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Ma pe rom ki ler ma tye ki cogo ngec, jami ma ki lwongo ni insects ki tamo ni gi pe ki jami ma konyo coo. I Anopheles gambiae, ecdysone steroid 20-hydroxyecdysone (20E) nen calo odongo me doro dongo pa tong ka ki yubu ki mon2 ki me kelo kare me nyom ka ki loko ki laco. ki nyom obedo kit me nywal ma pire tek, niang kit ma owinyo ma mon ma ki lwongo ni Anopheles gi keto kwede lanyut me hormone magi twero konyo me yubu yub manyen me gengo two atipa. Kany, wa nyutu ni tic me nywal ni ki doro ki jami ma ki lwongo ni co steroids ma pat pat kun wok ki i dul ma ki lwongo ni ecdysteroid-activating/inactivating enzymes. Wanongo two ma ki lwongo ni ecdysteroid-activating/inactivating enzymes. 3-dehydro-20E (3D20E), ma gwoko lunyodo kun juku tic pa mon me butu inge nywako buto ki tic ki dephosphorylation. Ki ngeyo ni, 3D20E loko bene okelo nyute pa gene ma gwoko dongo pa tong i kare me two Plasmodium, ma miyo yotkom pa mon ma kom gi lit ni pe balo E.20E. gamo lok me butu, ento miyo twero bot dano ma gi tye ka nyome me keto tong inge 20E-inhibiting kinases ni ki juku. Ngeyo pa gin ma ki lwongo ni male-specific insect steroid hormone ki tic ne me doro kit ma mon gi gamo kwede buto, nywal ki rwate ki Plasmodium nyutu ni twero dwoko piny rwom me nywal pa two atipa.
Two ma mako two atipa ki too tye ka mede odoco4 pi teko pa yat ma neko kwidi i kom ler ma ki lwongo ni Anopheles, ma obedo gin acel ma kelo kwidi two atipa i kom dano. Kit ma ler magi gi nyome kwede obedo gin ma kelo miti i kom yub me gengo two atipa pien mon gi nyome kicel keken5; miyo yub me nyom acel ni bedo ma pe ki nywalo twero bedo ki kero madit me dwoko piny wel ober i poto.
Mon gi doko goro me butu inge nongo jami ma ki lwongo ni steroid hormones ki bot coo. Kwan ma kitimo onyutu ni gin ma kelo peko me medo nyom tye 20-hydroxyecdysone (20E), gin ma ki lwongo ni steroid hormone ma ki ngeyo maber calo gin ma doro kit ma ler dongo kwede i kare me nywal. Kero pa coo me yubu ki cwalo 20 E odongo i Anocipheles ma pat pat dul pa dul ma ki lwongo ni Cellia7, ma ki poko i Afrika ki tye ki jami ma kelo two atipa, medo ki Anopheles gambiae. Man pire tek tutwal pien i kin dul ler magi mon bene gi kelo 20E inge camo remo acel acel, ki 20E aye doro yub me oogenesis (nen ref. 8). Ento, jami manok aye ki ngeyo i kom kit ma mon gi rwate kwede ki lanyut ma pat pat ma aa ki bot mon aryo. ecdysone (male transfer ki remo feeding induction) labongo balo kero gi me nyom. Ki lok adaa, ka 20E ma mon gi yubu okelo peko me butu, man obi kelo peko me nywal i kom mon ma gi miyo cam bot mon, gin ma pol kare tiime i kom obe magi5.
Lok ma twero bedo tye ni A. gambiae coo gi cwalo ecdysone ma ki loko i kom coo, ma weko tic ki yoo me nywal pa mon, ma kelo peko me nyom. Ento, kadi bed ni ler ma tye ki cogo tye ki steroid hormones mapol, calo estrogen ki androgen (ma ki neno i ref. 9), ki ngeyo wa, ki sterogenic-roids pe ki nongo gi i kom kwidi.
Wa keto yub me moko kit ma steroid hormones tye kwede i dul kom ma lacoo ma ki lwongo ni male male accessory gland (MAG) pa A. gambiae ma odongo me butu kun wa yenyo steroids ma twero loko. Nono tic ki jami ma ki lwongo ni high performance liquid chromatography kacel ki tandem mass spectrometry (HPLC-MS/MS) ma ka tic ki yoo ma pe ki tiyo kwede con, wa nongo edy (E2020) dul kom, ma moko adwogi macon. Ento, gin ma ki nyutu ni onwongo tye ki steroids ma ki lwongo ni oxidized phosphorylated steroids, ma rwate ki formula 3-dehydro-20E22P)12 (lanyut 1). Kit mukene tye 3-dehydro-20E (3D20E22P) ki 20E-phosphate (20E22P). HPLC-MS/MS signal intensity pa 3D20E22P onwongo tye rwom aryo me rwom ma malo loyo kit ma ki kwanyo kwede, 3D20E, ki rwom adek me rwom ma malo loyo pa E ki 20E (lanyut 1). Kadi bed ni i dul kom mukene ki dul kom ma piny (Labol 1). bene ki kwano ecdysteroids i kom coo ki mon ma ki umu nyen (<1 nino) ki onongo 3D20E ki 3D20E22P i MAG keken; E, 20E ki 20E22P onongo tye i kom coo ki mon (Extended Data Fig. 1b). Lok man nyutu ni A. gambiae coo madongo gi kelo rwom ma malo me loko hormone i MAGs gi ma pe ki yubu ki mon.
MAG ki LRT pa mon (ma tye iye atria, seminal vesicles, ki parovarium) ki kwanyo ki ikom coo ma mwaka gi tye 4 (nino 4) ki mon ma peya gunyomme ki mon (0.5, 3, ki 12 hpm). Ecdysone i dul kom magi ki kwano ki HPLC-MS (memered seminal vesicles, ±t; wel ma ki nongo ma pi adaa (FDR) ki yubu; *P < 0.05, **P < 0.01 . 3D20E: cawa 3 ki cawa 0.5, P = 0.035; 3D20E22P: cawa 3 ki cawa 0.5, P = 0.25; cawa 12 ki cawa 3, P = 0.0032; cawa 12 ki cawa 0.5, P = 0.015). obe. Ecdysone ki nyutu ki kala ma calo: E, lum lum; 20E, orange; 20E22P, purple; 3D20E, bulu; 3D20E22P, pink. Gin ma ki keto ni medo rwom me y-axis me nyutu rwom me ecdysone ma piny.
Me kwedo ka 3D20E22P ki 3D20E gi loko i kare me nyom, wa poko LRTs pa mon i kare ma pat pat inge nyom. Kadi bed ni ecdysone onongo pe ki nongo i kom mon ma peya gunyomme, waneno wel ma dit pa 3D20E22P i LRT cut cut inge nyom (05-hpm, ma nongo tye ka dwoko piny i kare ma okato. 3D20E omede matek (Fig. 1). Nono 3D20E ma ki yubu ki jami ma ki lwongo ni chemical synthesised 3D20E ma calo gin ma ki rwate, wa nongo ni rwom pa steroid hormone ni i mating LRTs onwongo tye 100-fold ma malo loyo 20E (Extended Data Table 1). Pi meno, 3D20E22P obedo ecdy ma lacoo ma ki cwalo bot dako LRT i kare me LRTs nyom, ki kit ma ki kwanyo kwede, 3D20E, doko madwong cut cut inge nyom. Man nyutu ni tic ma pire tek pi ecdysone i kom mon inge nyom.
Iyonge yubu RNA sequencing manyen (RNA-seq) dataset (Fig. 2a), kun kitiyo ki custom-built bioinformatics pipeline, wa yenyo ecdysone kinase (EcK), ecdysone oxidase (EO), ki ecdysone ma nyutu 20E-modified phosphatase genes ma ki nyutu i WPP. onongo gene acel ma twero bedo EPP ki gene aryo ma twero bedo EcK (EcK1 ki EcK2), ento onongo pe gitwero nongo gene maber ma twero bedo EO. Ki ngeyo ni, gene pa EPP acel acel onongo ki nyutu i rwom ma malo (98.9th percentile) i Gambian MAGs ento pe i LRTs pa mon (Fig. 2b), ma pe rwate ki kit ma onongo watamo kwede nicakke i kare ma dephopholation otimme i kom EcK2 2020 i dul kom pa mon ni. Pi meno, waye ni EPP pa lacoo twero bedo ka rwate i kare me nyom. Adaa, watiyo ki in vivo stable isotope labeling me umu protein pa mon inge nyom, enzyme ma kinongo ki MS i atrium pa mon (Fig. 2c ki Supplementary Table 1). Bedo pa EPP i MAG ki mon ma ki nyomo (RT LRT) bene pe ki moko kun kitiyo ki jami ma gwoko kom (Fig. 2d).
a, Gin ma ki yubu me yenyo jami ma kwako nywal pa coo ki mon pi gene ma nyutu EcKs, EOs, ki EPPs. Namba ma tye i nget lanyut ni nyutu wel coo ki mon ma ki yero i rwom acel acel. Kwedo man onyutu gene acel me EPP (EPP) ki gene acel me EcK (Ec1) ma ki nyutu i kom coo ki gene acel, acel (EcK2) ma ki nyutu i kom coo ki mon ento pe miyo gene ma ki lwongo ni EO gene.b, Heatmap ma poro gene ma ki nyutu i virgin (V) ki mating (M) Anopheles gambiae ki Anopheles albicans tissues.Spca, fertilization; MAGs, jami ma konyo i kom coo; dul kom mukene, medo ki cak, bwome, tyen, kom ma moo, ki jami ma i iye i kom coo ki mon, ki ic ​​pa mon. EcK2 ki nyutu i MAG ki atria me Gambia, kun EPP nonge i MAG.c keken, Proteomic analysis of male ejaculate group translocation into femaria at 33h, showing, showing 67 ma pol loyo. Mon ki dongo gi i cam ma tye ki 15N me keto (ki umu) protein weny. Co ma pe ki keto lanyut i kom gi ki nywako ki mon ma ki keto lanyut i kom gi, ki LRTs pa mon ki kwanyo gi i cawa 3, 12 ki 24 hpm pi temo jami ma konyo kom (nen Supplementary Table 1 pi rwom me jami ma ki lwongo ni ejaculatory list, EPP). Eck1 ki EcK2 kinongo gi i MAG pa coo ma peya gunyomme ki proteomic analysis pa dul kom magi.d, EPP onongo kinongo ki western blot i MAG ki LRT pa mon ma gunyomme, ento pe i mon ma peya gunyomme nyo co nyo kom mon mukene.Membranes onongo ki temo ki anti-inacting (EPP) ki anti-APP antiboes ma tye co. mon ma pwod pe gi nyome.Nen cal ma ki medo 1 pi ngec ma mako kama ki nongo iye gel. Western blots ki otimo tyen aryo ki adwogi ma rom.
Tic pa ecdysteroid phosphophosphatase pa EPP ki moko i nge keto ne i HPLC-MS/MS ki 3D20E22P ma ki kwanyo ki i MAG (Extended Data Fig. 2a). Medo ikom meno, i kare ma wa juku EPP ki RNA-mediated interference (RNAi), wa nongo ni tic pa phosphatase ma ki nywalo i tisuphase ni odok piny matek coo (Fig. 3a), ki mon ma ki nyomo ki EPP-silenced males gunyutu A ma nok pa dephosphorylated 3D20E (Fig. 3b) kede ma gene silencing (Extended Data Fig. 2b,c). I apoka poka, wan pe wa nongo alokaloka ma pire tek i 20E22P/20E ratio i kom ler acel-lu, ma twero bedo ni enme acel-lu ma mako 3D20E22P (Fig. 3b).
a, dwoko piny tic pa phosphatase i MAG ma oa ki i EPP silencing kun kitiyo ki double-stranded EPP RNA (dsEPP) onyo double-stranded GFP RNA (dsGFP) ma ki temo. Twenty MAG pools ki tiyo kwede i replicate acel acel (P = 0.0046, paired t-test, two-sided), ma ki nyutu ki males ma ki poko, ki Feb. EPP-silenced coo onongo tye ki rwom ma piny pa dephosphorylated 3D20E i 3 hpm (P = 0.0043, unpaired t-test, two-sided), kun 20E onongo pe ki balo (P = 0.063, unpaired). t-test, two-sided).Data ki nyutu calo mean ± sem ki i kin dul adek ma tye ki mon 13, 16 ki 19 acel acel.c, Mon ma ki nyomo ki co ma ki juku EPP onongo tye ki rwom ma malo me re-mating (P = 0.0002, Fisher's exact test, two-sided).Mon onongo ki diyo gi mukwongo me neno ni gitye ki nyom; I nge nino 2, ki lok kwed gi ki coo mukene ma tye ki ic ​​ma ki loko i kom gi me neno kit ma gi rwate kwede ki i kom gin ma ki lwongo ni quantitative PCR detection pa transgene.d, Mon ma ki miyo remo ki coo ma ki juku gi ki EPP onongo tye ki kero me nywal ma odok piny (P < 0.0001; Mann-Whitney test, two-sided) ki wel egg odok piny manok (P < 0.0008, = 088.000). Mann-Whitney test, two-sided) , ma nongo rwom me nywalo lutino onongo pe ki balo (P = 0.94, Fisher's exact test, two-sided).I kin dul weny, n nyutu wel jami ma ki kwanyo ma pe jenge i kom jami makwo.NS, pe pire tek.*P < 0.05, **P < 0.01, ***P < 0.00, *** P < 0.00 * P < 0.00.
Ma lubo, wa temo ka ce ecdysone dephosphorylation pire tek me kelo teko me nyom i mon. Ki ngeyo, mon ma gi nyome ki coo ma EPP-depleted gi dwoko nyom i rwom ma malo (44.9%) loyo mon ma ki temo (10.4%) ka ki keto gi i kom coo mukene (ma ki loko) (Fig. Wob bene odok piny ma pire tek). kero me nywal (Fig. 3d, tung lacuc) ki dwoko piny manok i wel tong ma mon magi gi keto (Fig. 3d, dyere), kun wel tong ma mon gi keto (lagam mukene ma kinongo ki i mon ki i nyom)) pe ki balo (Fig. 3d, tung lacuc). Ki neno ni EPP 2020 tye ki adwogi ma pat pat tic pa 3D20E ki EPP ma ki cwalo i kare me nyom twero bedo ki tic ma pire tek me juku miti pa mon me medde ki nyom, kit ma onongo ki keto i kom 20E me buto. Pi meno, hormone man ma mako coo bene gudo teko pa mon me nywal.
Ma lubo, wa poro tic pa 20E ki 3D20E i kom yat ma ki keto i kom mon ma gu dongo i buto kun wa tiyo ki 3D20E ma ki yubu ki jami ma ki lwongo ni chemical synthesised (Fig. 4a–c) ki 20E ma ki cato. Waneno ni 3D20E onongo tye maber loyo 20E i juku kero pa mon i kom mon ma gu dongo (Fig. 4a-c). 4d).Ki ngeyo, nucu pa rwom me 3D20E i LRT (1,066 pg inge keto yat ki 2,022 pg inge keto yat) okelo rwom pa mon ma pe gi twero tic ma onongo tye 20-fold ma malo loyo rwom me 20E (361 pg inge keto yat) i cawa 24 inge keto yat i kom dano 18 pot karatac inge nyom; Adwogi man rwate ki tam ni keto 20E i buto pe kelo kare me nyom, ki bene nyutu ni 3D20E obedo gin ma pire tek me neno ni wat i kin lunyodo ki lutino.3D20E bene onongo tye ka tic matek loyo 20E i temo keto tong i kom mon ma peya gunyomme (Fig. 4), ma nyutu ni rwom me keto tong tye maber. ma ki neno inge juku EPP manok onwongo tye pi bedo tye pa tic pa 3D20E ma pud ki kelo ki ikom jami ma mon ma okelo nyom.
(a,b) 3D20E ma ki yubu ki 20E (a) ki loko/tic maber atika (ngec ma ki nyutu calo mean ± sem ki i kom jami adek ma ki yubu ma pat pat) (b).c, Mass spectrum (nucu ma piny) rwate ki ecdysone ma kinongo i LRT pa mon ma ki nyomo (nucu ma malo, ka kiporo ki (E206). µg, P = 0.02; 0.21 µg, P < 0.0001; Fisher's exact test, two-sided) ki 10% ethanol (0.63 µg, P < 0.0001; 0.21 µg, P < 0.0001; Fisher's 0.0001), Fisher's 0.0001 onongo tye ma malo, 20-0. ma loyo jami ma ki temo i kom yat ma malo keken (0.63 μg, P = 0.0002; 0.21 μg, P = 0.54; Fisher's exact test, 2-sided).e, 3D20E injection okelo rwom me nywal ma malo i kom mon ma peya gunyomme ma loyo 10% ethanol (P = 0.000 μg, P = 0.54). 0.0001; 0.13 μg, P = 0.0003; Fisher's exact test, two-sided), kun 20E ka kiporo ki ma pe ki temo Only i rwom ma malo (0.21 μg, P = 0.022; 0.13 μg, P = 0.0823 Fisher's exact two-sided, 20D). rwom me nywalo ma malo loyo 20E i rwom ma malo (0.21 μg, P = 0.0019; 0.13 μg, P = 0.075; Fisher's exact test, two-sided).I kin jami weny, n nyutu wel jami ma ki kwanyo ma pe rwate ki kwo. ***P < 0.001, ****P < 0.001. Ngec ni aa ki i kom jami adek ma ki dwoko cen.
I kwan mukato, wa nongo ni keto steroid hormones i buto kelo nyutu pa MISO (Mating-Induced Stimulator of Oogenesis 11), gin ma gwoko mon ma gwoko A. gambiae ki two ma ki lwongo ni P. falciparum. Peko me yot kom ma oa ki i 13, two atipa ma neko dano loyo pien MISO pire tek bot Anopheles. kabedo ma tye ki two atipa, wamoko tamwa me moko ngo ma hormone 3D20E onyo 20E ma weko gene eni nyute. Wanongo ni kadi bed ni 20E ma ki keto i kom dano ni okelo nuclear hormone receptors (HR), calo HR3 ki HR4, ki steroid ma rwate, calo yolgkogenic genes V14, MISO onongo tye matek, 16 ma ki kelo ki 3D20E (Extended Data Fig. 3). Pi meno, lok me buto pa gin ma ki lwongo ni androgenic steroid hormone ni nen calo kelo yoo ma gwoko mon ki i peko ma bino pi two ma aa ki i kom ler makwo. Medo ikom meno, 3D20E weko isoforms me E receptor EcR, dwoko piny EcR-R, ki dwoko piny EcR-B ma kelo teko i kom gene mukene ma kelo nyom, medo ki HPX15, ma gudo nywal pa mon. Man twero gonyo peko me nyodo ma ki neno i kom mon ma gi nyome ki coo ma EPP-silented (Extended Data Fig. 3). Data magi nyutu ni tye yoo ma lubo ma ki tiyo kwede ki ecdyson aryo ma twero bedo i te tic pa mon.
Ma lubo, wa temo tic pa gene aryo me EcK ma ki nongo i yub me bioinformatics wa. Kwero EcK1 onyo EcK2 okelo too pa coo (Extended Data Fig. 4a), ma nyutu ni ecdysone phosphorylation, ki dong inactivation, pire tek pi kwo. Pien EcK2 onongo tye malo ki rwom ma malo loyo EcK1 ma kinongo i MAGs ki proteomics (Fig. 2b,c ki Supplementary Table 2), wa moko tic pa ecdysteroid kinase ne kun wa keto ne i 20E, ma okelo phosphorylation 20E22P (Extended Data Figure 2).4b).Ka kitiyo ki 3D20E macalo substrate, wa nongo ni pe ki nongo cal ma ki nongo ki i poto 3D20E22P (Extended Data Fig. 4c), ma nyutu ni 20E ma ka 3D20E twero bedo gin ma EcK2 maro.
Malube ki kwedo RNA-seq wa, EcK2 bene onongo tye ka nyute matek i LRT pa mon ma peya gunyomme, ma onongo ki juku iye inge nyom (Fig. 2b). Wamoko ngec magi ki wamoko ni nyute pa EcK2 onongo pe ki balo ki miyo remo (Extended Data Fig. 5a). Ka wa medo rwom me MS ma okwongo, wa nongo ni peak pa 20 EcK2 onongo tye macok ma rwate ki rwom ma malo pa 20E (cawa 22-26 inge camo remo; ngec ma ki medo cal 5b). Kwero EcK2 i kom mon ma pwod pe gunyomme okelo dongo pa 20E ki 20E22P tyen 3 i cawa 26 inge camo remo (cal ma ki medo 2c ki EcphoccphoK2 bene moko ni EcK2 20E ikom mon. Ki ngeyo ni, anyira ma pe ki EcK2 gubedo ki miti me buto (Extended Data Fig. 5d,e), ma medo nyutu ni tic pa mon me 20E pe kelo kare me nyom. Ento, mon magi onongo gitye ki wel ma malo me keto tong ka kiporo ki mon ma pe ki nyom, ma kato 30% me keto tong ic (Extended Data Fig. 5f). Ka ki keto Eck2 RNA (dsEcK2) ma ki lwongo ni double-stranded Eck2 RNA (dsEcK2) ingee miyo remo, nywalo latin pe otimme, i kare ma 20E ma malo pi camo remo odok piny. I kom lok, adwogi eni miiyo kony i kom labol ni 20E ma ki kelo i nge mato remo twero kelo nywalo latin keken, ento ka ki gengo nywalo latin (EcK2 twero bedo ni tye jami mukene) ki juku woko ki i nyom. Pe 20E onyo 3D20E ma ki keto i kom dano ma gengo EcK2 i kom anyira ma pe gunyomme (Extended Data Fig. 5g), ma nyutu ni jami mukene aye gengo kinase man. Ento, rwom me 20E inge miyo remo onongo pe romo kelo arem cwiny, ento onongo tye ka kelo peko ma malo 3D20E ma ki keto i yoo me buto.
Adwogi wa ni miyo ngec ma pire tek i kom kit ma A. gambiae twero nywalo kwede. Labol mo okati ma coo odongo me yubu 3D20E ma malo, ma obedo ecdysone ma ki loko pi coo ma miyo twero bot coo kun weko mon pe gi mede ki nyom. I kare acel-lu, vectors magi bene gu dongo yoo maber me yubu 3D20E. tic ki 3D20E i kom mon me gamo lok me buto pa coo-ma rwate ki EPP. Ki ngeyo wa, man aye labol mukwongo pa coo- ki mon-dominated steroid hormone system ma tiyo tic ma pat ki ma pire tek i kom kwidi.Tic pa ecdysone ma rwate ki coo dong ki moko ento pe ki nyutu maber. Me labolle, labol madit 18 nyutu ni tic magi pe nyutu ni tic magi nyutu ni tic magi 18 pe tye kakare. twero bedo ni ki tiimo ki 20E precursor E1. Ki ngeyo maber ni i Drosophila, monandry kicako ki lok me buto pa jami matino tino ma mako butu19,20 ma gikubbe ki neurons ma gi tiyo ki yoo me nywal pa dako kun wok ki i dul kom ma ki lwongo ni sex peptide receptors21,22.Tic mukene mitte me moko kit ma jami ma ki lwongo ni downstream signaling ma ki doro kwede ki A20D. mon ki me moko ka jami ma ki lwongo ni cascades ni ki twero gwoko gi i kin obe ki Drosophila.
Ki miiyo tic ma pire tek pa 3D20E i kom nywal pa mon ki kit ma gikwo kwede ma kinongo i kwan wa, yoo ma tero 3D20E me yubu ki tic kwede miyo kare manyen pi yoo me lweny i kom ober i anyim, ma calo kelo co ma pe ki nywal i yoo me tic ki jami ma pe ki nywal. 3D20E twero bedo ni odongo i kare ma A. gambiae ki dul ler mukene ma ki lwongo ni Cellia gunongo kero me mako remo gi me doko gin ma ki lwongo ni mating plugs, pien man miyo twero me tic ki jami mapol ma weko kom gi bedo maber. kabedo ma two atipa dwong iye, ma konyo i nywaro Plasmodium. Kit ma mon 20E ki nyutu ni tye ki adwogi madit i kom kwo ki dongo pa P. falciparum i kom ler ma mon ma ki lwongo ni Anopheles,24 yoo pa steroid hormone pa laco ki mon dong obedo gin ma pire tek i kin ler ki ler.
A. gambiae G3 strains ki gwoko gi i te jami ma ki lwongo ni insect conditions (26-28 °C, 65-80% relative humidity, 12:12 h light/dark photoperiod). Larvae ni ki miiyo cam me rec ma ki yubu ki buru (TetraMin Tropical Flakes, Koi Pellets ki Tetra Pond Sticks i rwom me 7:2A). obe ni ki miino gi cam ma miite ki 10% dextrose solution ki remo pa dano cabit ki cabit (jami ma ki kwano i remo). Obe ma pe ki nyomo ki nongo gi ki poko kin coo ki mon i kare me pupal ingee neno agiki ki microscopy. Co ma tye ki DsRed transgene dong ki tito gi con.
Temo me nyom ma ki diiyo ki timo kit ma ki waco kwede con. Pi nyom ma pe ki ngeyo, mon ma mwaka gi tye 4 ki gwoko gi i rwom me 1:3 ki co ma pe gunyomme pi dyewor aryo. Pi temo ma ki keto dsEPP i kom coo, co-caging orwate ki nino 3-4 inge keto yat, ka tic pa phosetaction onongo ki dwoko piny (Extended Data Fig. 2b).
Kom pa obe, kom ma odong (kom mukene), onyo kom weny ki poko i 100% methanol ki ki yubu ki gin ma ki lwongo ni beader (2 mm glass beads, 2,400 rpm, 90 sec). Wel kom ki dwong pa methanol onwongo tye kit man: kom mukene, i 500 μl; MAG, 50-100 80 μl; LRT pa mon, 25-50 80 μl. Gin ma opoto ni ki kwanyo methanol me aryo ki dwong pa methanol marom. Ki kwanyo jami ma odong i cell ni ki centrifugation. Methanol ma ki kwanyo ni ki rubu ki ki juku i te nitrogen flow, ki dong ki dwoko cen i dwonge ma lubo ni me 80% methanol i kom pii: μl, 50 μl; MAGs ki LRT pa mon, 30 μl.
Kamu kene ni ki kwano i kom gin ma ki lwongo ni mass spectrometer (ID-X, Thermo Fisher) ma ki keto i kom gin ma ki lwongo ni LC instrument (Vanquish, Thermo Fisher).5 μl me kamu kene ni ki keto i kom 3 μm, 100 × 4.6 mm column (Inspire C8, Dikma) ma ki gwoko i 25 °C The mobile ALC. (pii, 0.1% formic acid) ki B (acetonitrile, 0.1% formic acid). LC gradient onwongo tye kit man: 5% B pi dakika 1, ki dong omede me doko 100% B pi dakika 11. Iyonge dakika 8 i 100%, dwoko rwom me 5% B pi dakika 4. Kit ma mol onwongo tye 30 ml. min-1. Ionization i MS source ki tiimo ne ki lyeto electrospray ionization i kit maber ki marac.
Gin ma ki lwongo ni mass spectrometer ni pimo ngec ma tye i m/z ma cake ki 350 nio wa i 680 i 60,000 resolution i kit me MS ma opong. Ngec me MS/MS ki nongo ki i [M + H]+ (jami weny), [M - H2O + H]+ (jami weny), ki [M - H]- (jami ma ki yubu ki phosphorylated ma ki tiyo kwede me moko ni gin ma ki keto ni tye iye). pe tye rwom mo ma onongo tye. Me ngeyo ecdysteroids ma pe ki keto i tic, MS/MS data pi HPLC ducu ma tye ki >15% ma rwate ki wel ma ki kwano. Kwan kun kitiyo ki standard curves ma ki yubu ki rwom ma pe ki keto (20E, 3D20E) me kwano wel ma rwate nyo dilutions pa sampul acel ma ki keto i tic (me acel) me kwano wel ma rom ki ma kinongo i acel lacoo. Pi 3D20E, ki kwano wel ne kun kitiyo ki wel jami ma ki medo ni: [M + TFA]-, [M + COOH]-, [M + Na]+, [M + Cl]-, [M + NO3]-. Ki kwanyo ngec ki ki kwano kun kitiyo ki Tracefinder (version 4.1). Ki kwano ngec me MS/MS kun kitiyo ki MS/MS ma ki kwano kun kitiyo ki Xversion 4.4. spectra pa E, 20E ki 3D20E ki poro ki rwom ma rwate.3D20E22P ki kwano ki derivatization ki Girard's reagent.20E22P ki kwano ki m/z ratio.
3D20E22P onongo ki lonyo ki i MAG. Lonyo ni kitimo i kom rwom me kwan kun kitiyo ki ultra-performance liquid chromatograph (Acquity, Waters) ki quadrupole mass-based detector (QDa, Acquity, Waters) i te LC ma rom ki me HPLC-MS/MS ma ki kwanyo. Fraction ma ki kwanyo i kare ma ki rwate ki mz/z 3D20E22P onongo kinongo i kare acel ma onongo ki moko con. Kit ma jami ma ki kwanyo ni leng kwede ki temo ki HPLC-MS/MS kit ma kiloko kwede malo ni.
Ki kwanyo RNA weny ki i kom 10-12 ma gi nywalo lutino onyo dul kom mukene (ma pe ki wic) kun kitiyo ki TRI reagent (Thermo Fisher) malube ki cik pa layub. RNA ki tiyo kwede ki TURBO DNase (Thermo Fisher). cDNA ki yubu kun kitiyo ki Moloney murine leukemia reverse transcript virus (MLVRT; Fisher) ma lubu cik pa layub. Primers pi reverse transcription quantitative PCR (RT-qPCR; Extended Data Table 2) onongo ki coyo con24 onyo ki yubu kun kitiyo ki Primer-BLAST26, ki miiyo tam bot jami ma tye ki 70-150 bp i dit ki ma kwako exon-exon junctions onyo Primers adek ma pat pat ki i DNA ma ki kwanyo ki i sampul adek. gin ma ki lwongo ni biological replicates ni ki diluted tyen angwen i pii pi RT-qPCR. Kwantify ni ki timo i 15 μl replicate reactions ma tye ki 1× PowerUp SYBR Green Master Mix (Thermo Fisher), primers, ki 5 μl me diluted cDNA. Reactions ni ki timo i kom QuantStudio Pro-time real-time PCR system (Thermo Fisher) ki coko ki nyamo kun kitiyo ki Design and Analysis (version 2.4.3). Kit ma ki nyutu kwede i kwan man, wel ma ki rwate ki ribosomal gene RpL19 (AGAP004422), ma nyutu ne pi oloko tutwal ki remo 27 onyo mating 3 .
Kit me RNA ki temo kun kitiyo ki Agilent Bioanalyzer 2100 Bioanalyzer (Agilent). Illumina paired-end libraries ki yubu ki tic kwede i Broad Institute of MIT ki Harvard. Sequencing reads ki keto i kom A. gambiae genome (PEST strain, version 4.12) kun kitiyo ki HISAT (2020) ki bal. jami ma ki kwano. Kwan ma tye ki rwom me rwom (MAPQ) ma ki nongo <30 ki kwanyo kun kitiyo ki Samtools (version 1.3.1). Wel kwan ma ki keto i kom gene ki kwano kun kitiyo ki htseq-count (version 0.9.1) ki parameters ma ki keto. Wel ma ki kwano ma ki kwano ki ki kwano gene expression ma pat pat kun kitiyo ki package package DEQ2 1.28.1) i R (dul 4.0.3).
Gin ma ki lwongo ni Ecdysone-modifying gene candidates onongo ki nongo gi ki yenyo A. gambiae genome kun kitiyo ki PSI-BLAST algorithm (https://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/2.8.1/), kun kitiyo ki default values ​​Parameters with the following query sequence proteins: sequence proteins from Boccession from Boccession (A. NP_001038956.1), Musca domestica (Accession No. XP_005182020.1, XP_005175332.1 ki XP_011294434.1) ki Microplitis demolitor (Accession No. XP_0085526461461561565) ki bot B. mori (Accession No. NP_001036900), Drosophila melanogaster (Accession No. NP_651202), Apis mellifera (Accession No. XP_394838) ki Acyrthosiphon pisum (Accession No. XP_001947166); ki EPP ki bot B. mori (Accession No. XP_001947166) NP_001177919.1 ki NP_001243996.1) ki EO pa D. melanogaster (Accession No. NP_572986.1) (step 1). Next, filter hit on high RNAs expression (00>100) dul/kilobase exons pi million ma ki kwano (FPKM) onyo >85%) i dul kom ma ki lwongo ni reproductive tissue (female LRT onyo MAG) i Gambia (dul me 2). Me medo rwom me tic, wa yero enzymes ma ki nyutu i dul kom ma nywalo pa A. albimanus, dul ler ma ki lwongo ni anopheles ma pe yubu onyo cwalo ecdysone i kare me nyom. Gin ma ki lwongo ni genes ni ki kwanyo gi malube ki nyute ma nok (<100 FPKM onyo <85th percentile) i dul kom ma ki lwongo ni A. albimanus (A. 3). filter (step 4), genes ma ki yero omyera oti ki acel i kin jami ma lubo ni: (1) ma ki medo i nge nyom (P < 0.05) malube ki nyamo gene ma ki nyutu ma pat pat ki (2) i kom ma pe nywalo latin (< 85 % onyo <100 FPKM).
Wa loko yoo ma ki waco con ni 28,29,30 me nongo lanyut me jami makwo weny. Ki lok macek, Saccharomyces cerevisiae ma ki lwongo ni wild-type type II (YSC2, Sigma) ki temo i yeast nitrogen base (BD Difco, DF0335) ma tye ki (wt/vol) %2 gluse (275% Ggluse), Sigma, 275%. amino acid-free ki ammonium sulfate. gin ma ki pito) ki 5% 15N ammonium sulfate (NLM-713, >99%, Cambridge Isotope Laboratories) ma calo gin acel ma kelo nitrogen. Yeast onongo ki nongo ki i centrifugation ki ki miyo cam bot ler ma ki lwongo ni mosquito nio ka gu dongo. Medo ki fishmeal (0.5 mg300 per larvae) me gengo ler me angwen i lakalatwe me angwen. too. Mon keken aye ki tiyo kwede i yub me nyom ki coo ma pe ki keto nying gi me nyamo gin ma ki lwongo ni proteome pa coo ma ki keto i kare me nyom.
4-6 nino ma mwaka gi 15N-tagged virgin females ki diyo gi me butu ki age-matched untagged virgin males. Mating maber ki moko ni ki nongo mating plugs i te epifluorescence microscopy.I cawa 3, 12, ki 24 hpm, atria pa mon 45-55 ma ki keto i ammonium 50 μl bicarbonate buffer (pH 7.8) ki homogenized ki pestle. Homogenate ni ki keto i centrifuge ki supernatant ki rubu ki 50 μl me 0.1% RapiGest (186001860, Waters) i 50 mM ammonium bicarbonate. Supernatant ki pellet ma ki kwanyo ki i sampol acel acel ki cwalo i wi yamo ma ngic i ot yat me MacCoss i University of Washington, kan ma ki tyeko iye yubu kamu kene pi LC-MS/MS. Medo gin ma ki lwongo ni pellet ni i 50 μl me 0.1% RapiGest i 50 mM ammonium bicarbonate ki sonicate i pii. Ki pimo wel protein ma tye i pellet ki supernatant ki BCA assay, ki dwoko piny ki 5 MM (DTT; Sigma), ma ki keto i 15 mM iodoacetamide (Sigma) ki ki keto i 37 °C (1:0 50) pi cawa 1 ki trypsinization: trypsin:substrate ratio).RapiGest onongo ki lysed ki medo 200 mM HCl, ki dong ki gwoko i °C pi dakika 37 ki centrigation 14,000 rpm pi dakika 10 i 4 °C me kwanyo jami ma obale. Gin ma ki nyutu ni ki lwoko ki dul ma ki lwongo ni dual-mode solid-phase extraction (Oasis MCX cartridges, Waters) ki ki dwoko i 0.1% formic acid pi rwom me protein me agiki me 0.33 μg µl-1. G ma ki keto i kom gin ma ki lwongo ni virginly proteins ma ki kwanyo ki i MAG onongo ki kwanyo ki i MAG. coo. Ki kwano jami aryo ma ki dwoko cen pi lanyut acel acel. Lacen, 1 μg pa acel acel ki kwano kun kitiyo ki 25-cm fused silica 75-μm column ki 4-cm fused silica Kasil1 (PQ) frit trap ma ki keto ki Jupiter C12 reversed-phase resin (PQ) chromatography. Kamu kene ma ki kwanyo - MS/MS ki tiyo kwede i kom Q-Exactive HF mass spectrometer (Thermo Fisher) ki nanoACQUITY UPLC System (Waters). Data-related acquisition data ma ki nongo pi tic acel acel ki loko ne i mzML format kun kitiyo ki Proteowizard (version 3.0.2027) ki Comet (version 3.2028) i kom (233) FASTA database ma tye ki jami ma ki lwongo ni Anopheles gambiae (VectorBase version 54), Anopheles coluzzi Kitimo yenyo i kom Mali-NIH (VectorBase version 54), Saccharomyces cerevisiae (Uniprot, dwe me adek mwaka 2021), A. gambiae RNA-seq, ki jami adek ma ki ngeyo ma kwako jami ma kelo two. map-matched FDRs ki nongo kun kitiyo ki Percolator32 (version 3.05) ki threshold me 0.01, ki peptides ki keto gi kacel i protein identifications kun kitiyo ki protein parsimony i Limelight33 (version 2.2.0). Relative protein abundance onongo ki byeko kun kitiyo ki normalized spectral abundance factor (NSAF) ma ki kwano pi protein acel acel kit ma ki waco kwede con. ma rwate ki protein acel acel ki kwano i kom jami ma ki kwanyo ki i kom jami makwo ma pat pat. 15N labeling onyutu proteome pa dako, kadi bed ni protein manok ma pe ki keto nying i kome onongo ki nongo ki i kom anyira ma ki keto label i komgi. Wa coyo rwom me dwoko piny pa protein pa coo (1-5 spectra) i kom mon ma pe ki kwanyo ki i kom jami ma ki kwanyo ki i kom jami ma ki kwanyo ki i kom laco/co ma ki kwanyo ki iye kamu kene, ma calo adwogi me HPLC "tero". Gin ma ki nongo i kare mogo ma calo 'jami ma balo' ki i kom mon ma ki keto i kom gi ki keto gi i Supplementary Table 1.
Peptide aryo ma ki lwongo ni antigenic peptides, QTTDRVAPAPDQQQ (i kin isotype PA) ki MESDGTTPSGDSEQ (i kin isotype PA ki PB) i Genscript. Peptides aryo ni ki rubu gi kacel, ki dong ki keto gi i carrier protein KLH ki ki keto gi i New Zealand rabbits. Rabbits ni ki neko gi ingee keto injection me angwen, ki IGG onongo ki keto i kin gi purification.IgG ma aa ki i kom apwoyo ma ki lwongo ni EPP-specific rabbit ni ki tiyo kwede me medo western blotting.
Pi western blots, MAG (n = 10, kan ma n nyutu wel labol pa obe ma pe jenge i kom jami makwo) ki LRT ma mon (n = 30) ki bot coo ma mwaka gi tye 4 ki mon ma peya gunyomme ki mon (<10 inge nyom), Protein extraction buffer (50 mM Tris, 80; pH-10; sodium deoxycholate; 150 mM NaCl; 1 mM EDTA; 1× protease inhibitor cocktail (Roche)) ki medo iye pat pat. Kamu ma ki nyutu ni ki medo gii cut cut inge poko ne ki gin ma ki lwongo ni beader (2 mm glass beads, 2,400 rpm, 90 sec In soluble debrived ki kwanyo ki i 200 g). i 4 °C. Proteins ki kwano ki Bradford assay (Bio-Rad). I ngeye, 20 μg me MAG protein, 40 μg me LRT protein, ki 20 μg me residual bulk protein ki loko gi ki poko gi ki 10% Bis-Tris NuPAGE kun kitiyo ki MOPS buffer. Proteins ki tero gi i polylidene membranes kun kitiyo ki fluoridene membranes jami ma ki lwongo ni transfer system (Thermo Fisher). Membranes ki lwoko tyen aryo i 1× PBS-T (0.1% Tween-20 in PBS) ki dong ki gengo i Odyssey blocking buffer (Li-Cor) pi cawa 1 i 22°C. Membranes ki yenge dyewor i 4 °C ki custom rabbit anti-EPP polyclonal polyclonal antibody primary (in blocking:10070) buffer) ki rat anti-actin monoclonal primary antibody MAC237 (Abeam; 1:4,000). Membranes ki lwoko ki PBS-T ki dong ki keto i incubation ki secondary antibodies (donkey anti-rabbit 800CW ki goat anti-rat 680LT (Li-Cor), gin weng 1:2000, ma tye ki buffer ma tye ki 100%. SDS ki 0.2% Tween -20 pi cawa 1 i 22 °C. Gin ma ki lwongo ni Membranes ki lwoko ki PBS-T ki ki kwanyo cal ki Odyssey CLx scanner. Ki kwanyo cal ki ki tiyo kwede i Image Studio (version 5.2). Pe kinongo band mo ma rwate ki EPP-RA isoform (82 kDa).
Kabedo ma ki coyo iye EPP (ma calo isoform AGAP002463-RB ma tye ki histidine phosphatase domain, NCBI conserved domain search 34) ki EcK2 (AGAP002181) ki keto i pET-21a(+) plasmid (Novagen Millipore Sigma) ; primers tye i Extended Data Table 2. GS4 linkers aboro (i tandem) ki keto i nyim C-terminal 6xHis tag me pET-21a(+)-EcK2 construct. Recombinant proteins ki yubu kun kitiyo ki NEBExpress cell-free E. coli protein synthesis reaction (New England BioRecombinant proteins onongo kitiyo ki NEBExpress proteins). Ni spin columns (New England BioLabs).Dihydrofolate reductase (DHFR) control protein ni ki yubu kun kitiyo ki DNA template ki i NEBExpress Cell-Free E. coli Protein Synthesis Kit. Proteins ni ki gwoko i 50% glycerol i PBS i -20 °C pi dwe ma romo 3.
Tic pa EPP ki jami ma ki kwanyo ki i kom dano ki pimo kun kitiyo ki 4-nitrophenyl phosphate (pNPP; Sigma-Aldrich). Gin ma ki lwongo ni reaction buffer ni tye ki 25 mM Tris, 50 mM acetic acid, 25 mM Bis-Tris, 150 mM NaCl, 10 mM, ki mM ED. DTT. Gin ma ki lwongo ni tissue ni ki keto i reaction buffer ki cell debris ki kwanyo ki centrifugation. Yab reaction ni ki medo enzyme onyo tissue extract i reaction buffer ma tye ki 2.5 mg ml-1 pNPP. Reaction mixture ni ki keto i rwom me lyeto me ot i piny macol, ki wel pNP ma ki kwanyo ki i pPP ki kwano ki pimo rwom me mako ne i 405 nm i kare ma pat pat.
Pi tic pa EcK ma ki lwongo ni in vitro, gin ma ki lwongo ni protein ni ki keto i 0.2 mg 20E onyo 3D20E i 200 μl buffer (pH 7.5) ma tye ki 10 mM HEPES–NaOH, 0.1% BSA, 2 mM ATP ki 10 mM MgCl2 pi 27 °C 800 μl methanol, ki dong ki juku ne i -20 °C pi cawa 1, ki dong ki juku ne i 20,000 g pi dakika 10 i 4 °C. Gin ma odong ni ki kwano ki HPLC-MS/MS. Me juku lyeto pa jami ma ki tiyo kwede i dul ma ki temo ni, ki keto jami ni i PBS pi 20% g 95°C.
Pi tic pa EPP ma ki lwongo ni in vitro, gin ma ki lwongo ni protein ni ki keto i 3D20E22P (ma rom ki wel ma ki nongo i 18 pairs me MAG, ma ki yubu ki HPLC-MS/MS) i 100 μl buffer (pH 7.5) ma tye ki 25 mM Tris, 50 mM acetic acid, 20 mM BiMs, 50 mMs NaCl, 0.1 mM EDTA, ki 1 mM DTT pi cawa 3 i 27 °C. Ki juku tic ni ki medo 400 μl me methanol ki ki juku i -20 °C pi cawa 1, ki dong ki juku i 20,000 g pi dakika 10 i 4 °C. Ki kwanyo supernaze ki MSLC/MSLC.
Gin ma ki lwongo ni PCR pi EPP (362 bp), EcK1 (AGAP004574, 365 bp) ki EcK2 (556 bp) ki kwanyo ki i cDNA ma ki yubu ki i kom ler ma ki lwongo ni mixed-sex headless mosquito. Gin ma ki lwongo ni PCR fragment me eGFP control (495 bp) ki kwanyo ki i pCR-FPG ma ki waco con ni; Gin ma ki lwongo ni PCR primers ki keto gi i Extended Data Table 2. Gin ma ki lwongo ni PCR fragment ni ki keto i kin inverted T7 promoters i kom pL4440 plasmid. Plasmid constructs ki nongo ki i NEB 5-α competent E. coli (New England Biolabs) ki ki moko ki DNA sequencing ma peya kitiyo kwede (nen Supplement Data sequence 1P sequence in PL4440 plasmid). gin ma ki lwongo ni T7 promoter (Extended Data Table 2) ki tiyo kwede me medo gin ma ki keto ki i pL4440-based plasmid. Kit ma ki yubu kwede PCR ki moko ki agarose gel electrophoresis. dsRNA ki coyo ki i PCR templates kun kitiyo ki Megascript T7 Transcription Kit (Thermo Fisher) ki ki lonyo kit ma luyub jami owaco kwede ki lok ma ki waco con.
Pi keto dsRNA, 1,380 ng me dsRNA (dsGFP, dsEcK1, dsEcK2, dsEPP) ki keto i rwom me 10 ng nl-1 i thorax pa coo onyo mon madongo (Nanoject III, Drummond) i nge nino acel inge eclosion. Gene knockdown ki nongo ki i biological ma romo adek Kwanyo RNA, cDNA synthesis, ki RT-qPCR. Pi keto ecdysone, mon ma mwaka gi tye 4 nyo 6 ma pud pe kinywalo gi ki miyo remo ki 0.13, 0.21, onyo 0.63 μg me 20E onyo 3D20E (Norum III, Dmond, 21, 21. kit meno, malube ki kit ma ki yubu kwede onyo 6.3 ng nl-1. Ket 100 nl me 10% (vol/vol) ethanol i pii; 100 nl me 3D20E22P i 10% ethanol (ma rom ki 75% me wel ma ki nongo i pair me MAGs). Ki poko obe i dul ma ki keto iye yat.
Pi temo nywalo lutino, ki miyo cam bot mon ma mwaka gi tye 3 ki remo pa dano. Kwany oyot ma ki miyo cam manok onyo ma pe ki miyo cam. Malube ki kit ma ki miyo kwede, mon ki keto gi i cup ma pat pat pi dyewor angwen ma romo cawa 48 inge camo remo. Ki kwano tong gweno i te gin ma ki lwongo ni stereoscope (Stemi Zeimi, 508); pi mon ma gi nyome, tong ma odonyo i kom ler ma ki lwongo ni larvae onongo kitero calo ma twero nywal.
Pi temo nyom, ki weko mon pi nino 2 malube ki kit ma ki miiyo kwede me dongo teko me nyom, ki coo ma mwaka gi rom ki gi ki keto gi i ot acel. I nge dyewor aryo, mon ma ki nywalo gi ki poko ki genomic DNA ki gonyo ki freeze-thaw ki sonication ma tye ki m 10 Tris-HCl, 1 mM EDTA, ki 25 mM NaCl (pH 8.2). Gin ma ki nyutu ni ki gwoko ki Proteinase K (0.86 μg μl-1) pi dakika 15 i 55 °C, ki ingeye ki dakika 10 i 95 °C. Gin ma ki yubu ma ki kwanyo ki i PCR ki 10 dul kom ma ki lwongo ni chromosome; jami ma ki lwongo ni primers ki keto gi i Extended Data Table 2. Peko pa Y chromosome sequence nyutu ni pe tye rwate.
Pi dwoko rwom me nyom, mon ma ki keto i nyom ki teko ki temo pi bedo tye pa jami me nyom me moko kit me nyom ki ki weko nino 2 me dongo tek me nyom ka co pe, kit ma onongo kiloko kwede con 36 .Co ma tye ki DsRed transgenic sperm ki keto gi i ot pa mon i dyewor aryo, lacen onongo ki keto nyig yat i kom mon. ki kwanyo ki i kom mon, ki genomic DNA ki yubu kit ma kiloko kwede malo ni ki ki keto i qPCR me nongo DsRed transgene; jami ma ki lwongo ni primers ni ki keto gi i Extended Data Table 2. Peko pa DsRed transgene nyutu ni pe tye re-mating mo ma otimme.
3D20E ki yubu kit ma onongo kiloko kwede con 37 . Ki lok manok, 10 mg me 20E (Sigma-Aldrich) ki rubu i 10 ml me pii, ki medo ki 30 mg me platinum black (i kit ma buru, Sigma-Aldrich). O2 ma mol mot mot ki medo i kom gin ma ki rubu ni, ma ki rubu ne i rwom me lyeto me ot. Iyonge cawa 6, ki medo 30 mL me methanol me juku tic. Gin ma ki rubu ni ki keto i centrifuge me kwanyo jami ma kelo alokaloka. Gin ma odong ni ki juku ne me otwo i vacuo i rwom me lyeto me ot. Gin ma ki yubu ni ki juku i 10% ethanol ki keto methanol pi rwom me HPMS. (cake i 20E nio wa i 3D20E) onwongo tye 97% (Fig. 4b), ki MS spectrum pa 3D20E ma ki yubu ni orwate ki ma kinongo i kom mon ma ki nyomo (Fig. 4c).
Lok ma ki coyo ni tye ki ngec ma mako temo ma kitimo. GraphPad (version 9.0) ki tiyo kwede me timo Fisher's exact test, Mantel-Cox test, ki Student's t-test. Cochran-Mantel-Haenszel tests ki otimo kun kitiyo ki custom R script (ma nonge i https://github.com/haenszel. Data ma ki poko ni ki temo). pi bedo maber kun kitiyo ki Shapiro-Wilk test ki rwom me 0.05. Ka ngec ni opoto i rwom me rwate, kitimo Mann-Whitney test. Ki kwano ngec ma mako kwo kun kitiyo ki Mantel-Cox test. DESeq2 package (version 1.28.1) ki tiyo kwede me timo RNA-seq-level genetal analysis i kom horizontal bar. nyutu wel ma tye i kin piny. Wel ma pire tek me P = 0.05 ki tiyo kwede calo rwom me rwom pi jami weny ma ki temo.
Pi ngec mukene ikom yub me kwan, nen ripot me kwedo lok ma mako jami makwo ma ki keto i pwony man.
Ngec pa MS proteomic ki keto i ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) ki i PRIDE Partner Repository (https://www.ebi.ac.uk/pride/) ki ngec ma ki lwongo ni PXD032157.
Gin ma ki lwongo ni RNA-seq ni ki keto i Gene Expression Comprehensive Library (https://www.ncbi.nlm.nih.gov/geo/) i te rekod GSE198665.
Jami mukene ma ki nongo ki/nyo ma ki nyamo i kare me kwan man ki twero nongo ne ki bot lucoc ma rwate ka ki penyo gi. Lok man miyo ngec ma ki nongo ki iye.
De Loof, A. Ecdysteroids: Neglected insect sex steroids? Male: Black Box.Insect Science.13, 325–338 (2006).
Redfern, CPF 20-hydroxyecdysone ki dongo pa ic i Anopheles stephens.J. Insect Physiology.28, 97–109 (1982).