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ʻAʻole e like me nā vertebrates, ua manaʻo nui ʻia nā pepeke ʻaʻole i loaʻa nā hormones steroid sex biased kāne. Ma Anopheles gambiae, ʻike ʻia ua ulu ka ecdysone steroid 20-hydroxyecdysone (20E) e kāohi i ka ulu ʻana o ka hua manu i ka wā i hana ʻia e nā wahine2 a e hoʻoulu i kahi manawa paʻakikī male i ka wā i hoʻoili ʻia ai e nā kāne3. ʻOiai ʻo ka ulu ʻana o ka hua manu a me ka male ʻana he mau ʻano hānau koʻikoʻi, ʻo ka hoʻomaopopo ʻana pehea e hoʻohui ai nā makika Anopheles wahine i kēia mau hōʻailona hormonal e hiki ke hoʻomaʻamaʻa i ka hoʻolālā ʻana o nā papahana hoʻomalu malaria hou. Maanei, hōʻike mākou ua hoʻoponopono ʻia kēia mau hana hānau e nā steroids sex like ʻole ma o kahi pūnaewele paʻakikī o nā enzymes ecdysteroid-activating/inactivating. Ua ʻike mākou i kahi ecdysone oxidized kāne-kikoʻī, 3-dehydro-20E (3D20E), e pale ana i ka makua ma ka pani ʻana i ka hoʻokipa moekolohe wahine ma hope o ka hoʻoili moekolohe a me ka hoʻāla ʻana e ka dephosphorylation. ʻO ka mea nui, ua hoʻoulu pū ka hoʻoili 3D20E i ka hōʻike ʻana o nā genes reproductive e mālama i ka ulu ʻana o ka hua manu i ka wā o ka maʻi Plasmodium, e hōʻoia ana i ke olakino o nā wahine i loaʻa i ka maʻi. ʻAʻole hoʻoulu ka 20E i loaʻa i ka wahine i kahi moekolohe pane, akā ʻae i nā kānaka male e waiho i nā hua ma hope o ka pale ʻia ʻana o nā kinases 20E-inhibiting. ʻO ka ʻike ʻana o kēia hormone steroid pepeke kāne-kikoʻī a me kāna kuleana i ka hoʻoponopono ʻana i ka ʻae ʻana i ka moekolohe wahine, ka momona a me ka launa pū ʻana me Plasmodium e hōʻike ana i ka hiki ke hōʻemi i ka holomua hānau o nā makika e hoʻoili ana i ka malaria.
Ke piʻi hou nei nā hihia malaria a me nā make4 ma muli o ke kūʻē ʻana i nā pepeke pepeke i nā makika Anopheles, ʻo ia wale nō ka mea lawe i nā parasite malaria kanaka. ʻO ke ʻano o ka male ʻana o kēia mau makika he pahuhopu hoihoi loa ia no nā hana hoʻomalu malaria hou no ka mea hoʻokahi wale nō ka male ʻana o nā wahine5; ʻo ka hana ʻana i kēia hanana male hoʻokahi e maʻemaʻe loa he hiki nui ia e hōʻemi i ka heluna makika ma ke kula.
Ua lilo nā wāhine i mea hiki ʻole ke moe kolohe ma hope o ka loaʻa ʻana o nā hormones steroid kiʻekiʻe-titer mai nā kāne. Ua hōʻike ʻia nā haʻawina ʻo ke kumu no ka paʻakikī i ka male hou ʻana ʻo 20-hydroxyecdysone (20E), kahi hormone steroid i ʻike nui ʻia ʻo ia ka mea hoʻoponopono o ke kaʻina molting i ke kahua larval. Ua ulu pono ka hiki o nā kāne ke synthesize a hoʻoili i ka 20E i nā ʻano Anopheles ʻo ia kekahi o ka subgenus Cellia7, kahi i hoʻolaha ʻia ma ʻApelika a me nā mea hoʻoili malaria weliweli loa, me Anopheles gambiae. He mea koʻikoʻi kēia no ka mea i loko o kēia mau ʻano, hana pū nā wahine i ka 20E ma hope o kēlā me kēia ʻai koko, a hoʻokele ʻo 20E i ke kaʻina oogenesis (e ʻike i ka ref. 8). Eia naʻe, ʻaʻole ʻike nui ʻia e pili ana i ke ʻano o ka hoʻohui ʻana o nā wahine i nā hōʻailona mai ʻelua mau kumu like ʻole o ka ecdysone (ka hoʻoili kāne a me ka hoʻoulu ʻana i ka hānai koko) me ka ʻole o ka hoʻopilikia ʻana i ko lākou hiki ke moe. ʻO ka ʻoiaʻiʻo, inā hoʻoulu ka 20E i hana ʻia e nā wahine i ka hoʻomanawanui ʻole i ka moekolohe, e alakaʻi kēia i ka hānau ʻole ʻana i nā poʻe hānai puʻupaʻa, kahi ʻano maʻamau i kēia mau makika5.
ʻO kahi wehewehe kūpono paha, ʻo ia ka hoʻoili ʻana o nā kāne A. gambiae i kahi ecdysone kāne i hoʻololi ʻia, kahi e hoʻoulu ai i kahi cascade hōʻailona i loko o ke ala hānau wahine, e hopena ana i ka paʻa ʻole o ka male ʻana. Eia nō naʻe, ʻoiai he nui nā hormones steroid o nā vertebrates, e like me ka estrogen a me ka androgen (i loiloi ʻia ma ref. 9), i ko mākou ʻike, ʻaʻole i ʻike ʻia nā steroid androgenic-biased i nā pepeke.
Ua hoʻoholo mākou e hoʻoholo i ka papa inoa o nā hormones steroid i loko o ke kelepa kāne kāne (MAG) o A. gambiae i oʻo i ka moekolohe i ka ʻimi ʻana i nā steroid hoʻololi hiki. Ma ka hoʻohana ʻana i ka chromatography wai hana kiʻekiʻe i hui pū ʻia me ka tandem mass spectrometry (HPLC-MS/MS) ma mua o ke ʻano hana i hoʻohana ʻole ʻia ma mua, ua ʻike mākou i ka ecdysone (E) a me 20E i loko o kēia ʻiʻo, e hōʻoia ana i ka hopena ma mua. Eia naʻe, ua noho aliʻi ʻia ka laʻana e nā steroids phosphorylated oxidized, e kūlike me ke ʻano 3-dehydro-20E-22-phosphate (3D20E22P)12 (Kiʻi 1). ʻO nā ʻano ʻē aʻe e pili ana i ka 3-dehydro-20E (3D20E) a me 20E-22-phosphate (20E22P). ʻO ka ikaika hōʻailona HPLC-MS/MS o 3D20E22P he ʻelua kauoha o ka nui ma mua o kona ʻano dephosphorylated, 3D20E, a ʻekolu kauoha o ka nui ma mua o E a me 20E (Kiʻi 1). ʻOiai ma nā ʻāpana ʻē aʻe o ke kino. a me ke ala hānau haʻahaʻa (LRT; Extended Data Fig. 1a). Ua kālailai pū mākou i nā ecdysteroids i nā kāne a me nā wahine i pani hou ʻia (<1 lā) a ua ʻike ʻia ʻo 3D20E a me 3D20E22P wale nō ma MAG; Loaʻa ʻo E, 20E a me 20E22P i nā kāne ʻelua (Extended Data Fig. 1b). Hōʻike kēia mau ʻikepili e hana ana nā kāne makua ʻo A. gambiae i nā titers kiʻekiʻe o nā hormones hoʻololi i kā lākou MAGs ʻaʻole i synthesized ʻia e nā wahine.
Ua ʻoki ʻia ʻo MAG a me ka wahine LRT (me ka atria, nā vesicles seminal, a me ka parovarium) mai nā kāne puʻupaʻa 4 lā (4 lā) a me nā wahine puʻupaʻa a me nā wahine i hoʻopili ʻia (0.5, 3, a me 12 hpm). Ua kālailai ʻia ʻo Ecdysone i loko o kēia mau ʻiʻo e HPLC-MS/MS (mean ± sem; unpaired t-test, ʻelua ʻaoʻao, false discovery rate (FDR) i hoʻoponopono ʻia; NS, ʻaʻole koʻikoʻi; *P < 0.05, **P < 0.01 . 3D20E: 3 hola vs. 0.5 hola, P = 0.035; 12 hola vs. 3 hola, P = 0.0015; 12 hola vs. 0.5 hola, P = 0.030. 3D20E22P: 3 hola vs. 0.5 hola, P = 0.25; 12 hola vs. 3 hola, P = 0.0032; 12 hola vs. 0.5 hola, P = 0.015). Mai ʻekolu mau replicates olaola nā ʻikepili. Ua helu ʻia a ua hoʻohālikelike ʻia ka wahi kiʻekiʻe no kēlā me kēia ecdysone hoihoi e ka helu o nā makika. Hōʻike ʻia ʻo Ecdysone e ke kala penei: E, ʻōmaʻomaʻo; 20E, ʻalani; 20E22P, poni; 3D20E, uliuli; 3D20E22P, poni. Hoʻonui ka inset i ka unahi ma ka y-axis e hōʻike i nā pae ecdysone haʻahaʻa.
No ka noiʻi ʻana inā ua hoʻoili ʻia ʻo 3D20E22P a me 3D20E i ka wā o ka male ʻana, ua ʻoki mākou i nā LRT wahine i nā manawa like ʻole ma hope o ka male ʻana. ʻOiai ʻaʻole i loaʻa ka ecdysone i nā wahine puʻupaʻa, ua ʻike mākou i nā nui nui o 3D20E22P i loko o ka LRT ma hope koke iho o ka male ʻana (0.5 h post-mating, hpm), e emi ana i ka hala ʻana o ka manawa, ʻoiai ua piʻi nui nā pae 3D20E (Kiʻi 1). Ma ka hoʻohana ʻana i ka 3D20E i hana ʻia me ka kemika ma ke ʻano he maʻamau, ua hoʻoholo mākou he 100 mau manawa ke kiʻekiʻe o nā pae o kēia hormone steroid i nā LRT male ʻana ma mua o 20E (Extended Data Table 1). No laila, ʻo 3D20E22P ka ecdysone kāne nui i hoʻoili ʻia i ka LRT wahine i ka wā o ka male ʻana, a ʻo kona ʻano dephosphorylated, 3D20E, ua lilo i mea nui loa ma hope koke iho o ka male ʻana. Hōʻike kēia i kahi kuleana koʻikoʻi no ka ecdysone hope loa i ka biology wahine post-mating.
Ma hope o ka hoʻokumu ʻana i kahi ʻikepili RNA sequencing (RNA-seq) hou (Kiʻi 2a), me ka hoʻohana ʻana i kahi pipeline bioinformatics i kūkulu ʻia, ua ʻimi mākou i ka ecdysone kinase (EcK), ecdysone oxidase (EO), a me ka ecdysone encoding 20E-modified phosphatase gene. Ua hōʻike ʻia ka EPP) i loko o nā ʻiʻo hānau. Ua ʻike mākou i hoʻokahi gene EPP moho a me ʻelua mau gene EcK hiki (EcK1 a me EcK2), akā ʻaʻole hiki ke loaʻa kahi gene EO moho maikaʻi. ʻIke ʻia, ua hōʻike ʻia nā gene EPP pākahi ma nā pae kiʻekiʻe (98.9th percentile) ma Gambian MAGs akā ʻaʻole ma nā LRT wahine (Kiʻi 2b), kūʻē i kā mākou mau manaʻolana mai ka wā i hana ʻia ai ka dephosphorylation o 3D20E22P i loko o kēia ʻiʻo wahine. No laila, ke manaʻoʻiʻo nei mākou e hoʻoili ʻia paha ke kāne EPP i ka wā o ka male ʻana. ʻOiaʻiʻo, ua hoʻohana mākou i ka lepili isotope paʻa in vivo e uhi i ka protein wahine ma hope o ka male ʻana, kahi enzyme i ʻike ʻia e MS ma ka atrium wahine (Kiʻi 2c a me ka Papa Hoʻohui 1). ʻO ke alo o Ua hōʻoia pū ʻia ʻo EPP ma MAG a me ka wahine LRT i hoʻopili ʻia (akā ʻaʻole puʻupaʻa) me ka hoʻohana ʻana i nā antibodies kikoʻī (Kiʻi 2d).
a, He pipeline bioinformatics i kūkulu ʻia e ʻimi i nā ʻiʻo hānau o kēlā me kēia kāne no nā genes e hoʻopili ana iā EcKs, EOs, a me EPPs. Hōʻike nā helu ma ka ʻaoʻao o nā pua i ka helu o nā moho kāne a me nā wahine ma kēlā me kēia ʻanuʻu. Ua ʻike kēia loiloi i hoʻokahi gene EPP (EPP) a me hoʻokahi gene EcK (EcK1) i hōʻike ʻia i nā kāne, a me hoʻokahi gene EcK (EcK2) i hōʻike ʻia i nā kāne ʻelua akā ʻaʻole i hāʻawi i kahi gene moho EO. b, Heatmap e hoʻohālikelike ana i ka hōʻike ʻana o ka gene moho i nā ʻiʻo puʻupaʻa (V) a me ka male ʻana (M) Anopheles gambiae a me Anopheles albicans. Spca, fertilization; MAGs, nā glands kōkua i nā kāne; nā ʻāpana ʻē aʻe o ke kino, me nā umauma, nā ʻēheu, nā wāwae, nā kino momona, a me nā ʻōpū o loko i nā kāne ʻelua, a me nā ovaries i nā wahine. Ua hōʻike nui ʻia ʻo EcK2 ma MAG a me atria o Gambia, ʻoiai ʻo EPP i loaʻa wale ma MAG.c, Proteomic analysis o ka translocation hui ejaculate kāne i loko o ka atria wahine ma 3, 12 a me 24 hpm, e hōʻike ana i nā protein 67 nui loa. Ua hānai ʻia nā wahine ma kahi ʻai e loaʻa ana he 15N e lepili (a uhi) i nā protein āpau. Ua hui pū ʻia nā kāne i hōʻailona ʻole ʻia me nā wahine i hōʻailona ʻia, a ua ʻoki ʻia nā LRT wahine ma 3, 12 a me 24 hpm no ka nānā ʻana i ka proteomic (e ʻike i ka Papa Hoʻohui 1 no ka papa inoa piha o nā protein ejaculatory). Ua ʻike ʻia ʻo Inset, EPP, Eck1 a me EcK2 ma ka MAG o nā kāne puʻupaʻa ma o ka nānā ʻana i ka proteomic o kēia mau ʻiʻo.d, Ua ʻike ʻia ʻo EPP e ka blot komohana ma MAG a me LRT o nā wahine i hui pū ʻia, akā ʻaʻole i nā wahine puʻupaʻa a i ʻole nā ​​​​kāne a i ʻole ke koena o ke kino wahine. Ua hui pū ʻia nā membranes ua hoʻāʻo ʻia me nā anti-actin (kaohi hoʻouka) a me nā antibodies anti-EPP. He mau puʻupaʻa nā kāne āpau. E nānā i ke Kiʻi Hoʻohui 1 no ka ʻikepili kumu gel. Ua hana ʻia nā blots Komohana i ʻelua manawa me nā hopena like.
Ua hōʻoia ʻia ka hana ecdysteroid phosphophosphatase o EPP ma hope o ka hoʻoulu ʻia ʻana e HPLC-MS/MS me 3D20E22P i hoʻokaʻawale ʻia mai MAG (Extended Data Fig. 2a). Eia kekahi, i ka wā i hoʻopau ai mākou i ka EPP ma o ka RNA-mediated interference (RNAi), ua ʻike mākou i ka emi nui ʻana o ka hana phosphatase i loko o nā ʻiʻo hānau o kēia mau kāne (Fig. 3a), a ʻo nā wahine i hui pū ʻia me nā kāne i hoʻopau ʻia e EPP ua hōʻike i ka hapa haʻahaʻa o A o ka dephosphorylated 3D20E (Fig. 3b) ʻoiai ka hoʻopau hapa ʻana o ka gene (Extended Data Fig. 2b,c). I ka hoʻohālikelike ʻana, ʻaʻole mākou i ʻike i nā loli koʻikoʻi i ka ratio 20E22P/20E i nā makika like, kahi e hōʻike ai he kikoʻī ka enzyme no 3D20E22P (Fig. 3b).
a, Ua emi ka hana phosphatase ma MAG i hoʻokumu ʻia e ka hoʻopau ʻana o ka EPP me ka hoʻohana ʻana i ka EPP RNA pālua (dsEPP) a i ʻole nā ​​​​mana GFP RNA pālua (dsGFP). Ua hoʻohana ʻia he iwakālua mau loko MAG i kēlā me kēia replicate (P = 0.0046, paired t-test, ʻelua ʻaoʻao), i hōʻike ʻia e nā kiko kaʻawale. b, ʻO nā wahine i hui pū ʻia me nā kāne i hoʻopau ʻia e EPP he haʻahaʻa loa ka hapa o ka dephosphorylated 3D20E ma 3 hpm (P = 0.0043, unpaired t-test, ʻelua ʻaoʻao), ʻoiai ʻaʻole i hoʻopilikia ʻia nā pae 20E (P = 0.063, unpaired). hoʻāʻo-t, ʻaoʻao ʻelua). Hōʻike ʻia nā ʻikepili ma ke ʻano he awelika ± sem mai ʻekolu mau loko o 13, 16 a me 19 mau wahine. c, ʻO nā wahine i moe me nā kāne i hoʻopau ʻia e EPP he kiʻekiʻe loa ka nui o ka moe hou ʻana (P = 0.0002, hoʻāʻo pololei a Fisher, ʻaoʻao ʻelua). Ua koi mua ʻia nā wahine e moe e hōʻoia i ko lākou kūlana moe; 2 mau lā ma hope mai, ua hoʻokaʻaʻike ʻia lākou me nā kāne ʻē aʻe e lawe ana i ka sperm transgenic e loiloi i nā helu hoʻopili hou ʻana ma o ka ʻike PCR quantitative o ka transgene.d, ʻO nā wahine hānai koko i hui pū ʻia me nā kāne i hoʻopau ʻia e EPP ua emi nui ka momona (P < 0.0001; hoʻāʻo Mann-Whitney, ʻelua ʻaoʻao) a ua emi iki ka helu hua (P = 0.088, hoʻāʻo Mann-Whitney, ʻelua ʻaoʻao), ʻoiai ʻaʻole i hoʻopilikia ʻia ka nui o ka spawning (P = 0.94, hoʻāʻo pololei a Fisher, ʻelua ʻaoʻao). Ma nā panela āpau, hōʻike ʻo n i ka helu o nā laʻana makika kūʻokoʻa biologically.NS, ʻaʻole koʻikoʻi.*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.001.
ʻO ka mea aʻe, ua loiloi mākou inā he mea nui ka ecdysone dephosphorylation no ka hoʻoulu ʻana i ke kūʻē ʻana i ka male ʻana i nā wahine. ʻO ka mea nui, ʻo nā wahine i moe me nā kāne i hoʻopau ʻia e ka EPP ua moe hou i kahi alapine kiʻekiʻe loa (44.9%) ma mua o nā wahine kaohi (10.4%) i ka wā i hōʻike ʻia ai i nā kāne (transgenic) hou (Kiʻi 3c). Ua ʻike pū mākou i ka emi nui ʻana o ka momona (Kiʻi 3d, hema) a me ka emi iki ʻana o ka nui o nā hua i waiho ʻia e kēia mau wahine (Kiʻi 3d, waena), ʻoiai ʻaʻole i hoʻopilikia ʻia ka pakeneka o nā hua i waiho ʻia e nā wahine (ʻo kekahi pane i hoʻāla ʻia i nā wahine ma o ka male ʻana)) (Kiʻi 3d, ʻākau). Hāʻawi ʻia i ka ʻike kikoʻī o EPP no 3D20E22P, hōʻike kēia mau hopena e hiki i ka hoʻoulu ʻana o 3D20E e ka EPP i hoʻoili ʻia i ka wā o ka male ʻana ke loaʻa kahi kuleana koʻikoʻi i ka hoʻopau ʻana i ka ʻae ʻana o ka wahine i ka male hou ʻana, kahi ʻano i hāʻawi mua ʻia i ka hoʻoili moekolohe o 20E. No laila, hoʻopilikia nui kēia hormone kāne i ka momona o ka wahine.
ʻO ka mea aʻe, ua hoʻohālikelike mākou i nā hana o 20E a me 3D20E i nā hoʻokolohua injection i nā wahine puʻupaʻa i oʻo i ka moekolohe me ka hoʻohana ʻana i ka 3D20E i hana ʻia me ka kemika (Kiʻi 4a-c) a me 20E i loaʻa i ke kālepa. Ua ʻike mākou he ʻoi aku ka maikaʻi o 3D20E ma mua o 20E i ka pani ʻana i ka ʻike o ka wahine i ka male ʻana ma nā ʻano ʻelua (Kiʻi 4d). ʻO ka mea nui, ʻo ka hapalua o ka pae physiological o 3D20E ma ka LRT (1,066 pg post-injection vs. 2,022 pg post-mating) i hoʻoulu i kahi hapa o nā wahine refractory i 20-fold ʻoi aku ka kiʻekiʻe ma mua o ka pae physiological o 20E (361 pg post-injection) 24 mau hola ma hope o ka injection ma ke kiʻekiʻe loa o 18 pg post-mating; Papa ʻIkepili Hoʻonui 1). Kūlike kēia hopena me ka manaʻo ʻaʻole hiki i ka hoʻoili moekolohe o 20E ke kumu o nā wā paʻakikī o ka male ʻana, a kuhikuhi hou aku iā 3D20E ma ke ʻano he kumu nui i ka hōʻoia ʻana i ka pilina makua-keiki. Ua ʻoi aku ka ikaika o 3D20E ma mua o 20E i nā hoʻāʻo hoʻomoe hua manu i nā wahine puʻupaʻa (Kiʻi 4e), e hōʻike ana i ka helu maʻamau o ka hoʻomoe hua manu a mākou i ʻike ai ma hope o ka hoʻomaha ʻana o ka EPP hapa ma muli o ke alo o ka hana 3D20E i koe i hana ʻia e nā mea wahine i hoʻokomo ʻia e ka Mating.
(a,b) 3D20E i hana kemika ʻia mai 20E (a) me ka hoʻololi/pono kiʻekiʻe loa (ʻikepili i hōʻike ʻia ma ke ʻano he awelika ± sem mai ʻekolu mau hopena synthesis kūʻokoʻa) (b).c, ʻO ka spectrum Mass (hapa lalo) e kūlike pono me ka ecdysone i loaʻa i ka LRT wahine i hoʻopili ʻia (hapa luna).d, Hoʻohālikelike ʻia me 20E (0.63 µg, P = 0.02; 0.21 µg, P < 0.0001; Hoʻāʻo pololei a Fisher, ʻelua ʻaoʻao) a me 10% ethanol (0.63 µg, P < 0.0001; 0.21 µg, P < 0.0001; Hoʻāʻo pololei a Fisher, 2-ʻaoʻao), ʻoiai ʻoi aku ka kiʻekiʻe o 20E ma mua o ka mana ma nā dosis kiʻekiʻe wale nō (0.63 µg, P = 0.0002; 0.21 µg, P = 0.54; Hoʻāʻo pololei a Fisher, 2-ʻaoʻao).e, ʻO ka hoʻokomo ʻana o 3D20E i hoʻoulu ai i nā helu hānau kiʻekiʻe loa i nā wahine puʻupaʻa ma mua o 10% mau kaohi ethanol (0.21 µg, P < 0.0001; 0.13 µg, P = 0.0003; Hoʻāʻo pololei a Fisher, ʻelua ʻaoʻao), ʻoiai ʻo 20E i hoʻohālikelike ʻia me nā kaohi wale nō ma nā dosis kiʻekiʻe (0.21 µg, P = 0.022; 0.13 µg, P = 0.0823; Hoʻāʻo pololei a Fisher, ʻelua ʻaoʻao). Ua hoʻoulu ʻo 3D20E i nā helu hānau kiʻekiʻe loa ma mua o 20E ma nā dosis kiʻekiʻe (0.21 µg, P = 0.0019; 0.13 µg, P = 0.075; Hoʻāʻo pololei a Fisher, ʻelua ʻaoʻao). Ma nā panela āpau, hōʻike ʻo n i ka helu o nā laʻana makika kūʻokoʻa biologically.NS, ʻaʻole koʻikoʻi.*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.001. Mai nā kope ʻekolu nā ʻikepili.
Ma nā haʻawina i hala, ua hoʻoholo mākou ʻo ka hoʻoili moekolohe o nā hormones steroid e hoʻoulu ai i ka hōʻike ʻana o MISO (Mating-Induced Stimulator of Oogenesis 11), kahi gene hānau wahine e pale ana i nā wahine A. gambiae mai ka maʻi P. falciparum. Nā kumukūʻai olakino i hoʻokumu ʻia e 13, ka parasite malaria kanaka make loa. Ma muli o ke koʻikoʻi o MISO i ke olakino hānau o Anopheles ma nā wahi malaria-endemic, ua hoʻoholo mākou e hoʻoholo i ka hormone 3D20E a i ʻole 20E e hoʻoulu ai i ka hōʻike ʻana o kēia gene. Ua ʻike mākou ʻoiai ʻo ka injection 20E i hoʻoulu kūikawā a ʻoi aku paha i kekahi mau receptors hormone nuklea (HR), e like me HR3 a me HR4, a me nā pahuhopu steroid maʻamau, e like me nā genes yolkogenic Vg14, 15, 16, ʻoi aku ka ikaika o MISO i hoʻoulu ʻia e 3D20E (Extended Data Fig. 3). No laila, ʻo ka hoʻoili moekolohe o kēia hormone steroid androgenic e ʻike ʻia e hoʻoulu i nā ʻano hana e pale ai i nā wahine mai nā kumukūʻai i kau ʻia e ka maʻi parasitic. Eia kekahi, hoʻopilikia like ʻole ʻo 3D20E i nā isoforms ʻelua o ka ʻO ka mea hoʻokipa E EcR, e hoʻoulu ai iā EcR-A a kāohi iā EcR-B, a ʻoi aku ka ikaika o ka hoʻoulu ʻana i nā genes ʻē aʻe e hoʻoulu ai i ka male ʻana, me HPX15, ka mea e hoʻopilikia ai i ka momona wahine. Hiki i kēia ke wehewehe i ka hānau ʻole nui i ʻike ʻia i nā wahine i moe ʻia me nā kāne i hoʻopau ʻia e EPP (Extended Data Fig. 3). Hōʻike kēia mau ʻikepili i ke ola ʻana o nā ala ma lalo i hoʻāla ʻia e ʻelua mau hormones ecdysone e hiki ke hoʻokumu i ka hana kikoʻī o ka wahine.
ʻO ka mea aʻe, ua hoʻāʻo mākou i ka hana o nā ʻano ʻelua EcK i ʻike ʻia i loko o kā mākou pipeline bioinformatics. ʻO ka hoʻopau ʻana iā EcK1 a i ʻole EcK2 ua hopena i ka make nui i nā kāne (Extended Data Fig. 4a), e hōʻike ana he mea nui ka phosphorylation ecdysone, a no laila ka inactivation, no ke ola. No ka mea, ua hōʻike ʻia ʻo EcK2 ma nā pae kiʻekiʻe ma mua o EcK1 a ua ʻike ʻia i loko o MAGs e proteomics (Fig. 2b,c a me ka Papa Hoʻohui 2), ua hōʻoia mākou i kāna hana ecdysteroid kinase ma ka hoʻoulu ʻana me 20E, kahi i hopena ai i ka phosphorylation 20E22P (Extended Data Figure 2).4b). I ka hoʻohana ʻana iā 3D20E ma ke ʻano he substrate, ʻaʻole hiki iā mākou ke ʻike i ka huahana phosphorylated 3D20E22P (Extended Data Fig. 4c), e hōʻike ana he 20E ma mua o 3D20E paha ke pahuhopu makemake ʻia o EcK2.
Wahi a kā mākou loiloi RNA-seq, ua hōʻike nui ʻia ʻo EcK2 ma ka LRT o nā wahine puʻupaʻa, kahi i pio ai ma hope o ka male ʻana (Kiʻi 2b). Ua hōʻoia mākou i kēia mau ʻikepili a ua hoʻoholo ʻaʻole i hoʻopilikia ʻia ka hōʻike ʻana o EcK2 e ka hānai koko (Extended Data Fig. 5a). Ma ka hoʻonui ʻana i kā mākou mau hoʻokolohua MS mua, ua hoʻoholo mākou ua pili loa ka piko o 20E22P i ka piko o 20E (22-26 mau hola ma hope o ka ʻai ʻana i ke koko; Extended Data Fig. 5b). ʻO ka hoʻopau ʻana o EcK2 i nā wahine puʻupaʻa ua hopena i ka hoʻonui ʻana o 3-fold i ka lakio pili o 20E a i 20E22P ma 26 h ma hope o ka ʻai ʻana i ke koko (Extended Data Figures 2c a me 5c), e hōʻoia ana ua hoʻopili pū ʻo EcK2 i ka 20E i nā wahine. ʻO ka mea nui, ua mālama nā wahine puʻupaʻa i hoʻopau ʻia e EcK2 i ka ʻae piha ʻana i ka moekolohe (Extended Data Fig. 5d, e), e hōʻike hou ana ʻaʻole e hoʻoulu ka hana wahine o 20E i nā wā paʻakikī o ka male ʻana. Eia naʻe, ʻo kēia mau wahine ua hoʻonui nui ʻia ka nui o ka hoʻomoe hua manu i hoʻohālikelike ʻia me nā kaohi, me ka ʻoi aku ma mua o 30% o nā wahine puʻupaʻa e hoʻomoe ana i nā hua (Extended Data Fig. 5f). Inā ua hana ʻia nā inikini Eck2 RNA pālua (dsEcK2) ma hope o ka hānai ʻana i ke koko, ʻaʻole i hana ʻia ka spawning, ma ia manawa ua emi ka piko 20E ma muli o ka ʻai ʻana i ke koko. Ma keʻano holoʻokoʻa, kākoʻo kēia mau hopena i kahi kumu hoʻohālike i hana ʻia e 20E ma hope o ka omo ʻana i ke koko hiki ke hoʻoulu i ka spawning, akā i ka wā e pio ai ka poloka spawning (EcK2 a me nā mea ʻē aʻe paha) e ka male ʻana. ʻAʻole i kāohi nā inikini 20E a i ʻole 3D20E i ka hōʻike ʻana o EcK2 i nā wahine puʻupaʻa (Extended Data Fig. 5g), e hōʻike ana he mau mea ʻē aʻe e hoʻoponopono i ka pale ʻana o kēia kinase. Eia nō naʻe, ʻaʻole lawa nā pae 20E ma hope o ka hānai ʻana i ke koko e hoʻoulu ai i ka ʻoluʻolu ʻole o ka male ʻana, akā ua hoʻoulu maikaʻi ʻia e nā titers kiʻekiʻe o ka 3D20E i hoʻoili ʻia i ka moekolohe.
Hāʻawi kā mākou mau hopena i nā ʻike koʻikoʻi i nā ʻano hana e hoʻoponopono ana i ka holomua o ka hānau ʻana o A. gambiae. Ua puka mai kahi kumu hoʻohālike kahi i ulu ai nā kāne e synthesize i nā titers kiʻekiʻe o 3D20E, kahi ecdysone i hoʻololi ʻia e ke kāne e hōʻoiaʻiʻo ana i ka makua ma ka desensitizing wahine i ka male hou ʻana. I ka manawa like, ua hoʻomohala pū kēia mau vectors malaria i kahi ʻōnaehana kūpono e hoʻāla i ka 3D20E i nā wahine ma ke ʻano he pane i ka hoʻoili moekolohe o ka EPP kāne. I ko mākou ʻike, ʻo ia ka laʻana mua o kahi ʻōnaehana hormone steroid i hoʻomalu ʻia e ke kāne a me ka wahine e hana ana i kahi hana kū hoʻokahi a koʻikoʻi i nā pepeke. Ua hoʻolaha ʻia ka hana ecdysone kāne akā ʻaʻole i hōʻike maopopo ʻia. No ka laʻana, ʻo kahi kuhiakau i hōʻole nui ʻia 18 ʻo ia ka hana ʻia ʻana o kēia mau hana e ka 20E precursor E1. Ua ʻike maikaʻi ʻia ma Drosophila, hoʻāla ʻia ka monandry e ka hoʻoili moekolohe o nā peptides wahine liʻiliʻi19,20 e launa pū me nā neurons e hoʻopili ana i ka ʻōnaehana hānau wahine ma o nā mea loaʻa peptide wahine kikoʻī21,22. Pono ka hana hou e hoʻoholo i ka hōʻailona ma lalo nā kahe i kāohi ʻia e 3D20E i nā wahine A. gambiae a no ka hoʻoholo ʻana inā hiki ke mālama ʻia kēia mau kahe ma waena o nā makika a me Drosophila.
Ma muli o ke kuleana koʻikoʻi o 3D20E ma ka momona wahine a me ke ʻano i ʻike ʻia i kā mākou noiʻi, hāʻawi nā ala e alakaʻi ana i ka synthesis a me ka hoʻāla ʻana o 3D20E i nā manawa hou no nā hoʻolālā kaohi namu e hiki mai ana, e like me ka hanauna o nā kāne sterile hoʻokūkū i nā hoʻolālā ʻenehana pepeke sterile E hoʻohana no ka hoʻokuʻu hihiu a i ʻole e hoʻohālike i ka 3D20E i ka pāʻani puʻupaʻa. Ua ulu paha ka hana kāne-kikoʻī o 3D20E i ka wā i loaʻa ai iā A. gambiae a me nā ʻano Cellia ʻē aʻe ka hiki ke hoʻohui i kā lākou semen i loko o nā plugs mating, no ka mea e ʻae ai kēia i ka hoʻoili pono ʻana o ka nui o nā hormones a me nā enzymes hoʻāla hormone. I ka huli ʻana, ʻo ka hoʻomohala 3D20E e hoʻokō ana i ka monandry e hāʻawi i kahi ʻano no nā wahine (ma o ka hōʻike kiʻekiʻe o MISO) e makemake i ko lākou kūpono hānau ma nā wahi o ka nui o ka malaria, kahi e hāʻawi pololei ʻole ai i ka hoʻoili ʻana o Plasmodium. Ma muli o ka hōʻike ʻia ʻana o ka wahine 20E he hopena koʻikoʻi i ke ola a me ka ulu ʻana o P. falciparum i nā namu wahine Anopheles,24 ʻo nā ala hormone steroid kāne a me ka wahine i kēia manawa he mau ʻano koʻikoʻi o ka pilina namu-parasite.
Ua hānai ʻia nā ʻano A. gambiae G3 ma lalo o nā kūlana pepeke maʻamau (26-28 °C, 65-80% ka haʻahaʻa pili, 12:12 h photoperiod mālamalama/pouli). Ua hānai ʻia nā larvae me ka meaʻai iʻa pauka (TetraMin Tropical Flakes, Koi Pellets a me Tetra Pond Sticks ma ka lakio o 7:7:2). Ua hānai ʻia nā makika makua me ka 10% dextrose solution ad libitum a me ke koko kanaka i kēlā me kēia pule (e aʻo i nā ʻāpana koko). Ua loaʻa nā makika puʻupaʻa ma ka hoʻokaʻawale ʻana i nā kāne ma ke kahua pupal ma hope o ka nānā ʻana i nā wēlau e ka microscopy. Ua wehewehe mua ʻia nā kāne e lawe ana i ka transgene DsRed.
Ua hana ʻia nā hoʻokolohua hoʻoikaika male e like me nā kaʻina hana i wehewehe mua ʻia. No ka male kūlohelohe, ua mālama ʻia nā wahine puʻupaʻa 4 lā i ka lakio 1:3 me nā kāne puʻupaʻa makua no ʻelua pō. No nā hoʻokolohua i hoʻokomo ʻia ai nā kāne me dsEPP, ua kūlike ka co-caging me nā lā 3-4 ma hope o ka hoʻokomo ʻana, i ka wā i hoʻopau loa ʻia ai ka hana phosphatase (Extended Data Fig. 2b).
Ua ʻoki ʻia nā ʻiʻo makika, nā kupapaʻu i koe (koena o ke kino), a i ʻole ke kino holoʻokoʻa i loko o 100% methanol a ua homogenized me kahi beader (2 mm glass beads, 2,400 rpm, 90 kec). ʻO ka nui o nā ʻiʻo a me nā nui o ka methanol penei: ke koena o ke kino, 50 i loko o 1,000 µl; MAG, 50–100 80 µl; wahine LRT, 25–50 80 µl. Ua hoʻokomo ʻia ka precipitate i ka lua o ka methanol extraction me ka nui like o ka methanol. Ua wehe ʻia nā ʻōpala cell ma o ka centrifugation. Ua hui pū ʻia ka methanol mai nā extractions ʻelua a hoʻomaloʻo ʻia ma lalo o ke kahe nitrogen, a laila hoʻokuʻu hou ʻia i loko o nā nui aʻe o 80% methanol i loko o ka wai: ke koena o ke kino, 50 µl; MAGs a me ka wahine LRT, 30 µl.
Ua kālailai ʻia nā laʻana ma kahi spectrometer mass (ID-X, Thermo Fisher) i hoʻopili ʻia me kahi mea hana LC (Vanquish, Thermo Fisher). Ua hoʻokomo ʻia he 5 µl o ka laʻana ma luna o kahi kolamu 3 µm, 100 × 4.6 mm (Inspire C8, Dikma) i mālama ʻia ma 25 °C. ʻO nā pae mobile no ka LC ʻo A (wai, 0.1% formic acid) a me B (acetonitrile, 0.1% formic acid). Penei ke ʻano o ka gradient LC: 5% B no 1 minuke, a laila hoʻonui ʻia i 100% B ma luna o 11 mau minuke. Ma hope o 8 mau minuke ma 100%, e hoʻoponopono hou i ke kolamu ma 5% B no 4 mau minuke. ʻO ka nui o ke kahe ʻana he 0.3 ml min-1. Hoʻokō ʻia ka ionization i loko o ke kumu MS e ka ionization electrospray wela i nā ʻano maikaʻi a me nā ʻano maikaʻi ʻole.
Hoʻohana ka spectrometer mass i ka ʻikepili ma ka pae m/z mai 350 a 680 ma ka hoʻonā 60,000 ma ke ʻano MS piha. Ua loaʻa nā ʻikepili MS/MS ma [M + H]+ (nā pahuhopu āpau), [M - H2O + H]+ (nā pahuhopu āpau), a me [M - H]- (nā pahuhopu phosphorylated). Ua hoʻohana ʻia nā ʻikepili MS/MS e hōʻoia i nā waiwai ecdysone o nā pahuhopu i loaʻa ʻole ai ke kūlana maʻamau. No ka ʻike ʻana i nā ecdysteroids i hoʻopaʻa ʻole ʻia, ua kālailai ʻia nā ʻikepili MS/MS no nā piko HPLC āpau me ka nui o >15%. E helu me ka hoʻohana ʻana i nā piʻo maʻamau i hana ʻia mai nā kūlana maʻemaʻe (20E, 3D20E) e helu i nā nui a i ʻole nā ​​​​dilutions o hoʻokahi laʻana kikoʻī (nā pahuhopu ʻē aʻe āpau) e helu i ko lākou kūlike me nā nui i loaʻa i hoʻokahi kāne. No 3D20E, ua hana ʻia ka helu ʻana me ka hoʻohana ʻana i ka huina o kēia mau adducts: [M + TFA]-, [M + COOH]-, [M + Na]+, [M + Cl]-, [M + NO3]-. Ua unuhi ʻia a helu ʻia ka ʻikepili me ka hoʻohana ʻana iā Tracefinder (mana 4.1). Ua kālailai ʻia ka ʻikepili MS/MS me ka hoʻohana ʻana iā Xcalibur (mana 4.4). Ua hoʻohālikelike ʻia nā spectra MS o E, 20E a me 3D20E i nā kūlana like ʻole. Ua kālailai ʻia ʻo 3D20E22P ma o ka derivatization me ka reagent a Girard. Ua kālailai ʻia ʻo 20E22P e ka lakio m/z.
Ua hoʻomaʻemaʻe ʻia ʻo 3D20E22P mai MAG. Ua hana ʻia ka hoʻomaʻemaʻe ʻana ma kahi unahi analytical me ka hoʻohana ʻana i kahi chromatograph wai ultra-performance (Acquity, Waters) me kahi mea ʻike quadrupole mass-based (QDa, Acquity, Waters) ma lalo o nā kūlana LC like me ka loiloi HPLC-MS/MS. Ua hoʻāla ʻia ka hōʻiliʻili ʻāpana i ka wā i ʻike ʻia ai ka m/z e pili ana iā 3D20E22P i ka manawa paʻa like e like me ka mea i hoʻoholo mua ʻia. A laila ua nānā ʻia ka maʻemaʻe o nā hui i unuhi ʻia e HPLC-MS/MS e like me ka mea i wehewehe ʻia ma luna.
Ua unuhi ʻia ka RNA holoʻokoʻa mai 10-12 mau ʻiʻo hānau a i ʻole nā ​​​​​​ʻāpana ʻē aʻe o ke kino (ʻaʻohe poʻo) me ka hoʻohana ʻana i ka TRI reagent (Thermo Fisher) ma hope o nā kuhikuhi a ka mea hana. Ua mālama ʻia ka RNA me TURBO DNase (Thermo Fisher). Ua synthesized ʻia ka cDNA me ka hoʻohana ʻana i ka Moloney murine leukemia virus reverse transcriptase (M-MLV RT; Thermo Fisher) ma hope o nā kuhikuhi a ka mea hana. Ua paʻi mua ʻia nā Primers no ka reverse transcription quantitative PCR (RT-qPCR; Extended Data Table 2)24 a i hoʻolālā ʻia me ka hoʻohana ʻana iā Primer-BLAST26, me ka makemake i hāʻawi ʻia i nā huahana o 70-150 bp ka nui a me nā exon-exon junctions a i ʻole Primer pair primers e hoʻokaʻawale i nā exons. Ua hoʻoheheʻe ʻia nā laʻana cDNA mai ʻekolu a ʻehā mau replicates biological i ʻehā manawa i ka wai no RT-qPCR. Ua hana ʻia ka quantification ma 15 µl replicate replicate e loaʻa ana ka 1 × PowerUp SYBR Green Master Mix (Thermo Fisher), primers, a me 5 µl o ka cDNA diluted. Ua holo nā reactions ma kahi QuantStudio 6 Pro. Ua hōʻiliʻili ʻia a kālailai ʻia ka ʻōnaehana PCR manawa maoli (Thermo Fisher) a me ka ʻikepili me ka hoʻohana ʻana i ka Design and Analysis (mana 2.4.3). E like me ka mea i hōʻike ʻia ma kēia haʻawina, ua hoʻohālikelike ʻia nā nui pili i ka gene ribosomal RpL19 (AGAP004422), nona ka hōʻike ʻana ʻaʻole i loli nui me ka hānai koko 27 a i ʻole ka male ʻana 3.
Ua nānā ʻia ke ʻano o ka RNA me ka hoʻohana ʻana i kahi Agilent Bioanalyzer 2100 Bioanalyzer (Agilent). Ua hoʻomākaukau ʻia nā hale waihona puke Illumina paired-end a holo ʻia ma ka Broad Institute o MIT a me Harvard. Ua hoʻohālikelike ʻia nā heluhelu sequencing i ka genome A. gambiae (PEST strain, mana 4.12) me ka hoʻohana ʻana iā HISAT2 (mana 2.0.5) me nā palena paʻamau. Ua wehe ʻia nā heluhelu me nā helu mapping quality (MAPQ) <30 me ka hoʻohana ʻana iā Samtools (mana 1.3.1). Ua helu ʻia ka helu o nā heluhelu i hoʻopaʻa ʻia i nā genes me ka hoʻohana ʻana i ka htseq-count (mana 0.9.1) me nā palena paʻamau. Ua helu ʻia nā helu heluhelu maʻamau a ua kālailai ʻia ka hōʻike ʻana o ka gene differential me ka hoʻohana ʻana i ka pūʻolo DESeq2 (mana 1.28.1) ma R (mana 4.0.3).
Ua ʻike ʻia nā moho gene hoʻololi-Ecdysone ma ka ʻimi mua ʻana i ka genome A. gambiae me ka hoʻohana ʻana i ka algorithm PSI-BLAST (https://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/2.8.1/), me ka hoʻohana ʻana i nā waiwai paʻamau Nā Palena me nā moʻo protein nīnau penei: mai Bombyx mori (No. Accession NP_001038956.1), Musca domestica (No. Accession XP_005182020.1, XP_005175332.1 a me XP_011294434.1) a me Microplitis demolitor (No. Accession XP_008552646.1 a me XP_008552645.1) EcK mai B. mori (No. Accession NP_001036900), Drosophila melanogaster (No. Accession NP_651202), Apis mellifera (Helu Komo XP_394838) a me Acyrthosiphon pisum (Helu Komo XP_001947166); a me EPP mai B. mori (Helu Komo XP_001947166) NP_001177919.1 a me NP_001243996.1) a me EO o D. melanogaster (Helu Komo NP_572986.1) (kaʻina hana 1). A laila, kānana i nā hits ma muli o ka hōʻike mRNA kiʻekiʻe (>100 mau ʻāpana/kilobase exons no hoʻokahi miliona mau heluhelu i hoʻopaʻa ʻia (FPKM) a i ʻole >85%) i loko o nā ʻiʻo hānau (wahine LRT a i ʻole MAG) ma Gambia (kaʻina hana 2). No ka hoʻomaikaʻi ʻana i ke ʻano kikoʻī, ua koho mākou i nā enzymes moho i hōʻike ʻia i loko o ka ʻiʻo hānau o A. albimanus, kahi ʻano anopheles ʻaʻole i synthesize a hoʻoili paha i ka ecdysone i ka wā o ka male ʻana. Ua kānana ʻia nā genes moho ma muli o ka haʻahaʻa o ka hōʻike ʻana (<100 FPKM a i ʻole <85th percentile) i loko o ka ʻiʻo hānau o A. albimanus (kaʻanuʻu 3). Ma ke ʻano he kānana hope loa (kaʻanuʻu 4), pono nā genes moho e hoʻokō i ka liʻiliʻi o hoʻokahi o kēia mau mea: (1) i hoʻonui nui ʻia ma hope o ka male ʻana (P <0.05) e like me ka nānā ʻana o nā genes i hōʻike ʻokoʻa ʻia a me (2) i loko o nā ʻiʻo hānau ʻole (<85% a i ʻole <100 FPKM).
Ua hoʻololi mākou i nā ʻano hana i wehewehe mua ʻia 28,29,30 e hoʻokō i ka lepili isotopic holoʻokoʻa. I ka pōkole, ua hoʻāʻo ʻia ka Saccharomyces cerevisiae type II (YSC2, Sigma) i loko o ka yeast nitrogen base (BD Difco, DF0335) nona ka (wt/vol) 2% glucose (G7528, Sigma), 1.7% amino acid-free a me ka ammonium sulfate. culture medium) a me 5% 15N ammonium sulfate (NLM-713, >99%, Cambridge Isotope Laboratories) ma ke ʻano he kumu hoʻokahi o ka nitrogen. Ua hoʻihoʻi ʻia ka yeast ma o ka centrifugation a ua hānai ʻia nā larvae makika ad libitum a hiki i ka pupation. Hoʻohui ʻia me ka fishmeal (0.5 mg no 300 larvae) e pale i ka make ʻana o ka instar ʻehā. A laila ua hoʻohana ʻia nā wahine wale nō i nā hoʻokolohua male me nā kāne i lepili ʻole ʻia e kālailai i ka proteome kāne i hoʻoili ʻia i ka wā male.
Ua koi ʻia nā wahine puʻupaʻa 15N-tagged 4-6 lā e moe me nā kāne puʻupaʻa i hoʻohālikelike ʻia me ka makahiki. Ua hōʻoia ʻia ka male kūleʻa ma ka ʻike ʻana i nā plugs male ma lalo o ka microscopy epifluorescence. Ma 3, 12, a me 24 hpm, ua hoʻokaʻawale ʻia ka atria o 45-55 mau wahine i moe ʻia i loko o 50 µl o ka ammonium bicarbonate buffer (pH 7.8) a homogenized me kahi pestle. Ua centrifuged ʻia ka homogenate a ua hui pū ʻia ka supernatant me 50 µl o 0.1% RapiGest (186001860, Waters) i loko o 50 mM ammonium bicarbonate. Ua hoʻopaʻa ʻia ka supernatant a me ka pellet mai kēlā me kēia laʻana ma ka hau maloʻo a hoʻouna ʻia i ka pō i ka hale hana MacCoss ma ke Kulanui o Wakinekona, kahi i hoʻopau ʻia ai ka hoʻomākaukau ʻana o ka laʻana no LC-MS/MS. E hoʻokuʻu hou i ka pellet i loko o 50 µl o 0.1% RapiGest i loko o 50 mM ammonium bicarbonate a sonicate i loko o kahi ʻauʻau wai. Ua ana ʻia ka nui o ka protein o ka pellet a me ka supernatant e ka BCA assay, ua hoʻemi ʻia nā laʻana me 5 mM dithiothreitol (DTT; Sigma), alkylated me 15 mM iodoacetamide (Sigma) a ua hoʻomoʻa ʻia ma 37 °C (1:0 50) no 1 hola me ka trypsinization: trypsin: substrate ratio). Ua lysed ʻia ʻo RapiGest ma ka hoʻohui ʻana o 200 mM HCl, ukali ʻia e ka incubation ma 37 °C no 45 mau minuke a me ka centrifugation ma 14,000 rpm no 10 mau minuke ma 4 °C e wehe i nā ʻōpala. Ua holoi ʻia nā laʻana e ka dual-mode solid-phase extraction (Oasis MCX cartridges, Waters) a ua hoʻoulu hou ʻia i loko o 0.1% formic acid no ka hoʻohui hope loa o ka protein o 0.33 µg µl-1. Ua kālailai like ʻia nā proteomes MAG i hoʻopaʻa ʻole ʻia mai nā kāne puʻupaʻa. Ua kālailai ʻia ʻelua mau replicates analytical no kēlā me kēia laʻana. ʻO ka mea aʻe, ua kālailai ʻia 1 µg o kēlā me kēia me ka hoʻohana ʻana i kahi kolamu 25-cm fused silica 75-μm me kahi pahele frit Kasil1 (PQ) 4-cm i hoʻopiha ʻia me ka Jupiter C12 reversed-phase resin (Phenomenex) a me ka 180-minuke liquid chromatography. Nā ʻōkuhi laʻana - Ua holo ʻia ʻo MS/MS ma kahi spectrometer mass Q-Exactive HF (Thermo Fisher) me kahi ʻōnaehana nanoACQUITY UPLC (Waters). Ua hoʻololi ʻia nā ʻikepili loaʻa e pili ana i ka ʻikepili i hana ʻia no kēlā me kēia holo i ke ʻano mzML me ka hoʻohana ʻana iā Proteowizard (mana 3.0.20287) a me ka hoʻohana ʻana iā Comet31 (mana 3.2) e kūʻē i ka waihona ʻikepili FASTA e loaʻa ana nā moʻo protein mai Anopheles gambiae (VectorBase mana 54), Anopheles coluzzi Ua hana ʻia kahi hulina ma Mali-NIH (VectorBase mana 54), Saccharomyces cerevisiae (Uniprot, Malaki 2021), A. gambiae RNA-seq, a me nā unuhi ʻekolu-kiʻi o nā mea haumia kanaka i ʻike ʻia. Ua hoʻoholo ʻia nā FDR i kūlike i ka palapala ʻāina Peptide me ka hoʻohana ʻana iā Percolator32 (mana 3.05) me ka paepae o 0.01, a ua hōʻuluʻulu ʻia nā peptides i loko o nā ʻike protein me ka hoʻohana ʻana i ka parsimony protein ma Limelight33 (mana 2.2.0). Ua kuhi ʻia ka nui o ka protein pili me ka hoʻohana ʻana i ka normalized spectral abundance factor (NSAF) i helu ʻia no kēlā me kēia protein i kēlā me kēia holo e like me ka mea i wehewehe mua ʻia. Ua hoʻohālikelike ʻia ʻo NSAF e pili ana i kēlā me kēia protein ma nā laʻana mai ʻelua mau replicates biological like ʻole. Ua uhi pono ka lepili 15N i ka proteome wahine, ʻoiai ua hiki ke ʻike ʻia kahi liʻiliʻi o ka protein i lepili ʻole ʻia mai nā puʻupaʻa i lepili ʻia. Ua hoʻopaʻa mākou i ka ʻike ʻana o ka hōʻemi ʻana o ka protein kāne (1-5 spectra) i nā laʻana maka wahine wale nō i nā holo loea, kahi i holo ai nā laʻana maka ma hope o nā laʻana kāne/mating, ma muli o ka "lawe ʻana" o HPLC. ʻO nā protein i loaʻa i kekahi manawa he 'mea haumia' mai nā puʻupaʻa i lepili ʻia ua helu ʻia ma ka Papa Hoʻohui 1.
ʻElua mau peptides antigenic, ʻo QTTDRVAPAPDQQQ (i loko o ka isotype PA) a me MESDGTTPSGDSEQ (i loko o ka isotype PA a me PB) ma Genscript. Ua hui pū ʻia nā peptides ʻelua, a laila ua hoʻohui ʻia i ka protein lawe KLH a ua hoʻokomo ʻia i loko o nā lāpaki o Aotearoa. Ua pepehi ʻia nā lāpaki ma hope o ka hoʻokomo ʻehā, a ua hoʻokaʻawale ʻia ka IgG holoʻokoʻa e ka hoʻomaʻemaʻe affinity. Ua hoʻohana ʻia ʻo IgG mai ka lāpaki kikoʻī loa o EPP no ka blotting komohana hou aku.
No nā blots komohana, ʻo MAG (n = 10, kahi e hōʻike ai ʻo n i ka helu o nā laʻana makika kūʻokoʻa biologically) a me ka wahine LRT (n = 30) mai nā kāne puʻupaʻa 4 lā a me nā wahine puʻupaʻa a i ʻole nā ​​​​wahine i hoʻopili ʻia me ka ikaika (<10 post-mating), 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)) i hoʻohui ʻokoʻa ʻia. Ua homogenized koke ʻia nā laʻana ma hope o ka dissection me kahi beader (2 mm glass beads, 2,400 rpm, 90 sec). Ua wehe ʻia nā ʻōpala hiki ʻole ke hoʻoheheʻe ʻia e ka centrifugation ma 20,000 g ma 4 °C. Ua helu ʻia nā protein e Bradford assay (Bio-Rad). A laila, ua hoʻohui ʻia he 20 µg o ka protein MAG, 40 µg o ka protein LRT, a me 20 µg o ke koena o ka protein bulk. ua hoʻokaʻawale ʻia a hoʻokaʻawale ʻia e 10% Bis-Tris NuPAGE me ka hoʻohana ʻana i ka buffer MOPS. Ua hoʻoili ʻia nā protein i nā membrane polyvinylidene fluoride me ka hoʻohana ʻana i ka ʻōnaehana hoʻoili iBlot2 (Thermo Fisher). Ua holoi ʻia nā membrane i ʻelua manawa i loko o 1 × PBS-T (0.1% Tween-20 ma PBS) a laila ua ālai ʻia i loko o ka buffer Odyssey blocking (Li-Cor) no 1 hola ma 22 ° C. Ua lulu ʻia nā membrane i ka pō ma 4 ° C me ka rabbit maʻamau anti-EPP polyclonal primary antibody (1:700 i ka blocking buffer) a me ka rat anti-actin monoclonal primary antibody MAC237 (Abeam; 1:4,000). Ua holoi ʻia nā membrane me PBS-T a laila ua hoʻoulu ʻia me nā antibodies lua (donkey anti-rabbit 800CW a me ka goat anti-rat 680LT (Li-Cor), ʻelua 1:20,000) i loko o ka blocking buffer e loaʻa ana he 0.01% SDS a me 0.2% Tween -20 no 1 hola ma 22 °C. Ua holoi ʻia nā membrane me PBS-T a ua kiʻi ʻia me kahi scanner Odyssey CLx. Ua hōʻiliʻili ʻia a hana ʻia nā kiʻi ma Image Studio (mana 5.2). ʻAʻole i ʻike ʻia kahi hui kikoʻī e pili ana i ka isoform EPP-RA (82 kDa).
Ua cloned ʻia nā ʻāpana coding o EPP (e like me ka isoform AGAP002463-RB e loaʻa ana ka histidine phosphatase domain, NCBI conserved domain search 34) a me EcK2 (AGAP002181) i loko o ka pET-21a(+) plasmid (Novagen Millipore Sigma); Ua helu ʻia nā primers ma ka Papa ʻIkepili Hoʻonui 2. Ua hoʻokomo ʻia ʻewalu mau mea hoʻopili GS4 (ma ka tandem) ma mua o ka lepili C-terminal 6xHis o ka hana pET-21a(+)-EcK2. Ua hana ʻia nā protein Recombinant me ka hoʻohana ʻana i ka NEBExpress cell-free E. coli protein synthesis reaction (New England BioLabs). Ua hoʻomaʻemaʻe ʻia nā protein Recombinant me ka hoʻohana ʻana i nā kolamu NEBExpress Ni spin (New England BioLabs). Ua hana ʻia ka protein control Dihydrofolate reductase (DHFR) me ka hoʻohana ʻana i ka template DNA mai ka NEBExpress Cell-Free E. coli Protein Synthesis Kit. Ua mālama ʻia nā protein i loko o 50% glycerol ma PBS ma -20 °C a hiki i 3 mau mahina.
Ua ana ʻia ka hana Phosphatase o EPP a me nā ʻāpana ʻiʻo me ka hoʻohana ʻana i ka 4-nitrophenyl phosphate (pNPP; Sigma-Aldrich). Aia i loko o ka buffer reaction he 25 mM Tris, 50 mM acetic acid, 25 mM Bis-Tris, 150 mM NaCl, 0.1 mM EDTA, a me 1 mM DTT. Ua homogenized ʻia ka ʻiʻo i loko o ka buffer reaction a ua wehe ʻia nā ʻōpala cell ma o ka centrifugation. E hoʻomaka i ka hopena ma ka hoʻohui ʻana i ka enzyme a i ʻole ka ʻāpana ʻiʻo i ka buffer reaction e loaʻa ana he 2.5 mg ml-1 pNPP. Ua hoʻomoʻa ʻia ka hui hopena ma ka mahana o ka lumi i ka pōʻeleʻele, a ua helu ʻia ka nui o ka pNP i hoʻololi ʻia mai ka pNPP ma ke ana ʻana i ka absorbance ma 405 nm i nā manawa like ʻole.
No ka hana EcK in vitro, ua hoʻoulu ʻia ka protein me 0.2 mg 20E a i ʻole 3D20E i loko o 200 µl buffer (pH 7.5) e loaʻa ana he 10 mM HEPES–NaOH, 0.1% BSA, 2 mM ATP a me 10 mM MgCl2 no 2 h ma 27 °C. Ua hoʻōki ʻia ka hopena ma ka hoʻohui ʻana i 800 µl methanol, a laila ua hoʻomaʻalili ʻia ma -20 °C no 1 hola, a laila ua centrifuged ʻia ma 20,000 g no 10 mau minuke ma 4 °C. A laila ua kālailai ʻia ka supernatant e HPLC-MS/MS. No ka hoʻopau wela ʻana i nā protein i hoʻohana ʻia ma ka hui kaohi, ua hoʻoulu ʻia nā protein i loko o 50% glycerol ma PBS no 20 min ma 95 °C.
No ka hana EPP in vitro, ua hoʻoulu ʻia ka protein me 3D20E22P (like me ka nui i loaʻa i loko o 18 mau hui o MAG, i hoʻomaʻemaʻe ʻia e HPLC-MS/MS) i loko o 100 µl buffer (pH 7.5) nona ka 25 mM Tris, 50 mM acetic acid, 25 mM Bis-Tris, 150 mM NaCl, 0.1 mM EDTA, a me 1 mM DTT no 3 mau hola ma 27 °C. Ua hoʻōki ʻia ka hopena ma ka hoʻohui ʻana i 400 µl methanol a hoʻomaʻalili ʻia ma -20 °C no 1 hola, a laila centrifuged ma 20,000 g no 10 mau minuke ma 4 °C. Ua kālailai ʻia ka supernatant e HPLC-MS/MS.
Ua hoʻonui ʻia nā ʻāpana PCR no EPP (362 bp), EcK1 (AGAP004574, 365 bp) a me EcK2 (556 bp) mai ka cDNA i hoʻomākaukau ʻia mai nā kupapaʻu makika poʻo ʻole i hui pū ʻia. Ua hoʻonui ʻia ka ʻāpana PCR o ka mana eGFP (495 bp) mai ka pCR2.1-eGFP i wehewehe mua ʻia; Ua helu ʻia nā primer PCR ma ka Papa ʻIkepili Hoʻonui 2. Ua hoʻokomo ʻia ka ʻāpana PCR ma waena o nā mea hoʻolaha T7 i hoʻohuli ʻia ma ka plasmid pL4440. Ua hoʻihoʻi ʻia nā mea hana plasmid mai NEB 5-α competent E. coli (New England Biolabs) a ua hōʻoia ʻia e ka DNA sequencing ma mua o ka hoʻohana ʻana (e ʻike i ka ʻIkepili Hoʻohui 1 no ke kaʻina hoʻokomo). Ua hoʻohana ʻia nā Primers i kūlike i ka mea hoʻolaha T7 (Papa ʻIkepili Hoʻonui 2) e hoʻonui i ka mea hoʻokomo mai ka plasmid i hoʻokumu ʻia ma pL4440. Ua hōʻoia ʻia ka nui o ka huahana PCR e ka electrophoresis gel agarose. Ua unuhi ʻia ʻo dsRNA mai nā template PCR me ka hoʻohana ʻana i ka Megascript T7 Transcription Kit (Thermo Fisher) a hoʻomaʻemaʻe ʻia e like me nā kuhikuhi a ka mea hana me nā hoʻololi i wehewehe mua ʻia.
No ka inikini dsRNA, ua hoʻokomo ʻia he 1,380 ng o dsRNA (dsGFP, dsEcK1, dsEcK2, dsEPP) ma kahi nui o 10 ng nl-1 i loko o ka thorax o nā kāne makua a wahine paha (Nanoject III, Drummond) i loko o 1 lā ma hope o ka eclosion. Ua hoʻoholo ʻia nā pae knockdown Gene ma ka liʻiliʻi he ʻekolu mau replicates biological ma o ka RNA extraction, cDNA synthesis, a me RT-qPCR. No ka inikini ecdysone, ua hoʻokomo ʻia nā wahine puʻupaʻa 4 lā a i ʻole 6 lā i hānai ʻia i ke koko me 0.13, 0.21, a i ʻole 0.63 µg o 20E a i ʻole 3D20E (Nanoject III, Drummond) ma nā nui o 1.3, 2.1, kēlā me kēia, ma muli o ka hoʻolālā hoʻokolohua a i ʻole 6.3 ng nl-1. E hoʻokomo i 100 nl o 10% (vol/vol) ethanol i loko o ka wai; 100 nl o 3D20E22P i loko o 10% ethanol (like me 75% o ka nui i loaʻa i loko o kahi pālua o MAGs). Ua hoʻokaʻawale ʻia nā makika i ka hui injection.
No nā hoʻāʻo hānau ʻana, ua hānai ʻia nā wahine 3 lā i ke koko kanaka ad libitum. Wehe i nā makika i hānai hapa ʻia a hānai ʻole ʻia paha. Ma muli o ka mālama ʻana, ua waiho ʻia nā wahine i loko o nā kīʻaha hānau kaʻawale no ʻehā pō ma ka liʻiliʻi he 48 mau hola ma hope o ka ʻai ʻana i ke koko. Ua helu ʻia nā hua ma lalo o kahi stereoscope (Stemi 508, Zeiss); no nā wahine i moe ʻia, ua manaʻo ʻia he momona nā hua i puka i loko o nā larvae.
No nā hoʻokolohua male ʻana, ua ʻae ʻia nā wahine ma ka liʻiliʻi he 2 mau lā ma muli o ka lāʻau lapaʻau e hoʻomohala i ke kūʻē ʻana i ka male ʻana, a ua hoʻokomo ʻia nā kāne hihiu i kūlike i ka makahiki i loko o ka hale like. ʻElua pō ma hope mai, ua ʻoki ʻia nā vesicles wahine i hoʻoulu ʻia a ua hoʻokuʻu ʻia ka DNA genomic e ka freeze-thaw a me ka sonication i loko o kahi buffer e loaʻa ana he 10 mM Tris-HCl, 1 mM EDTA, a me 25 mM NaCl (pH 8.2). Ua hoʻoulu ʻia nā laʻana me Proteinase K (0.86 µg µl-1) no 15 mau minuke ma 55 °C, a ukali ʻia e 10 mau minuke ma 95 °C. Ua hoʻoheheʻe ʻia nā hoʻomākaukau DNA genomic maka i 10-fold a ua hoʻokau ʻia i ka ʻike ʻana o qPCR o nā moʻo chromosome Y; ua helu ʻia nā primers ma ka Extended Data Table 2. ʻO ka nele o ka moʻo chromosome Y e hōʻike ana ʻaʻohe male ʻana.
No nā hoʻāʻo hoʻopili hou ʻana, ua nānā ʻia nā wahine i hoʻopili ikaika ʻia no ke alo o nā plugs hoʻopili e hōʻoia i ke kūlana hoʻopili a ʻae ʻia i 2 mau lā e hoʻomohala i ka refractoriness i ka hoʻopili ʻana i ka loaʻa ʻole o nā kāne, e like me ka mea i wehewehe mua ʻia 36. A laila ua hoʻokomo ʻia nā kāne e lawe ana i ka sperm transgenic DsRed i loko o nā hale wahine. ʻElua pō ma hope mai, ua ʻoki ʻia nā vesicles fertilizing mai nā wahine, a ua hoʻomākaukau ʻia ka DNA genomic e like me ka mea i wehewehe ʻia ma luna a ua kau ʻia i ka ʻike qPCR o ka transgene DsRed; ua helu ʻia nā primers ma ka Extended Data Table 2. ʻO ka loaʻa ʻole o ka transgene DsRed i hōʻike ʻaʻole i hana ʻia ka hoʻopili hou ʻana.
Ua hoʻohui ʻia ʻo 3D20E e like me ka mea i wehewehe mua ʻia 37. I ka pōkole, ua hoʻoheheʻe ʻia he 10 mg o 20E (Sigma-Aldrich) i loko o 10 ml o ka wai, a ukali ʻia e ka hoʻohui ʻana o 30 mg o ka platinum ʻeleʻele (ma ke ʻano pauka, Sigma-Aldrich). Ua hoʻomoʻa mau ʻia kahi kahawai mālie o O2 i loko o ka hui hopena, kahi i hoʻoulu ʻia ma ka mahana o ka lumi. Ma hope o 6 mau hola, ua hoʻohui ʻia he 30 mL o ka methanol e hoʻōki i ka hopena. Ua centrifuged ʻia ka hui e wehe i nā ʻāpana catalyst. Ua hoʻoheheʻe ʻia ka supernatant a maloʻo i loko o ka vacuum ma ka mahana o ka lumi. Ua hoʻoheheʻe ʻia ka huahana hopena maloʻo i loko o 10% ethanol a me methanol no ka injection no ka loiloi HPLC-MS/MS. ʻO ka helu hoʻololi (mai 20E a i 3D20E) ma kahi o 97% (Kiʻi 4b), a ua kūlike ka MS spectrum o ka 3D20E i hoʻohui ʻia me ka mea i loaʻa i nā wahine i hoʻopili ʻia (Kiʻi 4c).
Aia i loko o ka moʻolelo nā kikoʻī kikoʻī o nā hoʻokolohua helu i hana ʻia. Ua hoʻohana ʻia ʻo GraphPad (mana 9.0) e hana i ka hoʻāʻo pololei a Fisher, ka hoʻāʻo Mantel-Cox, a me ka hoʻāʻo t-Student. Ua hana ʻia nā hoʻāʻo Cochran-Mantel-Haenszel me ka hoʻohana ʻana i kahi palapala R maʻamau (loaʻa ma https://github.com/duopeng/mantelhaen.test). Ua hoʻāʻo ʻia ka hoʻolaha ʻikepili no ka maʻamau me ka hoʻohana ʻana i ka hoʻāʻo Shapiro-Wilk me ka paepae koʻikoʻi o 0.05. I ka wā i hāʻule ai ka ʻikepili i ka hoʻāʻo maʻamau, ua hana ʻia ka hoʻāʻo Mann-Whitney. Ua kālailai ʻia nā ʻikepili ola me ka hoʻohana ʻana i ka hoʻāʻo Mantel-Cox. Ua hoʻohana ʻia ka pūʻolo DESeq2 (mana 1.28.1) e hana i ka loiloi hōʻike ʻokoʻa pae gene RNA-seq. Hōʻike ka pā pae ma ka pakuhi i ka median. Ua hoʻohana ʻia kahi waiwai koʻikoʻi o P = 0.05 ma ke ʻano he paepae no nā hoʻāʻo āpau.
No ka ʻike hou aku e pili ana i ka hoʻolālā noiʻi, e ʻike i ka hōʻuluʻulu manaʻo o ka Nature Research Report i hoʻopili ʻia i kēia ʻatikala.
Ua waiho ʻia ka ʻikepili proteomic MS i loko o ka ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) ma o ka PRIDE Partner Repository (https://www.ebi.ac.uk/pride/) me ka mea hōʻike ʻikepili PXD032157.
Ua waiho ʻia ka ʻikepili RNA-seq i loko o ka Gene Expression Comprehensive Library (https://www.ncbi.nlm.nih.gov/geo/) ma lalo o ka moʻolelo serial GSE198665.
Hiki ke loaʻa nā ʻikepili hou aʻe i hana ʻia a/a i ʻole i kālailai ʻia i ka wā o ke aʻo ʻana o kēia manawa mai nā mea kākau pili ma ke noi kūpono. Hāʻawi kēia ʻatikala i ka ʻikepili kumu.
ʻO De Loof, A. Ecdysteroids: Nā steroid moe kolohe pepeke i hoʻowahāwahā ʻia? Kāne: Black Box.Insect Science.13, 325–338 (2006).
ʻO Redfern, CPF 20-hydroxyecdysone a me ka hoʻomohala ʻana o ka ovarian ma Anopheles stephens.J. Insect Physiology.28, 97–109 (1982).