ku veleka ka xisati na mahanyelo lama hlawuriweke eka dyondzo ya hina, tindlela leti yisaka eka ku hlanganisiwa na ku tirhisiwa ka 3D20E ti nyika swivandlanene leswintshwa eka tindlela ta nkarhi lowu taka to lawula switsotswana, ku fana na ku tumbuluxiwa ka ta xinuna leti phikizanaka leti nga riki na switsotswana eka tindlela ta thekinoloji ya switsotswana leswi nga riki na switsongwatsongwana Tirhisa ku humesiwa ka nhova kumbe ku tekelela 3D20E eka ntlangu wa nhwana.The ntirho wo karhi wa xinuna wa 3D20E wu nga ha va wu humelerile loko A. gambiae na tinxaka tin’wana ta Cellia ti kume vuswikoti byo coagulate mbewu ya tona ya vununa ku va ti mating plugs, tanihileswi leswi swi pfumelelaka ku hundziseriwa hi ndlela leyinene ka nhlayo leyikulu ya tihomoni na ti-enzyme leti tirhisaka tihomoni.Hi ku tlhelela, ku hundzuka ka 3D20E loku tirhisaka monandry ku nyika endlelo eka ta xisati (hi ku tirhisa ku paluxiwa lokukulu ka MISO) ku tsakela ka tona ku ringanela ka ku veleka eka tindhawu leti nga na ku hangalaka lokukulu ka malariya, leswi hi ndlela leyi nga kongomangiki swi hoxaka xandla eka ku tluleriwa ka Plasmodium.Hi ku tekela enhlokweni leswaku 20E ya xisati yi kombisiwile ku va na vuyelo lebyikulu eka ku hanya na ku kula ka P. falciparum eka switsotswana swa xisati swa Anopheles,24 tindlela ta tihomoni ta steroid ta xinuna na ta xisati sweswi i swiyenge swa nkoka swa ku tirhisana ka switsotswana na switsotswana.
Tinxaka ta A. gambiae G3 ti fuyiwile ehansi ka swiyimo swa switsotswana swa ntolovelo (26-28 °C, 65-80% relative humidity, 12:12 h light/dark photoperiod).Ti larvae ti phameriwile swakudya swa tihlampfi leswinga powder (TetraMin Tropical Flakes, Koi Pellets na Tetra Pond Sticks hi ratio ya 7:7:2).Adult switsotswana swi phameriwile ad libitum 10% dextrose solution na ngati ya munhu ya vhiki na vhiki (swiphemu swa ngati ya dyondzo).Switsotswana leswi nga riki na nhwana swi kumiwile hi ku hambanyisa rimbewu eka xiteji xa pupal endzhaku ko kambela makumu hi microscopy.Swixinuna leswi rhwalelaka DsRed transgene swi hlamuseriwile khale.
Swikambelo swa ku hlanganisa hi ku sindzisiwa swi endliwile kuya hi ti protocol leti hlamuseriweke khale.Eka ku hlangana ka ntumbuluko, ta xisati ta masiku ya 4 ta virgin ti hlayisiwile eka ratio ya 1:3 na ta xinuna ta virgin leti vupfeke hi tlhelo ra rimbewu kuringana vusiku byimbirhi.Eka swikambelo laha ta xinuna ti cheriweke dsEPP, co-caging yi fambisana na masiku ya 3-4 endzhaku ka ku cheriwa, loko ntirho wa phosphatase wu miyetiwile hi xitalo (Extended Data Xifaniso xa 2b).
Tinyama ta switsotswana, mintsumbu leyi saleke (miri hinkwawo), kumbe miri hinkwawo wu tsemiwile kuva wuva 100% methanol naswona wu homogenized hi beader (2 mm glass beads, 2,400 rpm, 90 sec).Tissue amounts na ti volumes ta methanol aswiri hi ndlela leyi landzelaka: miri hinkwawo, 50 eka 1,000 μl; MAG, 50–100 80 μl ya xirhendzevutani; female LRT, 25–50 80 μl.Precipitate yi endliwile ku humesiwa ka methanol ka vumbirhi hi volume leyi fanaka ya methanol.Cell debris yi susiwile hi centrifugation.Methanol kusuka eka ku humesiwa loku havumbirhi bya yona yi hlanganisiwile kutani yi omisiwa ehansi ka ku khuluka ka nitrogen, kutani yi tlhela yi suspended eka ti volumes leti landzelaka ta 80% methanol ematini: miri hinkwawo, 50 μl ya xirhendzevutani; MAGs na LRT ya xisati, 30 μl.
Tisampulu ti kamberiwile eka mass spectrometer (ID-X, Thermo Fisher) leyi hlanganisiweke na xitirhisiwa xa LC (Vanquish, Thermo Fisher).5 μl ya xikombiso yi cheriwile eka kholomo ya 3 μm, 100 × 4.6 mm (Inspire C8, Dikma) leyi hlayisiweke eka 25 °C.Swiphemu leswi fambafambaka swa LC a swi ri A (mati, 0.1%). formic acid) na B (acetonitrile, 0.1% formic acid).LC gradient a yi ri hi ndlela leyi landzelaka: 5% B ku ringana 1 wa timinete, kutani yi engeteriwa ku ya eka 100% B eka 11 wa timinete.Endzhaku ka 8 wa timinete eka 100%, tlhela u ringanisela kholomo eka 5% B ku ringana 4 wa timinete.Nhlayo ya ku khuluka a yi ri 0.3 ml min-1.Ionization eka xihlovo xa MS i ku endliwa hi ku hisiwa ka electrospray ionization eka tindlela letinene na to biha.
Mass spectrometer yi pima datha eka m/z range kusuka eka 350 kuya eka 680 eka 60,000 resolution eka full MS mode.Data ya MS/MS yi kumiwile eka [M + H]+ (swikongomiso hinkwaswo), [M - H2O + H]+ (swikongomiso hinkwaswo), na [M - H]- (swikongomiso leswi phosphorylated).Data ya MS/MS yi tirhisiwile ku tiyisisa swivumbeko swa ecdysone swa swikongomelo leswi eka swona ku nga riki na mpimanyeto a yi kumeka.Ku kuma ti ecdysteroids leti nga kongomisiwangiki, datha ya MS/MS ya tinhlohlorhi hinkwato ta HPLC leti nga na >15% wa ku tala ka xiringaniso yi kamberiwile.Ku pima nhlayo hi ku tirhisa ti curve ta ntolovelo leti tumbuluxiweke ku suka eka swipimelo swo basa (20E, 3D20E) ku hlayela nhlayo leyi heleleke kumbe ku hungutiwa ka xikombiso xin’we xo karhi (swikongomiso swin’wana hinkwaswo) ku hlayela ku ringana ka tona na nhlayo leyi kumekaka eka male yin’we.For 3D20E, ku pima nhlayo ku endliwile hi ku tirhisa nhlayo ya ti adducts leti landzelaka: [M + TFA]-, [M + COOH]-, [M + Na]+, [M + Cl]-, [M + NO3]-.Data yi humesiwile no pimiwa hi ku tirhisa Tracefinder (version 4.1).Data ya MS/MS yi kamberiwile hi ku tirhisa Xcalibur (version 4.4).The MS spectra of E, 20E na 3D20E swi pimanisiwile na swipimelo leswi faneleke.3D20E22P yi kamberiwile hi derivatization hi Girard’s reagent.20E22P yi kamberiwile hi m/z ratio.
3D20E22P yi basisiwile kusuka eka MAG.Ku basisiwa ku endliwile eka xikalo xa nxopaxopo hiku tirhisa ultra-performance liquid chromatograph (Acquity, Waters) leyingana quadrupole mass-based detector (QDa, Acquity, Waters) ehansi ka swiyimo swa LC leswi fanaka na nxopaxopo wa HPLC-MS/MS.Ku hlengeletiwa ka swiphemu swi hlohleteriwile loko m/z yi fambelana na 3D20E22P yi kumiwile hi nkarhi wo khoma lowu fanaka na lowu a wu kumiwile khale.Ku basa ka swihlanganisi leswi humesiweke ku tlhele ku kamberiwa hi HPLC-MS/MS tanihilaha swi hlamuseriweke hakona laha henhla.
RNA hinkwayo yi humesiwile kusuka eka 10-12 wa ti tissues to veleka kumbe swiphemu swin’wana swa miri (leswi nga riki na nhloko) hiku tirhisa TRI reagent (Thermo Fisher) hiku landzelela swiletelo swa muendli.RNA yi tshunguriwile hi TURBO DNase (Thermo Fisher).cDNA yi endliwile hiku tirhisa Moloney murine leukemia virus reverse transcriptase (M-MLV RT; Thermo Fisher) endzhaku ka swiletelo swa muendli.Tiprimer ta reverse transcription quantitative PCR (RT-qPCR; Extended Data Table 2) ti kandziyisiwile khale24 kumbe ti endliwile hi ku tirhisa Primer-BLAST26, hi ku tsakela loku nyikiweke swikumiwa swa 70-150 bp hi vukulu na ku hlanganisa ti exon-exon junctions kumbe ti Primer pair primers ti hambanyisa ti exons.cDNA tisampulu ku suka eka tisampulu tinharhu ku ya eka mune ta biological ti cheriwile ka mune ematini ku endlela RT-qPCR.Ku hlayiwa ka nhlayo ku endliwile eka 15 μl ya ti replicate reactions leti nga na 1× PowerUp SYBR Green Master Mix (Thermo Fisher), ti primers, na 5 μl ya diluted cDNA.Ti reactions ti fambisiwa eka QuantStudio 6 Pro real-time PCR system (Thermo Fisher) naswona data yi hlengeletiwile no xopaxopiwa hiku tirhisa Design na Nxopaxopo (version 2.4.3).Hilaha swi kombisiweke hakona eka ndzavisiso lowu, nhlayo ya xiringaniso yi normalised eka ribosomal gene RpL19 (AGAP004422), leyi ku paluxiwa ka yona ku nga cincangiki ngopfu hi ku dyisa ngati 27 kumbe ku hlangana 3 .
Khwalithi ya RNA yi kamberiwile hi ku tirhisa Agilent Bioanalyzer 2100 Bioanalyzer (Agilent).Tilayiburari ta Illumina paired-end ti lulamisiwile no fambisiwa eBroad Institute of MIT na Harvard.Ku hlayiwa ka ntlhandlamano ku ringanisiwile na genome ya A. gambiae (PEST strain, version 4.12) hi ku tirhisa HISAT2 (version 2.0.5) na ti parameters.Reads with default quality (MAPQ) scores <30 yi susiwile hiku tirhisa Samtools (version 1.3.1).Nhlayo ya ti reads leti mepiweke eka ti genes yi hlayiwile hiku tirhisa htseq-count (version 0.9.1) na ti parameters ta default.Normalized read counts yi hlayiwile naswona differential gene expression yi xopaxopiwile hiku tirhisa DESeq2 package (version 1.28.1) eka R (version 4.0.3).
Ecdysone-modifying gene candidates yi tiviwile hi ku rhanga hi ku lavisisa genome ya A. gambiae hi ku tirhisa algorithm ya PSI-BLAST (https://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/2.8.1/), hi ku tirhisa mimpimo ya ntolovelo Tipharamitha leti nga na ntlhandlamano wa tiphrotheyini ta xivutiso leti landzelaka: ku suka eka Bombyx mori (Accession No. NP_001038956.1), Musca domestica (Nomboro ya Nhlanganelo XP_005182020.1, XP_005175332.1 na XP_011294434.1) na xitirhisiwa xo herisa Microplitis (Nomboro ya Nhlanganelo XP_008552646.1 na XP_008552645.1)) EcK kusuka eka B. mori (Nomboro ya Nhlanganelo NP_001036900), Drosophila melanogaster (Nomboro ya Nhlanganelo NP_651202), Apis mellifera (Nomboro ya Nhlanganelo XP_394838) na Acyrthosiphon pisum (Nomboro ya Nhlanganelo XP_001947166); na EPP ku suka eka B. mori (Accession No. XP_001947166) NP_001177919.1 na NP_001243996.1) na EO ya D. melanogaster (Accession No. NP_572986.1) (goza 1).Endzhaku, ku hlasela ka sefo loku sekeriweke eka ku paluxiwa ka mRNA ya le henhla (>100 swiphemu/kilobase exons hi miliyoni ya ku hlaya loku mepiweke (FPKM) kumbe >85%) eka tinyama ta ku veleka (LRT ya xisati kumbe MAG) eGambia (goza ra 2 ). Ku antswisa ku kongoma, hi hlawurile ti enzymes ta nkandziyiso leti tlhelaka ti kombisiwa eka tinyama ta ku veleka ta A. albimanus, muxaka wa anopheles lowu nga endliki kumbe ku hundzisela ecdysone hi nkarhi wa ku hlangana.Tijini ta vahlawuriwa ti sefiwile hi ku ya hi ku paluxiwa ka le hansi (<100 FPKM kumbe <85th percentile) eka tinyama ta ku veleka ta A. albimanus (goza ra 3).As a final filter (step 4), tijini ta nkandziyiso ti lava ku enerisa xin’we xa leswi landzelaka: (1) leswi lawuriwaka swinene endzhaku ka ku hlangana (P < 0.05) kuya hi nxopaxopo wa tijini leti kombisiweke hindlela yohambana na (2) eka tinyama letinga veriki (< 85 % kumbe <100 FPKM).
Hi cincile tindlela leti hlamuseriweke khale 28,29,30 ku fikelela ku lebula ka isotopic ka xitsongwatsongwana hinkwaxo.Hi ku komisa, muxaka wa nhova wa Saccharomyces cerevisiae muxaka wa II (YSC2, Sigma) wu kamberiwile eka yeast nitrogen base (BD Difco, DF0335) leyi nga na (wt/vol) 2% glucose (G7528, Sigma), 1.7%. leyi nga riki na amino acid na ammonium sulfate. culture medium) na 5% 15N ammonium sulfate (NLM-713, >99%, Cambridge Isotope Laboratories) tani hi xihlovo xin’we xa nitrogen.Yeast yi vuyisiwile hi centrifugation naswona switsotswana swa switsotswana swi phameriwile ad libitum kufikela loko swi endliwa pupation.Ku engeteriwa hi fishmeal (0.5 mg hi 300 wa switsotswana) ku sivela instar ya vumune mortality.I ta xisati ntsena endzhaku ka sweswo ti tirhisiwile eka swikambelo swa ku hlangana na ta xinuna leti nga lebuliwangiki ku kambisisa proteome ya xinuna leyi hundziseriweke hi nkarhi wa ku hlangana.
4-6 wa masiku ya 15N-tagged virgin ya xisati yi boheke ku hlangana na malembe lawa ya fambelanaka na untagged virgin males.Ku hlangana loku humelelaka ku tiyisisiwile hiku kuma ti mating plugs ehansi ka epifluorescence microscopy.Eka 3, 12, na 24 hpm, ti atria ta 45-55 wa ta xisati leti hlanganisiweke ti tsemiwile eka 50 μl ya ammonium bicarbonate buffer (pH 7.8) naswona yi homogenized hi pestle.Homogenate yi centrifuged naswona supernatant yi hlanganisiwile na 50 μl ya 0.1% RapiGest (186001860, Waters) eka 50 mM ammonium bicarbonate.Supernatant na pellet kusuka eka xikombiso xin’wana na xin’wana swi snap frozen eka dry ice kutani swi rhumeriwa vusiku hinkwabyo kuya eka MacCoss laboratory e University of Washington, laha ku lulamisiwa ka xikombiso xa LC-MS/MS ku hetiweke.Resuspend pellet eka 50 μl ya 0.1% RapiGest eka 50 mM ammonium bicarbonate na sonicate eka water bath.Nhlayo ya ti protein ta pellet na supernatant yi pimiwe hi BCA assay, tisampulu ti hungutiwile hi 5 mM dithiothreitol (DTT; Sigma), alkylated na 15 mM iodoacetamide (Sigma) naswona yi pfaleriwile eka 37 °C (1:0 50) kuringana 1 wa awara na trypsinization: trypsin:substrate ratio).RapiGest yi lysed hiku engeteriwa ka 200 mM HCl, ku landzela ku pfaleriwa eka 37 °C kuringana 45 wa timinete na centrifugation eka 14,000 rpm kuringana 10 wa timinete eka 4 °C ku susa swilo leswinga sala.Tisampulu ti hlantswiwile hi dual-mode solid-phase extraction (Oasis MCX cartridges, Waters) naswona titlhela ti suspended eka 0.1% formic acid eka nhlayo yohetelela ya protein ya 0.33 μg μl-1.Unlabeled MAG proteomes ti kamberiwile hiku fanana kusuka eka virgin males.Ti replicates timbirhi ta analytical ti kamberiwile eka xikombiso xin’wana na xin’wana.Endzhaku ka sweswo, 1 μg ya yin’wana na yin’wana yi kamberiwile hiku tirhisa 25-cm fused silica 75-μm column na 4-cm fused silica Kasil1 (PQ) frit trap leyi pakiweke hi Jupiter C12 reversed-phase resin (Phenomenex) na 180-minute liquid chromatography. Ti digests ta xikombiso – MS/MS yi fambisiwa eka Q-Exactive HF mass spectrometer (Thermo Fisher) na nanoACQUITY UPLC System (Waters).Data ya ku kuma leyi fambelanaka na data leyi endliweke eka ku tsutsuma kun’wana na kun’wana yi hundzuriwile eka xivumbeko xa mzML hiku tirhisa Proteowizard (version 3.0.20287) na hiku tirhisa Comet31 (version 3.2) ku lwisana na database ya FASTA leyingana ti protein ntlhandlamano ku suka eka Anopheles gambiae (VectorBase version 54), Anopheles coluzzi Ku lavisisa ku endliwile eka Mali-NIH (VectorBase version 54), Saccharomyces cerevisiae (Uniprot, Dzivamisoko 2021), A. gambiae RNA-seq, na vuhundzuluxi bya tifureme tinharhu ta swilo leswi tivekaka swa swilo leswi thyakeke swa vanhu.Ti-FDR leti ringanisiweke hi mepe wa peptide ti kumiwile hi ku tirhisa Percolator32 (version 3.05) leyi nga na threshold ya 0.01, na ti peptides ti hlengeletiwile eka ku tiveka ka ti protein hiku tirhisa protein parsimony eka Limelight33 (version 2.2.0).Ku tala ka ti protein leti yelanaka ku ringanyetiwe hiku tirhisa normalized spectral abundance factor (NSAF) leyi hlayiweke eka protein yin’wana na yin’wana eka ku tsutsuma kun’wana na kun’wana tani hileswi swi hlamuseriweke khale.NSAF loko yi pimanisiwa na protein yin’wana na yin’wana yi ringanisiwile eka tisampulu timbirhi to hambana ta biological replicates.15N labeling yi humelerile ku fihla proteome ya xisati, hambi leswi nhlayo yintsongo ya ti protein letinga lebuliwangiki yinga voniwa kusuka eka vanhwanyana lavanga lebuliwa.Hi rhekhodile ku kumiwa ka kuhunguteka ka ti protein ta xinuna (1-5 spectra) eka tisampulu ta xisati letinga swekiwanga ntsena eka ti technical runs, laha tisampulu to ka ti nga swekiwanga ti fambisiwa endzhaku ka tisampulu ta xinuna/mating, tani hi mbuyelo wa HPLC “carry over”.Occasional proteins found as ‘swilo leswi thyakeke’ kusuka eka vanhwanyana lava tsariweke swi xaxametiwile eka Tafula ro Engetela ra 1.
Ti antigenic peptides timbirhi, QTTDRVAPAPDQQQ (endzeni ka isotype PA) na MESDGTTPSGDSEQ (endzeni ka isotype PA na PB) eka Genscript.Ti peptides timbirhi ti hlanganisiwile, kutani ti hlanganisiwa na carrier protein KLH kutani ti cheriwa eka mimpfundla ya New Zealand.Mimpfundla yi endliwile magandzelo endzhaku ka ku cheriwa ka vumune, naswona IgG hinkwayo yi hambanisiwile hi affinity purification.IgG kusuka eka mbyana leyi kongomisiweke swinene eka EPP yitirhisiwile eka ku ya emahlweni na western blotting.
Eka ti western blots, MAG (n = 10, laha n yi yimelaka nhlayo ya tisampulu ta switsotswana leti tiyimeleke hi tlhelo ra biologically) na LRT ya xisati (n = 30) kusuka eka ta xinuna ta virgin ta masiku ya 4 na ta xisati letinga virgin kumbe letinga sindzisiwa (<10 endzhaku ka ku hlangana), 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)) yi cheriwile hiku hambana.Tisampulu ti homogenized hiku hatlisa endzhaku ka dissection hi beader (2 mm glass beads, 2,400 rpm, 90 sec).Insoluble debris yi susiwile hi 20,000 g eka 4 °C.Ti protein ti pimiwile hi Bradford assay (Bio-Rad).Kutani, 20 μg ya MAG protein, 40 μg ya LRT protein, na 20 μg ya residual bulk protein swi denatured no hambanisiwa hi 10% Bis-Tris NuPAGE hiku tirhisa MOPS buffer.Ti protein ti hundziseriwe eka ti polyvinylidene fluoride membranes hiku tirhisa iBlot2 transfer system (Thermo Fisher).Ti membranes ti hlantswiwile kambirhi eka 1× PBS-T (0.1% Tween-20 eka PBS) kutani ti pfaleriwa eka Odyssey blocking buffer (Li-Cor) kuringana 1 wa awara eka 22°C.Ti membranes ti rhurhumerisiwile vusiku hinkwabyo eka 4 °C hi custom rabbit anti-EPP polyclonal primary antibody (1:700 eka blocking buffer) na rat anti-actin monoclonal primary antibody MAC237 (Abeam; 1:4,000).Ti membranes ti hlantswiwile hi PBS-T kutani ti pfaleriwa na ti antibodies ta vumbirhi (donkey anti-rabbit 800CW na goat anti-rat 680LT (Li-Cor), hinkwaswo 1:20,000) eka blocking buffer leyingana 0.01% SDS na 0.2%. Tween -20 kuringana 1 wa awara eka 22 °C.Ti membranes ti hlantswiwile hi PBS-T naswona ti tekiwile swifaniso hi Odyssey CLx scanner.Swifaniso swi hlengeletiwile no lulamisiwa eka Image Studio (version 5.2).Band yokarhi leyi fambelanaka na EPP-RA isoform (82 kDa) ayi voniwanga.
Tindzhawu ta khodi ta EPP (tanihi isoform AGAP002463-RB leyi nga na domain ya histidine phosphatase, NCBI conserved domain search 34) na EcK2 (AGAP002181) ti cloned eka pET-21a(+) plasmid (Novagen Millipore Sigma) ; ti primers ti xaxametiwile eka Extended Data Table 2.Ti GS4 linkers ta tsevu (hi tandem) ti nghenisiwile emahlweni ka C-terminal 6xHis tag ya pET-21a(+)-EcK2 construct.Ti protein leti hlanganisiweke nakambe ti endliwile hiku tirhisa NEBExpress cell-free E. coli protein synthesis reaction (New England BioLabs).Ti protein leti hlanganisiweke nakambe ti basisiwile hiku tirhisa NEBExpress Ni spin columns (New England BioLabs).Dihydrofolate reductase (DHFR) control protein yi endliwile hiku tirhisa DNA template kusuka eka NEBExpress Cell-Free E. coli Protein Synthesis Kit.Ti protein ti hlayisiwile eka 50% glycerol eka PBS eka -20 °C kuringana 3 wa tinhweti.
Ntirho wa phosphatase wa EPP na tissue extracts wu pimiwe hiku tirhisa 4-nitrophenyl phosphate (pNPP; Sigma-Aldrich).Buffer ya reaction ayiri na 25 mM Tris, 50 mM acetic acid, 25 mM Bis-Tris, 150 mM NaCl, 0.1 mM EDTA, na 1 mM DTT.Tissue yi homogenized eka reaction buffer na cell debris yi susiwile hi centrifugation.Sungula reaction hiku chela enzyme kumbe tissue extract eka reaction buffer leyingana 2.5 mg ml-1 pNPP.Nkatsakanyo wa reaction wu pfaleriwile eka mahiselo ya kamara emunyameni, naswona nhlayo ya pNP leyi hundzuriweke kusuka eka pNPP yi pimiwile hiku pima absorbance eka 405 nm hi minkarhi yohambana hambana.
Eka ntirho wa in vitro EcK, protein yi pfaleriwile na 0.2 mg 20E kumbe 3D20E eka 200 μl buffer (pH 7.5) leyingana 10 mM HEPES–NaOH, 0.1% BSA, 2 mM ATP na 10 mM MgCl2 kuringana 2 h eka 27 °C .Ku cinca cinca ku yimisiwile hiku cheriwa 800 μl methanol, kutani yi titimela eka -20 °C kuringana 1 wa awara, kutani yi centrifuged eka 20,000 g kuringana 10 wa timinete eka 4 °C.Kutani supernatant yi kamberiwile hi HPLC-MS/MS.Ku heat-inactivate ti proteins leti tirhisiweke eka ntlawa wa vulawuri, ti proteins ti pfaleriwile eka 50% glycerol eka PBS kuringana 20 min eka 95°C.
Eka ntirho wa in vitro EPP, protein yi pfaleriwile na 3D20E22P (leyi ringanaka na nhlayo leyi kumekaka eka 18 wa ti pairs ta MAG, leyi basisiweke hi HPLC-MS/MS) eka 100 μl buffer (pH 7.5) leyingana 25 mM Tris, 50 mM acetic acid, 25 mM Bis-Tris, 150 mM NaCl, . 0.1 mM EDTA, na 1 mM DTT kuringana 3 wa tiawara eka 27 °C.Reaction yi yimisiwile hiku cheriwa 400 μl methanol kutani yi titimela eka -20 °C kuringana 1 wa awara, kutani yi centrifuged eka 20,000 g kuringana 10 wa timinete eka 4 °C.Supernatant yi kamberiwile hi HPLC-MS/MS.
Swiphemu swa PCR swa EPP (362 bp), EcK1 (AGAP004574, 365 bp) na EcK2 (556 bp) swi antswisiwile kusuka eka cDNA leyi lulamisiweke kusuka eka mintsumbu ya switsotswana leswinga na tinhloko ta rimbewu leri pfanganisiweke.Xiphemu xa PCR xa vulawuri bya eGFP (495 bp) xi antswisiwile kusuka eka pCR2.1-eGFP leyi hlamuseriweke khale; Ti PCR primers ti xaxametiwile eka Extended Data Table 2.Xiphemu xa PCR xi nghenisiwile exikarhi ka ti inverted T7 promoters eka pL4440 plasmid.Ti plasmid constructs ti vuyisiwile kusuka eka NEB 5-α competent E. coli (New England Biolabs) naswona ti tiyisisiwile hi DNA sequencing loko tinga se tirhisiwa (vona Supplementary Data 1 eka insert sequence).Primers yi fambelanisiwile na T7 promoter (Extended Data Table 2) yi tirhisiwile ku kurisa insert kusuka eka pL4440-based plasmid.PCR product size yi tiyisisiwile hi agarose gel electrophoresis.dsRNA yi tsariwile kusuka eka PCR templates hiku tirhisa Megascript T7 Transcription Kit (Thermo Fisher) naswona yi basisiwile kuya hi swiletelo swa muendli hi ku cinca loku hlamuseriweke khale.
Eka ku cheriwa ka dsRNA, 1,380 ng ya dsRNA (dsGFP, dsEcK1, dsEcK2, dsEPP) yi cheriwile hi nhlayo ya 10 ng nl-1 eka thorax ya va xinuna lavakulu kumbe va xisati (Nanoject III, Drummond) ku nga si hela siku rin’we endzhaku ka eclosion.Ti levele ta gene knockdown ti kumiwile eka kwalomu ka ti biological replicates tinharhu hi RNA ku humesiwa, ku endliwa ka cDNA, na RT-qPCR.Eka ku cheriwa ka ecdysone, ta xisati ta masiku ya 4 kumbe nhwana wa masiku ya 6 leti dyiweke ngati ti cheriwile 0.13, 0.21, kumbe 0.63 μg ya 20E kumbe 3D20E (Nanoject III, Drummond) eka nhlayo ya 1.3, 2.1, . hi ku landzelelana, ku ya hi dizayini ya xikambelo kumbe 6.3 ng nl-1.Inject 100 nl ya 10% (vol/vol) ethanol ematini; 100 nl ya 3D20E22P eka 10% ethanol (leswi ringanaka na 75% wa nhlayo leyi kumekaka eka mpatswa wa MAGs).Switsotswana swi averiwe hi ku landzelelana eka ntlawa wa ku cheriwa.
Eka swikambelo swaku tswala, ta xisati ta masiku ya 3 ti phameriwile ad libitum eka ngati ya munhu.Susa switsotswana leswi dyiweke xiphemu kumbe leswinga dyiwangiki.Kuya hi vutshunguri, ta xisati ti vekiwile eka ti spawning cups to hambana kuringana vusiku bya mune kwalomu ka 48 wa tiawara endzhaku ka swakudya swa ngati.Matandza ya hlayiwile ehansi ka stereoscope (Stemi 508, Zeiss); eka ta xisati leti hlanganisiweke, tandza leri tshovekaka ri va switsotswana a ri tekiwa ri ri leri noneke.
Eka swikambelo swa ku hlangana, ta xisati ti pfumeleriwile kwalomu ka masiku ya 2 kuya hi vutshunguri kuva ti hluvukisa ku lwisana na ku hlangana, naswona ta xinuna leti ringanaka na malembe ya muxaka wa nhova endzhaku ti nghenisiwile eka khege yin’we.Endzhaku ka vusiku byimbirhi, ti vesicles ta xisati leti nonisiweke ti tsemiwile naswona genomic DNA yi humesiwile hi freeze-thaw na sonication eka buffer leyingana 10 mM Tris-HCl, . 1 mM EDTA, na 25 mM NaCl (pH 8.2).Tisampulu ti pfaleriwile na Proteinase K (0.86 μg μl-1) kuringana 15 wa timinete eka 55 °C, ku landzela 10 wa timinete eka 95 °C.Malunghiselelo ya crude genomic DNA ya cheriwile 10-fold naswona ya endliwile qPCR detection ya Y chromosome sequences; ti primers ti xaxametiwile eka Extended Data Table 2.Ku pfumaleka ka ntlhandlamano wa ti chromosome ta Y swikomba ku pfumaleka ka ku hlangana.
Eka swikambelo swa re-mating, ta xisati leti force-mated ti kamberiwile kuva tiri na ti mating plugs ku tiyisisa xiyimo xa mating naswona ti pfumeleriwile 2 wa masiku kuva ti hluvukisa refractoriness eka mating loko kungari na ta xinuna, tani hileswi swinga hlamuseriwa khale 36 .Males carrying DsRed transgenic sperm were then introduced into female cages.Endzhaku ka vusiku byimbirhi, ti fertilizing vesicles ti nghenisiwile yi tsemiwile kusuka eka ta xisati, naswona genomic DNA yi lulamisiwile tani hileswi swinga hlamuseriwa laha henhla naswona yi endliwile qPCR detection ya DsRed transgene; ti primers ti xaxametiwile eka Extended Data Table 2.Ku pfumaleka ka DsRed transgene swikombe leswaku akuri hava ku hlangana nakambe loku humeleleke.
3D20E yi endliwile tani hileswi swinga hlamuseriwa khale 37 .Hiku komisa, 10 mg ya 20E (Sigma-Aldrich) yi pfanganisiwile na 10 ml ya mati, ku landzela ku engeteriwa ka 30 mg ya platinum black (hi xivumbeko xa powder, Sigma-Aldrich).A gentle stream of O2 was continuely bubbled into the reaction mixture, which was stirred at room temperature.Endzhaku ka 6 wa tiawara, 30 mL ya methanol yi cheriwile ku yimisa reaction.Nkatsakanyo wu centrifuged ku susa ti catalyst particles.Supernatant yi pfaleriwile kufikela yi oma eka vacuum eka mahiselo ya kamara.Xiendliwa xa reaction lexi omisiweke xi pfanganisiwile eka 10% ethanol na methanol kuva xi cheriwa eka HPLC-MS/MS analysis.The conversion rate (kusuka eka 20E kuya eka 3D20E) ayiri kwalomu ka 97% (Fig. 4b), naswona MS spectrum ya 3D20E leyi endliweke yi fambelana na leyi kumekaka eka ta xisati leti hlanganisiweke (Fig. 4c).
Ntsheketo wu na vuxokoxoko byo karhi bya swikambelo swa tinhlayo leswi endliweke.GraphPad (version 9.0) yi tirhisiwile ku endla xikambelo xa Fisher lexi kongomeke, xikambelo xa Mantel-Cox, na Student’s t-test.Swikambelo swa Cochran-Mantel-Haenszel swi endliwile hi ku tirhisa tsalwa ra R ra ntolovelo (leri kumekaka eka https://github.com/duopeng/mantelhaen.test).Ku hangalasiwa ka datha ku kamberiwile ntolovelo hi ku tirhisa xikambelo xa Shapiro-Wilk lexi nga na mpimo wa nkoka wa 0.05.Loko datha yi tsandzekile eka xikambelo xa ntolovelo, ku endliwile xikambelo xa Mann-Whitney.Data ya ku pona yi kamberiwile hi ku tirhisa xikambelo xa Mantel-Cox.Phakeji ya DESeq2 (vuhundzuluxeri bya 1.28.1) yi tirhisiwile ku endla nxopaxopo wa ku paluxiwa ka ku hambana ka xiyimo xa tijini ta RNA-seq.Barhi ya horizontal eka girafu yi yimela median.A nkoka wa nkoka wa P = 0.05 wu tirhisiwile tanihi threshold eka swikambelo hinkwaswo.
Ku kuma vuxokoxoko byo tala hi pulani ya dyondzo, vona xitlhokovetselo xa Nature Research Report lexi hlanganisiweke na xihloko lexi.
Data ya proteomic ya MS yi vekiwile eka ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) hi ku tirhisa Vuhlayiselo bya Mutirhisani wa PRIDE (https://www.ebi.ac.uk/pride/) na xihlawulekisi xa dataset PXD032157.
Dataset ya RNA-seq yi vekiwile eka Gene Expression Comprehensive Library (https://www.ncbi.nlm.nih.gov/geo/) ehansi ka rhekhodo ya serial GSE198665.
Tidathaseti to engetela leti endliweke na/kumbe leti xopaxopiweke hi nkarhi wa dyondzo ya sweswi ti nga kumiwa eka vatsari lava yelanaka loko va komberiwa hi ndlela leyi twalaka.Athikili leyi yi nyika datha ya xihlovo.
De Loof, A. Ecdysteroids: Ti-steroid ta rimbewu ta switsotswana leti honisiweke?Wa xinuna: Bokisi ra Ntima.Sayense ya Switsotswana.13, 325–338 (2006).
Redfern, CPF 20-hydroxyecdysone na ku kula ka ovary eka Anopheles stephens.J. Ntivo-vutomi bya Switsotswana.28, 97–109 (1982).