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Mosiyana ndi zamoyo zokhala ndi msana, tizilombo timaganiziridwa kuti tilibe mahomoni a steroid ogonana omwe amasankha amuna. Mu Anopheles gambiae, ecdysone steroid 20-hydroxyecdysone (20E) ikuwoneka kuti yasintha kuti ilamulire kukula kwa dzira ikapangidwa ndi akazi2 komanso kuyambitsa nthawi yogonana yosakhazikika ikasamutsidwa ndi amuna3. Popeza kukula kwa dzira ndi kukwatirana ndi makhalidwe ofunikira obereketsa, kumvetsetsa momwe udzudzu wachikazi wa Anopheles umagwirizanirana ndi zizindikiro za mahomoni izi kungathandize kupanga mapulogalamu atsopano oletsa malungo. Apa, tikuwulula kuti ntchito zobereketsa izi zimayendetsedwa ndi ma steroids osiyanasiyana ogonana kudzera mu netiweki yovuta ya ma enzymes oyambitsa/oletsa a ecdysteroid. Tapeza ecdysone yokhazikika ya amuna, 3-dehydro-20E (3D20E), yomwe imateteza makolo mwa kutseka kulandira kwa akazi pambuyo pa kusamutsidwa ndi kuyatsidwa ndi dephosphorylation. Chodziwika bwino, kusamutsidwa kwa 3D20E kunayambitsanso kuwonetsedwa kwa majini obereketsa omwe amasunga kukula kwa dzira panthawi ya matenda a Plasmodium, kuonetsetsa thanzi la akazi omwe ali ndi kachilomboka. 20E yochokera kwa akazi siimabweretsa Kuyankha pogonana, koma kumalola kuti anthu okwatirana aziikira mazira pambuyo poti ma kinase a 20E-inhibiting alepheretsedwa. Kuzindikira mahomoni a steroid a amuna awa komanso udindo wake pakulamulira momwe akazi amalandirira, kubereka komanso kuyanjana ndi Plasmodium kukuwonetsa kuthekera kochepetsa kupambana kwa kubereka kwa udzudzu wofalitsa malungo.
Milandu ya malungo ndi imfa zikuwonjezekanso4 chifukwa cha kukana mankhwala ophera tizilombo mu udzudzu wa Anopheles, womwe ndi wokhawo womwe umayambitsa matenda a malungo kwa anthu. Kukwerana kwa udzudzu uwu ndi cholinga chokopa kwambiri pa njira zatsopano zothana ndi malungo chifukwa akazi amakwerana kamodzi kokha5; kupangitsa kuti kukwerana kamodzi kokha kukhale kopanda udzudzu kungakhale ndi kuthekera kwakukulu kochepetsa kuchuluka kwa udzudzu m'munda.
Azimayi amalephera kugonana akalandira mahomoni a steroid ochokera kwa amuna. Kafukufuku wasonyeza kuti chomwe chimayambitsa vuto pakuberekana ndi 20-hydroxyecdysone (20E), mahomoni a steroid omwe amadziwika bwino kuti ndi owongolera kayendedwe ka molting mu gawo la mphutsi. Kuthekera kwa amuna kupanga ndi kusamutsa 20E kwasintha makamaka mu mitundu ya Anopheles yomwe ili m'gulu la Cellia7, yomwe imapezeka ku Africa ndipo imaphatikizapo ma vector oopsa kwambiri a malungo, kuphatikizapo Anopheles gambiae. Izi ndizofunikira kwambiri chifukwa m'mitundu iyi akazi amapanganso 20E pambuyo pa chakudya chilichonse chamagazi, ndipo 20E imayendetsa kayendedwe ka oogenesis (onani ref. 8). Komabe, palibe chodziwika bwino cha momwe akazi amalumikizira zizindikiro kuchokera ku magwero awiri osiyanasiyana a ecdysone (kusamutsa amuna ndi kuyambitsa magazi) popanda kusokoneza luso lawo lokwatira. Ndipotu, ngati 20E yopangidwa ndi akazi imayambitsa kusalolera kugonana, izi zimabweretsa kusabereka kwa anamwali, khalidwe lofala kwambiri mwa udzudzu uwu5.
Kufotokozera komwe kungatheke ndi kwakuti amuna a A. gambiae amasamutsa ecdysone yosinthidwa ya amuna, yomwe imayambitsa kufalikira kwa zizindikiro m'njira yoberekera ya akazi, zomwe zimapangitsa kuti kusakhazikika kwa kugonana kukhalepo. Komabe, ngakhale kuti nyama zokhala ndi vertebrate zili ndi mahomoni ambiri a steroid, monga estrogen ndi androgen (yomwe yawunikidwa mu ref. 9), monga momwe tikudziwira, ma steroids ogwirizana ndi androgenic sanapezeke mwa tizilombo.
Tinayamba kufufuza kuchuluka kwa mahomoni a steroid mu gland ya amuna okhwima m'thupi (MAG) pofunafuna ma steroids osinthika omwe angatheke. Pogwiritsa ntchito chromatography yamadzimadzi yogwira ntchito bwino kwambiri pamodzi ndi tandem mass spectrometry (HPLC-MS/MS) m'malo mwa njira yosagwiritsidwa ntchito kwenikweni, tinapeza ecdysone (E) ndi 20E mu minofu iyi, kutsimikizira zotsatira zam'mbuyomu. Komabe, chitsanzocho chinali cholamulidwa ndi ma steroids oxidized phosphorylated, mogwirizana ndi fomula 3-dehydro-20E-22-phosphate (3D20E22P)12 (Chithunzi 1). Mitundu ina ikuphatikizapo 3-dehydro-20E (3D20E) ndi 20E-22-phosphate (20E22P). Mphamvu ya chizindikiro cha HPLC-MS/MS ya 3D20E22P inali yayikulu kawiri kuposa mawonekedwe ake a dephosphorylated, 3D20E, ndi yayikulu katatu kuposa ya E ndi 20E (Chithunzi 1). Ngakhale m'mbali zina za thupi ndi njira yoberekera ya m'munsi (LRT; Extended Data Fig. 1a). Tinafufuzanso ma ecdysteroids mwa amuna ndi akazi omwe atsekedwa kumene (osakwana tsiku limodzi) ndipo tinapeza 3D20E ndi 3D20E22P mu MAG yokha; E, 20E ndi 20E22P zinalipo mwa amuna ndi akazi onse (Extended Data Fig. 1b). Deta iyi ikusonyeza kuti amuna akuluakulu a A. gambiae amapanga mahomoni ambiri osintha mu MAG awo omwe sapangidwa ndi akazi.
MAG ndi LRT yachikazi (kuphatikiza atria, seminal vesicles, ndi parovarium) zinachotsedwa kwa amuna azaka zapakati pa 4 (masiku 4) ndi akazi azaka zapakati pa 0.5, 3, ndi 12 hpm). Ecdysone m'maselo awa idawunikidwa ndi HPLC-MS/MS (average ± sem; unpaired t-test, two-sided, false discovery rate (FDR) yokonzedwa; NS, si yofunika; *P < 0.05, **P < 0.01 . 3D20E: maola 3 vs. maola 0.5, P = 0.035; maola 12 vs. maola 3, P = 0.0015; maola 12 vs. maola 0.5, P = 0.030. 3D20E22P: maola 3 vs. maola 0.5, P = 0.25; maola 12 vs. maola 3, P = 0.0032; Maola 12 poyerekeza ndi maola 0.5, P = 0.015). Deta ikuchokera ku ma replicate atatu a zamoyo. Malo okwera kwambiri a ecdysone iliyonse yofunikira adawerengedwa ndikusinthidwa ndi kuchuluka kwa udzudzu. Ecdysone imayimiridwa ndi mtundu motere: E, wobiriwira; 20E, lalanje; 20E22P, wofiirira; 3D20E, wabuluu; 3D20E22P, pinki. Chiwonetserochi chimawonjezera sikelo pa y-axis kuti chiwonetse kuchuluka kwa ecdysone kotsika.
Kuti tifufuze ngati 3D20E22P ndi 3D20E zimasamutsidwa panthawi yogonana, tinasanthula ma LRT achikazi nthawi zosiyanasiyana pambuyo pogonana. Ngakhale kuti ecdysone sinapezeke mwa anamwali, tinaona kuchuluka kwakukulu kwa 3D20E22P mu LRT nthawi yomweyo titagonana (maola 0.5 pambuyo pogonana, hpm), kuchepa pakapita nthawi, pomwe kuchuluka kwa 3D20E kunakwera kwambiri (Chithunzi 1). Pogwiritsa ntchito 3D20E yopangidwa ndi mankhwala monga muyezo, tinapeza kuti kuchuluka kwa mahomoni a steroid mu ma LRT ogonana kunali kokwera kuposa 20E (Table Extended Data Table 1). Chifukwa chake, 3D20E22P ndiye ecdysone yayikulu yachimuna yomwe imasamutsidwa ku LRT yachikazi panthawi yogonana, ndipo mawonekedwe ake a dephosphorylated, 3D20E, amakhala ochuluka kwambiri atangokwatirana. Izi zikusonyeza udindo wofunikira wa ecdysone yomaliza mu biology yachikazi pambuyo pogonana.
Pambuyo popanga deta yatsopano ya RNA sequencing (RNA-seq) (Chithunzi 2a), pogwiritsa ntchito njira yopangira bioinformatics, tinafufuza ecdysone kinase (EcK), ecdysone oxidase (EO), ndi ecdysone encoding 20E-modified phosphatase gene.EPP) imawonetsedwa m'maselo obereketsa. Tinapeza jini imodzi ya EPP yoyenera ndi majini awiri omwe angakhale a EcK (EcK1 ndi EcK2), koma sitinathe kupeza jini yabwino ya EO yoyenera. Chodziwika bwino n'chakuti, majini a EPP pawokha adawonetsedwa pamlingo wapamwamba (98.9th percentile) mu Gambian MAGs koma osati mu ma LRT a akazi (Chithunzi 2b), mosiyana ndi zomwe tinkayembekezera popeza dephosphorylation ya 3D20E22P idachitika m'thupi la akazi ili. Chifukwa chake, tikukhulupirira kuti EPP ya amuna ikhoza kusamutsidwa panthawi yogonana. Zoonadi, tidagwiritsa ntchito zilembo za isotope zokhazikika mu vivo kuti tibise puloteni ya akazi pambuyo pogonana, enzyme yomwe idadziwika ndi MS mu atrium ya akazi (Chithunzi 2c ndi Gome Lowonjezera 1). Kupezeka kwa EPP mu MAG ndi LRT yachikazi yokwatiwa (koma osati yachikazi) kunatsimikiziridwanso pogwiritsa ntchito ma antibodies enaake (Chithunzi 2d).
a, Njira yopangira bioinformatics yopangidwa mwapadera kuti ifufuze minofu yoberekera ya mtundu uliwonse wa majini omwe amalemba ma EcK, ma EO, ndi ma EPP. Manambala omwe ali pafupi ndi mivi akuwonetsa chiwerengero cha amuna ndi akazi omwe akufuna pa sitepe iliyonse. Kusanthula kumeneku kunapeza jini limodzi la EPP (EPP) ndi jini limodzi la EcK (EcK1) lomwe limawonetsedwa mwa amuna, ndi jini limodzi la EcK (EcK2) lomwe limawonetsedwa mwa amuna ndi akazi onse koma silipereka jini la EO lomwe limasankhidwa.b, Mapu owunikira poyerekeza mawonekedwe a jini la candidate mu namwali (V) ndi mating (M) Anopheles gambiae ndi Anopheles albicans minofu.Spca, feteleza; MAGs, ma glands owonjezera mwa amuna; ziwalo zina za thupi, kuphatikizapo mabere, mapiko, miyendo, matupi amafuta, ndi ziwalo zamkati mwa amuna ndi akazi, komanso mazira mwa akazi. EcK2 imapezeka kwambiri mu MAG ndi atria ku Gambia, pomwe EPP imapezeka mu MAG.c yokha, kusanthula kwa Proteomic kwa kusintha kwa ejaculate group kupita ku atria ya akazi pa 3, 12 ndi 24 hpm, kuwonetsa mapuloteni 67 ochulukirapo. Akazi adaleredwa pazakudya zokhala ndi 15N kuti alembe (ndi kuphimba) mapuloteni onse. Anyamata osatchulidwa adakwatiwa ndi akazi okhala ndi ma tag, ndipo ma LRT a akazi adadulidwa pa 3, 12 ndi 24 hpm kuti afufuze proteomic (onani Supplementary Table 1 kuti mupeze mndandanda wathunthu wa mapuloteni otulutsa ejaculatory). Inset, EPP, Eck1 ndi EcK2 zidapezeka mu MAG ya amuna osaphunzira posanthula proteomic ya minofu iyi.d, EPP idapezeka ndi western blot mu MAG ndi LRT ya akazi osaphunzira, koma osati mwa akazi osaphunzira kapena amuna kapena akazi ena onse. thupi. Ma nembanemba anafufuzidwa nthawi imodzi ndi anti-actin (loading control) ndi ma antibodies a anti-EPP. Amuna onse ndi anamwali. Onani Chithunzi Chowonjezera 1 kuti mudziwe zambiri za gwero la gel. Ma Western blots adachitika kawiri ndipo zotsatira zake zinali zofanana.
Ntchito ya ecdysteroid phosphophosphatase ya EPP idatsimikiziridwa pambuyo pa kulowetsedwa ndi HPLC-MS/MS ndi 3D20E22P yochotsedwa ku MAG (Extended Data Fig. 2a). Kuphatikiza apo, pamene tidaletsa EPP ndi RNA-mediated interference (RNAi), tidazindikira kuchepa kwakukulu kwa ntchito ya phosphatase m'maselo obereketsa a amuna awa (Chithunzi 3a), ndipo akazi omwe adagonana ndi amuna omwe adaletsa EPP adawonetsa kwambiri. Gawo lochepa la dephosphorylated 3D20E (Chithunzi 3b) ngakhale kuti majini ena adaletsa pang'ono (Extended Data Fig. 2b,c). Mosiyana ndi zimenezi, sitinapeze kusintha kwakukulu mu chiŵerengero cha 20E22P/20E mu udzudzu womwewo, zomwe zingasonyeze kuti enzyme ndi yeniyeni ya 3D20E22P (Chithunzi 3b).
a, Kuchepa kwa phosphatase mu MAG komwe kumachitika chifukwa cha kuletsa kwa EPP pogwiritsa ntchito njira zowongolera za EPP RNA (dsEPP) kapena GFP RNA (dsGFP) zomwe zimakhala ndi zingwe ziwiri. Ma pool a MAG makumi awiri adagwiritsidwa ntchito mu replicate iliyonse (P = 0.0046, paired t-test, two-side), yomwe imayimiridwa ndi madontho osiyana.b, Akazi omwe adakwatirana ndi amuna omwe adaletsa EPP anali ndi gawo lochepa kwambiri la dephosphorylated 3D20E pa 3 hpm (P = 0.0043, unpaired t-test, two-side), pomwe milingo ya 20E sinakhudzidwe (P = 0.063, unpaired). mayeso a t, mbali ziwiri). Deta imaperekedwa ngati apakati ± sem kuchokera m'madziwe atatu a akazi 13, 16 ndi 19 aliyense. c, Akazi omwe adakwatiwa ndi amuna omwe adatsekedwa ndi EPP anali ndi kuchuluka kwakukulu kwa kukwatiwanso (P = 0.0002, mayeso enieni a Fisher, mbali ziwiri). Akazi adakakamizidwa koyamba kukwatiwa kuti atsimikizire kuti ali paubwenzi; Patatha masiku awiri, adakumana ndi amuna ena omwe anali ndi umuna wosinthika kuti awone kuchuluka kwa kukwerana kwa kachilomboka pogwiritsa ntchito PCR yochuluka pozindikira transgene.d, akazi odyetsedwa magazi omwe adakwatiwa ndi amuna omwe adatsekedwa ndi EPP anali ndi kubereka kochepa kwambiri (P < 0.0001; Mann-Whitney test, two-side) ndi dzira lochepa pang'ono (P = 0.088, Mann-Whitney test, two-side), pomwe kuchuluka kwa kubereka sikunakhudzidwe (P = 0.94, Fisher's eni eni test, two-side). M'magawo onse, n ikuyimira chiwerengero cha zitsanzo za udzudzu wodziyimira pawokha.NS, sikofunikira.*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.001.
Kenako, tinayang'ana ngati ecdysone dephosphorylation ndi yofunika kwambiri pakulimbikitsa kukana kugonana mwa akazi. Chodziwika bwino n'chakuti, akazi omwe anakwatiwa ndi amuna omwe atha EPP anakwatiwanso pafupipafupi kwambiri (44.9%) kuposa akazi olamulira (10.4%) akakumana ndi amuna ena (omwe asintha majini) (Chithunzi 3c). Tinaonanso kuchepa kwakukulu kwa kubereka (Chithunzi 3d, kumanzere) ndi kuchepa pang'ono kwa mazira omwe anayikidwa ndi akazi awa (Chithunzi 3d, pakati), pomwe kuchuluka kwa mazira omwe anayikidwa ndi akazi (mayankho ena omwe anaperekedwa mwa akazi chifukwa cha kukwatiwa )) sikunakhudzidwe (Chithunzi 3d, kumanja). Popeza EPP ya 3D20E22P yawonedwa, zotsatirazi zikusonyeza kuti kuyambika kwa 3D20E ndi EPP yomwe imasamutsidwa panthawi yokwatiwa kungakhale ndi gawo lofunikira pakuletsa kulandiridwa kwa akazi kuti akwerenso, khalidwe lomwe kale linkaganiziridwa kuti linayambitsidwa ndi kusamutsa kugonana kwa 20E. Chifukwa chake, mahomoni awa a amuna amakhudzanso kwambiri kubereka kwa akazi.
Kenako, tinayerekeza ntchito za 20E ndi 3D20E mu kuyesa jakisoni mwa anamwali okhwima pogonana pogwiritsa ntchito 3D20E yopangidwa ndi mankhwala (Chithunzi 4a–c) ndi 20E yogulitsidwa. Tinaona kuti 3D20E inali yothandiza kwambiri kuposa 20E poletsa chidwi cha akazi pa kugonana pamlingo wonse (Chithunzi 4d). Chodziwika bwino n'chakuti, theka la mulingo wa thupi wa 3D20E mu LRT (1,066 pg pambuyo pa jakisoni vs. 2,022 pg pambuyo pa kugonana) linayambitsa gawo la akazi okana jekeseni lomwe linali lalikulu nthawi 20 kuposa mulingo wa thupi wa 20E (361 pg pambuyo pa jakisoni) maola 24 pambuyo pa jakisoni pa mulingo wapamwamba kwambiri wa 18 pg pambuyo pa kugonana; Gome Lowonjezera la Deta 1). Zotsatirazi zikugwirizana ndi lingaliro lakuti kusamutsa kugonana kwa 20E sikuyambitsa nthawi yogonana yokana kugonana, ndipo zikuwonetsanso kuti 3D20E ndi chinthu chachikulu pakutsimikizira ubale wa kholo ndi mwana. 3D20E inalinso yogwira ntchito kwambiri kuposa 20E pakuyesa kuyikira mazira mwa akazi osakwatiwa (Chithunzi 4e), zomwe zikusonyeza kuti kuchuluka kwabwinobwino kwa kuyikira mazira komwe tidawona titatha kuletsa pang'ono EPP kunali chifukwa cha kukhalapo kwa ntchito yotsala ya 3D20E yomwe imachitikabe ndi zinthu zachikazi zomwe zimayambitsa kugonana.
(a,b) 3D20E yopangidwa ndi mankhwala kuchokera ku 20E (a) yokhala ndi kusintha/kugwira ntchito bwino kwambiri (deta yoperekedwa ngati mean ± sem kuchokera ku machitidwe atatu odziyimira pawokha a synthesis) (b).c, Mass spectrum (theka la pansi) ikugwirizana ndendende ndi ecdysone yomwe imapezeka mu LRT yachikazi yolumikizidwa (theka lapamwamba).d, Poyerekeza ndi 20E (0.63 µg, P = 0.02; 0.21 µg, P < 0.0001; Mayeso enieni a Fisher, mbali ziwiri) ndi 10% ethanol (0.63 µg, P < 0.0001; 0.21 µg, P < 0.0001; Mayeso enieni a Fisher, mbali ziwiri), pomwe 20E inali yayikulu kwambiri kuposa yolamulira yokha pamlingo wapamwamba (0.63 µg, P = 0.0002; 0.21 µg, P = 0.54; Mayeso enieni a Fisher, 2-sided).e, jakisoni wa 3D20E unayambitsa kuchuluka kwa kubereka kwa akazi osakwatiwa kuposa 10% zowongolera za ethanol (0.21 µg, P < 0.0001; 0.13 µg, P = 0.0003; Mayeso enieni a Fisher, mbali ziwiri), pomwe 20E poyerekeza ndi zowongolera Pa mlingo wapamwamba (0.21 µg, P = 0.022; 0.13 µg, P = 0.0823; Mayeso enieni a Fisher, mbali ziwiri).3D20E inayambitsa kuchuluka kwa kubereka kwapamwamba kwambiri kuposa 20E pa mlingo wapamwamba (0.21 µg, P = 0.0019; 0.13 µg, P = 0.075; Mayeso enieni a Fisher, mbali ziwiri). M'magawo onse, n ikuyimira chiwerengero cha zitsanzo za udzudzu wodziyimira pawokha. NS, sikofunikira.*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.001. Deta imachokera ku ma kopi atatu.
Mu kafukufuku wakale, tinapeza kuti kusamutsa mahomoni a steroid pogonana kumayambitsa kutulutsidwa kwa MISO (Mating-Induced Stimulator of Oogenesis 11), jini yoberekera yachikazi yomwe imateteza akazi a A. gambiae ku matenda a P. falciparum. Ndalama zaumoyo zomwe zimayambitsidwa ndi 13, tizilombo toyambitsa matenda toopsa kwambiri pa thanzi la anthu. Popeza kufunika kwa MISO pa thanzi la kubereka la Anopheles m'madera omwe malungo amapezeka, tinaganiza zopeza kuti ndi mahomoni ati a 3D20E kapena 20E omwe amayambitsa kutulutsidwa kwa jini iyi. Tinapeza kuti ngakhale jakisoni wa 20E makamaka kapena mwamphamvu kwambiri unayambitsa ma receptors ena a nyukiliya (HR), monga HR3 ndi HR4, ndi zolinga za steroid zomwe zili pansi pake, monga majini a yolkogenic Vg14, 15, 16, MISO inayambitsidwa kwambiri ndi 3D20E (Extended Data Fig. 3). Chifukwa chake, kusamutsa kwa mahomoni a androgenic steroid awa kumawoneka kuti kumayambitsa njira zomwe zimateteza akazi ku zovuta zomwe zimayambitsidwa ndi matenda opatsirana. Kuphatikiza apo, 3D20E imakhudza mosiyanasiyana ma isoform onse awiri a E receptor EcR, kuyambitsa EcR-A ndikuletsa EcR-B, komanso kuyambitsa kwambiri majini ena omwe amayambitsa kukwatirana, kuphatikizapo HPX15, yomwe imakhudza kubereka kwa akazi. Izi zitha kufotokoza kusabereka kwakukulu komwe kumawoneka mwa akazi omwe amagwirizana ndi amuna omwe alibe EPP (Extended Data Fig. 3). Deta iyi ikuwonetsa kukhalapo kwa njira zotsikira zomwe zimayendetsedwa makamaka ndi mahomoni awiri a ecdysone omwe angayambitse ntchito yokhudzana ndi kugonana.
Kenako, tinayesa ntchito ya majini awiri a EcK omwe adapezeka mu njira yathu ya bioinformatics. Kuletsa EcK1 kapena EcK2 kunapangitsa kuti amuna azifa kwambiri (Extended Data Fig. 4a), zomwe zikusonyeza kuti ecdysone phosphorylation, motero kuletsa kugwira ntchito, ndikofunikira kuti munthu apulumuke. Chifukwa EcK2 idawonetsedwa pamlingo wapamwamba kuposa EcK1 ndipo idapezeka mu MAGs ndi proteomics (Chithunzi 2b,c ndi Supplementary Table 2), tinatsimikizira ntchito yake ya ecdysteroid kinase mwa kuiyika mu 20E, zomwe zidapangitsa kuti phosphorylation 20E22P (Extended Data Figure 2).4b). Pogwiritsa ntchito 3D20E ngati substrate, sitinathe kuzindikira 3D20E22P (Extended Data Fig. 4c), zomwe zikusonyeza kuti 20E m'malo mwa 3D20E ikhoza kukhala cholinga cha EcK2.
Malinga ndi kusanthula kwathu kwa RNA-seq, EcK2 idawonekeranso kwambiri mu LRT ya akazi osakwatiwa, komwe idazimitsidwa atakwatirana (Chithunzi 2b). Tinatsimikizira izi ndipo tinapeza kuti kufotokozedwa kwa EcK2 sikunakhudzidwe ndi kudya magazi (Chithunzi Chowonjezera cha Deta 5a). Powonjezera zoyeserera zathu zoyambirira za MS, tinapeza kuti kuchuluka kwa 20E22P kunali kogwirizana kwambiri ndi kuchuluka kwa 20E (maola 22-26 mutadya magazi; Chithunzi Chowonjezera cha Deta 5b). Kuletsa kwa EcK2 mwa akazi osakwatiwa kunapangitsa kuti chiwerengero cha 20E mpaka 20E22P chiwonjezeke katatu pa maola 26 mutadya magazi (Chithunzi Chowonjezera cha Deta 2c ndi 5c), kutsimikizira kuti EcK2 imapanganso phosphorylates 20E mwa akazi. Chodziwika bwino, anamwali osakwatiwa a EcK2 adasungabe mphamvu zonse zogonana (Chithunzi Chowonjezera cha Deta 5d,e), zomwe zikusonyeza kuti kupanga kwa akazi kwa 20E sikupangitsa kuti kugonana kusagwirizane. nthawi. Komabe, akazi awa anali ndi kuchuluka kwakukulu kwa mazira omwe amaika mazira poyerekeza ndi olamulira, ndi oposa 30% a anamwali omwe amaika mazira (Extended Data Fig. 5f). Ngati jakisoni wa Eck2 RNA (dsEcK2) wokhala ndi zingwe ziwiri unachitidwa pambuyo poyamwitsa magazi, kubereka sikunachitike, pomwe chiwopsezo cha 20E chifukwa chodya magazi chinachepa. Ponseponse, zotsatirazi zikuthandizira chitsanzo chomwe 20E idapanga pambuyo poyamwa magazi chingayambitse kubereka, koma pokhapokha ngati choletsa kubereka (EcK2 ndi zina) chazimitsidwa ndi kukwerana. Si jakisoni wa 20E kapena 3D20E womwe sunaletse kufalikira kwa EcK2 mwa anamwali (Extended Data Fig. 5g), zomwe zikusonyeza kuti zinthu zina zimayambitsa kuletsa kwa kinase iyi. Komabe, kuchuluka kwa 20E pambuyo poyamwitsa magazi sikunali kokwanira kuyambitsa kusasangalala kwa kukwerana, koma kunayambitsidwa bwino ndi kuchuluka kwa 3D20E yotumizidwa mwa kugonana.
Zotsatira zathu zikupereka chidziwitso chofunikira pa njira zomwe zimawongolera kupambana kwa kubereka kwa A. gambiae. Chitsanzo chaonekera pomwe amuna asintha kuti apange ma titers apamwamba a 3D20E, ecdysone yosinthidwa ya amuna yomwe imatsimikizira kubereka mwa kuchepetsa chidwi cha akazi kuti apitirize kukwatirana. Nthawi yomweyo, ma vector a malungo awa apanganso njira yothandiza yoyatsira 3D20E mwa akazi poyankha kusamutsa kwa kugonana kwa EPP ya amuna. Monga momwe tikudziwira, ichi ndi chitsanzo choyamba cha dongosolo la mahomoni a steroid lomwe limayang'aniridwa ndi amuna ndi akazi likuchita ntchito yapadera komanso yofunika kwambiri mwa tizilombo. Ntchito ya ecdysone ya amuna yaganiziridwa koma sinawonetsedwe bwino. Mwachitsanzo, lingaliro losatsimikizika kwambiri 18 ndilakuti ntchito izi zitha kuchitidwa ndi 20E precursor E1. Ndikodziwika bwino kuti mu Drosophila, monandry imayambitsidwa ndi kusamutsa kwa kugonana kwa ma peptide ang'onoang'ono a kugonana19,20 omwe amalumikizana ndi ma neuron omwe amasunga njira yoberekera ya akazi kudzera mu ma receptor enaake a peptide a kugonana21,22. Ntchito yowonjezera ikufunika kuti mudziwe zomwe zili pansi pake. ma cascade owonetsa omwe amayendetsedwa ndi 3D20E mwa akazi a A. gambiae ndikuwona ngati ma cascade awa angasungidwe pakati pa udzudzu ndi Drosophila.
Popeza 3D20E ndi yofunika kwambiri pa kubereka kwa akazi ndi khalidwe lawo lomwe lapezeka mu kafukufuku wathu, njira zomwe zikutsogolera ku 3D20E synthesis ndi activation zimapereka mwayi watsopano wa njira zowongolera udzudzu mtsogolo, monga kupanga amuna opikisana osabala mu njira zaukadaulo wa tizilombo tosabala Kugwiritsa ntchito potulutsa tizilombo tachilengedwe kapena kutsanzira 3D20E mu virgin play. Ntchito ya 3D20E ya amuna mwina idasintha pamene A. gambiae ndi mitundu ina ya Cellia adapeza mphamvu yolumikizira umuna wawo m'mapulagi okwatirana, chifukwa izi zimathandiza kusamutsa bwino mahomoni ambiri ndi ma enzymes omwe amachititsa mahomoni. Kuphatikiza apo, kusintha kwa 3D20E komwe kumagwiritsa ntchito monandry kumapereka njira kwa akazi (kudzera mu MISO) kuti athandize thanzi lawo lobereka m'madera omwe ali ndi matenda ambiri a malungo, zomwe zimathandizira mwachindunji kufalikira kwa Plasmodium. Popeza 20E ya akazi yawonetsedwa kuti ili ndi zotsatira zazikulu pa kupulumuka ndi kukula kwa P. falciparum mu udzudzu wachikazi wa Anopheles,24 njira za mahomoni a steroid a amuna ndi akazi tsopano ndi zinthu zofunika kwambiri pakuyanjana kwa udzudzu ndi tizilombo.
Mitundu ya A. gambiae G3 inaleredwa pansi pa mikhalidwe yokhazikika ya tizilombo (26-28 °C, chinyezi cha 65-80%, nthawi ya kuwala/mdima ya maola 12:12). Mphutsi zinadyetsedwa chakudya cha nsomba cha ufa (TetraMin Tropical Flakes, Koi Pellets ndi Tetra Pond Sticks mu chiŵerengero cha 7:7:2). Udzudzu waukulu unadyetsedwa ndi madzi a 10% dextrose ndi magazi a munthu sabata iliyonse (zigawo za magazi zophunziridwa). Udzudzu wa anamwali unapezeka mwa kusiyanitsa amuna ndi akazi pa siteji ya pupal atatha kufufuza malekezero pogwiritsa ntchito microscopy. Amuna omwe ali ndi DsRed transgene afotokozedwa kale.
Kuyesa kukakamiza kugonana kunachitidwa motsatira njira zomwe zafotokozedwa kale. Pa kugonana kwachilengedwe, akazi a masiku 4 omwe anali anamwali anasungidwa mu chiŵerengero cha 1:3 ndi amuna omwe anali anamwali okhwima kugonana kwa masiku awiri. Pa kuyesa komwe amuna anabayidwa ndi dsEPP, co-caging inagwirizana ndi masiku 3-4 pambuyo pa jakisoni, pomwe ntchito ya phosphatase inaletsedwa kwambiri (Extended Data Fig. 2b).
Minofu ya udzudzu, mitembo yotsala (thupi lonse), kapena thupi lonse linadulidwa kukhala 100% methanol ndipo linasinthidwa ndi beader (2 mm galasi mikanda, 2,400 rpm, masekondi 90). Kuchuluka kwa minofu ndi kuchuluka kwa methanol kunali motere: thupi lonse, 50 mu 1,000 µl; MAG, 50–100 80 µl; LRT yachikazi, 25–50 80 µl. Mpweya wochepa unachotsedwa kachiwiri ndi kuchuluka komweko kwa methanol. Zinyalala za maselo zinachotsedwa pogwiritsa ntchito centrifugation. Methanol kuchokera ku zochotsedwa zonse ziwiri idaphatikizidwa ndikuumitsidwa pansi pa nayitrogeni, kenako inabwezeretsedwanso m'mavoliyumu otsatirawa a 80% methanol m'madzi: thupi lonse, 50 µl; MAG ndi LRT yachikazi, 30 µl.
Zitsanzo zinasanthulidwa pa chipangizo choyezera kuchuluka kwa madzi (ID-X, Thermo Fisher) cholumikizidwa ndi chipangizo cha LC (Vanquish, Thermo Fisher). 5 µl ya chitsanzo inalowetsedwa pa 3 µm, 100 × 4.6 mm column (Inspire C8, Dikma) yomwe inasungidwa pa 25 °C. Magawo oyenda a LC anali A (madzi, 0.1% formic acid) ndi B (acetonitrile, 0.1% formic acid). LC gradient inali motere: 5% B kwa mphindi imodzi, kenako inawonjezeka kufika 100% B kwa mphindi 11. Pambuyo pa mphindi 8 pa 100%, gwirizanitsaninso column pa 5% B kwa mphindi 4. Kuthamanga kwa madzi kunali 0.3 ml min-1. Ionization mu MS source imachitika ndi heated electrospray ionization mu njira zabwino ndi zoipa.
Masekondi oyesera kuchuluka kwa shuga (mass spectrometer) amayesa deta mu m/z kuyambira 350 mpaka 680 pa resolution ya 60,000 mu MS mode yonse. Deta ya MS/MS idapezeka pa [M + H]+ (zolinga zonse), [M - H2O + H]+ (zolinga zonse), ndi [M - H]- (zolinga zonse za phosphorylated). Deta ya MS/MS idagwiritsidwa ntchito kutsimikizira katundu wa ecdysone wa zolinga zomwe sizinali muyezo. Kuti tipeze ma ecdysteroids osayang'aniridwa, deta ya MS/MS ya ma HPLC onse okhala ndi kuchuluka kwa >15% idasanthulidwa. Kuyeza pogwiritsa ntchito ma curve okhazikika opangidwa kuchokera ku miyezo yoyera (20E, 3D20E) kuti tiwerengere kuchuluka konse kapena kuchepetsedwa kwa chitsanzo chimodzi (zolinga zina zonse) kuti tiwerengere kufanana kwawo ndi kuchuluka komwe kwapezeka mwa mwamuna m'modzi. Pa 3D20E, kuyeza kunachitika pogwiritsa ntchito kuchuluka kwa zowonjezera izi: [M + TFA]-, [M + COOH]-, [M + Na]+, [M + Cl]-, [M + NO3]-. Deta idachotsedwa ndikuyesedwa pogwiritsa ntchito Tracefinder (mtundu 4.1). Deta ya MS/MS idasanthulidwa pogwiritsa ntchito Xcalibur (mtundu 4.4). Ma spectra a MS a E, 20E ndi 3D20E adayerekezeredwa ndi miyezo yofananira. 3D20E22P idasanthulidwa pogwiritsa ntchito reagent ya Girard. 20E22P idasanthulidwa ndi chiŵerengero cha m/z.
3D20E22P inayeretsedwa kuchokera ku MAG. Kuyeretsa kunachitika pamlingo wowunikira pogwiritsa ntchito chromatograph yamadzimadzi ya ultra-performance (Acquity, Waters) yokhala ndi chowunikira cha quadrupole mass-based (QDa, Acquity, Waters) pansi pa mikhalidwe yofanana ya LC monga kusanthula kwa HPLC-MS/MS. Kusonkhanitsa magawo kunayambitsidwa pamene m/z yofanana ndi 3D20E22P inapezeka panthawi yomweyo yosungira monga momwe idadziwidwira kale. Kuyera kwa mankhwala ochotsedwawo kunayang'aniridwa ndi HPLC-MS/MS monga tafotokozera pamwambapa.
RNA yonse inachotsedwa m'maselo 10-12 obereketsa kapena ziwalo zina za thupi (zopanda mutu) pogwiritsa ntchito reagent ya TRI (Thermo Fisher) potsatira malangizo a wopanga. RNA inachiritsidwa ndi TURBO DNase (Thermo Fisher). cDNA inapangidwa pogwiritsa ntchito Moloney murine leukemia virus reverse transcriptase (M-MLV RT; Thermo Fisher) potsatira malangizo a wopanga. Ma primers a reverse transcription quantitative PCR (RT-qPCR; Extended Data Table 2) adasindikizidwa kale24 kapena adapangidwa pogwiritsa ntchito Primer-BLAST26, ndipo zokonda zimaperekedwa kuzinthu za 70-150 bp kukula kwake ndi kufalikira kwa exon-exon junctions kapena Primer pair primers zosiyana ma exons. cDNA zitsanzo kuchokera ku ma biological replicates atatu mpaka anayi adachepetsedwa kanayi m'madzi a RT-qPCR. Kuchuluka kunachitika mu ma replica reactions 15 µl okhala ndi 1 × PowerUp SYBR Green Master Mix (Thermo Fisher), ma primers, ndi 5 µl ya diluted cDNA. Ma reactions adayendetsedwa pa Dongosolo la QuantStudio 6 Pro la PCR nthawi yeniyeni (Thermo Fisher) ndi deta zinasonkhanitsidwa ndi kufufuzidwa pogwiritsa ntchito Design and Analysis (version 2.4.3). Monga momwe zasonyezedwera mu kafukufukuyu, kuchuluka kwa majini a ribosomal RpL19 (AGAP004422), omwe mawonekedwe ake sanasinthe kwambiri podyetsa magazi 27 kapena kugonana 3.
Ubwino wa RNA unayang'aniridwa pogwiritsa ntchito Agilent Bioanalyzer 2100 Bioanalyzer (Agilent). Malaibulale a Illumina paired-end adakonzedwa ndikuyendetsedwa ku Broad Institute of MIT ndi Harvard. Kuwerenga kotsatizana kunalumikizidwa ndi genome ya A. gambiae (mtundu wa PEST, mtundu wa 4.12) pogwiritsa ntchito HISAT2 (mtundu wa 2.0.5) ndi magawo okhazikika. Kuwerenga komwe kuli ndi ma mapping quality (MAPQ) scores <30 kunachotsedwa pogwiritsa ntchito Samtools (mtundu wa 1.3.1). Chiwerengero cha kuwerenga komwe kumalumikizidwa ku majini chinawerengedwa pogwiritsa ntchito htseq-count (mtundu wa 0.9.1) ndi magawo okhazikika. Kuwerenga kokhazikika kunawerengedwa ndipo kufotokozera kwa majini kosiyana kunafufuzidwa pogwiritsa ntchito phukusi la DESeq2 (mtundu wa 1.28.1) mu R (mtundu wa 4.0.3).
Ma gene osintha a Ecdysone adapezeka poyamba pofufuza genome ya A. gambiae pogwiritsa ntchito njira ya PSI-BLAST (https://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/2.8.1/), pogwiritsa ntchito ma default values. Ma Parameters okhala ndi ma query protein sequences otsatirawa: kuchokera ku Bombyx mori (Accession No. NP_001038956.1), Musca domestica (Accession No. XP_005182020.1, XP_005175332.1 ndi XP_011294434.1) ndi Microplitis demolitor (Accession No. XP_008552646.1 ndi XP_008552645.1) EcK kuchokera ku B. mori (Accession No. NP_001036900), Drosophila melanogaster (Accession No. NP_651202), Apis mellifera (Accession No. XP_394838) ndi Acyrthosiphon pisum (Accession No. XP_001947166); ndi EPP kuchokera ku B. mori (Accession No. XP_001947166) NP_001177919.1 ndi NP_001243996.1) ndi EO ya D. melanogaster (Accession No. NP_572986.1) (gawo 1). Kenako, fyuluta imagunda kutengera kuchuluka kwa mRNA (>100 fragments/kilobase exons pa miliyoni mapped reads (FPKM) kapena >85%) mu minofu yoberekera (female LRT kapena MAG) ku Gambia (gawo 2). Kuti tiwongolere kudziwika bwino, tinasankha ma enzyme omwe amapezekanso mu minofu yoberekera ya A. albimanus, mtundu wa anopheles womwe supanga kapena kusamutsa ecdysone panthawi yoberekera. Majini omwe adasankhidwa adasefedwa kutengera kutsika kwa mawu (<100 FPKM kapena <85th percentile) mu minofu yoberekera ya A. albimanus (gawo 3). Monga fyuluta yomaliza (gawo 4), majini omwe adasankhidwa ayenera kukwaniritsa chimodzi mwa izi: (1) kukwera kwambiri pambuyo poberekera (P < 0.05) malinga ndi kusanthula kwa majini omwe adawonetsedwa mosiyana ndi (2) mu minofu yosaberekera (< 85% kapena <100 FPKM).
Tinasintha njira zomwe tafotokoza kale 28,29,30 kuti tikwaniritse zolemba za isotopic za chamoyo chonse. Mwachidule, mtundu wa Saccharomyces cerevisiae wa mtundu wachiwiri (YSC2, Sigma) unayesedwa mu yisiti nitrogen base (BD Difco, DF0335) yokhala ndi (wt/vol) 2% glucose (G7528, Sigma), 1.7% amino acid-free ndi ammonium sulfate. culture medium) ndi 5% 15N ammonium sulfate (NLM-713, >99%, Cambridge Isotope Laboratories) ngati gwero lokha la nayitrogeni. Yisiti inapezekanso pogwiritsa ntchito centrifugation ndipo mphutsi za udzudzu zinadyetsedwa ad libitum mpaka zitakula. Zowonjezera ndi ufa wa nsomba (0.5 mg pa mphutsi 300) kuti tipewe kufa kwachinayi. Akazi okha ndi omwe adagwiritsidwa ntchito poyesa kukwatiwa ndi amuna osalembedwa kuti afufuze proteome yamphongo yomwe idasamutsidwa panthawi yokwatiwa.
Azimayi azaka 4-6 omwe ali ndi zilembo 15N adakakamizidwa kukwatirana ndi amuna azaka zosakwana 15 omwe sanatchulidwe. Kugonana kopambana kunatsimikiziridwa pozindikira mapulagi okwatirana pansi pa microscopy ya epifluorescence. Pa 3, 12, ndi 24 hpm, atria ya akazi okwatirana 45-55 idadulidwa mu 50 µl ya ammonium bicarbonate buffer (pH 7.8) ndikusinthidwa ndi pestle. Homogenate idayikidwa pakati ndipo supernatant idasakanizidwa ndi 50 µl ya 0.1% RapiGest (186001860, Waters) mu 50 mM ammonium bicarbonate. Supernatant ndi pellet kuchokera ku chitsanzo chilichonse zidachotsedwa mu ayezi wouma ndikutumizidwa usiku wonse ku labotale ya MacCoss ku University of Washington, komwe kukonzekera chitsanzo cha LC-MS/MS kudatha. Bwezeretsani pellet mu 50 µl ya 0.1% RapiGest mu 50 mM. Ammonium bicarbonate ndi sonicate m'madzi osambira. Kuchuluka kwa mapuloteni mu pellet ndi supernatant kunayesedwa ndi BCA assay, zitsanzo zinachepetsedwa ndi 5 mM dithiothreitol (DTT; Sigma), alkylated ndi 15 mM iodoacetamide (Sigma) ndikuyikidwa pa 37 °C (1:0 50) kwa ola limodzi ndi trypsinization: trypsin: substrate ratio). RapiGest inayesedwa mwa kuwonjezera 200 mM HCl, kutsatiridwa ndi incubation pa 37 °C kwa mphindi 45 ndikuzunguliridwa pa 14,000 rpm kwa mphindi 10 pa 4 °C kuti achotse zinyalala. Zitsanzo zinatsukidwa ndi dual-mode solid-phase extraction (Oasis MCX cartridges, Waters) ndikubwezeretsedwanso mu 0.1% formic acid kuti pakhale kuchuluka komaliza kwa mapuloteni a 0.33 µg µl-1. Ma proteome a MAG osalembedwa adasanthulidwa mofananamo kuchokera ku virgin. Amuna. Ma kopi awiri owunikira adawunikidwa pa chitsanzo chilichonse. Kenako, 1 µg ya chilichonse idawunikidwa pogwiritsa ntchito 25-cm fused silica 75-μm column yokhala ndi 4-cm fused silica Kasil1 (PQ) frit trap yodzaza ndi Jupiter C12 reversed-phase resin (Phenomenex) ndi 180-minute liquid chromatography. Zitsanzo za digest - MS/MS idayendetsedwa pa Q-Exactive HF mass spectrometer (Thermo Fisher) yokhala ndi nanoACQUITY UPLC System (Waters). Deta yokhudzana ndi kupeza deta yomwe idapangidwa pa run iliyonse idasinthidwa kukhala mtundu wa mzML pogwiritsa ntchito Proteowizard (mtundu 3.0.20287) ndikugwiritsa ntchito Comet31 (mtundu 3.2) motsutsana ndi database ya FASTA yokhala ndi mapuloteni ochokera ku Anopheles gambiae (VectorBase mtundu 54), Anopheles coluzzi Kufufuza kunachitika pa Mali-NIH (VectorBase mtundu 54), Saccharomyces cerevisiae (Uniprot, Marichi 2021), A. gambiae RNA-seq, ndi matembenuzidwe atatu a zodetsa zodziwika bwino za anthu. Ma FDR ogwirizana ndi mapu a Peptide adapezeka pogwiritsa ntchito Percolator32 (mtundu 3.05) wokhala ndi malire a 0.01, ndipo ma peptide adasonkhanitsidwa kukhala ma protein identification pogwiritsa ntchito protein parsimony mu Limelight33 (mtundu 2.2.0). Kuchuluka kwa mapuloteni ogwirizana kunayesedwa pogwiritsa ntchito normalized spectral abundance factor (NSAF) yowerengedwa pa puloteni iliyonse pa nthawi iliyonse monga momwe tafotokozera kale. NSAF yokhudzana ndi puloteni iliyonse inawerengedwa pa avareji kuchokera ku zitsanzo ziwiri zosiyana zamoyo. 15N labeling inabisa proteome yachikazi bwino, ngakhale kuti pang'ono puloteni yosalembedwapo inapezeka kuchokera kwa anamwali olembedwapo. Tinalemba kupezeka kwa kuchepa kwa mapuloteni a amuna (1-5 spectra) m'zitsanzo zachikazi zosaphika zokha mu njira zaukadaulo, komwe zitsanzo zosaphika zinkayendetsedwa pambuyo pa zitsanzo za amuna/kukwatirana, chifukwa cha HPLC "kunyamula". Mapuloteni ena omwe amapezeka ngati 'zodetsa' kuchokera kwa anamwali olembedwapo alembedwa mu Supplementary Table 1.
Ma peptide awiri a antigenic, QTTDRVAPAPDQQQ (mkati mwa isotype PA) ndi MESGTTTPSGDSEQ (mkati mwa isotype PA ndi PB) mu Genscript. Ma peptide awiriwa adaphatikizidwa, kenako adalumikizidwa ku protein yonyamula KLH ndikubayidwa mu akalulu aku New Zealand. Akalulu adaperekedwa nsembe atabayidwa kachinayi, ndipo IgG yonse idachotsedwa mwa kuyeretsa affinity. IgG kuchokera ku kalulu wodziwika bwino wa EPP idagwiritsidwa ntchito pochotsa ma blotting akumadzulo.
Pa ma blots akumadzulo, MAG (n = 10, pomwe n ikuyimira chiwerengero cha zitsanzo za udzudzu wodziyimira pawokha) ndi LRT yachikazi (n = 30) kuchokera kwa amuna azaka 4 ndi akazi azaka 4 ndi akazi azaka 10 kapena 15 (<10 pambuyo pa kugonana), 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)) idawonjezedwa padera. Zitsanzo zidasinthidwa nthawi yomweyo mutaduladula ndi beader (2 mm glass beads, 2,400 rpm, 90 sec). Zinyalala zosasungunuka zidachotsedwa ndi centrifugation pa 20,000 g pa 4 °C. Mapuloteni adayesedwa ndi Bradford assay (Bio-Rad). Kenako, 20 µg ya mapuloteni a MAG, 40 µg ya mapuloteni a LRT, ndi 20 µg ya mapuloteni otsala. Zinachotsedwa ndipo zinalekanitsidwa ndi 10% Bis-Tris NuPAGE pogwiritsa ntchito MOPS buffer. Mapuloteni anasamutsidwa ku polyvinylidene fluoride membranes pogwiritsa ntchito iBlot2 transfer system (Thermo Fisher). Ma membranes anatsukidwa kawiri mu 1 × PBS-T (0.1% Tween-20 mu PBS) kenako anatsekedwa mu Odyssey blocking buffer (Li-Cor) kwa ola limodzi pa 22°C. Ma membranes anagwedezeka usiku wonse pa 4 °C ndi antibody yapadera ya rabbit anti-EPP polyclonal primary (1:700 mu blocking buffer) ndi antibody ya makoswe anti-actin monoclonal primary MAC237 (Abeam; 1:4,000). Ma membranes anatsukidwa ndi PBS-T kenako anaikidwa ndi ma antibodies achiwiri (donkey anti-rabbit 800CW ndi goat anti-rat 680LT (Li-Cor), onse 1:20,000) mu blocking buffer yokhala ndi 0.01% SDS ndi 0.2% Tween -20 kwa ola limodzi pa 22 °C. Ma nembanemba adatsukidwa ndi PBS-T ndikujambulidwa ndi Odyssey CLx scanner. Zithunzi zidasonkhanitsidwa ndikukonzedwa mu Image Studio (mtundu 5.2). Mzere winawake wofanana ndi isoform ya EPP-RA (82 kDa) sunapezeke.
Magawo olembera a EPP (monga isoform AGAP002463-RB okhala ndi histidine phosphatase domain, NCBI conserved domain search 34) ndi EcK2 (AGAP002181) adapangidwa kukhala pET-21a(+) plasmid (Novagen Millipore Sigma); Ma primer alembedwa mu Extended Data Table 2. Ma linker asanu ndi atatu a GS4 (ogwirizana) adayikidwa C-terminal 6xHis tag isanapangidwe ndi pET-21a(+)-EcK2. Mapuloteni ophatikizana adapangidwa pogwiritsa ntchito NEBEExpress cell-free E. coli protein synthesis reaction (New England BioLabs). Mapuloteni ophatikizana adayeretsedwa pogwiritsa ntchito NEBEExpress Ni spin columns (New England BioLabs). Puloteni yowongolera ya Dihydrofolate reductase (DHFR) idapangidwa pogwiritsa ntchito template ya DNA kuchokera ku NEBEExpress Cell-Free E. coli Protein Synthesis Kit. Mapuloteni adasungidwa mu 50% glycerol mu PBS pa -20 °C kwa miyezi itatu.
Ntchito ya phosphatase ya EPP ndi zotulutsa minofu inayesedwa pogwiritsa ntchito 4-nitrophenyl phosphate (pNPP; Sigma-Aldrich). Chida chosungiramo zinthu chinali ndi 25 mM Tris, 50 mM acetic acid, 25 mM Bis-Tris, 150 mM NaCl, 0.1 mM EDTA, ndi 1 mM DTT. Minofu inasinthidwa kukhala homogenized mu reaction buffer ndipo zinyalala za maselo zinachotsedwa pogwiritsa ntchito centrifugation. Yambitsani reaction powonjezera enzyme kapena tissue extract ku reaction buffer yokhala ndi 2.5 mg ml-1 pNPP. Chisakanizo cha reaction chinayikidwa mu chipinda kutentha mumdima, ndipo kuchuluka kwa pNP komwe kunasinthidwa kuchokera ku pNPP kunayesedwa poyesa kuyamwa pa 405 nm nthawi zosiyanasiyana.
Pa ntchito ya EcK mu vitro, puloteniyi inayikidwa mu incubator ndi 0.2 mg 20E kapena 3D20E mu 200 µl buffer (pH 7.5) yokhala ndi 10 mM HEPES–NaOH, 0.1% BSA, 2 mM ATP ndi 10 mM MgCl2 kwa maola awiri pa 27 °C. Yankho linayimitsidwa powonjezera 800 µl methanol, kenako kuziziritsidwa pa -20 °C kwa ola limodzi, kenako kuyikidwa mu centrifuge pa 20,000 g kwa mphindi 10 pa 4 °C. Kenako supernatant inasanthulidwa ndi HPLC-MS/MS. Kuti mapuloteni omwe amagwiritsidwa ntchito mu gulu lolamulira atenthetse, mapuloteniwo anaikidwa mu 50% glycerol mu PBS kwa mphindi 20 pa 95 °C.
Pa ntchito ya EPP mu vitro, puloteniyi inayikidwa mu 3D20E22P (yofanana ndi kuchuluka komwe kunapezeka mu ma pairs 18 a MAG, oyeretsedwa ndi HPLC-MS/MS) mu 100 µl buffer (pH 7.5) yokhala ndi 25 mM Tris, 50 mM acetic acid, 25 mM Bis-Tris, 150 mM NaCl, 0.1 mM EDTA, ndi 1 mM DTT kwa maola atatu pa 27 °C. Kuchitapo kanthu kunayimitsidwa powonjezera 400 µl methanol ndikuziziritsidwa pa -20 °C kwa ola limodzi, kenako nkuyikidwa mu centrifuge pa 20,000 g kwa mphindi 10 pa 4 °C. Supernatant idawunikidwa ndi HPLC-MS/MS.
Zidutswa za PCR za EPP (362 bp), EcK1 (AGAP004574, 365 bp) ndi EcK2 (556 bp) zinakulitsidwa kuchokera ku cDNA yokonzedwa kuchokera ku mitembo ya udzudzu yopanda mitu ya amuna ndi akazi. Chidutswa cha PCR cha eGFP control (495 bp) chinakulitsidwa kuchokera ku pCR2.1-eGFP yomwe yafotokozedwa kale; Ma PCR primers alembedwa mu Extended Data Table 2. Chidutswa cha PCR chinayikidwa pakati pa ma inverted T7 promoters pa pL4440 plasmid. Ma plasmid constructs adapezeka kuchokera ku NEB 5-α competent E. coli (New England Biolabs) ndipo adatsimikiziridwa ndi DNA sequencing musanagwiritse ntchito (onani Supplementary Data 1 kuti mupeze insert sequence). Ma primers ofanana ndi T7 promoter (Extended Data Table 2) adagwiritsidwa ntchito kukulitsa insert kuchokera ku pL4440-based plasmid. Kukula kwa mankhwala a PCR kunatsimikiziridwa ndi agarose gel electrophoresis.dsRNA idalembedwa kuchokera ku ma tempuleti a PCR pogwiritsa ntchito Megascript T7 Transcription Kit (Thermo Fisher) ndikuyeretsedwa malinga ndi malangizo a wopanga ndi zosintha zomwe zafotokozedwa kale.
Pa jakisoni wa dsRNA, 1,380 ng ya dsRNA (dsGFP, dsEcK1, dsEcK2, dsEPP) idabayidwa pamlingo wa 10 ng nl-1 m'chifuwa cha amuna kapena akazi akuluakulu (Nanoject III, Drummond) mkati mwa tsiku limodzi kuchokera pamene eclosion idachitika. Kuchuluka kwa majini kunapezeka m'ma replica atatu achilengedwe mwa kuchotsa RNA, kupanga cDNA, ndi RT-qPCR. Pa jakisoni wa ecdysone, akazi a masiku 4 osabadwa kapena a masiku 6 osabadwa omwe amamwa magazi adabayidwa ndi 0.13, 0.21, kapena 0.63 µg ya 20E kapena 3D20E (Nanoject III, Drummond) pamlingo wa 1.3, 2.1, motsatana, kutengera kapangidwe kake koyesera kapena 6.3 ng nl-1. Jakisoni 100 nl ya 10% (vol/vol) ethanol m'madzi; 100 nl ya 3D20E22P mu 10% ethanol (yofanana ndi 75% ya kuchuluka komwe kumapezeka mu ma MAG awiri). Udzudzu unaperekedwa mwachisawawa ku gulu lobayidwa.
Pa mayeso obereketsa ana, akazi a masiku atatu ankapatsidwa magazi a anthu popanda kudyetsedwa. Chotsani udzudzu wodyetsedwa pang'ono kapena wosadyetsedwa. Kutengera ndi chithandizo, akazi ankayikidwa m'makapu osiyana obereketsa ana kwa masiku anayi osachepera maola 48 atatha kudya magazi. Mazira ankawerengedwa pogwiritsa ntchito stereoscope (Stemi 508, Zeiss); kwa akazi obereketsa ana, mazira omwe anaswa mphutsi ankaonedwa kuti ndi a chonde.
Pa mayeso oti akwatire, akazi analoledwa masiku osachepera awiri kutengera chithandizocho kuti asamakwatire, ndipo amuna azaka zakuthengo omwe anali ofanana ndi msinkhu wawo analowetsedwa m'khola lomwelo. Patatha masiku awiri, ma vesicles achikazi omwe anali ndi umuna anadulidwa ndipo DNA ya genomic inatulutsidwa mwa kuzizira ndi sonication mu buffer yokhala ndi 10 mM Tris-HCl, 1 mM EDTA, ndi 25 mM NaCl (pH 8.2). Zitsanzo zinayikidwa ndi Proteinase K (0.86 µg µl-1) kwa mphindi 15 pa 55 °C, kutsatiridwa ndi mphindi 10 pa 95 °C. Kukonzekera kwa DNA ya genomic yopanda kanthu kunachepetsedwa ka 10 ndipo kunayesedwa kuti qPCR izindikire ma chromosome a Y; ma primers alembedwa mu Extended Data Table 2. Kusakhalapo kwa ma chromosome a Y kumasonyeza kuti palibe mating.
Pa mayeso obwerezabwereza a kugonana, akazi okakamizidwa anayesedwa kuti awone ngati pali ma plug okwatirana kuti atsimikizire momwe kugonana kulili ndipo analola masiku awiri kuti ayambe kukana kugonana popanda amuna, monga momwe tafotokozera kale. 36. Amuna omwe anali ndi umuna wa DsRed transgenic analowetsedwa m'mabokosi a akazi. Patatha masiku awiri, ma vesicles obereketsa anadulidwa kuchokera kwa akazi, ndipo DNA ya majini inakonzedwa monga tafotokozera pamwambapa ndipo anayesedwa kuti adziwe qPCR ya DsRed transgene; ma primer alembedwa mu Extended Data Table 2. Kusakhalapo kwa DsRed transgene kunasonyeza kuti palibe kukwatirana komwe kunachitika.
3D20E idapangidwa monga momwe tafotokozera kale 37. Mwachidule, 10 mg ya 20E (Sigma-Aldrich) idasungunuka mu 10 ml ya madzi, kutsatiridwa ndi kuwonjezera 30 mg ya platinum wakuda (mu mawonekedwe a ufa, Sigma-Aldrich). Mtsinje wofatsa wa O2 unapitilizidwa mosalekeza mu chisakanizo cha reaction, chomwe chinasunthidwa kutentha kwa chipinda. Patatha maola 6, 30 mL ya methanol idawonjezedwa kuti iyimitse reaction. Chisakanizocho chinayikidwa pakati kuti chichotse tinthu ta catalyst. Supernatant idasanduka nthunzi kukhala youma mu vacuo kutentha kwa chipinda. Chogulitsa chouma cha reaction chinasungunuka mu 10% ethanol ndi methanol kuti chilowetsedwe mu HPLC-MS/MS analysis. Kuchuluka kwa kusintha (kuchokera pa 20E mpaka 3D20E) kunali pafupifupi 97% (Chithunzi 4b), ndipo MS spectrum ya 3D20E yopangidwayo inali yofanana ndi yomwe imapezeka mwa akazi okwatiwa (Chithunzi 4c).
Nkhaniyi ili ndi tsatanetsatane wa mayeso a ziwerengero omwe adachitika. GraphPad (mtundu 9.0) idagwiritsidwa ntchito poyesa mayeso enieni a Fisher, mayeso a Mantel-Cox, ndi mayeso a Student's t. Mayeso a Cochran-Mantel-Haenszel adachitika pogwiritsa ntchito script ya R yapadera (yomwe ikupezeka pa https://github.com/duopeng/mantelhaen.test). Kugawa deta kudayesedwa kuti kukhale koyenera pogwiritsa ntchito mayeso a Shapiro-Wilk okhala ndi malire ofunikira a 0.05. Pamene deta idalephera mayeso ofunikira, mayeso a Mann-Whitney adachitika. Deta yopulumuka idasanthulidwa pogwiritsa ntchito mayeso a Mantel-Cox. Phukusi la DESeq2 (mtundu 1.28.1) lidagwiritsidwa ntchito poyesa kusanthula kwa kusiyana kwa majini a RNA-seq. Mzere wopingasa pa graph ukuyimira wapakati. Mtengo wofunikira wa P = 0.05 udagwiritsidwa ntchito ngati malire a mayeso onse.
Kuti mudziwe zambiri za kapangidwe ka kafukufukuyu, onani chidule cha Nature Research Report cholumikizidwa ndi nkhaniyi.
Deta ya MS proteomic idasungidwa mu ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) kudzera mu PRIDE Partner Repository (https://www.ebi.ac.uk/pride/) ndi chizindikiro cha deta PXD032157.
Deta ya RNA-seq imasungidwa mu Gene Expression Comprehensive Library (https://www.ncbi.nlm.nih.gov/geo/) pansi pa zolemba za serial GSE198665.
Ma data ena opangidwa ndi/kapena kusanthula panthawi ya kafukufukuyu angapezeke kwa olemba omwe akugwirizana nawo ngati pakufunika kutero. Nkhaniyi ikupereka deta yochokera.
De Loof, A. Ecdysteroids: Ma steroid ogonana ndi tizilombo omwe sanasamalidwe? Amuna: Black Box. Sayansi ya Tizilombo.13, 325–338 (2006).
Redfern, CPF 20-hydroxyecdysone ndi chitukuko cha mazira mu Anopheles stephens.J. Tizilombo toyambitsa matenda.28, 97–109 (1982).