Kupeza ndi kugawa zakudya kumaphatikiza kufunafuna zakudya za tizilombo ndi mbiri ya moyo. Pofuna kubweza zofooka za zakudya zinazake pazigawo zosiyanasiyana za moyo, tizilombo tingapeze zakudyazi kudzera mu chakudya chowonjezera, mwachitsanzo, podya zotulutsa za nyama zomwe zimakhala ndi vertebrate mu njira yotchedwa puddles. Udzudzu wotchedwa Anopheles arabiani umawoneka kuti suli ndi zakudya zokwanira ndipo, motero, umafuna zakudya kuti zigwiritsidwe ntchito kagayidwe kachakudya komanso kubereka. Cholinga cha kafukufukuyu chinali kuwona ngati An. arabiensis akugwedezeka pa mkodzo wa ng'ombe kuti apeze zakudya zimathandizira kuti pakhale mbiri ya moyo.
Onetsetsani kuti ndi yotetezeka. Arabiensis adakopeka ndi fungo la mkodzo watsopano, wa maola 24, wa maola 72, ndi wa maola 168, komanso akazi ofunafuna ndi kupatsidwa magazi (omwe amadya maola 48 pambuyo pa chakudya cha magazi) adayesedwa mu Y-tube olfactometer, ndipo akazi apakati adayesedwa kuti ayese kubereka. Kenako kusanthula kwa mankhwala ndi electrophysiological kunagwiritsidwa ntchito kuzindikira mankhwala ogwira ntchito mu mkodzo wa ng'ombe m'magulu onse anayi azaka. Zosakaniza zopangidwa za mankhwala ogwira ntchito zidayesedwa mu Y-tube ndi mayeso akumunda. Kuti afufuze mkodzo wa ng'ombe ndi urea womwe uli ndi nayitrogeni ngati zakudya zowonjezera za matenda a malungo, magawo odyetsera ndi makhalidwe a mbiri ya moyo adayesedwa. Chiŵerengero cha udzudzu wamkazi ndi kuchuluka kwa mkodzo wa ng'ombe ndi urea zomwe zimayamwa zidayesedwa. Pambuyo podyetsa, akazi adayesedwa kuti apulumuke, kuuluka molumikizidwa, ndi kubereka.
Fufuzani magazi ndi chakudya cha wolandirayo. Mu kafukufuku wa labotale ndi m'munda, Aarabu anakopeka ndi fungo lachilengedwe komanso lopangidwa la mkodzo watsopano ndi wokalamba wa ng'ombe. Akazi apakati sanasamale ndi momwe mkodzo wa ng'ombe umayankhira pamalo oberekera. Akazi omwe amafunafuna ndi kuyamwa magazi amayamwa mkodzo wa ng'ombe ndi urea ndipo amagawa zinthuzi malinga ndi mbiri ya moyo wawo ngati chifukwa cha momwe thupi lawo limakhalira pouluka, kupulumuka, kapena kubereka.
Kupeza ndi kufalitsa mkodzo wa ng'ombe wa Anopheles kuti ukhale ndi makhalidwe abwino a moyo. Kudyetsa mkodzo wowonjezera wa ng'ombe kumakhudza mphamvu ya ng'ombe mwachindunji mwa kuwonjezera moyo watsiku ndi tsiku komanso kuchuluka kwa ng'ombe, komanso mwanjira ina mwa kusintha zochita za ndege ndipo motero ziyenera kuganiziridwa m'mafano amtsogolo.
Kupeza ndi kugawa zakudya kumaphatikiza kufunafuna zakudya ndi mbiri ya moyo [1,2,3]. Tizilombo timatha kusankha ndikupeza chakudya ndikuchita kudyetsa koyenera kutengera kupezeka kwa chakudya ndi zofunikira za michere [1, 3]. Kugawa kwa zakudya kumadalira njira ya moyo ndipo kungayambitse zofunikira zosiyanasiyana za ubwino ndi kuchuluka kwa zakudya m'magawo osiyanasiyana a moyo wa tizilombo [1, 2]. Pofuna kubweza zofooka za michere inayake, tizilombo titha kupeza michere iyi kudzera mu kudyetsa kowonjezera, monga pamatope, ndowe zosiyanasiyana ndi zotulutsa za nyama zowongoka, ndi nyama zowola, njira yotchedwa mabowo [2]. Ngakhale mitundu yosiyanasiyana ya gulugufe ndi njenjete imafotokozedwa makamaka, mabowo othirira madzi amapezekanso m'magulu ena a tizilombo, ndipo kukopeka ndi kudya zinthu zamtunduwu kumatha kukhala ndi zotsatirapo zazikulu pa thanzi ndi makhalidwe ena a moyo [2, 4, 5, 6] ,7]. Udzudzu wa malungo Anopheles gambiae sensu lato (sl) umatuluka ngati munthu wamkulu 'wosadya bwino' [8], kotero kuthirira kumatha kukhala ndi gawo lofunikira m'makhalidwe ake a mbiri ya moyo, koma khalidweli mpaka pano yanyalanyazidwa. Kugwiritsa ntchito kusonkhezera ngati njira yowonjezera kudya zakudya zofunika kwambiri m'gulu lofunikali kuyenera kuganiziridwa bwino chifukwa izi zitha kukhala ndi zotsatirapo zazikulu pa matenda.
Kudya nayitrogeni mwa udzudzu wachikulire wa Anopheles kumakhala kochepa chifukwa cha kuchuluka kwa ma calories omwe amatengedwa kuchokera ku mphutsi komanso kusagwiritsa ntchito bwino chakudya chamagazi [9]. Ann.gambiae sl yachikazi nthawi zambiri imakwaniritsa izi powonjezera ndi chakudya chowonjezera chamagazi [10, 11], motero kuyika anthu ambiri pachiwopsezo chotenga matendawa ndikuyika udzudzu pachiwopsezo chachikulu chodya. Kapenanso, udzudzu ungagwiritse ntchito kudyetsa kowonjezera kwa ndowe za m'chiuno kuti upeze mankhwala a nayitrogeni omwe amathandizira kusintha ndi kuuluka, monga momwe tizilombo tina tawonetsera [2]. Pachifukwa ichi, kukopa kwamphamvu komanso kosiyana kwa mtundu umodzi mwa abale mkati mwa An. Gulu la mitundu ya Gambian sl, Anopheles arabinis, mkodzo wa ng'ombe watsopano komanso wokalamba [12,13,14], ndikosangalatsa. Anopheles arabinis ndi wokonda mwayi pa zomwe amakonda ndipo amadziwika kuti amagwirizana ndi kudya ng'ombe. Mkodzo wa ng'ombe ndi gwero lolemera la mankhwala a nayitrogeni, ndipo urea imapanga 50-95% ya nayitrogeni yonse mu mkodzo watsopano [15, 16]. Pamene mkodzo wa ng'ombe ukukalamba, tizilombo toyambitsa matenda timagwiritsa ntchito zinthuzi kuti tichepetse kuuma kwa mankhwala a nayitrogeni mkati mwa maola 24 [15]. Chifukwa cha kuchuluka kwa ammonia mwachangu, komwe kumakhudzana ndi kuchepa kwa nayitrogeni wachilengedwe, tizilombo toyambitsa matenda a alkalophilic (ambiri mwa iwo amapanga mankhwala oopsa kwa udzudzu) timakula [15], omwe mwina ndi akazi a Ann.arabiensis omwe amakonda kwambiri mkodzo ali ndi maola 24 kapena kuchepera [13, 14].
Mu kafukufukuyu, Ans wolandira ndi wopatsidwa magazi anafunidwa. Pa nthawi yoyamba ya gonadotropin, arabiensis inayesedwa kuti ipeze mankhwala a nayitrogeni, kuphatikizapo urea, mwa kusakaniza mkodzo. Kenako, mayesero angapo adachitika kuti awone momwe udzudzu wamkazi amagawira chakudya ichi kuti chikhale ndi moyo wabwino, kubereka komanso kufunafuna chakudya china. Pomaliza, fungo la mkodzo wa ng'ombe watsopano ndi wokalamba linayesedwa kuti lidziwe ngati izi zinapereka zizindikiro zodalirika za An wolandira ndi wopatsidwa magazi. Pofufuza kwawo chakudya ichi, arabiensis adapeza mgwirizano wa mankhwala kumbuyo kwa kukongola kosiyana komwe kunawonedwa. Zosakaniza zopangidwa ndi fungo la mankhwala osasunthika (VOCs) omwe adapezeka mu mkodzo wa maola 24 zinawunikidwanso pansi pa mikhalidwe yamunda, kukulitsa zotsatira zomwe zapezeka pansi pa mikhalidwe ya labotale ndikuwonetsa momwe fungo la mkodzo wa ng'ombe limakhudzira mikhalidwe yosiyanasiyana ya thupi. Kukopa udzudzu. Zotsatira zomwe zapezeka zimatsimikizira kuti An. arabiensis amapeza ndikugawa mankhwala a nayitrogeni omwe amapezeka mu mkodzo wa nyama zam'mimba kuti akhudze mawonekedwe a mbiri ya moyo. Zotsatirazi zikukambidwa pankhani ya zotsatira zomwe zingachitike chifukwa cha matenda komanso momwe zingagwiritsidwire ntchito poyang'anira ndi kuwongolera ventricles.
Anopheles arabicans (mtundu wa Dongola) adasungidwa pa 25 ± 2 °C, 65 ± 5% RH ndi kuwala kwa maola 12:12. Mphutsi zinaleredwa m'mathireyi apulasitiki (20 cm × 18 cm × 7 cm) odzazidwa ndi madzi osungunuka ndikudyetsedwa chakudya cha nsomba cha Tetramin® (Tetra Werke, Melle, DE). Ma pupae adasonkhanitsidwa m'makapu 30 ml (Nolato Hertila, Åstorp, SE) kenako adasamutsidwira ku zimbudzi za Bugdorm (30 cm × 30 cm × 30 cm; MegaView Science, Taichung, Taiwan) kuti alole kuti akuluakulu atuluke. Akuluakulu adapatsidwa yankho la 10% la sucrose ad libitum mpaka masiku 4 atangotuluka (dpe), pomwe akazi omwe akufunafuna alendo adapatsidwa chakudya nthawi yomweyo asanayambe kuyesa, kapena adaphedwa ndi madzi osungunuka usiku wonse asanayambe kuyesa, monga tafotokozera pansipa. Akazi omwe adagwiritsidwa ntchito poyesa machubu othamanga adaphedwa ndi njala kwa nthawi yochepa. Maola 4-6 ndi madzi nthawi yomweyo. Pofuna kukonzekera udzudzu woyamwa magazi kuti ufufuze za matenda ena, akazi 4 a dpe adapatsidwa magazi a nkhosa opanda ma fibrotic (Håtunalab, Bro, SE) pogwiritsa ntchito njira yodyetsera nembanemba (Hemotek Discovery Workshops, Accrington, UK). Akazi odzaza kwambiri adasamutsidwira m'mabokosi osiyanasiyana ndipo adapatsidwa chakudya mwachindunji, monga tafotokozera pansipa, kapena 10% sucrose ad libitum kwa masiku atatu mayeso asanachitike. Akazi omalizawa adagwiritsidwa ntchito poyesa matenda a flight tube ndipo adasamutsidwira ku labotale, kenako adamwa madzi osungunuka ad libitum kwa maola 4-6 mayeso asanachitike.
Kuyesa kudya kunagwiritsidwa ntchito poyesa kuchuluka kwa mkodzo ndi urea mwa akazi achikulire achiarabu. Akazi omwe amafunafuna alendo komanso omwe amadyetsedwa magazi anapatsidwa zakudya zokhala ndi mkodzo wa ng'ombe watsopano komanso wokalamba wochepetsedwa ndi 1%, kuchuluka kwa urea kosiyanasiyana, ndi zowongolera ziwiri (10% sucrose ndi madzi) kwa maola 48. Kuphatikiza apo, utoto wa chakudya (1 mg ml-1 xylene cyanide FF; CAS 2650-17-1; Sigma-Aldrich, Stockholm, SE) unawonjezedwa muzakudyazo ndipo unaperekedwa mu 4 × 4 matrix mu machubu a 250 µl microcentrifuge (Aoxygen Scientific, Union City, CA, US; Chithunzi 1A) Dzazani mpaka m'mphepete (~300 µl). Kuti mupewe mpikisano pakati pa udzudzu ndi zotsatira zomwe zingachitike chifukwa cha utoto, ikani udzudzu 10 mu mbale yayikulu ya Petri (12 cm m'mimba mwake ndi 6 cm kutalika; Semadeni, Ostermundigen, CH; Chithunzi 1A) mumdima wonse pa 25 ± 2 cm °C ndi 65 ± 5% poyerekeza. chinyezi. Kuyesera kumeneku kunabwerezedwanso nthawi 5 mpaka 10. Pambuyo podya zakudya, udzudzu unayikidwa pa -20 °C mpaka kufufuzidwa kwina.
Yang'anani mkodzo wa ng'ombe ndi urea womwe umayamwa ndi ng'ombe yaikazi ya Anopheles arabianus ndi yoyamwa magazi. Mu kuyesa kudyetsa (A), udzudzu wamkazi unapatsidwa zakudya zokhala ndi mkodzo watsopano ndi wakale wa ng'ombe, kuchuluka kosiyanasiyana kwa urea, sucrose (10%), ndi madzi osungunuka (H2O). Akazi ofunafuna ng'ombe (B) ndi odyetsedwa magazi (C) adamwa sucrose yambiri kuposa zakudya zina zilizonse zomwe zidayesedwa. Dziwani kuti akazi ofunafuna ng'ombe adamwa mkodzo wa ng'ombe wa maola 72 wosakwana mkodzo wa ng'ombe wa maola 168 (B). Avereji ya nayitrogeni yonse (± kupotoka kwachizolowezi) ya mkodzo ikuimiridwa mu chithunzi. Akazi ofunafuna ng'ombe (D, F) ndi oyamwa magazi (E, G) amamwa urea motengera mlingo. Avereji ya voliyumu yopumira (D, E) yokhala ndi mayina osiyanasiyana a zilembo inali yosiyana kwambiri (ANOVA ya njira imodzi pogwiritsa ntchito kusanthula kwa Tukey's post hoc; p < 0.05). Mipiringidzo yolakwika ikuyimira cholakwika cha mean (BE). Mzere wowongoka wolunjika ukuyimira mzere wowongolera wa log-linear (F, G)
Kuti chakudya chitulutsidwe, udzudzu unayikidwa m'machubu a 1.5 ml a microcentrifuge okhala ndi 230 µl ya madzi osungunuka ndipo minofu inasokonekera pogwiritsa ntchito pestle yotayidwa ndi mota yopanda zingwe (VWR International, Lund, SE), kenako centrifugation inayikidwa pa 10 krpm kwa mphindi 10. Supernatant (200 µl) inasamutsidwira ku microplate ya 96-well (Sigma-Aldrich) ndipo absorbance (λ620) inapezeka pogwiritsa ntchito spectrophotometer-based microplate reader (SPECTROStar® Nano, BMG Labtech, Ortenberg, DE) nm). Kapenanso, udzudzu unaphwanyidwa mu 1 ml ya madzi osungunuka, 900 µl yomwe inasamutsidwira ku cuvette kuti iwunikenso spectrophotometric (λ 620 nm; UV 1800, Shimadzu, Kista, SE). Kuti muyeze kuchuluka kwa chakudya chomwe mumadya, njira yokhazikika inakonzedwa mwa kusakaniza serial kuti ipereke 0.2 µl mpaka 2.4. µl ya 1 mg ml-1 xylene cyanide. Kenako, kuchuluka kwa utoto wodziwika bwino kunagwiritsidwa ntchito kudziwa kuchuluka kwa chakudya chomwe udzudzu uliwonse unadya.
Deta ya voliyumu idasanthulidwa pogwiritsa ntchito kusanthula kwa njira imodzi kwa kusiyana (ANOVA) kutsatiridwa ndi kufananiza kwa Tukey kwa post hoc pairwise (JMP Pro, v14.0.0, SAS Institute Inc., Cary, NC, US, 1989–2007). Kusanthula kwa Linear regression kunafotokoza kuchuluka kwa urea komwe kumadalira kuchuluka kwa udzudzu ndipo kunayerekeza mayankho pakati pa udzudzu wofunafuna ndi woyamwa magazi (GraphPad Prism v8.0.0 ya Mac, GraphPad Software, San Diego, CA, US).
Pafupifupi 20 µl ya zitsanzo za mkodzo kuchokera ku gulu lililonse la zaka zinamangidwa pa Chromosorb® W/AW (10 mg 80/100 mesh, Sigma Aldrich) ndipo zinaikidwa mu makapisozi a tin (8 mm × 5 mm). Makapisozi anaikidwa m'chipinda choyaka cha CHNS/O analyzer (Flash 2000, Thermo Fisher Scientific, Waltham, MA, US) kuti adziwe kuchuluka kwa nayitrogeni mu mkodzo watsopano ndi wakale malinga ndi njira ya wopanga. Nayitrogeni yonse (g N l-1) inayesedwa kutengera kuchuluka kwa urea komwe kumadziwika komwe kumagwiritsidwa ntchito ngati muyezo.
Kuti muwone momwe zakudya zimakhudzira kupulumuka kwa akazi omwe akufunafuna magazi komanso omwe amayamwa magazi, udzudzu unayikidwa payekhapayekha m'mbale zazikulu za Petri (12 cm m'mimba mwake ndi 6 cm kutalika; Semadeni) ndi dzenje lophimbidwa ndi maukonde m'chivindikiro (3 cm m'mimba mwake) ndi Kupereka mpweya wabwino ndi chakudya. Zakudya zinaperekedwa mwachindunji pambuyo pa 4 dpe ndipo zinaphatikizapo 1% mkodzo watsopano komanso wokalamba wa ng'ombe, kuchuluka kwa urea kanayi, ndi zowongolera ziwiri, 10% sucrose ndi madzi. Chakudya chilichonse chinayikidwa pa tampon ya mano (DAB Dental AB, Upplands Väsby, SE) mu syringe ya 5 ml (Thermo Fisher Scientific, Gothenburg, SE), chopukutiracho chinachotsedwa, ndikuyikidwa pamwamba pa mbale ya petri (chithunzi 1).1A). Sinthani zakudya zanu tsiku lililonse. Sungani labu monga tafotokozera pamwambapa. Udzudzu wopulumuka unawerengedwa kawiri patsiku, pomwe udzudzu wakufa unatayidwa mpaka udzudzu womaliza utafa (n = 40 pa chithandizo chilichonse). Kupulumuka kwa udzudzu wodyetsedwa zakudya zosiyanasiyana kunawunikidwa pogwiritsa ntchito ma curve opulumuka a Kaplan-Meyer ndi mayeso a log-rank kuti ayerekezere kufananiza kugawa kwa moyo pakati pa zakudya (IBM SPSS Statistics 24.0.0.0).
Chipangizo chopangira udzudzu chopangidwa mwapadera chochokera kwa Attisano et al.[17], chopangidwa ndi ma panel a acrylic owoneka bwino okwana 5 mm (10 cm mulifupi x 10 cm kutalika x 10 cm kutalika) opanda ma panel akutsogolo ndi kumbuyo (Chithunzi 3: pamwamba). Cholumikizira chozungulira chokhala ndi chubu choyima chopangidwa ndi mzati wa gasi chromatography (0.25 mm id; 7.5 cm L) chokhala ndi malekezero omangiriridwa ku singano ya tizilombo yopachikidwa pakati pa maginito awiri a neodymium 9 cm kutali. Chubu chopingasa chopangidwa ndi chinthu chomwecho (6.5 cm L) chinagawa chubu choyima pakati kuti chipange mkono womangiriridwa ndi mkono womwe unali ndi chidutswa chaching'ono cha zojambulazo za aluminiyamu ngati chizindikiro cholepheretsa kuwala.
Akazi omwe anali ndi njala kwa maola 24 anapatsidwa chakudya chomwe chili pamwambapa kwa mphindi 30 asanapatsidwe mankhwala. Udzudzu wa akazi womwe unadyetsedwa mokwanira unayikidwa pa ayezi kwa mphindi 2-3 ndipo unalumikizidwa ku zikhomo za tizilombo pogwiritsa ntchito sera (Joel Svenssons Vaxfabrik AB, Munka Ljungby, SE) kenako unamangidwa m'manja mwa machubu opingasa. Flying Mill. Kusintha kwa ndege iliyonse kunalembedwa ndi deta yolembedwa mwapadera, kenako kusungidwa ndikuwonetsedwa pogwiritsa ntchito pulogalamu ya PC-Lab 2000™ (v4.01; Velleman, Gavere, BE). Mphero yoyendetsera ndege inayikidwa m'chipinda cholamulidwa ndi nyengo (12 h:12 h, kuwala: mdima, 25 ± 2 °C, 65 ± 5% RH).
Kuti tiwone momwe ndege imayendera, mtunda wonse wouluka (m) ndi chiwerengero chonse cha zochitika zotsatizana zouluka zinawerengedwa pa ola limodzi pa nthawi ya maola 24. Kuphatikiza apo, mtunda wapakati womwe akazi amauluka unayerekezeredwa pa chithandizo ndikuwunika pogwiritsa ntchito njira imodzi ya ANOVA ndi kusanthula kwa Tukey pambuyo pa hoc (JMP Pro, v14.0.0, SAS Institute Inc.), komwe mtunda wapakati unkaonedwa ngati chosinthika chodalira, pomwe chithandizo ndi chinthu chodziyimira pawokha. Kuphatikiza apo, chiwerengero chapakati cha maulendo chimawerengedwa mu mphindi 10 zowonjezera.
Kuti aone momwe zakudya zimakhudzira kubereka kwa An.arabiensis, akazi asanu ndi mmodzi (4 dpe) adasamutsidwira mwachindunji ku zimbudzi za Bugdorm (30 cm × 30 cm × 30 cm) atatenga magazi kenako adapereka zakudya zoyesera kwa maola 48 monga tafotokozera pamwambapa. Zakudya zinachotsedwa ndipo makapu oberekera (30 ml; Nolato Hertila) odzazidwa ndi 20 ml ya madzi osungunuka adaperekedwa tsiku lachitatu kwa maola 48, kusintha maola 24 aliwonse. Bwerezani njira iliyonse yodyera nthawi 20-50. Mazira adawerengedwa ndikulembedwa pa zimbudzi zilizonse zoyesera. Zitsanzo zazing'ono za mazira zidagwiritsidwa ntchito poyesa kukula ndi kutalika kwa mazira pawokha (n ≥ 200 pa chakudya chilichonse) pogwiritsa ntchito maikulosikopu ya Dialux-20 (DM1000; Ernst Leitz Wetzlar, Wetzlar, DE) yokhala ndi Leica Camera (DFC) 320 R2; Leica Microsystems Ltd., DE). Mazira otsalawo anasungidwa m'chipinda cholamulidwa ndi nyengo pansi pa mikhalidwe yokhazikika yolerera kwa maola 24, ndipo chitsanzo cha mphutsi yoyamba yomwe yangotuluka kumene (n ≥ 200 pa chakudya chilichonse) chinayesedwa, monga tafotokozera pamwambapa. Chiwerengero cha mazira ndi kukula kwa mazira ndi mphutsi zinayerekezeredwa pakati pa chithandizo ndi kugwiritsa ntchito njira imodzi ya ANOVA ndi kusanthula kwa Tukey pambuyo pa hoc (JMP Pro, v14.0.0, SAS Institute Inc.).
Mkodzo wotuluka m'mutu (osapitirira ola limodzi pambuyo poyesa), mkodzo wokalamba wa maola 24, maola 72 ndi maola 168 unasonkhanitsidwa kuchokera ku zitsanzo zomwe zinasonkhanitsidwa kuchokera ku ng'ombe za Zebu, Arsi races. Kuti zikhale zosavuta, zitsanzo za mkodzo zinasonkhanitsidwa m'mawa kwambiri ng'ombe zikadali m'khola. Zitsanzo za mkodzo zinasonkhanitsidwa kuchokera kwa anthu 10 ndipo 100-200 ml ya chitsanzo chilichonse chinasamutsidwira ku matumba ophikira a polyamide (Toppits Cofresco, Frischhalteprodukte GmbH and Co., Minden, DE) mu polyamide ya malita atatu yokhala ndi chivindikiro mu ng'oma zapulasitiki za vinyl chloride. Zotuluka m'mutu kuchokera ku chitsanzo chilichonse cha mkodzo wa ng'ombe zinasonkhanitsidwa mwachindunji (mwatsopano) kapena mutakhwima kutentha kwa chipinda kwa maola 24, maola 72 ndi maola 168, mwachitsanzo chitsanzo chilichonse cha mkodzo chinali choyimira gulu lililonse la msinkhu.
Pa kusonkhanitsa ma volatile a headspace, njira yotsekedwa idagwiritsidwa ntchito kufalitsa mpweya wosefedwa ndi kaboni (100 ml min-1) kudzera mu thumba la polyamide kupita ku column yotsatsira kwa maola 2.5 pogwiritsa ntchito pampu ya vacuum ya diaphragm (KNF Neuberger, Freiburg, DE). Monga chowongolera, kusonkhanitsa ma headspace kunachitika kuchokera ku thumba lopanda kanthu la polyamide. Column yotsatsira idapangidwa ndi chubu cha Teflon (5.5 cm x 3 mm id) chokhala ndi 35 mg ya Porapak Q (50/80 mesh; Waters Associates, Milford, MA, US) pakati pa ma plug a ubweya wagalasi. Musanagwiritse ntchito, column idatsukidwa ndi 1 ml ya n-hexane yowonjezeredwa (Merck, Darmstadt, DE) ndi 1 ml ya pentane (99.0% pure solvent GC grade, Sigma Aldrich). Ma volatile otsatsira adachotsedwa ndi 400 μl ya pentane. Ma headspace adasonkhanitsidwa pamodzi kenako nkusungidwa pa -20°C mpaka atagwiritsidwa ntchito nthawi ina. kusanthula.
Mayankho a machitidwe a An omwe amafunafuna komanso kudya magazi. Ma extracts a volatile ochokera ku mkodzo watsopano, wa maola 24, maola 72, ndi maola 168 adawunikidwa kuti apeze ma volatile ochokera ku udzudzu wa Arabidopsis pogwiritsa ntchito chubu chowongolera chagalasi [18]. Kuyeseraku kunachitika panthawi ya ZT 13-15, nthawi yayitali ya ntchito ya An yofunafuna kunyumba. Arab [19]. Chubu chowongolera chagalasi (80 cm × 9.5 cm id) chidawunikidwa ndi kuwala kofiira kwa 3 ± 1 lx kuchokera pamwamba. Kuyenda kwa mpweya wosefedwa ndi wonyowa ndi makala (25 ± 2 °C, 65 ± 2% chinyezi) kudadutsa mayeso a bioassay pa 30 cm s-1. Mpweya umadutsa m'ma screens angapo achitsulo chosapanga dzimbiri, ndikupanga kuyenda kwa laminar ndi kapangidwe kofanana ka ma plume. Chotsukira mano (4 cm × 1 cm; L:D; DAB Dental AB), chopachikidwa pa coil ya 5 cm kumapeto kwa mphepo olfactometer, yokhala ndi kusintha kwa stimulator mphindi 5 zilizonse. Pofufuza, 10 μl ya chotsitsa chilichonse cha mutu, chochepetsedwa 1:10, chinagwiritsidwa ntchito ngati choyambitsa. Kuchuluka kofanana kwa pentane kunagwiritsidwa ntchito ngati chowongolera. Udzudzu wofunafuna kapena woyamwa magazi payekhapayekha unayikidwa m'mabokosi otulutsira munthu aliyense maola 2-3 asanayambe kuyesa. Khola lotulutsira linayikidwa kumbali ya mphepo ya olfactometer, ndipo udzudzu unaloledwa kuzolowera kwa mphindi 1, kenako valavu ya gulugufe ya khola inatsegulidwa kuti itulutse. Kukopa chithandizo kapena kulamulira kunasanthulidwa ngati kuchuluka kwa udzudzu womwe unakumana ndi gwero mkati mwa mphindi 5 kuchokera pamene linatulutsidwa. Chotsitsa chilichonse cha mutu ndi kulamulira kosasunthika kunabwerezedwanso nthawi zosachepera 30, ndipo kuti tipewe zotsatira za tsiku limodzi, kuchuluka komweko kwa chithandizo ndi kulamulira kunayesedwa tsiku lililonse loyesera. Fufuzani mayankho kuchokera kwa wolandira ndi omwe amadya magazi. Magulu a Arabic vs headspace anasanthulidwa pogwiritsa ntchito nominal logistic regression kutsatiridwa ndi kufananiza pawiri kwa ma odd ratios (JMP Pro, v14.0.0, SAS Institute Inc.).
Yankho la An pakukula kwa ng'ombe. Zotulutsa za mkodzo wa ng'ombe zatsopano komanso zakale zinasanthulidwa m'makhola a Bugdorm (30 cm × 30 cm × 30 cm; MegaView Science). Makapu apulasitiki (30 mL; Nolato Hertila) odzazidwa ndi madzi osungunuka a 20 mL anapereka gawo loyambira kubereka ndipo anaikidwa m'makona osiyana a khola, mtunda wa masentimita 24. Makapu ochizira anasinthidwa ndi 10 μl ya chotsitsa chilichonse cha mutu pa dilution ya 1:10. Kuchuluka kofanana kwa pentane kunagwiritsidwa ntchito kusintha chikho chowongolera. Makapu ochizira ndi owongolera anasinthidwa pakati pa kuyesera kulikonse kuti ayang'anire zotsatira za malo. Akazi khumi omwe amadyetsedwa magazi anatulutsidwa m'makhola oyesera pa ZT 9-11 ndipo mazira m'makhola anawerengedwa patatha maola 24. Njira yowerengera index ya kubereka ndi iyi: (chiwerengero cha mazira omwe anaikidwa mu chikho chochizira - chiwerengero cha mazira omwe anaikidwa mu chikho chowongolera) / (chiwerengero chonse cha mazira omwe anaikidwa). Chithandizo chilichonse chinabwerezedwa kasanu ndi katatu.
Kusanthula kwa gasi chromatographic ndi electron antenna pattern detection (GC-EAD) ya An.arabiensis yachikazi kunachitika monga momwe tafotokozera kale [20]. Mwachidule, zotulutsa zatsopano za headspace volatile zinalekanitsidwa pogwiritsa ntchito Agilent Technologies 6890 GC (Santa Clara, CA, US) yokhala ndi column ya HP-5 (30 m × 0.25 mm id, 0.25 μm film thickness, Agilent Technologies). ndi mkodzo wokalamba. Haidrojeni idagwiritsidwa ntchito ngati gawo loyenda ndi avareji ya kuyenda kwa 45 cm s-1. Chitsanzo chilichonse (2 μl) chidabayidwa kwa masekondi 30 munjira yopanda kugawanika ndi kutentha kolowera kwa 225 °C. Kutentha kwa uvuni wa GC kudakonzedwa kuyambira 35 °C (kugwira kwa mphindi 3) mpaka 300 °C (kugwira kwa mphindi 10) pa 10 °C min-1. Mu splitter ya GC effluent, 4 psi ya nayitrogeni idawonjezedwa ndikugawidwa 1:1 mu Gerstel 3D/2 low dead volume cross (Gerstel, Mülheim, DE) pakati pa chowunikira cha flame ionization ndi EAD. GC effluent capillary ya EAD idadutsa mu mzere wosinthira wa Gerstel ODP-2, womwe umatsata kutentha kwa uvuni wa GC kuphatikiza 5 °C, mu chubu chagalasi (10 cm × 8 mm), komwe kudasakanizidwa ndi mpweya wosefedwa ndi kaboni (1.5 l min−1). Antena idayikidwa 0.5 cm kuchokera potulukira chubu. Udzudzu uliwonse unkaimira udzudzu umodzi wobwerezabwereza, ndipo kwa udzudzu wofunafuna malo okhala, udzudzu wobwerezabwereza katatu unachitidwa pa mkodzo wa msinkhu uliwonse.
Kuzindikira mankhwala ogwiritsira ntchito mphamvu m'mitsempha ya mkodzo wa ng'ombe watsopano ndi wokalamba pogwiritsa ntchito GC-MS yophatikizana ndi mass spectrometer (GC-MS; 6890 GC ndi 5975 MS; Agilent Technologies) kuti ipange mayankho a antenna mu kusanthula kwa GC-EAD, ikugwira ntchito mu electron impact ionization mode pa 70 eV. GC inali ndi HP-5MS UI-coated fused silica capillary column (60 m × 0.25 mm inner diameter, 0.25 μm film thickness) pogwiritsa ntchito helium ngati gawo loyenda ndi avareji ya linear flow rate ya 35 cm s-1. Chitsanzo cha 2 μl chinalowetsedwa pogwiritsa ntchito ma injector settings omwewo ndi kutentha kwa uvuni monga momwe zinalili pa kusanthula kwa GC-EAD. Ma compounds adazindikirika kutengera nthawi yawo yosungira (Kovát index) ndi mass spectra poyerekeza ndi laibulale yapadera ndi laibulale ya NIST14 (Agilent). Ma compounds odziwika adatsimikiziridwa polowetsa miyezo yeniyeni (Fayilo Yowonjezera 1: Table S2). Kuti mudziwe kuchuluka, heptyl acetate (10 ng, 99.8% kuyera kwa mankhwala, Aldrich) idabayidwa ngati muyezo wakunja.
Kuwunika mphamvu ya chisakanizo cha fungo lopangidwa ndi mankhwala ogwiritsidwa ntchito m'thupi omwe amapezeka mu mkodzo watsopano ndi wokalamba kuti akope Ans.arabiensis yemwe amafunafuna komanso kuyamwa magazi, pogwiritsa ntchito olfactometer ndi njira yomweyi monga momwe zilili pamwambapa. Zosakaniza zopangidwa zinatsanzira kapangidwe ndi kuchuluka kwa mankhwala mu zosakaniza zosakanikirana za mkodzo watsopano, wa maola 24, wa maola 48, wa maola 72, ndi wa maola 168 (Chithunzi 5D-G; Fayilo Yowonjezera 1: Gome S2). Kuti muwunike, gwiritsani ntchito 10 μl ya 1:100 dilution ya chisakanizo chonse chopangidwa, ndi kuchuluka konse kotulutsidwa kuyambira pafupifupi 140-2400 ng h-1, kuti muwone kukongola kwa udzudzu wolandira ndi kuyamwa magazi. Pambuyo pake, mayesowa amachitika pazosakaniza zonse, momwe zosakaniza zochotsera za mankhwala amodzi a chisakanizo chonsecho zimachotsedwa. Fufuzani mayankho kuchokera kwa Ans wolandira ndi womwa magazi. Zosakaniza zachiarabu vs zopangidwa ndi zochotsera zinasanthulidwa pogwiritsa ntchito nominal logistic regression kutsatiridwa ndi kufananiza kwa awiriawiri kwa odd. ma ratio (JMP Pro, v14.0.0, SAS Institute Inc.).
Kuti tiwone ngati mkodzo wa ng'ombe ungakhale ngati chizindikiro cha malo okhala udzudzu wa malungo, mkodzo wa ng'ombe watsopano komanso wokalamba, womwe unasonkhanitsidwa monga tafotokozera pamwambapa, ndipo madzi anaikidwa m'zidebe zodzaza ndi malita atatu (100 ml) ndikuyikidwa mu misampha ya nyambo. (BG-HDT version; BioGents, Regensburg, DE). Misampha khumi inayikidwa mtunda wa mamita 50 m'malo odyetserako ziweto, mtunda wa mamita 400 kuchokera kumudzi (Silay, Ethiopia, 5°53´24´´N, 37°29´24´´E) ndipo palibe ng'ombe, m'malo obereketserako okhazikika ndi m'midzi. Misampha isanu inatenthedwa kuti iyerekezere kukhalapo kwa wolandirayo, pomwe misampha isanu sinatenthedwe. Malo aliwonse ochiritsira amazunguliridwa usiku uliwonse kwa masiku asanu onse. Chiwerengero cha udzudzu chomwe chinagwidwa mu misampha yolumikizidwa ndi mkodzo wazaka zosiyanasiyana chinayerekezeredwa pogwiritsa ntchito njira yosinthira ndi kugawa kwa beta binomial (JMP Pro, v14.0.0, SAS Institute Inc.).
Mumudzi womwe uli pafupi ndi tawuni ya Maki, m'chigawo cha Oromia, ku Ethiopia (8° 11′ 08″ N, 38° 81′ 70″ E; Chithunzi 6A). Kafukufukuyu adachitika pakati pa mwezi wa Ogasiti ndi pakati pa mwezi wa Seputembala asanapereke mankhwala otsala amkati pachaka, pamodzi ndi nyengo yayitali yamvula. Mabanja asanu (20–50 m kutali) omwe ali kunja kwa mudzi adasankhidwa kuti achite kafukufukuyu (Chithunzi 6A). Njira zomwe zidagwiritsidwa ntchito posankha nyumbazo zinali: ziweto siziloledwa kulowa m'nyumba, kuphika m'nyumba (kutengera nkhuni kapena makala) sikuloledwa (osachepera nthawi yoyeserera), komanso nyumba zokhala ndi anthu awiri okha, ogona m'malo osapha tizilombo. pansi pa ukonde wa udzudzu wochiritsidwa. Chivomerezo cha makhalidwe abwino chaperekedwa ndi Institutional Research Ethics Review Board (IRB/022/2016) ya Faculty of Natural Sciences (CNS-IRB), Addis Ababa University, motsatira malangizo omwe adakhazikitsidwa ndi World Medical Association Declaration of Helsinki. Chilolezo kuchokera kwa mutu uliwonse wa banja chinapezedwa mothandizidwa ndi ogwira ntchito yowunikira zaumoyo. Njira yonseyi ikuvomerezedwa ndi akuluakulu am'deralo pamlingo wa chigawo ndi ward ('kebele'). Kapangidwe ka kuyeseraku kanatsatira kapangidwe ka 2 × 2 Latin square, momwe zosakaniza zopangidwa ndi zowongolera zinaperekedwa m'nyumba zolumikizidwa usiku woyamba ndikusinthidwa pakati pa nyumba usiku wotsatira woyeserera. Njirayi idabwerezedwanso kakhumi. Kuphatikiza apo, kuti ayese kuchuluka kwa udzudzu m'nyumba zosankhidwa, misampha ya CDC idayikidwa kuti iyende usiku asanu motsatizana kumayambiriro, pakati ndi kumapeto kwa mayeso amunda nthawi yomweyo ya tsiku.
Chosakaniza chopangidwa chokhala ndi mankhwala asanu ndi limodzi olimbikitsa chilengedwe chinasungunuka mu heptane (97.0% solvent GC grade, Sigma Aldrich) ndipo chinatulutsidwa pa 140 ng h-1 pogwiritsa ntchito chotulutsira thonje la thonje [20]. Chotulutsira thonjecho chinalola kuti mankhwala onse atulutsidwe mofanana panthawi yonse yoyesera ya maola 12. Heptane inagwiritsidwa ntchito ngati chowongolera. Botololo linapachikidwa pafupi ndi malo olowera a msampha wopepuka wa Centers for Disease Control and Prevention (CDC) (John W. Hock Company, Gainesville, FL, US; Chithunzi 6A). Misamphayo inapachikidwa 0.8 - 1 mita pamwamba pa nthaka, pafupi ndi phazi la bedi, ndipo wodzipereka anagona pansi pa ukonde wa udzudzu wosachiritsidwa ndipo anachitidwa ntchito pakati pa 18:00 ndi 06:30. Udzudzu womwe unagwidwa ndi kugonana ndi momwe thupi lilili (wosadyetsedwa, wodyetsedwa, woyembekezera pang'ono, komanso woyembekezera [21] pambuyo pake unafufuzidwa pogwiritsa ntchito kusanthula kwa polymerase chain reaction (PCR) kuti adziwe mtundu womwe umadziwika kuti A. gambiae sl. Ziwalo za malo ogwirira ntchito [23]. Mu kafukufuku wa m'munda, kutsekereza msampha wa nyumba zopatukana kunasanthulidwa pogwiritsa ntchito chitsanzo chodziwika bwino cha logistic fit, komwe kukopa kunali kusintha kodalira ndipo chithandizo (synthetic mixture vs control) chinali zotsatira zokhazikika (JMP® 14.0. 0. SAS Institute Inc.). Pano, tikupereka lipoti la χ2 ndi p-values ​​​​kuchokera ku mayeso a likelihood ratio.
Unikani ngati ndi yotetezeka. Arabiensis adatha kupeza mkodzo, womwe ndi gwero lake lalikulu la nayitrogeni, urea, mwa kudyetsa mwachindunji, mkati mwa maola 48 kuchokera pamene adapatsidwa kwa masiku 4 pambuyo pa (dpe) kuyesa kwa amayi ofuna chithandizo komanso kudyetsa magazi (Chithunzi 1A). Akazi onse omwe akufuna chithandizo komanso omwe amayamwa magazi adamwa sucrose yambiri kuposa zakudya zina zilizonse kapena madzi (F(5,426) = 20.15, p < 0.0001 ndi F(5,299) = 56.00, p < 0.0001, motsatana; Chithunzi 1B,C). Kuphatikiza apo, akazi omwe akufuna chithandizo adadya zochepa mu mkodzo pa maola 72 poyerekeza ndi mkodzo pa maola 168 (Chithunzi 1B). Atapatsidwa zakudya zokhala ndi urea, akazi omwe akufuna chithandizo adamwa urea wambiri pa 2.69 mM poyerekeza ndi kuchuluka kwina konse ndi madzi, pomwe sizikusiyana ndi 10% sucrose (F(10,813) = 15.72, p < 0.0001; Chithunzi 1D). Izi zinali zosiyana ndi momwe akazi omwe amadya magazi amayankhira, omwe nthawi zambiri amadya zakudya zambiri zokhala ndi urea kuposa madzi, ngakhale kuti ndi zochepa kwambiri kuposa 10% sucrose (F(10,557) = 78.35, p < 0.0001; Chithunzi 1).1E). Kuphatikiza apo, poyerekeza pakati pa mikhalidwe iwiri ya thupi, akazi omwe ali ndi phlebotomist adamwa urea wambiri kuposa akazi omwe akufunafuna alendo pamlingo wotsika kwambiri, ndipo akazi awa adamwa kuchuluka kofanana kwa urea pamlingo wapamwamba (F(1,953) = 78.82, p < 0.0001; Chithunzi 1F, G). Ngakhale kuti kudya zakudya zokhala ndi urea kumawoneka kuti kuli ndi phindu labwino kwambiri (Chithunzi 1D,E), akazi m'mikhalidwe yonse ya thupi adatha kusintha kuchuluka kwa urea komwe kumayamwa pamlingo wonse wa urea mwanjira yolunjika (Chithunzi 1F,G). ).Mofananamo, udzudzu umawoneka kuti umalamulira kuyamwa kwa nayitrogeni mwa kulamulira kuchuluka kwa mkodzo womwe umayamwa, chifukwa kuchuluka kwa nayitrogeni mu mkodzo kumaonekera mu kuchuluka kwa kuyamwa (Chithunzi 1B, C ndi B insets).
Kuti awone momwe mkodzo ndi urea zimakhudzira udzudzu womwe umafunafuna ndi kuyamwa magazi, akazi adapatsidwa mkodzo wazaka zonse zinayi (watsopano, maola 24, maola 72, ndi maola 168 atachotsedwa) komanso kuchuluka kwa urea, komanso madzi osungunuka ndi 10% sucrose adagwiritsidwa ntchito ngati chowongolera (Chithunzi 2A). Kusanthula kumeneku kwa kupulumuka kunawonetsa kuti zakudya zinali ndi zotsatirapo zazikulu pa kupulumuka konse kwa akazi omwe akufunafuna (mkodzo: χ2 = 108.5, df = 5, p < 0.0001; urea: χ2 = 122.8, df = 5, p < 0.0001; Chithunzi 2B, C) ndi akazi omwe amadya magazi (mkodzo: χ2 = 93.0, df = 5, p < 0.0001; urea: χ2 = 137.9, df = 5, p < 0.0001; Chithunzi 2D,E). Mu zoyeserera zonse, akazi omwe adapatsidwa mkodzo, urea, ndi madzi anali ndi chiwerengero chochepa cha moyo poyerekeza ndi akazi omwe adapatsidwa chakudya cha sucrose (Chithunzi 2B-E). Akazi omwe adapatsidwa mkodzo watsopano ndi wokalamba adawonetsa kuchuluka kosiyana kwa moyo, pomwe omwe adapatsidwa mkodzo wokalamba wa maola 72 (p = 0.016) anali ndi mwayi wochepa kwambiri wopulumuka (Chithunzi 2B). Kuphatikiza apo, akazi omwe adapatsidwa mkodzo wa 135 mM adapulumuka nthawi yayitali kuposa momwe amalamulira madzi (p < 0.04) (Chithunzi 2C). Poyerekeza ndi madzi, akazi omwe adapatsidwa mkodzo watsopano ndi mkodzo wa maola 24 adapulumuka nthawi yayitali (p = 0.001 ndi p = 0.012, motsatana; Chithunzi 2D), pomwe akazi omwe adapatsidwa mkodzo wa maola 72 adapulumuka nthawi yayitali kuposa omwe adapatsidwa mkodzo waufupi watsopano ndi mkodzo wazaka 24 (p < 0.0001 ndi p = 0.013, motsatana; Chithunzi 2D). Atapatsidwa 135 mM urea, akazi omwe adapatsidwa magazi adapulumuka nthawi yayitali kuposa onse kuchuluka kwina kwa urea ndi madzi (p < 0.013; Chithunzi 2E).
Kupulumuka kwa udzudzu wa ng'ombe ndi wa ng'ombe woyamwa magazi wotchedwa Anopheles arabinis. Mu kafukufuku wa bioassay (A), udzudzu wa ng'ombe unapatsidwa chakudya chokhala ndi mkodzo watsopano ndi wakale, kuchuluka kosiyanasiyana kwa urea, sucrose (10%) ndi madzi osungunuka (H2O). Kupulumuka kwa udzudzu wofunafuna (B, C) ndi woyamwa magazi (D, E) kunalembedwa maola 12 aliwonse mpaka akazi onse odyetsedwa mkodzo (B, D) ndi urea (C, E), ndi olamulira, Sucrose ndi madzi, atafa.
Mtunda wonse ndi chiwerengero cha maulendo omwe adapezeka mu mayeso a mphero yowuluka kwa maola 24 zinali zosiyana pakati pa udzudzu wofunafuna ndi woyamwa magazi, zomwe sizinawonetse kuchuluka kwa ntchito yowuluka (Chithunzi 3). Udzudzu wofunafuna womwe unkapereka mkodzo watsopano kapena sucrose ndi madzi unawonetsa njira zosiyanasiyana zowuluka (Chithunzi 3), pomwe akazi amadya mkodzo watsopano amakhala otanganidwa kwambiri m'mawa, pomwe omwe amadyetsedwa maola 24 ndi 168 Udzudzu wodya mkodzo unali ndi njira zosiyanasiyana zowuluka ndipo makamaka unali wozungulira tsiku lililonse. Udzudzu wachikazi womwe unkapereka sucrose kapena mkodzo wa maola 72 unawonetsa kugwira ntchito mkati mwa maola 24, pomwe akazi omwe ankapereka madzi anali otanganidwa kwambiri mkati mwa nthawiyo. Udzudzu wodya sucrose unali ndi ntchito zambiri usiku ndi m'mawa kwambiri, pomwe omwe ankadya mkodzo wa maola 72 anali ndi kuchepa kwa ntchito mkati mwa maola 24 (Chithunzi 3).
Kuuluka kwa mbalame yaikazi ya Anopheles arabinis yomwe ikufuna magazi yomwe ikudya mkodzo wa ng'ombe ndi urea. Mu mayeso a mphero yowuluka, udzudzu wamkazi yemwe amadya mkodzo wa ng'ombe watsopano komanso wokalamba, kuchuluka kosiyanasiyana kwa urea, sucrose (10%), ndi madzi osungunuka (H2O) kunalumikizidwa ku mikono yopingasa, yozungulira momasuka (pamwambapa). Kwa mbalame yaikazi yofuna magazi (kumanzere) ndi yoyamwa magazi (kumanja), mtunda wonse ndi chiwerengero cha maulendo ouluka pa ola lililonse pa chakudya chilichonse kwa maola 24 zinalembedwa (zakuda: imvi; zopepuka: zoyera). Mtunda wapakati ndi chiwerengero chapakati cha maulendo akuwonetsedwa kumanja kwa graph ya zochitika za circadian. Mipiringidzo yolakwika ikuyimira cholakwika cha mean. Kusanthula kwa ziwerengero onani zolemba
Kawirikawiri, ntchito yonse youluka ya akazi ofuna alendo inatsatira njira yofanana ndi ya mtunda wouluka kwa maola 24. Mtunda wapakati wouluka unakhudzidwa kwambiri ndi zakudya zomwe adadya (F(5, 138) = 28.27, p < 0.0001), ndipo akazi ofuna alendo omwe adamwa mkodzo kwa maola 72 adayenda mtunda wautali kwambiri poyerekeza ndi zakudya zina zonse (p < 0.0001), ndipo udzudzu wodyetsedwa sucrose unayenda mtunda wautali kuposa udzudzu watsopano (p = 0.022) ndi mkodzo wa maola 24 (p = 0.022). Mosiyana ndi njira youluka yomwe yafotokozedwa ndi zakudya za mkodzo, akazi ofuna alendo omwe adadya urea adawonetsa ntchito youluka mosalekeza kwa maola 24, zomwe zidafika pachimake mkati mwa theka lachiwiri la gawo lamdima (Chithunzi 3). Ngakhale kuti njira yogwirira ntchito inali yofanana, akazi ofuna alendo omwe adadya urea adawonjezera kwambiri mtunda wouluka kutengera kuchuluka kwa madzi omwe adamwa (F(5, 138) = 1310.91, p < 0.0001). Akazi omwe amapatsidwa kuchuluka kulikonse kwa urea adauluka nthawi yayitali kuposa akazi omwe amapatsidwa madzi kapena sucrose (p < 0.03).
Ntchito yonse youluka ya udzudzu woyamwa magazi inali yokhazikika ndipo inapitirira kwa maola 24 pa zakudya zonse, ndi kuchuluka kwa ntchito ya mkodzo mkati mwa theka lachiwiri la nthawi yamdima kwa akazi omwe amadya madzi komanso akazi omwe amadya atsopano komanso azaka 24 (chithunzi 3). Ngakhale kuti kudya mkodzo kunakhudza kwambiri mtunda wapakati wouluka mwa akazi omwe amadya magazi (F(5, 138) = 4.83, p = 0.0004), zakudya za urea sizinatero (F(5, 138) = 1.36, p = 0.24). Ndi mkodzo wina ndi zakudya zowongolera (zatsopano, p = 0.0091; maola 72, p = 0.0022; maola 168, p = 0.001; sucrose, p = 0.0017; dH2O, p = 0.036).
Zotsatira za kudya mkodzo ndi urea pa zinthu zoberekera zinayesedwa mu mayeso a bioassays a mazira (Chithunzi 4A) ndipo zinafufuzidwa malinga ndi kuchuluka kwa mazira omwe amaikira mkazi aliyense, kukula kwa dzira, ndi mphutsi zoyamba kumene zomwe zaswa. Chiwerengero cha mazira omwe amaikira. Akazi achiarabu omwe amadya mkodzo amasiyana malinga ndi zakudya (F(5,222) = 4.38, p = 0.0008; Chithunzi 4B). Akazi amadya mkodzo kwa maola 24, chakudya cha m'magazi chomwe chimayakira mazira ambiri kuposa akazi omwe amadya zakudya zina za mkodzo ndipo anali ofanana ndi omwe amadya sucrose (Chithunzi 4B). Momwemonso, kukula kwa mazira omwe amaikira akazi omwe amadya mkodzo kumasiyana malinga ndi zakudya (F(5, 209) = 12.85, p < 0.0001), pomwe mkodzo wa maola 24 ndi akazi omwe amadya sucrose amayakira mazira akuluakulu kwambiri kuposa akazi omwe amadya madzi, pomwe mazira a akazi omwe amadya mkodzo kwa maola 168 anali ochepa kwambiri (Chithunzi 4C). Kuphatikiza apo, zakudya za mkodzo zinakhudza kwambiri kukula kwa mphutsi (F(5, 187) = 7.86, p < 0.0001), ndi mphutsi zazikulu kwambiri zomwe zimatuluka m'mazira omwe amayikidwa ndi akazi azaka 24 ndi 72 omwe amamwa mkodzo kuposa mazira omwe amaikidwa ndi mphutsi za mazira. Akazi omwe amamwa mkodzo kwa maola 168 ndi omwe amamwa mkodzo kwa maola 168 (Chithunzi 4D).
Kubereka kwa Anopheles arabinis yaikazi yomwe imadya mkodzo wa ng'ombe ndi urea. Udzudzu wa akazi womwe umadya magazi unapatsidwa zakudya zokhala ndi mkodzo wa ng'ombe watsopano komanso wokalamba, kuchuluka kosiyanasiyana kwa urea, sucrose (10%), ndi madzi osungunuka (H2O) kwa maola 48 musanayike mu bioassays ndikupeza magawo oikira mazira. Maola 48 (A). Nambala ya dzira (B, E), kukula kwa dzira (C, F) ndi kukula kwa mphutsi (D, G) zinakhudzidwa kwambiri ndi zakudya zomwe zinaperekedwa (mkodzo wa ng'ombe: BD; urea: EG). Njira za parameter iliyonse yoyesedwa pogwiritsa ntchito mayina osiyanasiyana a zilembo zinali zosiyana kwambiri (ANOVA ya njira imodzi pogwiritsa ntchito kusanthula kwa Tukey pambuyo pa hoc; p < 0.05). Mipiringidzo yolakwika ikuyimira cholakwika cha mean
Monga gawo lalikulu la nayitrogeni mu mkodzo, urea, ikaperekedwa ngati chakudya kwa akazi odyetsedwa m'magazi, idakhudza kwambiri magawo oberekera m'maphunziro onse. Chiwerengero cha mazira omwe amayi amaika omwe amadyetsedwa urea, atatha kudya m'magazi, kutengera kuchuluka kwa urea (F(11, 360) = 4.69; p < 0.0001), akazi amadya kuchuluka kwa urea pakati pa 134 µM ndi 1.34 mM amaika mazira ambiri (Chithunzi 4E). Akazi omwe amadyetsedwa kuchuluka kwa urea kwa 134 µM kapena kupitirira apo amaika mazira akuluakulu kuposa akazi omwe amadyetsedwa m'madzi (F(10, 4245) = 36.7; p < 0.0001; Chithunzi 4F), ndi kukula kwa mphutsi, ngakhale kuti zimakhudzidwa ndi kuchuluka kofanana kwa urea mwa amayi (F(10, 3305) = 37.9; p < 0.0001) kunali kosiyana kwambiri (Chithunzi 4G).
Kukopa konse kwa zotulutsa za mkodzo wa ng'ombe zomwe zimafunafuna ng'ombe. Arabiensis yomwe idawunikidwa mu chubu chagalasi olfactometer (Chithunzi 5A) idakhudzidwa kwambiri ndi zaka za mkodzo (χ2 = 15.9, df = 4, p = 0.0032; Chithunzi 5B). Kusanthula pambuyo pake kunawonetsa kuti fungo la mkodzo wokalamba pa maola 24 limayambitsa kuchuluka kwakukulu kwa kukopa poyerekeza ndi mankhwala ena onse (maola 72: p = 0.0060, maola 168: p = 0.012, pentane: p = 0.00070), Kupatula fungo la mkodzo watsopano (p = 0.13; Chithunzi 5B). Ngakhale kuti kukoka konse kwa udzudzu woyamwa magazi ku fungo la mkodzo sikunali kosiyana kwambiri (χ2 = 8.78, df = 4, p = 0.067; Chithunzi 5C), akazi awa adapezeka kuti ndi okongola kwambiri ku zotulutsa za mkodzo wa headspace poyerekeza ndi mkodzo wa maola 72 wokalamba poyerekeza ku zowongolera (p = 0.0066; Chithunzi 5C).
Mayankho a khalidwe ku fungo la mkodzo wa ng'ombe wachilengedwe komanso wopangidwa pofufuza Anopheles arabianus wolandirayo ndi magazi. Chithunzi cha galasi chubu olfactometer (A). Kukopa kwa zotulutsa zosasunthika za mutu kuchokera ku mkodzo wa ng'ombe watsopano komanso wokalamba kupita ku udzudzu wolandirayo (B) ndi woyamwa magazi (C). Pezani momwe Lord An amachitira. Zotulutsa za mutu zimachotsedwa ku mkodzo wa ng'ombe watsopano (D), maola 24 (E), maola 72 (F), ndi maola 168 (G) wokalamba zikuwonetsedwa. Zotsatira za electron antenna detection (EAD) zikuwonetsa kusintha kwa mphamvu poyankha mankhwala ogwira ntchito m'mutu omwe achotsedwa ku gasi chromatograph ndipo apezeka ndi chowunikira cha flame ionization (FID). Sikelo yoyezera imayimira amplitude yoyankha (mV) motsutsana ndi nthawi yosungira (s). Katundu ndi kuchuluka kwa kumasulidwa (µg h-1) kwa mankhwala omwe amagwira ntchito m'thupi akuwonetsedwa. Asterisk imodzi (*) imasonyeza kuyankha kotsika kokhazikika. Asterisk ziwiri (**) zimasonyeza mayankho osaberekanso. Pezani wolandirayo (H) ndi (I) An.arabiensis ali ndi mawonekedwe osiyanasiyana osakanikirana ndi fungo la mkodzo watsopano ndi wakale wa ng'ombe. Avereji ya udzudzu wokokedwa ndi mayina osiyanasiyana a zilembo inali yosiyana kwambiri (ANOVA ya njira imodzi pogwiritsa ntchito kusanthula kwa Tukey pambuyo pa hoc; p < 0.05). Mipiringidzo yolakwika ikuyimira cholakwika cha sikelo
Mayi Ann.arabiensis, maola 72 ndi maola 120 atadya magazi, panthawi yobereka, palibe chomwe chinawonetsedwa chokonda cha zotulutsa za mkodzo watsopano komanso wakale wa ng'ombe poyerekeza ndi zowongolera za pentane (χ2 = 3.07, p > 0.05; Fayilo yowonjezera 1: Chithunzi S1).
Kwa akazi a Ann.arabiensis, kusanthula kwa GC-EAD ndi GC-MS kunapeza mankhwala asanu ndi atatu, asanu ndi limodzi, atatu ndi atatu omwe amagwira ntchito m'thupi (Chithunzi 5D-G). Ngakhale kuti kusiyana kwa kuchuluka kwa mankhwala omwe adayambitsa mayankho amagetsi kunawonedwa, ambiri mwa mankhwalawa analipo mu chotsitsa chilichonse cha headspace volatile chomwe chinasonkhanitsidwa kuchokera ku mkodzo watsopano komanso wakale. Chifukwa chake, pa chotsitsa chilichonse, mankhwala okhawo omwe adapanga yankho la thupi kuchokera ku tinyanga ta akazi pamwamba pa malire ndi omwe adaphatikizidwa mu kusanthula kwina.
Kuchuluka kwa kutulutsidwa kwa mankhwala ophatikizika m'malo osungiramo zinthu m'mutu kunakwera kuchoka pa 29 µg h-1 mu mkodzo watsopano kufika pa 242 µg h-1 mu mkodzo wokalamba wa maola 168, makamaka chifukwa cha p-cresol ndi m-formaldehyde Phenol imawonjezeka komanso phenol. Mosiyana ndi zimenezi, kuchuluka kwa kutulutsidwa kwa mankhwala ena, monga 2-cyclohexen-1-one ndi decanal, kunachepa ndi msinkhu wokulirapo wa mkodzo, zomwe zimagwirizana ndi kuchepa kwa mphamvu ya chizindikiro (kuchuluka) mu chromatogram (Chithunzi 5D)-G kumanzere) ndi mayankho a thupi ku mankhwala awa (Chithunzi 5D-G kumanja).
Ponseponse, chisakanizo chopangidwacho chinali ndi chiŵerengero chofanana chachilengedwe cha mankhwala ogwira ntchito omwe amapezeka mu zotulutsa zosasunthika za mkodzo watsopano ndi wokalamba (Chithunzi 5D–G) ndipo sizikuwoneka kuti zimabweretsa chidwi chachikulu pakufufuza wolandirayo (χ2 = 8.15, df = 4, p = 0.083; Chithunzi 5H) kapena udzudzu woyamwa magazi (χ2 = 4.91, df = 4, p = 0.30; Chithunzi 5I). Komabe, kufananiza kwa mankhwala pambuyo pa hoc awiriawiri kunawonetsa kuti udzudzu wofunafuna wolandirayo unali wokopa kwambiri ku chisakanizo chopangidwa cha mkodzo wa maola 24 poyerekeza ndi zowongolera za pentane (p = 0.0086; Chithunzi 5H).
Kuti muwone momwe zigawo za munthu aliyense zimagwirira ntchito mu zosakaniza zopangidwa ndi mkodzo wa maola 24, zosakaniza zisanu ndi chimodzi zochotsera zinayesedwa poyerekeza ndi zosakaniza zonse mu kuyesa kwa Y-tube, momwe mankhwala amodzi amachotsedwa. Kwa udzudzu wofunafuna wolandira, kuchotsa mankhwala amodzi kuchokera mu zosakaniza zonse kunakhudza kwambiri mayankho a khalidwe (χ2 = 19.63, df = 6, p = 0.0032; Fayilo yowonjezera 1: Chithunzi S2A), zosakaniza zonse zochotsera zinali zokongola kuposa Zochepa kuposa zosakanikirana kwathunthu. Mosiyana ndi zimenezi, kuchotsa mankhwala amodzi kuchokera mu zosakaniza zopangidwa kwathunthu sikunakhudze momwe udzudzu woyamwa magazi umayankhira (χ2 = 11.38, df = 6, p = 0.077), kupatula decanal, zomwe zinapangitsa kuti pakhale milingo yotsika poyerekeza ndi zosakaniza zonse. Kukopa (p = 0.022; Fayilo Yowonjezera 1: Chithunzi S2B).
Mumudzi womwe uli ndi malungo ku Ethiopia, mphamvu ya mkodzo wa ng'ombe wopangidwa ndi mankhwala osakaniza maawa 24 pokopa udzudzu m'munda inayesedwa kwa masiku khumi (Chithunzi 6A). Udzudzu wonse 4,861 unagwidwa ndi kuzindikirika, womwe 45.7% unali Anthropus.gambiae sl, 18.9% unali Anopheles pharoensis ndipo 35.4% unali Culex spp. (Fayilo yowonjezera 1: Table S1). Anopheles arabinis ndiye membala yekhayo wa mtundu wa An.Gambian womwe unapezeka pogwiritsa ntchito PCR analysis. Pa avareji, udzudzu 320 unagwidwa usiku uliwonse, panthawiyi misampha yokhala ndi chambo chosakaniza chopangidwa inagwira udzudzu wambiri kuposa misampha yolumikizidwa popanda kusakaniza (χ2(0, 3196) = 170.0, p < 0.0001). Misampha yopanda chambo inayikidwa pa usiku uliwonse wa masiku asanu olamulira kumayambiriro, pakati, ndi kumapeto kwa mayeso. Chiwerengero chofanana cha udzudzu chinagwidwa pa awiriawiri aliwonse. za misampha, zomwe zikusonyeza kuti palibe tsankho pakati pa nyumba (χ2(0, 1665) = 9 × 10-13, p > 0.05) ndipo palibe kuchepa kwa chiwerengero cha anthu panthawi yophunzira. Poyerekeza ndi misampha yoletsa, chiwerengero cha udzudzu womwe unagwidwa mu misampha yokhala ndi zosakaniza zopangidwa chinawonjezeka kwambiri: kufunafuna munthu (χ2(0, 2107) = 138.7, p < 0.0001), kudya magazi kwaposachedwa (χ2(0, 650) = 32.2, p < 0.0001) ndi mimba (χ2(0, 228) = 6.27, p = 0.0123; Fayilo yowonjezera 1: Table S1). Izi zikuwonekeranso mu chiwerengero chonse cha udzudzu womwe unagwidwa: kufunafuna munthu > kuyamwa magazi > pakati > pakati > amuna.
Kuwunika kwa munda kwa momwe fungo la mkodzo wa ng'ombe wopangidwa limagwirira ntchito kwa maola 24. Mayeso a m'munda adachitika kum'mwera chapakati pa Ethiopia (mapu), pafupi ndi tawuni ya Maki (ikani), pogwiritsa ntchito msampha wopepuka wa Centers for Disease Control (CDC) (kumanja) m'nyumba zophatikizana, ndi kapangidwe ka sikweya ya ku Latin (chithunzi cha mlengalenga) (A). Ma phototraps a CDC opangidwa ndi fungo amakopa ndikugwira ang'onoang'ono a Anopheles arabesques (B), koma osati Anopheles farroes (C), mwanjira ina, zotsatira zake zimatengera momwe thupi limakhalira. Kuphatikiza apo, misampha iyi idagwira kuchuluka kwakukulu kwa udzudzu wa Culex womwe umakhala nawo. (D) Poyerekeza ndi kulamulira. Mipiringidzo kumanzere ikuyimira avareji ya udzudzu womwe wagwidwa m'mipiringidzo iwiri ya fungo (yobiriwira) ndi misampha yolamulira (yotseguka) (N = 10), pomwe mipiringidzo kumanja ikuyimira avareji ya kusankha m'mipiringidzo iwiri ya kuwongolera (yotseguka; N = 5). ). Ma asterisks akuwonetsa kuchuluka kwa ziwerengero (*p = 0.01 ndi ***p < 0.0001)
Mitundu itatuyi inagwidwa mosiyana mu misampha yokhala ndi zosakaniza zopangidwa. Kufunafuna wolandira (χ2(1, 1345) = 71.7, p < 0.0001), kudya magazi (χ2(1, 517) = 16.7, p < 0.0001) ndi mimba (χ2(1, 180) = 6.11, p = 0.0134) a .arabiensis anagwidwa mumsampha womwe unatulutsa zosakaniza zopangidwa (Chithunzi 6B), pomwe kuchuluka kwa An sikunasiyane. Pharoensis m'magawo osiyanasiyana a thupi kunapezeka (Chithunzi 6C). Pa Culex, kuwonjezeka kwakukulu kwa chiwerengero cha udzudzu wofunafuna alendo kunapezeka mu misampha yomwe inali ndi chokoka ndi zosakaniza zopangidwa (χ2(1,1319) = 12.6, p = 0.0004; Chithunzi 6D), poyerekeza ndi misampha yowongolera.
Misampha ya nyambo yomwe ili kunja kwa malo omwe angabzalire pakati pa malo oberekera ndi madera akumidzi ku Ethiopia idagwiritsidwa ntchito poyesa ngati udzudzu wa malungo umagwiritsa ntchito fungo la mkodzo wa ng'ombe ngati chizindikiro cha malo okhala. Popanda zizindikiro za mkodzo wa ng'ombe, kutentha, komanso popanda fungo la mkodzo wa ng'ombe, palibe udzudzu womwe unagwidwa (Fayilo yowonjezera 1: Chithunzi S3). Komabe, pakakhala kutentha kwambiri ndi fungo la mkodzo wa ng'ombe, udzudzu wachikazi wa malungo unakokedwa ndi kugwidwa, ngakhale pang'ono, mosasamala za msinkhu wa mkodzo (χ2(5, 25) = 2.29, p = 0.13; Fayilo yowonjezera 1: Chithunzi S3). Mosiyana ndi zimenezi, njira zowongolera madzi sizinagwire udzudzu wa malungo pa kutentha kwambiri (Fayilo yowonjezera 1: Chithunzi S3).
Udzudzu wa malungo umapeza ndikugawa mankhwala okhala ndi nayitrogeni kudzera mu chakudya chobwezera mkodzo wa ng'ombe (monga, matope) kuti uwonjezere makhalidwe a moyo, mofanana ndi tizilombo tina [2, 4, 24, 25, 26]. Mkodzo wa ng'ombe ndi chinthu chopezeka mosavuta chomwe chimagwirizana kwambiri ndi malo opumulirako a tizilombo toyambitsa matenda a malungo, monga makola a ng'ombe ndi zomera zazitali pafupi ndi nyumba zakumidzi ndi malo oberekera. Udzudzu wachikazi umapeza chinthuchi mwa fungo ndipo umatha kuwongolera kuyamwa kwa mankhwala a nayitrogeni mu mkodzo, kuphatikizapo urea, gawo lalikulu la nayitrogeni mu mkodzo [15, 16]. Kutengera ndi momwe udzudzu wachikazi ulili, michere mu mkodzo imaperekedwa kuti iwonjezere ntchito youluka ndi kupulumuka kwa udzudzu wachikazi wofunafuna malo, komanso kupulumuka ndi kubereka kwa anthu omwe amadya magazi panthawi yoyamba ya gonadotropic. Chifukwa chake, kusakaniza mkodzo kumachita gawo lofunika kwambiri pazakudya kwa tizilombo toyambitsa matenda a malungo omwe amakhala otsekedwa ngati akuluakulu osadya bwino [8], chifukwa kumapatsa udzudzu wachikazi mphamvu yopezera mankhwala ofunikira a nayitrogeni mwa kudya zakudya zochepa. Kupeza kumeneku kuli ndi zotsatirapo zazikulu za matenda, pamene akazi amawonjezera nthawi ya moyo, zochita ndi kubereka, zonse zomwe zimakhudza mphamvu ya vector. Kuphatikiza apo, khalidweli lingakhale cholinga cha mapulogalamu oyang'anira vector amtsogolo.