Ukufunyanwa nokusasazwa kwezondlo kudibanisa ukutya kwezinambuzane kunye neempawu zembali yobomi. Ukulungisa ukusilela kwezondlo ezithile kumanqanaba ahlukeneyo obomi, izinambuzane zingazifumana ezi zondlo ngokutya okongezelelweyo, umzekelo, ngokutya ukukhupha izilwanyana ezinomqolo kwinkqubo eyaziwa ngokuba yi-puddles. I-mosquito i-Anopheles arabiani ibonakala ingondlekanga kwaye, ke ngoko, ifuna izondlo zombini i-metabolism kunye nokuzala. Injongo yolu phononongo yayikukuvavanya ukuba i-An. arabiensis agitation kumchamo wenkomo ukuze kufunyanwe izondlo iyayiphucula na iimpawu zembali yobomi.
Qinisekisa ukuba ikhuselekile. I-arabiensis yatsalwa livumba lomchamo wenkomo omtsha, ohlala iiyure ezingama-24, iiyure ezingama-72, kunye nomchamo wenkomo omdala weeyure ezili-168, kunye neemazi ezifuna umninimzi kwaye zityiswe igazi (iyure ezingama-48 emva kokutya kwegazi) zalinganiswa kwi-olfactometer ye-Y-tube, kwaye iimazi ezikhulelweyo zavavanywa ukuze kuvavanywe ukuzala. Uhlalutyo oludibeneyo lweekhemikhali kunye ne-electrophysiological lwasetyenziswa ukuchonga iikhompawundi eziphilayo kumchamo wenkomo kuzo zonke iindidi ezine zeminyaka. Imixube yokwenziwa yeekhompawundi eziphilayo yavavanywa kwi-Y-tube kunye novavanyo lwasendle. Ukuphanda umchamo wenkomo kunye nekhompawundi yayo ephambili ye-urea equlethe i-nitrogen njengezondlo ezongezelelweyo ze-malaria vectors, iiparameter zokutya kunye neempawu zembali yobomi zalinganiswa. Umlinganiselo weengcongconi eziziimazi kunye nobungakanani bomchamo wenkomo kunye ne-urea efunxiweyo zavavanywa. Emva koko, iimazi zavavanywa ukuze ziphile, zibhajwe, kwaye zizalwe.
Khangela igazi kunye nokondla komninimzi. Kwizifundo zelebhu nasentsimini, ama-Arabhu atsaleleka kwivumba lendalo nelokwenziwa lomchamo wenkomo omtsha nowalupheleyo. Iimazi ezikhulelweyo azizange zikhathalele iimpendulo zomchamo wenkomo kwiindawo zokuzala. Iimazi ezifuna umninimzi nezifunxa igazi zifunxa umchamo wenkomo kunye ne-urea kwaye zabela ezi zixhobo ngokwembali yobomi njengomsebenzi wesimo somzimba sokubhabha, ukusinda, okanye ukuzala.
Ukufunyanwa kunye nokusasazwa komchamo wenkomo yi-Anopheles arabinis ukuze kuphuculwe iimpawu zobomi. Ukondla umchamo wenkomo okongezelelweyo kuchaphazela amandla eevector ngokuthe ngqo ngokwandisa ukusinda kwemihla ngemihla kunye noxinano lwevector, kwaye ngokungathanga ngqo ngokutshintsha umsebenzi wokubhabha kwaye ke ngoko kufuneka kuqwalaselwe kwiimodeli ezizayo.
Ukufunyanwa nokusasazwa kwezondlo kudibanisa ukutya kwezinambuzane kunye neempawu zembali yobomi [1,2,3].Izinambuzane ziyakwazi ukukhetha nokufumana ukutya kwaye zenze ukondla okuhlawulelwayo ngokusekelwe ekufumanekeni kokutya kunye neemfuno zezondlo [1, 3].Ukusasazwa kwezondlo kuxhomekeke kwinkqubo yembali yobomi kwaye kunokukhokelela kwiimfuno ezahlukeneyo zomgangatho wokutya kunye nobungakanani kumanqanaba obomi ahlukeneyo ezinambuzane [1, 2].Ukubuyisela ukusilela kwezondlo ezithile, izinambuzane zingazifumana ezi zondlo ngokutya okongezelelweyo, njengodaka, indle eyahlukeneyo kunye nokuphuma kwezilwanyana ezinomqolo, kunye nenyama eyomileyo, inkqubo eyaziwa ngokuba ngamadama [2].Nangona iintlobo ngeentlobo zebhabhathane kunye ne-moth zichazwa ngokuyintloko, imingxunya yokunkcenkceshela ikwakho nakwezinye iintlobo zezinambuzane, kwaye ukutsala nokutya kwezi ntlobo zezixhobo kunokuba nefuthe elibalulekileyo kwimpilo nakwezinye iimpawu zembali yobomi [2, 4, 5, 6] ,7].I-malaria mosquito Anopheles gambiae sensu lato (sl) ivela njengomntu omdala 'ongondlekanga' [8], ngoko ke ukunkcenkceshela kunokudlala indima ebalulekileyo kwiimpawu zayo zembali yobomi, kodwa le ndlela yokuziphatha ukuza kuthi ga ngoku ayikhathalelwanga. Ukusetyenziswa kokuvuselela njengendlela yokwandisa ukutya okunesondlo kwesi sixhobo sibalulekileyo kufuna ingqalelo njengoko oku kunokuba nemiphumo ebalulekileyo ye-epidemiological.
Ukungenisa i-nitrogen kwiingcongconi ze-Anopheles ezindala eziziimazi kunqongophele ngenxa yokugcinwa kweekhalori eziphantsi ezithathwe kwinqanaba le-larval kunye nokusetyenziswa kakubi kokutya kwegazi [9]. I-Ann.gambiae sl yebhinqa idla ngokuyilungisa le nto ngokongeza ukutya okongezelelweyo kwegazi [10, 11], ngaloo ndlela ibeka abantu abaninzi emngciphekweni wokufumana esi sifo kwaye ibeke iingcongconi emngciphekweni omkhulu wokutya. Ngaphandle koko, iingcongconi zingasebenzisa ukondla okongezelelweyo kwendle yezilwanyana ezinomqolo ukuze zifumane iikhompawundi ze-nitrogen eziphucula ukuziqhelanisa nokuhambahamba, njengoko kuboniswe zezinye izinambuzane [2]. Kule meko, ukutsala okuqinileyo nokucacileyo kolunye uhlobo lwezilwanyana ezizalwa kunye kwi-An. I-complex ye-Gambian sl species, i-Anopheles arabinis, umchamo wenkomo omtsha nowalupheleyo [12,13,14], inomdla. I-Anopheles arabinis inomdla kakhulu kwizinto ezithandwa ngumninimzi kwaye yaziwa ngokunxulumana nokutya iinkomo. Umchamo wenkomo sisixhobo esityebileyo kwiikhompawundi ze-nitrogen, apho i-urea ibalwa kwi-50-95% ye-nitrogen iyonke kumchamo omtsha [15, 16].Njengoko umchamo wenkomo ukhula, iintsholongwane zisebenzisa ezi zixhobo ukunciphisa ubunzima beekhompawundi zenitrogen kwiiyure ezingama-24 [15]. Ngenxa yokwanda ngokukhawuleza kwe-ammonia, enxulunyaniswa nokwehla kwe-nitrogen ephilayo, iintsholongwane ze-alkalophilic (ezininzi zazo ezivelisa iikhompawundi ezinetyhefu kwiingcongconi) ziyachuma [15], ezinokuba yi-Ann.arabiensis yowesifazane ethandwa kakhulu ngumchamo oneminyaka engama-24 okanye ngaphantsi [13, 14].
Kolu phononongo, kwakhangelwa i-Ans eyondliwe ngumninimzi kunye negazi. Ngexesha lomjikelo wayo wokuqala we-gonadotropin, i-arabiensis yavavanywa ukuze kufunyanwe iikhompawundi ze-nitrogen, kuquka i-urea, ngokuxuba umchamo. Okulandelayo, kwenziwa uthotho lweemvavanyo zokuvavanya indlela iingcongconi eziziinkozo ezisasaza ngayo esi sixhobo sesondlo esinokubakho ukuze kuphuculwe ukusinda, ukuzala kunye nokutya ukutya. Okokugqibela, ivumba lomchamo wenkomo omtsha nowalupheleyo lavavanywa ukuze kuqinisekiswe ukuba ezi zinto zibonelela ngezikhokelo ezithembekileyo ze-An eyondliwe ngumninimzi kunye negazi. Ekukhangelweni kwabo kwesi sixhobo sesondlo esinokubakho, i-arabiensis yafumanisa ulwalamano lweekhemikhali ngasemva kokutsala okwahlukileyo okubonwayo. Imixube yevumba elenziweyo yeekhompawundi ze-organic eziguquguqukayo (ii-VOC) ezichongiweyo kumchamo osele ukhulile iiyure ezingama-24 yavavanywa ngakumbi phantsi kweemeko zasentsimini, yandisa iziphumo ezifunyenwe phantsi kweemeko zelebhu kwaye ibonisa impembelelo yevumba lomchamo wenkomo kwiimeko ezahlukeneyo ze-physiological. Ukutsalwa kwengcongconi. Iziphumo ezifunyenweyo ziqinisekisa ukuba i-An. arabiensis ifumana kwaye isasaze iikhompawundi ze-nitrogen ezifumaneka kumchamo wezilwanyana ezinethambo lomqolo ukuze ichaphazele iimpawu zembali yobomi. Ezi ziphumo zixoxwa kumxholo weziphumo ezinokubakho ze-epidemiological kunye nendlela ezinokusetyenziswa ngayo ukujonga nokulawula i-vector.
Ii-Anopheles arabicans (uhlobo lweDongola) zigcinwe kwi-25 ± 2 °C, 65 ± 5% RH kunye nomjikelo wokukhanya: omnyama weeyure eziyi-12:12. Iimbungu zikhuliswe kwiitreyi zeplastiki (20 cm × 18 cm × 7 cm) zizaliswe ngamanzi acociweyo kwaye zondliwa ukutya kweentlanzi zeTetramin® (Tetra Werke, Melle, DE). Ii-pupae ziqokelelwe kwiikomityi ezingama-30 ml (Nolato Hertila, Åstorp, SE) zaze zadluliselwa kwiikheyiji zeBugdorm (30 cm × 30 cm × 30 cm; MegaView Science, Taichung, Taiwan) ukuvumela ukuba abantu abadala bavele. Abantu abadala banikwe isisombululo se-sucrose se-10% ngokukhawuleza de kube ziintsuku ezi-4 emva kokuvela (dpe), apho iimazi ezifuna umninimzi zinikwa ukutya kwangoko ngaphambi kovavanyo, okanye zalamba ubusuku bonke ngamanzi acociweyo ngaphambi kovavanyo, njengoko kuchaziwe ngezantsi. Iimazi ezisetyenziselwa uvavanyo lwetyhubhu yokubhabha zalamba kuphela. Iiyure ezi-4-6 ngamanzi ngokukhawuleza. Ukulungiselela iingcongconi ezifunxa igazi kwiimvavanyo zebhayoloji ezilandelayo, iimazi ezi-4 ze-dpe zinikwe igazi legusha elisusa iifibrotic (Håtunalab, Bro, SE) kusetyenziswa inkqubo yokondla nge-membrane (Hemotek Discovery Workshops, Accrington, UK). Iimazi ezixineneyo ngokupheleleyo emva koko zadluliselwa kwiikheyiji ezahlukeneyo kwaye zanikwa ukutya ngokuthe ngqo, njengoko kuchaziwe ngezantsi, okanye i-10% ye-sucrose ngokukhawuleza kangangeentsuku ezi-3 ngaphambi kovavanyo oluchazwe ngezantsi. Iimazi zokugqibela zasetyenziselwa uvavanyo lwebhayoloji yetyhubhu yokubhabha zaza zadluliselwa kwilebhu, zaza zanikwa amanzi acocekileyo ngokukhawuleza kangangeeyure ezi-4-6 ngaphambi kovavanyo.
Uvavanyo lokutyisa lusetyenziselwe ukulinganisa ukusetyenziswa komchamo kunye ne-urea kwimazi endala yase-An.Arab. Iimazi ezifuna umninimzi nezondliwa ngegazi zinikwe ukutya okuqulethe umchamo wenkomo omtsha nosele ukhulile oyi-1%, ubuninzi obahlukeneyo be-urea, kunye neendlela ezimbini zokulawula (10% sucrose namanzi) kangangeeyure ezingama-48. Ukongeza, umbala wokutya (1 mg ml-1 xylene cyanide FF; CAS 2650-17-1; Sigma-Aldrich, Stockholm, SE) wongezwa kukutya kwaye wanikezelwa kwi-matrix ye-4 × 4 kwiityhubhu ze-microcentrifuge ze-250 µl (Aoxygen Scientific, Union City, CA, US; Umfanekiso 1A) Zalisa ukuya emaphethelweni (~300 µl). Ukuze uphephe ukhuphiswano phakathi kweengcongconi kunye nemiphumo enokubakho yombala wedayi, beka iingcongconi ezili-10 kwisitya esikhulu sePetri (12 cm ububanzi kunye ne-6 cm ukuphakama; Semadeni, Ostermundigen, CH; Umfanekiso 1A) ebumnyameni obupheleleyo kwi-25 ± 2 cm °C kunye ne-65 ± 5% ngokuhambelanayo. ukufuma. Ezi mvavanyo ziphindwe izihlandlo ezi-5 ukuya kwezili-10. Emva kokuchatshazelwa kukutya, iingcongconi zibekwe kwi--20 °C de kube luhlalutyo olongezelelweyo.
Khangela umchamo wenkomo kunye ne-urea efunxwa ngumninimzi kunye nemazi efunxwa igazi i-Anopheles arabianus. Kwisilingo sokondla (A), iingcongconi ezingamabhinqa zinikwe ukutya okuquka umchamo wenkomo omtsha nosele ukhulile, amanqanaba ahlukeneyo e-urea, i-sucrose (10%), kunye namanzi acociweyo (H2O). Iingcongconi ezifuna umninimzi (B) kunye nemazi ezondliwe ngegazi (C) zifunxwe i-sucrose engaphezulu kunalo naluphi na olunye uhlobo lokutya oluvavanyiweyo. Qaphela ukuba iimazi ezifuna umninimzi zifunxwe ngumchamo wenkomo weeyure ezingama-72 ngaphantsi komchamo wenkomo weeyure ezili-168 (B). Umxholo ophakathi we-nitrogen iyonke (± ukuphambuka okuqhelekileyo) womchamo umelwe kwi-inset. Iingcongconi ezifuna umninimzi (D, F) kunye nemazi ezifunxwa igazi (E, G) zifunxwe yi-urea ngendlela exhomekeke kwidosi. Ubungakanani obuphakathi obuphefumliweyo (D, E) obunamagama oonobumba ahlukeneyo bahluke kakhulu omnye komnye (i-ANOVA yendlela enye kusetyenziswa uhlalutyo lwe-post hoc lukaTukey; p < 0.05). Iibha zeempazamo zimele impazamo eqhelekileyo ye-mean (BE). Umgca othe tye onamachaphaza umele umgca wokubuyela umva we-log-linear (F, G)
Ukuze kukhululwe ukutya okufunxiweyo, iingcongconi zafakwa ngazinye kwiityhubhu ze-microcentrifuge eziyi-1.5 ml eziqulethe i-230 µl yamanzi acociweyo kwaye izicubu zaphazamiseka kusetyenziswa i-pestle elahlwayo kunye ne-cordless motor (VWR International, Lund, SE), kulandele i-centrifugation kwi-10 krpm imizuzu eli-10. I-supernatant (200 µl) yadluliselwa kwi-microplate engama-96-well (Sigma-Aldrich) kwaye i-absorbance (λ620) yafunyanwa kusetyenziswa i-spectrophotometer-based microplate reader (SPECTROStar® Nano, BMG Labtech, Ortenberg, DE) nm). Kungenjalo, iingcongconi zagaywa kwi-1 ml yamanzi acociweyo, i-900 µl yayo yadluliselwa kwi-cuvette ukuze kuhlalutywe i-spectrophotometric (λ 620 nm; UV 1800, Shimadzu, Kista, SE). Ukuze kulinganiswe ukutya okutyiwayo, kwalungiselelwa i-standard curve ngokuxutywa ngokulandelelana ukuze kuvelise i-0.2 µl ukuya kwi-2.4. µl ye-1 mg ml-1 xylene cyanide. Emva koko, kwasetyenziswa ubuninzi be-optical be-dye concentrations eyaziwayo ukuze kuchongwe ubungakanani bokutya ingcongconi nganye etyiweyo.
Idatha yomthamo ihlalutywe kusetyenziswa uhlalutyo lwendlela enye lokwahluka (ANOVA) olulandelwe luthelekiso lukaTukey lwe-post hoc pairwise (JMP Pro, v14.0.0, SAS Institute Inc., Cary, NC, US, 1989–2007). Uhlalutyo lokubuyela umva olunemigca luchaze ukungenisa kwe-urea okuxhomekeke kuxinzelelo kwaye luthelekise iimpendulo phakathi kweengcongconi ezifuna umninimzi kunye nezo zifunxa igazi (GraphPad Prism v8.0.0 yeMac, GraphPad Software, San Diego, CA, US).
Malunga ne-20 µl yeesampuli zomchamo kwiqela ngalinye leminyaka zibotshelelwe kwiChromosorb® W/AW (10 mg 80/100 mesh, Sigma Aldrich) zaza zafakwa kwiikhaphu zetin (8 mm × 5 mm). Iikhaphu zifakwe kwigumbi lokutsha le-CHNS/O analyzer (Flash 2000, Thermo Fisher Scientific, Waltham, MA, US) ukuze kuchongwe umxholo we-nitrogen kumchamo omtsha nowalupheleyo ngokwemigaqo yomenzi. I-nitrogen iyonke (g N l-1) yalinganiswa ngokusekelwe kumanqanaba e-urea aziwayo asetyenziswa njengomgangatho.
Ukuvavanya impembelelo yokutya ekufumaneni umninimzi kunye nokumunca igazi, iingcongconi zafakwa ngazinye kwizitya ezinkulu zePetri (12 cm ububanzi kunye ne-6 cm ukuphakama; Semadeni) kunye nomngxuma ogqunywe ngemesh kwisivalo (3 cm ububanzi) kunye nokubonelela ngomoya kunye nokutya. Ukutya kwanikwa ngqo emva kwe-4 dpe kwaye kwaquka i-1% yomchamo wenkomo omtsha nowalupheleyo, amanqanaba amane e-urea, kunye neendlela ezimbini zokulawula, i-10% ye-sucrose kunye namanzi. Ukutya ngakunye kwafakwa ngepayipi kwi-dental tampon (DAB Dental AB, Upplands Väsby, SE) efakwe kwisirinji ye-5 ml (Thermo Fisher Scientific, Gothenburg, SE), i-plunger yasuswa, yaza yabekwa phezu kwesitya sepetri (umfanekiso 1).1A). Tshintsha ukutya kwakho yonke imihla. Gcina ilebhu njengoko kuchaziwe apha ngasentla. Iingcongconi ezisindileyo zabalwa kabini ngemini, ngelixa iingcongconi ezifileyo zalahlwa de kwafa ingcongconi yokugqibela (n = 40 ngonyango ngalunye). Ukusinda kweingcongconi ezityiwa kwiindlela ezahlukeneyo zokutya kwahlalutywa ngokwezibalo kusetyenziswa ii-Kaplan-Meyer survival curves kunye uvavanyo lwe-log-rank ukuthelekisa uthelekiso lokusasazwa kokusinda phakathi kokutya (IBM SPSS Statistics 24.0.0.0).
Isixhobo sokubhabha seengcongconi esenziwe ngokwezifiso esisekelwe ku-Attisano et al.[17], esenziwe ngeephaneli ze-acrylic ezicacileyo ezinobukhulu obuyi-5 mm (ububanzi obuyi-10 cm x ubude obuyi-10 cm x ukuphakama okuyi-10 cm) ngaphandle kweephaneli zangaphambili nangasemva (Umzobo 3: phezulu). Indibano ye-pivot enetyhubhu ethe nkqo eyenziwe ngekholamu ye-gas chromatography (0.25 mm id; 7.5 cm L) eneziphelo ezincamatheliswe kwinaliti yezinambuzane exhonywe phakathi kweemagnethi ze-neodymium ezimbini ezikude nge-9 cm. Ityhubhu ethe tye eyenziwe ngezinto ezifanayo (6.5 cm L) yahlula ityhubhu ethe nkqo ukuze yenze ingalo ebotshelelweyo kunye nengalo eyayiphethe iqhekeza elincinci le-aluminium foil njengomqondiso wokuphazamisa ukukhanya.
Iimazi ezilambileyo iiyure ezingama-24 zanikwa oku kutya kungentla imizuzu engama-30 ngaphambi kokuba zithintelwe. Iimazi ezifuywayo ngokupheleleyo zaza zafakwa i-anesthesia nganye emkhenkceni imizuzu emi-2-3 zaza zaqhotyoshelwa kwiiphini zezinambuzane nge-beeswax (uJoel Svenssons Vaxfabrik AB, uMunka Ljungby, SE) zaza zabotshelelwa kwiingalo zeetyhubhu ezithe tye. I-Flying Mill. Utshintsho ngendiza nganye lwarekhodwa yi-data logger eyenzelwe wena, emva koko yagcinwa kwaye yaboniswa kusetyenziswa isoftware ye-PC-Lab 2000™ (v4.01; Velleman, Gavere, BE). I-flight mill yabekwa kwigumbi elilawulwa yimozulu (iiyure ezili-12:12, ukukhanya: mnyama, iiyure ezingama-25 ± 2 °C, iipesenti ezingama-65 ± 5% ze-RH).
Ukuze kubonwe indlela esebenza ngayo inqwelo moya, umgama opheleleyo ohamba ngenqwelo moya (m) kunye nenani lilonke lemisebenzi yokubhabha elandelelanayo ibalwa ngeyure kwixesha leeyure ezingama-24. Ukongeza, umgama oqhelekileyo ohamba ngenqwelo moya ngabasetyhini ngabanye uthelekiswe kunyango kwaye wahlalutywa kusetyenziswa uhlalutyo lwe-ANOVA oluhamba ngendlela enye kunye noTukey's post hoc (JMP Pro, v14.0.0, SAS Institute Inc.), apho umgama oqhelekileyo wawuthathwa njengotshintsho oluxhomekeke kuye, ngelixa unyango luyinto ezimeleyo. Ukongeza, inani eliphakathi leemijikelo libalwa ngokwenyuka kwemizuzu eli-10.
Ukuvavanya impembelelo yokutya ekusebenzeni kokuzala kwe-An.arabiensis, iimazi ezintandathu (4 dpe) zidluliselwe ngqo kwiikheyiji zeBugdorm (30 cm × 30 cm × 30 cm) emva kokuqokelelwa kwegazi zaza zanikwa ukutya kovavanyo iiyure ezingama-48 njengoko kuchaziwe apha ngasentla. Emva koko ukutya kwasuswa kwaye kwanikwa iikomityi zokuzala (30 ml; Nolato Hertila) ezizaliswe yi-20 ml yamanzi acociweyo ngosuku lwesithathu kangangeeyure ezingama-48, zitshintshwa rhoqo emva kweeyure ezingama-24. Phinda indlela yokutya nganye izihlandlo ezingama-20-50. Amaqanda abalwa kwaye arekhodwa kwikheyiji nganye yovavanyo. Iisampuli zamaqanda zasetyenziselwa ukuvavanya ubungakanani obuphakathi kunye notshintsho lobude bamaqanda ngamanye (n ≥ 200 ngokutya ngakunye) kusetyenziswa imakroskopu yeDialux-20 (DM1000; Ernst Leitz Wetzlar, Wetzlar, DE) exhotyiswe ngeLeica Camera (DFC) 320 R2; Leica Microsystems Ltd., DE). Amaqanda aseleyo agcinwe kwigumbi elilawulwa yimozulu phantsi kweemeko zokukhulisa eziqhelekileyo iiyure ezingama-24, kwaye isampuli encinci ye-1st instar larvae esandul' ukuvela (n ≥ 200 ngokutya ngakunye) yalinganiswa, njengoko kuchaziwe apha ngasentla. Inani lamaqanda kunye nobukhulu bamaqanda kunye ne-larvae zathelekiswa phakathi konyango kunye nokusebenzisa i-ANOVA kunye nohlalutyo lwe-post hoc lukaTukey (JMP Pro, v14.0.0, SAS Institute Inc.).
Iintsholongwane zeHeadspace ezivela kwisampuli entsha (iyure e-1 emva kokuthathwa kwesampulu), umchamo omdala weeyure ezingama-24, iiyure ezingama-72 kunye neeyure ezili-168 zaqokelelwa kwiisampuli eziqokelelwe kwiinkomo zeZebu, kwiintlobo zeArsi. Ukuze kube lula, iisampuli zomchamo zaqokelelwa kusasa ngelixa iinkomo zisesesibayeni. Iisampuli zomchamo zaqokelelwa kwizilwanyana ezili-10 kwaye i-100-200 ml yesampuli nganye yadluliselwa kwiingxowa zokubhaka zepolyamide nganye (iToppits Cofresco, Frischhalteprodukte GmbH and Co., Minden, DE) kwi-3 l yepolyamide enesiciko kwiidrama zeplastiki zevinyl chloride. Iintsholongwane zeHeadspace ezivela kwisampuli nganye yomchamo wenkomo zaqokelelwa ngokuthe ngqo (kutsha) okanye emva kokuvuthwa kubushushu begumbi iiyure ezingama-24, iiyure ezingama-72 kunye neeyure ezili-168, oko kukuthi, isampuli nganye yomchamo yayimele iqela ngalinye leminyaka.
Kwingqokelela ye-headspace volatiles, inkqubo ye-closed-loop isetyenzisiwe ukujikeleza umjelo wegesi ohluziweyo wekhabhoni (100 ml min-1) ngebhegi ye-polyamide ukuya kwikholamu yokufunxa kangangeeyure ezi-2.5 ngokusebenzisa ipompo yokufunxa i-diaphragm (KNF Neuberger, Freiburg, DE). Njengolawulo, ukuqokelelwa kwe-headspace kwenziwe kwibhegi ye-polyamide engenanto. Ikholamu yokufunxa yenziwe nge-Teflon tube (5.5 cm x 3 mm id) equlethe i-35 mg ye-Porapak Q (50/80 mesh; Waters Associates, Milford, MA, US) phakathi kweeplagi zoboya beglasi. Ngaphambi kokusetyenziswa, ikholamu yahlanjwa nge-1 ml ye-n-hexane ephinde yahluzwa (Merck, Darmstadt, DE) kunye ne-1 ml yepentane (99.0% solvent GC grade, Sigma Aldrich). Ii-volatiles ezifunxiweyo zasuswa nge-400 μl yepentane. Iingqokelela ze-Headspace zahlanganiswa zaza zagcinwa kwi--20°C de zisetyenziswe ixesha elide. uhlalutyo.
Iimpendulo zokuziphatha ze-An-host-seeking kunye ne-blood-eating-blood extracts An.Headspace volatile extracts eziqokelelwe kumchamo omtsha, iiyure ezingama-24, iiyure ezingama-72, kunye neeyure ezili-168 ubudala zihlalutywe ukuze kufunyanwe i-volatile extracts ezivela kwiingcongconi ze-Arabidopsis kusetyenziswa i-olfactometer yeglasi ethe tye [18]. Olu vavanyo lwenziwe ngexesha le-ZT 13-15, ixesha eliphezulu lomsebenzi ka-An wokufuna ikhaya. IsiArabhu [19]. I-olfactometer yeglasi (80 cm × 9.5 cm id) ikhanyiswe nge-3 ± 1 lx yokukhanya okubomvu okuvela phezulu. Ukuhamba komoya ocociweyo nofumileyo ngamalahle (25 ± 2 °C, 65 ± 2% ukufuma) kudlule uvavanyo lwe-bioassay kwi-30 cm s-1. Umoya udlula kuthotho lwezikrini zentsimbi engagqwaliyo, okwenza ukuhamba kwe-laminar kunye nesakhiwo se-plume esifanayo. I-Dental tampon dispenser (4 cm × 1 cm; L:D; DAB Dental AB), ixhonywe kwi-coil ye-5 cm kwicala elijonge emoyeni le- i-olfactometer, enotshintsho lwesikhuthazi rhoqo emva kwemizuzu emi-5. Uhlalutyo, i-10 μl yesicatshulwa ngasinye sentloko, esixutywe yi-1:10, sisetyenzisiwe njengesikhuthazi. Isixa esilinganayo se-pentane sisetyenzisiwe njengolawulo. Iingcongconi ezifuna umninimzi okanye ezifunxa igazi zafakwa kwiikheyiji zokukhululwa ngazinye iiyure ezi-2-3 ngaphambi kokuqala kovavanyo. Ikheyiji yokukhululwa yabekwa kwicala lomoya we-olfactometer, kwaye iingcongconi zavunyelwa ukuba ziqhele umzuzu o-1, emva koko ivalvu yebhabhathane yekheyiji yavulwa ukuze ikhululeke. Ukutsala unyango okanye ulawulo kwahlalutywa njengomlinganiselo weengcongconi ezadibana nomthombo ngaphakathi kwemizuzu emi-5 yokukhululwa. Isicatshulwa ngasinye sentloko kunye nolawulo oluguquguqukayo lwaphindaphindwa ubuncinane amaxesha angama-30, kwaye ukuze kuthintelwe iziphumo zalo naluphi na usuku olunye, inani elifanayo lonyango kunye nolawulo lwavavanywa ngosuku ngalunye lovavanyo. Funa iimpendulo ezivela kumninimzi kunye negazi. Iiseti ze-Arabhu ngokuchasene ne-headspace zahlalutywa kusetyenziswa i-nominal logistic regression elandelwa kuthelekiso oluphindwe kabini lwe-odd ratios (JMP Pro, v14.0.0, iSAS Institute Inc.).
Impendulo ka-An yokuzalela. Izicatshulwa zentloko ezivela kumchamo wenkomo omtsha nowalupheleyo zihlalutywe kwiikheyiji zeBugdorm (30 cm × 30 cm × 30 cm; MegaView Science). Iikomityi zeplastiki (30 mL; Nolato Hertila) ezizaliswe yi-20 mL yamanzi acociweyo zanika isiseko sokuzalela kwaye zabekwa kwiikona ezichaseneyo zekheyiji, ezingama-24 cm ukusuka kwenye. Iikomityi zonyango zilungiswe nge-10 μl yesicatshulwa ngasinye sentloko kwindawo yokuhluza nge-1:10 dilution. Isixa esilinganayo sepentane sasetyenziswa ukulungisa indebe yokulawula. Iikomityi zonyango kunye nolawulo zatshintshiselwana phakathi kovavanyo ngalunye ukuze kulawulwe iziphumo zesikhundla. Iimazi ezilishumi ezondliwe ngegazi zakhululwa kwiikheyiji zovavanyo kwi-ZT 9-11 kwaye amaqanda kwiikomityi abalwa emva kweeyure ezingama-24. Ifomyula yokubala isalathisi sokuzalela yile: (inani lamaqanda abekwe kwikomityi yonyango - inani lamaqanda abekwe kwikomityi yokulawula) / (inani lilonke lamaqanda abekwe). Unyango ngalunye luphindwe izihlandlo ezisi-8.
Uhlalutyo lwe-gas chromatographic kunye ne-electron antenna pattern detection (GC-EAD) lwe-An.arabiensis yebhinqa lwenziwe njengoko kuchaziwe ngaphambili [20]. Ngamafutshane, ii-extracts ezintsha ze-headspace volatile zahlulwe kusetyenziswa i-Agilent Technologies 6890 GC (Santa Clara, CA, US) exhotyiswe ngekholamu ye-HP-5 (30 m × 0.25 mm id, ubukhulu befilimu ye-0.25 μm, i-Agilent Technologies). kunye nomchamo ogugayo. I-hydrogen isetyenziswe njengesigaba sokuhamba ngesantya sokuhamba esiphakathi kwe-45 cm s-1. Isampulu nganye (2 μl) yafakwa imizuzwana engama-30 kwimo yokuqhekeka kunye nobushushu bokungena obuyi-225 °C. Ubushushu be-oven ye-GC bucwangciswe ukusuka kwi-35 °C (ukubamba imizuzu emi-3) ukuya kwi-300 °C (ukubamba imizuzu eli-10) kwi-10 °C min-1. Kwi-GC effluent splitter, kongezwa i-4 psi ye-nitrogen kwaye yahlulwahlulwa ngo-1:1 kwi-Gerstel 3D/2 low dead volume cross (Gerstel, Mülheim, DE) phakathi kwe-flame ionization detector kunye ne-EAD. I-GC effluent capillary ye-EAD yadluliselwa kumgca wokudlulisa we-Gerstel ODP-2, olandelela ubushushu be-GC oven kunye ne-5 °C, kwityhubhu yeglasi (10 cm × 8 mm), apho yaxutywa nomoya ohluziweyo we-carbon (1.5 l min−1). I-antenna yabekwa I-0.5 cm ukusuka ekuphumeni kombhobho. Ingcongconi nganye yayinomlinganiselo omnye, kwaye kwingcongconi ezifuna umninimzi, ubuncinane iingcongconi ezintathu zenziwe kwiisampuli zomchamo zobudala ngabunye.
Ukuchongwa kweekhompawundi eziphilayo kwiindawo eziqokelelweyo zomchamo weenkomo omtsha nowalupheleyo kusetyenziswa i-GC edibeneyo kunye ne-mass spectrometer (GC-MS; 6890 GC kunye ne-5975 MS; Agilent Technologies) ukuze kuvele iimpendulo ze-antenna kuhlalutyo lwe-GC-EAD, olusebenza kwimo ye-electron impact ionization kwi-70 eV. I-GC yayixhotyiswe nge-HP-5MS UI-coated fused silica capillary column (60 m × 0.25 mm inner diameter, 0.25 μm film thickness) kusetyenziswa i-helium njengesigaba esihambayo esine-avareji ye-linear flow rate ye-35 cm s-1. Isampulu ye-2 μl yafakwa kusetyenziswa useto olufanayo lwe-injector kunye nobushushu be-oven njengohlalutyo lwe-GC-EAD. Iikhompawundi zachongwa ngokusekelwe kwixesha lazo lokugcina (i-Kovát index) kunye ne-mass spectra xa kuthelekiswa nelayibrari eyenzelwe wena kunye nelayibrari ye-NIST14 (Agilent). Iikhompawundi ezichongiweyo zaqinisekiswa ngokufaka imigangatho yokwenyani (Ifayile eyongezelelweyo 1: Itheyibhile S2). Ukuze kulinganiswe ubungakanani, i-heptyl i-acetate (10 ng, ubumsulwa beekhemikhali obuyi-99.8%, i-Aldrich) yafakwa njengomgangatho wangaphandle.
Ukuvavanya ukusebenza kakuhle komxube wevumba elenziweyo oquka iikhompawundi eziphilayo ezichongiweyo kumchamo omtsha nowadala ukuze kutsalwe i-Ans.arabiensis efuna umninimzi kwaye emunca igazi, kusetyenziswa i-olfactometer kunye neprotocol efanayo nengasentla. Iikhompawundi ezenziweyo zilinganise ukwakheka kunye nobungakanani beekhompawundi kwi-mixed headspace volatile extracts yomchamo omtsha, weeyure ezingama-24, weeyure ezingama-48, weeyure ezingama-72, kunye noweeyure ezili-168 ubudala (Umfanekiso 5D-G; Ifayile eyongezelelweyo 1: Itheyibhile S2). Uhlalutyo, sebenzisa i-10 μl ye-1:100 dilution yomxube owenziwe ngokupheleleyo, kunye nezinga lokukhululwa lilonke eliqala malunga ne-140-2400 ng h-1, ukuvavanya ukutsala komninimzi kunye neengcongconi ezimunca igazi. Emva koko, uvavanyo lwenziwa kwimixube epheleleyo, apho imixube esusayo yeekhompawundi enye yomxube opheleleyo isuswa khona. Funa iimpendulo kwi-host kunye ne-Ans ezityiswa igazi. I-Arab vs i-synthetic kunye ne-subtract mixtures zihlalutywe kusetyenziswa i-nominal logistic regression elandelwa kuthelekiso oluphindwe kabini lwe-odd ii-ratios (JMP Pro, v14.0.0, SAS Institute Inc.).
Ukuvavanya ukuba umchamo wenkomo ungasebenza njengendawo yokuhlala yezinambuzane zemalaria, umchamo wenkomo omtsha nowalupheleyo, oqokelelwe njengoko kuchaziwe apha ngasentla, kwaye amanzi afakwa kwiibhakethi ezi-3 l ezifakwe i-mesh (100 ml) aze afakwe kwiibathi zeentlanzi. (Inguqulelo ye-BG-HDT; BioGents, Regensburg, DE). Iibathi ezilishumi zibekwe kude nge-50 m edlelweni, i-400 m ukusuka kuluntu lwasezilalini (Silay, Ethiopia, 5°53´24´´N, 37°29´24´´E) kwaye akukho zinkomo, kwiindawo zokuzalela ezisisigxina nakwiilali. Iibathi ezintlanu zifudunyezwe ukuze kuxelwe ukuba kukho umninimzi, ngelixa iibathi ezintlanu zishiywe zingafudunyezwanga. Indawo nganye yonyango ijikeleziswa ubusuku ngabunye ubusuku obuhlanu. Amanani eengcongconi abanjwe kwiibathi ezifakwe umchamo wobudala obahlukeneyo athelekiswa kusetyenziswa i-logistic regression ngokusasazwa kwe-beta binomial (JMP Pro, v14.0.0, SAS Institute Inc.).
Kwilali ehlala i-malaria kufutshane nedolophu yaseMaki, kummandla wase-Oromia, e-Ethiopia (8° 11′ 08″ N, 38° 81′ 70″ E; Umfanekiso 6A). Olu phononongo lwenziwe phakathi kuka-Agasti noSeptemba ngaphambi kokuba kutshizwe inkunkuma yonyaka, kunye nexesha elide lemvula. Iindlu ezintlanu (ezingama-20–50 m ukusuka komnye) ezikwicala lale lali zikhethwe kolu phononongo (Umzobo 6A). Imigaqo esetyenzisiweyo ukukhetha izindlu yayikukuba: akukho zilwanyana zivumelekileyo endlwini, akukho kupheka ngaphakathi (ukutsala iinkuni okanye amalahle) kuvunyelwe (ubuncinane ngexesha lovavanyo), kunye nezindlu ezinabantu ababini kuphela, abalala kwimithi yokubulala izinambuzane. phantsi kwenethi yeengcongconi enyangiweyo. Imvume yokuziphatha inikwe yiBhodi yoHlolo lweeNqobo zoPhando lweZiko (IRB/022/2016) yeFakhalthi yeSayensi yeNdalo (CNS-IRB), kwiYunivesithi yaseAddis Ababa, ngokuhambelana nezikhokelo ezimiselwe yiWorld Medical Association Declaration yaseHelsinki. Imvume evela kwintloko yekhaya nganye ifunyenwe ngoncedo lwabasebenzi bolwandiso lwezempilo. Yonke le nkqubo iyavunywa lulawulo lwasekuhlaleni kwinqanaba lesithili nelewadi ('kebele'). Uyilo lovavanyo lulandele uyilo lwesikwere sesiLatini esingu-2 × 2, apho umxube wokwenziwa kunye nolawulo lwabelwa izindlu ezidibeneyo ngobusuku bokuqala kwaye lwatshintshiselwa phakathi kwezindlu ngobusuku bovavanyo olulandelayo. Le nkqubo yaphindwa izihlandlo ezilishumi. Ukongeza, ukuze kuqikelelwe umsebenzi weengcongconi kwizindlu ezikhethiweyo, imigibe yeCDC yamiselwa ukuba isebenze ubusuku obuhlanu obulandelelanayo ekuqaleni, phakathi nasekupheleni kovavanyo lwasentsimini ngexesha elifanayo losuku.
Umxube owenziweyo oqulethe iikhompawundi ezintandathu ezisebenzayo emzimbeni wanyibilikiswa kwi-heptane (97.0% solvent GC grade, Sigma Aldrich) waza wakhululwa kwi-140 ng h-1 kusetyenziswa i-cotton wick dispenser [20]. I-wick dispenser ivumele zonke iikhompawundi ukuba zikhutshwe ngokulinganayo kulo lonke uvavanyo lweeyure ezili-12. I-Heptane yasetyenziswa njengolawulo. Ibhotile yaxhonywa ecaleni kwendawo yokungena kwi-Centers for Disease Control and Prevention (CDC) light trap (John W. Hock Company, Gainesville, FL, US; Umfanekiso 6A). Iikhompawundi zaxhonywa kwi-0.8 - 1 m ngaphezu komhlaba, kufutshane nonyawo lwebhedi, kwaye ivolontiya lalala phantsi kwenethi yezinambuzane enganyangwanga kwaye yasebenza phakathi kwe-18:00 kunye ne-06:30. Iingcongconi ezibanjwe sisini kunye nesimo somzimba (ezingatyiwayo, ezityiwayo, ezikhulelweyo, nezikhulelweyo [21] emva koko zahlolwa kusetyenziswa uhlalutyo lwe-polymerase chain reaction (PCR) ukuchonga uhlobo oluchongiweyo ngokwemo ye-A. gambiae sl. Amalungu e-complex [23]. Kwisifundo sasentsimini, ukubanjwa kweemathiriyeli zezindlu ezidibeneyo kuhlalutywe kusetyenziswa imodeli yokulingana kwezinto ezisetyenziswayo, apho ukutsala kwakuyi-dependent variable kwaye unyango (umxube we-synthetic vs control) yayiyi-fixed effect (JMP® 14.0. 0. SAS Institute Inc.). Apha, sixela i-χ2 kunye ne-p-values ​​​​kuvavanyo lwe-likelihood ratio.
Vavanya ukuba ikhuselekile na. I-arabiensis ikwazile ukufumana umchamo, umthombo wayo oyintloko we-nitrogen, i-urea, ngokutya ngokuthe ngqo, kwiiyure ezingama-48 emva kokusetyenziswa kwiintsuku ezi-4 emva kovavanyo lokutya lwabasetyhini (dpe) lokufuna umninimzi kunye novavanyo lokutya lwabasetyhini oluncelisayo (Umzobo 1A). Zombini iimazi ezifuna umninimzi kunye nezifunxa igazi zifunxe i-sucrose eninzi kakhulu kunalo naluphi na olunye uhlobo lokutya okanye amanzi (F(5,426) = 20.15, p < 0.0001 kunye ne-F(5,299) = 56.00, p < 0.0001, ngokulandelanayo; Umzobo 1B,C). Ngaphezu koko, iimazi ezifuna umninimzi zitye kancinci kumchamo kwiiyure ezingama-72 xa kuthelekiswa nomchamo kwiiyure ezili-168 (Umzobo 1B). Xa zinikwa ukutya okuqulethe i-urea, iimazi ezifuna umninimzi zifunxe inani elikhulu le-urea kwi-2.69 mM xa kuthelekiswa nazo zonke ezinye iindawo kunye namanzi, ngelixa zingahlukaniswa kwi-10% ye-sucrose (F(10,813) = 15.72, p < 0.0001; Umfanekiso 1D). Oku kwakungafani nempendulo yabasetyhini abondliwe ngegazi, ababedla ngokufunxa ukutya okune-urea kakhulu kunamanzi, nangona bengaphantsi kakhulu kwi-10% ye-sucrose (F(10,557) = 78.35, p < 0.0001; Umfanekiso 1).1E). Ngaphezu koko, xa kuthelekiswa phakathi kwezi meko zimbini zomzimba, abafazi abane-phlebotomized bafunxa i-urea engaphezulu kunabafazi abafuna umninimzi kumanqanaba aphantsi, kwaye la mabhinqa afunxa izixa ezifanayo ze-urea kumanqanaba aphezulu (F(1,953)= 78.82, p < 0.0001; Umfanekiso 1F, G). Ngelixa ukutya okutya okune-urea kubonakala kunexabiso elifanelekileyo (Umzobo 1D,E), abafazi kuzo zombini iimeko zomzimba bakwazile ukuguqula ubungakanani be-urea efunxwe kulo lonke uluhlu lwamanqanaba e-urea ngendlela ye-log-linear (Umzobo 1F,G). ).Ngokunjalo, iingcongconi zibonakala zilawula ukufunxwa kwazo yi-nitrogen ngokulawula ubungakanani bomchamo ofunxwayo, njengoko ubungakanani be-nitrogen kumchamo bubonakala kubungakanani bomchamo ofunxwayo (Umfanekiso 1B, C kunye no-B insets).
Ukuvavanya imiphumo yomchamo kunye ne-urea ekusindeni kweengcongconi ezifuna umninimzi kunye nezifunxa igazi, iimazi zondliwa umchamo wazo zonke iminyaka emine (ezintsha, iiyure ezingama-24, iiyure ezingama-72, kunye neeyure ezili-168 emva kokutsalwa) kunye noluhlu lwamanqanaba e-urea, kunye namanzi acociweyo kunye ne-10% ye-sucrose esetyenziswa njengolawulo (Umfanekiso 2A). Olu hlalutyo lokusinda lubonise ukuba ukutya kunempembelelo ebalulekileyo ekusindeni ngokubanzi kwiimazi ezifuna umninimzi (umchamo: χ2 = 108.5, df = 5, p < 0.0001; urea: χ2 = 122.8, df = 5, p < 0.0001; Umzobo 2B, C) kunye neemazi ezondliwa ngegazi (umchamo: χ2 = 93.0, df = 5, p < 0.0001; urea: χ2 = 137.9, df = 5, p < 0.0001; Umfanekiso 2D,E).Kuzo zonke iimvavanyo, abafazi abatya umchamo, i-urea, kunye namanzi babenezinga lokusinda eliphantsi kakhulu xa kuthelekiswa nabasetyhini abatya ukutya okune-sucrose (Umfanekiso 2B-E). Abafazi abafuna umbungazi abatya umchamo omtsha nowomileyo babonise izinga lokusinda elahlukileyo, abo batya umchamo obolileyo weeyure ezingama-72 (p = 0.016) benethuba eliphantsi lokusinda (Umfanekiso 2B). Ngaphezu koko, abafazi abafuna umbungazi abatya i-urea eyi-135 mM baphile ixesha elide kunolawulo lwamanzi (p < 0.04) (Umfanekiso 2C). Xa kuthelekiswa namanzi, abafazi abatya umchamo omtsha kunye nomchamo weeyure ezingama-24 baphile ixesha elide (p = 0.001 kunye ne-p = 0.012, ngokulandelelana; Umfanekiso 2D), ngelixa abafazi abatya umchamo weeyure ezingama-72 baphile ixesha elide kunabo batya umbungu omfutshane omtsha kunye nomchamo weeyure ezingama-24 ubudala (p < 0.0001 kunye ne-p = 0.013, ngokulandelelana; Umfanekiso 2D). Xa betya i-urea eyi-135 mM, abafazi abatya igazi baphile ixesha elide kunabo bonke ezinye iindawo ezixineneyo ze-urea namanzi (p < 0.013; Umfanekiso 2E).
Ukusinda kwe-Anopheles arabinis yebhinqa elihlala kunye nelifunxa igazi esitya umchamo wenkomo kunye ne-urea. Kwi-bioassay (A), iingcongconi ezibhinqileyo zinikwe ukutya okuquka umchamo wenkomo omtsha nowalupheleyo, amanqanaba ahlukeneyo e-urea, i-sucrose (10%) kunye namanzi acociweyo (H2O). Ukusinda kwe-host-seeking (B, C) kunye ne-blood-sucking (D, E) iingcongconi kwarekhodwa rhoqo emva kweeyure ezili-12 de zonke iimazi zityiswe umchamo (B, D) kunye ne-urea (C, E), kunye nolawulo, i-Sucrose kunye namanzi, zifile.
Umgama opheleleyo kunye nenani lemijikelo echongiweyo kuvavanyo lwe-flight mill kwixesha leeyure ezingama-24 lahlukile phakathi kweengcongconi ezifuna umninimzi kunye nezifunxa igazi, ezibonise umsebenzi omncinci wokubhabha ngokubanzi (Umzobo 3). Iingcongconi ezifuna umninimzi ezinike umchamo omtsha nowalupheleyo okanye i-sucrose namanzi zibonise iipatheni zokubhabha ezahlukeneyo (Umzobo 3), apho iimazi ezitya umchamo omtsha zisebenza ngakumbi ekuzeni kokusa, ngelixa ezo zondliwayo ii-24 kunye ne-168 ubudala Iingcongconi ezitya umchamo zibonise iipatheni ezahlukeneyo zokubhabha kwaye ikakhulu zazihlala emini. Iingcongconi ezibhinqileyo ezinike i-sucrose okanye umchamo weeyure ezingama-72 zibonise umsebenzi kulo lonke ixesha leeyure ezingama-24, ngelixa iimazi ezinike amanzi zisebenza ngakumbi phakathi kwexesha. Iingcongconi ezitya i-sucrose zibonise amanqanaba aphezulu omsebenzi ebusuku nakusasa, ngelixa ezo zitya umchamo weeyure ezingama-72 ubudala zibone ukwehla okuzinzileyo komsebenzi kwiiyure ezingama-24 (Umfanekiso 3).
Ukusebenza kokubhabha kwemazi efuna igazi efunxa igazi i-Anopheles arabinis etya umchamo wenkomo kunye ne-urea. Kuvavanyo lwe-flight mill, iingcongconi ezifunxa igazi ezitya umchamo wenkomo omtsha nosele ukhulile, amazinga ahlukeneyo e-urea, i-sucrose (10%), kunye namanzi acociweyo (H2O) axhonywe kwiingalo ezithe tye, ezijikelezayo ngokukhululekileyo (ngasentla). Kwimazi efuna igazi (ekhohlo) kunye nemazi efunxa igazi (ekunene), umgama uwonke kunye nenani lokubhabha ngeyure yokutya ngakunye kwixesha leeyure ezingama-24 kubhalwe (mnyama: ngwevu; ukukhanya: mhlophe). Umgama ophakathi kunye nenani eliqhelekileyo leentshukumo ziboniswe ngasekunene kwegrafu yomsebenzi we-circadian. Iibha zeempazamo zimele impazamo eqhelekileyo ye-mean. Uhlalutyo lwezibalo jonga umbhalo
Ngokubanzi, umsebenzi wokubhabha jikelele weemazi ezifuna umninimzi ulandela iphethini efana naleyo yomgama wokubhabha kwixesha leeyure ezingama-24. Umgama ophakathi wokubhabha uchatshazelwe kakhulu kukutya okutyiweyo (F(5, 138) = 28.27, p < 0.0001), kwaye iimazi ezifuna umninimzi ezityiweyo iiyure ezingama-72 zomchamo zibhabha umgama omde kakhulu xa kuthelekiswa nazo zonke ezinye iindlela zokutya (p < 0.0001), kwaye iingcongconi ezitya i-sucrose zibhabha ixesha elide kuneengcongconi ezintsha (p = 0.022) kunye nomchamo oneminyaka engama-24 (p = 0.022). Ngokwahlukileyo kwindlela yokusebenza yokubhabha echazwe kukutya komchamo, iimazi ezifuna umninimzi ezitya i-urea zibonise umsebenzi wokubhabha oqhubekayo kwixesha leeyure ezingama-24, zifikelela kwinqanaba eliphezulu ngexesha lesiqingatha sesibini sesigaba sobumnyama (Umzobo 3). Nangona iipateni zomsebenzi zazifana, iimazi ezifuna umninimzi ezitya i-urea zandisa kakhulu umgama wokubhabha ophakathi ngokuxhomekeke kuxinzelelo olufunxiweyo (F(5, 138) = 1310.91, p < 0.0001). Ukufuna umninimzi Iimazi ezifunxwe naluphi na uxinaniso lwe-urea zibhabha ixesha elide kuneemazi ezifunxwe ngamanzi okanye i-sucrose (p < 0.03).
Umsebenzi wokubhabha ngokubanzi weengcongconi ezifunxa igazi ubuzinzile kwaye uqhubeka iiyure ezingama-24 kuzo zonke iindlela zokutya, kunye nomsebenzi womchamo owandisiweyo kwisiqingatha sesibini sexesha lobumnyama kwiimazi ezifunxa amanzi kunye nakwizimazi ezifunxa zisandula ukufunxwa kwaye zineminyaka engama-24 ubudala (umfanekiso 3). Nangona ukutya komchamo kuchaphazele kakhulu umgama ophakathi wokubhabha kwiimazi ezifunxa igazi (F(5, 138) = 4.83, p = 0.0004), ukutya kwe-urea akuzange (F(5, 138) = 1.36, p = 0.24). kunye nomnye umchamo kunye nokutya okulawulayo (okutsha, p = 0.0091; iiyure ezingama-72, p = 0.0022; iiyure ezili-168, p = 0.001; sucrose, p = 0.0017; dH2O, p = 0.036).
Iziphumo zokutya komchamo kunye ne-urea kwiiparameter zokuzala zihlolwe kwiimvavanyo zebhayoloji ezibeka amaqanda (Umfanekiso 4A) kwaye ziphandwe ngokwenani lamaqanda abekwe yimazi nganye, ubungakanani beqanda, kunye ne-larvae yokuqala esandul' ukuqanduselwa. Inani lamaqanda abekwe. Iimazi zama-Arabhu ezondliwe ngomchamo zahluka ngokutya (F(5,222) = 4.38, p = 0.0008; Umzobo 4B). Iimazi zondliwe ngomchamo weeyure ezingama-24, isidlo segazi esibeka amaqanda amaninzi kakhulu kunamabhinqa azondliwe ezinye iindlela zokutya komchamo kwaye zazifana nezo zondliwe nge-sucrose (Umzobo 4B). Ngokufanayo, ubungakanani bamaqanda abekwe ziimazi ezondliwe ngomchamo bahluka ngokutya (F(5, 209) = 12.85, p < 0.0001), kunye nomchamo weeyure ezingama-24 kunye neemazi ezondliwe nge-sucrose zibeka amaqanda amakhulu kakhulu kunamabhinqa azondliwe ngamanzi, ngelixa amaqanda ezimazi ezondliwe ngomchamo weeyure ezili-168 ayemancinci kakhulu (Umzobo 4C). Ukongeza, ukutya komchamo kuchaphazele kakhulu ubungakanani be-larval (F(5, 187) = 7.86, p < 0.0001), kunye neembungu ezinkulu kakhulu eziphuma kumaqanda ezibekwa ziimazi ezitya umchamo ezineminyaka engama-24 kunye nengama-72 ubudala kunamaqanda ezibekwa kwiimbungu ezitya umchamo. Iimazi ezitya amanzi kunye nezotya umchamo ezitya iiyure ezili-168 (Umfanekiso 4D).
Ukusebenza kokuzala kwe-Anopheles arabinis yemazi etya umchamo wenkomo kunye ne-urea. Iingcongconi zemazi ezondliwe ngegazi zondliwe ukutya okuquka umchamo wenkomo omtsha nowalupheleyo, amazinga ahlukeneyo e-urea, i-sucrose (10%), kunye namanzi acociweyo (H2O) iiyure ezingama-48 ngaphambi kokufaka uvavanyo lwebhayoloji kunye nokufumana ii-substrates zokubeka amaqanda. Iiyure ezingama-48 (A). Inani leqanda (B, E), ubungakanani beqanda (C, F) kunye nobukhulu bee-larvae (D, G) zichaphazeleke kakhulu kukutya okubonelelweyo (umchamo wenkomo: BD; urea: EG). Iindlela zeparameter nganye ezilinganiswe kusetyenziswa amagama oonobumba ahlukeneyo zahluke kakhulu kwenye (i-ANOVA yendlela enye isebenzisa uhlalutyo lwe-post hoc lukaTukey; p < 0.05). Iimpazamo zimele impazamo eqhelekileyo ye-mean
Njengenxalenye ephambili ye-nitrogen kumchamo, i-urea, xa inikwa ukutya kwabasetyhini abatyiswa igazi, ichaphazele kakhulu iiparameter zokuzala kuzo zonke izifundo. Inani lamaqanda abekwa ngabafazi abatyiswa i-urea, emva kokutya kwegazi, kuxhomekeke kuxinzelelo lwe-urea (F(11, 360) = 4.69; p < 0.0001), abasetyhini abatyiswa uxinano lwe-urea phakathi kwe-134 µM kunye ne-1.34 mM babeka amaqanda amaninzi ( Umfanekiso 4E ). Abafazi abatyiswa uxinano lwe-urea lwe-134 µM okanye ngaphezulu babeka amaqanda amakhulu kunabafazi abatyiswa ngamanzi ( F(10, 4245) = 36.7; p < 0.0001; Umfanekiso 4F), kunye nobukhulu be-larval, nangona bechaphazeleka kuxinzelelo olufanayo lwe-urea koomama ( F(10, 3305) = 37.9; p < 0.0001) beluguquguquka ngakumbi ( Umfanekiso 4G ).
Ukutsalela ngokubanzi kwiimveliso zomchamo zentloko yenkomo ezifuna umninimzi. I-arabiensis ehlolwe kwi-olfactometer yeglasi (Umzobo 5A) ichaphazeleke kakhulu bubudala bomchamo (χ2 = 15.9, df = 4, p = 0.0032; Umzobo 5B). Uhlalutyo lwasemva kwexesha lubonise ukuba ivumba lomchamo elidala kwiiyure ezingama-24 libangele amanqanaba aphezulu okutsala xa kuthelekiswa nazo zonke ezinye iindlela zonyango (iiyure ezingama-72: p = 0.0060, iiyure ezili-168: p = 0.012, pentane: p = 0.00070), Ngaphandle kwevumba lomchamo omtsha (p = 0.13; Umzobo 5B). Nangona ukutsalela ngokubanzi kweengcongconi ezifunxa igazi kwivumba lomchamo kwakungahlukanga kakhulu (χ2 = 8.78, df = 4, p = 0.067; Umzobo 5C), ezi zilwanyana zifunyaniswe zinomtsalane ngakumbi kwiimveliso zomchamo zentloko xa kuthelekiswa nomchamo osele uneminyaka engama-72 xa kuthelekiswa ulawulo (p = 0.0066; Umfanekiso 5C).
Iimpendulo zokuziphatha kwivumba lomchamo wenkomo wendalo nowokwenziwa ekufuneni i-Anopheles arabianus eyondliwayo negazi. I-Schematic ye-olfactometer yeglasi (A). Ukutsalwa kwe-headspace volatile extracts kumchamo wenkomo omtsha nowalupheleyo ukuya kwi-host (B) kunye neengcongconi ezifunxa igazi (C). Fumana impendulo ye-tentacle ye-Lord An. I-Headspace extracts ehlukaniswe kumchamo wenkomo omtsha (D), iiyure ezingama-24 (E), iiyure ezingama-72 (F), kunye neeyure ezili-168 (G) zibonisiwe. I-electron antenna detection (EAD) traces ibonisa utshintsho lwe-voltage ekuphenduleni kwi-bioactive compounds kwindawo yentloko ekhutshwe kwi-gas chromatograph kwaye ifunyenwe yi-flame ionization detector (FID). I-scale bar imele i-response amplitude (mV) ngokuchasene nexesha lokugcina (s). Iipropati kunye namazinga okukhululwa (µg h-1) kwee-bioactive compounds zibonisiwe. I-asterisk enye (*) ibonisa impendulo ehambelanayo ye-low-amplitude. I-asterisks ezimbini (**) zibonisa iimpendulo ezingaphindi ziphindeke. Fumana i-host (H) kunye ne-blood-sucking (I) I-An.arabiensis ineempembelelo ezahlukeneyo kwimixube yokwenziwa yevumba lomchamo wenkomo omtsha nowalupheleyo. Umlinganiselo ophakathi weengcongconi ezitsalwa ngamagama oonobumba ahlukeneyo wahluke kakhulu omnye komnye (i-ANOVA yendlela enye isebenzisa uhlalutyo lukaTukey lwe-post hoc; p < 0.05). Iimpazamo zimele impazamo eqhelekileyo yesikali
Imazi i-Ann.arabiensis, emva kweeyure ezingama-72 kunye neeyure ezili-120 emva kokutya igazi, ngexesha lokuzala, akukho kukhetha kubonisiwe kwiincasa ze-volatible ze-headspace ezivela kumchamo wenkomo omtsha nowalupheleyo xa kuthelekiswa nolawulo lwe-pentane (χ2 = 3.07, p > 0.05; Ifayile eyongezelelweyo 1: Umfanekiso S1).
Kwi-Ann.arabiensis yabasetyhini, uhlalutyo lwe-GC-EAD kunye ne-GC-MS luchonge iikhompawundi ezisibhozo, ezintandathu, ezintathu kunye nezintathu ze-bioactive (Umfanekiso 5D-G). Nangona umahluko kwinani leekhompawundi ezibangele iimpendulo ze-electrophysiological ubonwe, uninzi lwale khompawundi lwalukho kwi-headspace volatile extract nganye eqokelelwe kumchamo omtsha nowalupheleyo. Ke ngoko, kwi-extract nganye, kuphela iikhompawundi ezivelise impendulo yomzimba kwi-antennae yabasetyhini engaphezulu komda ezibandakanyiweyo kuhlalutyo olongezelelweyo.
Izinga lokukhululwa kwe-variable iyonke yee-compounds ezisebenzayo kwi-headspace collection linyuke ukusuka kwi-29 µg h-1 kumchamo omtsha ukuya kwi-242 µg h-1 kumchamo osele ukhulile kangangeeyure ezili-168, ikakhulu ngenxa ye-p-cresol kunye ne-m-formaldehyde Phenol iyanda kunye ne-phenol. Ngokwahlukileyo koko, amazinga okukhululwa kwezinye ii-compounds, ezifana ne-2-cyclohexen-1-one kunye ne-decanal, ancipha ngokukhula komchamo, nto leyo ehambelana nokwehla okubonakalayo kobunzulu besignali (ubuninzi) kwi-chromatogram (Umzobo 5D)-G left panel) kunye neempendulo zomzimba kwezi compounds (Umzobo 5D-G left panel).
Ngokubanzi, umxube wokwenziwa wawunomlinganiselo wendalo ofanayo weekhompawundi ezisebenzayo eziphilayo ezichongiweyo kwiincindi eziguquguqukayo zeendawo zomchamo ezintsha nezindala (Umzobo 5D–G) kwaye awuzange ubonakale ubangela umdla omkhulu ekufuneni umsingathi (χ2 = 8.15, df = 4, p = 0.083; Umzobo 5H) okanye iingcongconi ezifunxa igazi (χ2 = 4.91, df = 4, p = 0.30; Umzobo 5I). Nangona kunjalo, uthelekiso lwangemva kwe-hoc phakathi konyango lubonise ukuba iingcongconi ezifuna umsingathi zazinomtsalane kakhulu kumxube wokwenziwa womchamo ondala weeyure ezingama-24 xa kuthelekiswa nolawulo lwe-pentane (p = 0.0086; Umzobo 5H).
Ukuvavanya indima yamacandelo omntu ngamnye kwimixube yokwenziwa yomchamo weeyure ezingama-24 ubudala, imixube emithandathu yokukhupha ihlolwe ngokuchasene nemixube epheleleyo kwi-Y-tube assay, apho imixube nganye isusiwe. Kwiingcongconi ezifuna umninimzi, ukukhupha imixube nganye kumxube opheleleyo kube nefuthe elikhulu kwiimpendulo zokuziphatha (χ2 = 19.63, df = 6, p = 0.0032; Ifayile eyongezelelweyo 1: Umfanekiso S2A), yonke imixube yokukhupha yayinomtsalane ngakumbi kuneMincinci kunexutywe ngokupheleleyo. Ngokwahlukileyo koko, ukususwa kwemixube nganye kumxube wokwenziwa ngokupheleleyo akuzange kuchaphazele iimpendulo zokuziphatha zeengcongconi ezifunxa igazi (χ2 = 11.38, df = 6, p = 0.077), ngaphandle kwe-decanal, eyabangela amanqanaba aphantsi xa kuthelekiswa nomxube opheleleyo. Ukutsala (p = 0.022; Ifayile eyongezelelweyo 1: Umfanekiso S2B).
Kwilali yase-Ethiopia exhaphake kakhulu yimalariya, ukusebenza kakuhle komxube wokwenziwa womchamo wenkomo osebenza iiyure ezingama-24 ekutsaleni iingcongconi phantsi kweemeko zasemasimini kuhlolwe ubusuku obulishumi (Umzobo 6A). Iingcongconi ezingama-4,861 zifunyenwe zaza zachongwa, apho ama-45.7% ayeyi-Anthropus.gambiae sl, ama-18.9% ayeyi-Anopheles pharoensis kwaye ama-35.4% ayeyi-Culex spp. (Ifayile eyongezelelweyo 1: Itheyibhile S1). I-Anopheles arabinis yeyona kuphela ilungu le-An.Gambian species complex echongiweyo ngohlalutyo lwe-PCR. Ngokomyinge, iingcongconi ezingama-320 zibanjwe ngobusuku, ngelo xesha iingcongconi ezineengcongconi ezidityanisiweyo zibambe iingcongconi ezininzi kuneengcongconi ezidityanisiweyo ngaphandle komxube (χ2(0, 3196) = 170.0, p < 0.0001). Iingcongconi ezingadityanisiweyo zibekwe kubusuku ngabunye bolawulo obuhlanu ekuqaleni, phakathi, nasekupheleni kovavanyo. Inani elifanayo leengcongconi libanjwe kwisibini ngasinye. yemigibe, ebonisa ukungabikho kocalucalulo phakathi kwezindlu (χ2(0, 1665) = 9 × 10-13, p > 0.05) kwaye akukho kuncipha kwenani labemi ngexesha lesifundo. Xa kuthelekiswa nemigibe yokulawula, inani leengcongconi ezibanjwe kwimigibe equlethe umxube owenziweyo landa kakhulu: ukufuna ummkeli (χ2(0, 2107) = 138.7, p < 0.0001), ukondliwa ngegazi kutshanje (χ2(0, 650) = 32.2, p < 0.0001) kunye nokukhulelwa (χ2(0, 228) = 6.27, p = 0.0123; Ifayile eyongezelelweyo 1: Itheyibhile S1). Oku kukwabonakala kwinani lilonke leengcongconi ezibanjiweyo: ukufuna ummkeli > ukumunca igazi > ukhulelwe > ukhulelwe kancinci > inkunzi.
Uvavanyo lwasendle lokusebenza komxube wevumba lomchamo wenkomo owenziweyo iiyure ezingama-24. Uvavanyo lwasendle lwenziwe kumzantsi-mbindi we-Ethiopia (imephu), kufutshane nedolophu yaseMaki (faka), kusetyenziswa i-Centers for Disease Control (CDC) light trap (ekunene) kwizindlu ezidibeneyo, kunye noyilo lwesikwere saseLatin (umfanekiso we-aerial) (A). Ii-phototraps ze-CDC ezifakwe ivumba zitsala kwaye zibamba ii-Anopheles arabesques (B) zabasetyhini, kodwa hayi ii-Anopheles farroes (C), ngendlela eyahlukileyo, isiphumo esixhomekeke kwimeko yomzimba. Ukongeza, ezi ngxaki zibambe inani elongezelelekileyo lee-host Culex mosquitoes. (D) Xa kuthelekiswa nolawulo. Iibhari ezisekhohlo zimele i-avareji yokukhetha iingcongconi ezibanjwe ngababini be-odorant bait (eluhlaza) kunye ne-control (evulekileyo) traps (N = 10), ngelixa iibhari ezisekunene zimele i-avareji yokukhetha i-avareji kwi-avareji ye-control traps (evulekileyo; N = 5). Ii-asterisks zibonisa amanqanaba okubaluleka kwezibalo (*p = 0.01 kunye ***p < 0.0001)
Ezi ntlobo zintathu zibanjwe ngokwahlukileyo kwiimigibe ezinemixube yokwenziwa. Ukukhangela umsingathi (χ2(1, 1345) = 71.7, p < 0.0001), ukondla ngegazi (χ2(1, 517) = 16.7, p < 0.0001) kunye nokukhulelwa (χ2(1, 180) = 6.11, p = 0.0134) i-.arabiensis yayibanjwe kumgibe ikhupha umxube wokwenziwa (Umzobo 6B), ngelixa ubungakanani be-An bungahlukanga. I-Pharoensis kwiimeko ezahlukeneyo ze-physiological zifunyenwe (Umzobo 6C). KwiCulex, kuphela ukwanda okukhulu kwinani leengcongconi ezifuna abasingathi kufunyenwe kwiimigibe ezifakwe umxube wokwenziwa (χ2(1,1319) = 12.6, p = 0.0004; Umzobo 6D), xa kuthelekiswa nemigibe yokulawula.
Imigibe yezithambiso ezifumaneka ngaphandle kweendawo ezinokubakho phakathi kweendawo zokuzalela kunye neendawo zasemaphandleni e-Ethiopia yasetyenziswa ukuvavanya ukuba iingcongconi ze-malaria zisebenzisa ivumba lomchamo wenkomo njengesixhobo sokuhlala kwindawo yazo. Ukuba akukho zimpawu zomchamo wenkomo, ubushushu, kwaye nokuba kukho okanye kungekho vumba lomchamo wenkomo, akukho ngcongconi zibanjwe (Ifayile eyongezelelweyo 1: Umfanekiso S3). Nangona kunjalo, xa kukho ubushushu obuphezulu kunye nevumba lomchamo wenkomo, iingcongconi ze-malaria eziziimazi zatsalwa zaza zabanjwa, nangona zincinci, ngaphandle kobudala bomchamo (χ2(5, 25) = 2.29, p = 0.13; Ifayile eyongezelelweyo 1: Umfanekiso S3). Ngokwahlukileyo koko, ulawulo lwamanzi aluzange lubambe iingcongconi ze-malaria kubushushu obuphezulu (Ifayile eyongezelelweyo 1: Umfanekiso S3).
Iingcongconi zeMalaria zifumana kwaye zisasaze iikhompawundi ezine-nitrogen ngokuzinika imali kumchamo wenkomo (oko kukuthi, amachibi) ukuze ziphucule iimpawu zobomi, ezifana nezinye izinambuzane [2, 4, 24, 25, 26]. Umchamo wenkomo sisixhobo esifumaneka ngokulula esivuselelekayo esinxulunyaniswa neendawo zokuphumla zezilwanyana ezithwala i-malaria, njengeendawo zokugcina iinkomo kunye nezityalo ezinde kufutshane namakhaya asemaphandleni kunye neendawo zokuzala. Iingcongconi zabasetyhini zifumana esi sixhobo ngokuvumba kwaye ziyakwazi ukulawula ukufunxwa kweekhompawundi ezine-nitrogen kumchamo, kubandakanya i-urea, eyona nto iphambili ene-nitrogen kumchamo [15, 16]. Ngokuxhomekeke kwimeko yomzimba wengcongconi yabasetyhini, izondlo kumchamo zabelwe ukuphucula umsebenzi wokubhabha kunye nokusinda kweengcongconi zabasetyhini ezifuna umninimzi, kunye neempawu zokusinda kunye nokuzala zabantu abondliwe ngegazi ngexesha lomjikelo wokuqala we-gonadotropic. Ke ngoko, ukuxuba umchamo kudlala indima ebalulekileyo yesondlo kwiingcongconi ze-malaria ezivaliweyo njengabantu abadala abangondlekanga [8], njengoko kubonelela iingcongconi zabasetyhini ngamandla okufumana iikhompawundi ezibalulekileyo ze-nitrogen ngokuzibandakanya ekutyeni okunomngcipheko omncinci. Olu fumaniso lunemiphumo ebalulekileyo ye-epidemiological, njengoko iimazi zonyusa umsebenzi wazo wokubhabha. ixesha lokuphila, umsebenzi kunye nemveliso yokuzala, konke oku kuchaphazela amandla evektha. Ngaphezu koko, le ndlela yokuziphatha isenokuba yeyona nto iphambili kwiinkqubo zolawulo lwevektha kwixesha elizayo.