The role of grass volatiles on oviposition site selection by Anopheles arabiensis and Anopheles coluzzii

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1 Asmre et l. Mlr J (27) 6:65 DOI.86/s z Mlri Journl RESEARCH Open Aess The role of grss voltiles on oviposition site seletion y Anopheles riensis n Anopheles oluzzii Yelfwgsh Asmre,2, Shron R. Hill 3, Rihr J. Hopkins 4, Hte Tekie n Rikr Ignell 3* Astrt Bkgroun: The reproutive suess n popultion ynmis, of Anopheles mlri mosquitoes is strongly influene y the oviposition site seletion of grvi femles. Mosquitoes selet oviposition sites t ifferent sptil sles, strting with seleting hitt in whih to serh. This stuy utilizes the ssoition of lrvl unne in the fiel with nturl reeing hitts, ominte y vrious types of wil grsses, s proxy for oviposition site seletion y grvi mosquitoes. Moreover, the role of olftory ues emnting from these hitts in the ttrtion n oviposition stimultion of femles ws nlyse. Methos: The ensity of Anopheles lrve in reeing sites ssoite with Ehinohlo pyrmilis, Ehinohlo stgnin, Typh ltifoli n Cyperus ppyrus, ws smple n the lrve ientifie to speies level. Hespe voltile extrts of the grsses were ollete n use to ssess ehviourl ttrtion n oviposition stimultion of grvi Anopheles riensis n Anopheles oluzzii mosquitoes in win tunnel n two-hoie oviposition ssys, respetively. The ility of the mosquitoes to ifferentite mong the grss voltile extrts ws teste in multi-hoie tent ssys. Results: Anopheles riensis lrve were the most unnt speies foun in the vrious grss-ssoite hitts. The lrvl ensities esrie hierrhil istriution, with Poee (Ehinohlo pyrmilis n Ehinohlo stgnin)-ssoite hitt sites emonstrting higher ensities thn tht of Typh-ssoite sites, n where lrve were sent from Cyperus-ssoite sites. This hierrhy ws mintine y grvi An. riensis n An. oluzzii mosquitoes in ttrtion, oviposition n multi-hoie ssys to grss voltile extrts. Conlusions: The emonstrte hierrhil preferene of grvi An. oluzzii n An. riensis for grss voltiles inites tht vegettion ues ssoite with lrvl hitts re instrumentl in the oviposition site hoie of the mlri mosquitoes. Ientifying voltile ues from grsses tht moulte grvi mlri mosquito ehviours hs istint potentil for the evelopment of tools to e use in future monitoring n ontrol methos. Keywors: Anopheles riensis, Anopheles oluzzii, Oviposition, Hitt, Seletion, Olftion, Attrtion Bkgroun Oviposition site seletion y grvi Anopheles mlri mosquitoes is key moment in the reproutive suess of the iniviul, n thus the popultion ynmis of the speies [, 2]. Consequently, the serh for n *Corresponene: rikr.ignell@slu.se 3 Deprtment of Plnt Protetion Biology, Unit of Chemil Eology, Sweish University of Agriulturl Sienes, Alnrp, Sween Full list of uthor informtion is ville t the en of the rtile oviposition site hs importnt implitions with regr to the ontrol of mlri vetors [2]. When insets selet oviposition sites, they mke hoies on inresingly fine sptil sles, strting with seleting hitt in whih to serh [3 5]. In the se of egg-lying mosquitoes, they my hve to leve hitt in whih they hve een quiring loo mels, in orer to enter hitt ontining egg-lying sites. These hitts iffer in physil, hemil n iologil hrteristis proviing ues for the serhing femle, iretly influening the The Author(s) 27. This rtile is istriute uner the terms of the Cretive Commons Attriution 4. Interntionl Liense ( whih permits unrestrite use, istriution, n reproution in ny meium, provie you give pproprite reit to the originl uthor(s) n the soure, provie link to the Cretive Commons liense, n inite if hnges were me. The Cretive Commons Puli Domin Deition wiver ( puliomin/zero/./) pplies to the t me ville in this rtile, unless otherwise stte.

2 Asmre et l. Mlr J (27) 6:65 Pge 2 of 9 istriution, survivl n unne of mosquito lrvl popultions [6 ]. The usge of n oviposition site y mosquitoes is epenent on their reltive position in the lnspe [8, ], visul ues [2, 3], wter vpour plumes [4], semiohemil ues ssoite with wter oies [5 8] n the physil prmeters of the wter [6, 7]. The most proutive nturl lrvl hitt types for Anopheles gmie/anopheles oluzzii n Anopheles riensis re trnsient pules [], often surroune y short grsses [7, 9, 9, 2]. Both of these mjor vetors in su-shrn Afri hve lso een reore in more stle wter oies, suh s the littorl zone of lkes n in swmps [, 9, 2 23]. Vegettion often popultes these wetln hitts [, 9, 23], n An. gmie/an. oluzzii n An. riensis re ommonly foun mongst ttils (Typh spp.; Typhee) n llis grsses (Psplum spp.; Poee) [24 26]. In ontrst, hitts populte y rees (Phrgmites spp.; Poee) n ppyrus (Cyperus ppyrus; Cyperee) generlly proue low numers of mosquitoes [9, 27, 28], proly ue to the nturl oil proution of these speies tht reues lrvl survivorship [29, 3]. Hene, grsses pper to ply n importnt role in the nturl reeing site seletion of An. riensis n An. gmie/an. oluzzii. Yet, the influene of nturl grsses n other emergent vegettion on the oviposition site seletion y grvi femle Anopheles mosquitoes is not lerly unerstoo. Moreover, the nture of the voltiles emitte from wil grsses n how they ffet the ehviour of An. riensis n An. gmie/ An. oluzzii hs not een investigte to te. The ojetive of this stuy ws to investigte nopheline lrvl ourrene n unne in nturl reeing hitts populte y four wil grss speies: ntelope grss, Ehinohlo pyrmilis (Poee); hippo grss, Ehinohlo stgnin (Poee); ommon ttil; Typh ltifoli (Typhee); n ppyrus ree, C. ppyrus (Cyperee), n to orrelte the ehviourl response of grvi An. riensis n An. oluzzii to the nturl voltiles ollete from these grsses. The implitions for nopheline eology n vetor mngement re isusse. Methos Anopheles lrvl ensity in hitts with emergent grss speies Stuy sites n smpling proeure Anopheles lrvl smpling ws me in potentil reeing hitts t the southern littorl region of, n wetlns jent to, Lke Tn, Ethiopi ( 37 N, 37 2 E; 83 m ove se level). The limte of the stuy re is typil of semi-ri regions lose to the equtor, with high iurnl temperture vrition etween ytime extremes of 3 C to night time lows of 6 C, ut minly vries etween 2 n 27 C. Rinfll is on verge 44 mm per yer, flling in one riny seson from My to Otoer, with pek uring July August [3]. During the El Niño event of 24 25, this region experiene severe rought, with n overll reution in rinfll of on verge 5% [32], whih h rsti effet on Anopheles mosquito popultions. For this reson, lrve of ll stges, rther thn first instrs lone, were ollete one in erly Septemer n gin in lte Septemer, in n ttempt to smple uring the min prolifertion perio of mosquitoes in the stuy re. Cyperus ppyrus n T. ltifoli re mong the ominnt grsses in eep wter oies of the lkeshore, wheres E. pyrmilis n E. stgnin preominntly re foun t the ege of the lkeshore or in wetlns jent to the lke. In the stuy re, su-sites ominte y eh iniviul grss speies were selete. In eh su-site, seprte smplings of lrve (tehnil replites) were me using stnr 35 ml ipper [33]. The ollete Anopheles lrve were ounte n reore for eh lrvl hitt ssoite with the ifferent grss speies. Of the ollete lrve, % were preserve in 7% ethnol for susequent ientifition to speies using stnr PCR nlysis [34]. Dt nlysis The t from the lrvl survey were sujete to univrite generl liner moel (GLM), using the sttistil softwre IBM SPSS Sttistis for Winows, Version 2.. Signifint ifferenes etween mens were etermine t α =.5 n post ho multiple omprisons mong the grsses were me using the Tukey s HSD test. Behviourl response of grvi mosquitoes to grss voltiles Hespe oour olletion Freshly ut grss ( g), inluing the vegettive n reproutive prts, ws enlose in Teflon g (Toppits, Cofreso, Germny). A hrol-filtere ontinuous irstrem ( l min ) ws rwn y Personl Air Smpler (PAS-5, Spetrex, Rewoo City, CA, USA) over the grss onto n ertion olumn for 2 h. Aertion olumns were me of Teflon tuing (4.5 m 3 mm i..), holing 5 mg Porpk Q (5/8 mesh; Wters Assoites, Milfor, MA, USA) etween glss wool plugs. The olumns were rinse with ml re-istille n-hexne (Merk, Drmstt, Germny) efore use. Asore voltiles were elute with 3 µl re-istille n-hexne. Oour olletions from eh grss speies were poole seprtely n then store in sele glss pillry tues t 2 C until use for ehviourl experiments.

3 Asmre et l. Mlr J (27) 6:65 Pge 3 of 9 Mosquito rering Anopheles riensis (Dongol strin) n An. oluzzii (Sukoko strin) were kept t 27 ± C, 7 ± 5% RH, n t 2 h:2 h light:rk photoperio. Lrve were rere in plsti trys (22 m 34 m m) fille with l istille wter, n fe powere Tetrmin fish foo (Tetrwerke, Melle, Germny) ily. Pupe (8 ) were ple in BugDorm- inset ges (3 m 3 m 3 m; Meg View Siene, Tiwn) for ult emergene. Ault mles n femles were kept together n provie liitum ess to % surose solution. For olony mintenne, femle mosquitoes were loo fe on e-firinte sheep loo vi memrne-feeing system (Disover Workshops, Arington, UK). Eggs were li in 3 ml plsti ups (Nolto Hertil, Sween) fille with istille wter, n then trnsferre to lrvl trys for hthing. For experiments, femle mosquitoes, 4 ys post-emergene, were llowe ess to sheep loo (Håtunl, Bro, Sween) from n rtifiil feeer (Hemotek Disovery Workshops, Arington, UK) for 3 h. Engorge femles 6 8 h post-loo mel were then trnsferre to new ge until use for experiments. Win tunnel iossy Attrtion of An. riensis n An. oluzzii to the hespe oour extrts of the four grss speies ws nlyse in win tunnel ssy [35]. Cotton rolls (DAB Dentl AB, Upplns Väsy, Sween) were use s ispensers, n the mount of extrt pipette onto the ispensers orrespone to the mount of voltiles relese uring.4,.4, 4, n 2 min from the iniviul grss speies. An equivlent mount of n-hexne ws use s ontrol. Both tretment n ontrol ispensers were suspene from 5 m wire oil t the upwin en of the win tunnel. Ten iniviul femle mosquitoes, 48 h post-loo mel, were trnsferre to relese ge 2 h prior to experiments. The hmers were then ple in the ownwin en of the win tunnel, where the insets were llowe 5 min to pt, efore the utterfly vlve of the ge ws opene for their relese. Attrtion to either tretment or ontrol ws nlyse s the proportion of mosquitoes tht me soure ontt within min fter relese. Eh relese rte for eh grss voltile extrt n the ontrol ws replite ten times. Oviposition iossy The oviposition response of An. riensis n An. oluzzii to the voltile extrts of the four grss speies ws nlyse in BugDorm- inset ges kept in limte-ontrolle room t 25 C, 7 ± 5% RH, n t 2 h:2 h light:rk photoperio. Plsti ups (3 ml; Nolto Hertil) fille with ml istille wter provie the oviposition sustrte, n were lote in opposite orners of the ge, 2 m from eh wll. The tretment ups were onitione with one of the wil grss voltile extrts, in the sme mounts use in the win tunnel iossy. An equivlent mount of n-hexne ws use s ontrol. Tretment n ontrol ups were exhnge in etween eh experiment. Ten mosquitoes, 48 h postloo mel, were relese into the experimentl ges t 8: :, n the numer of eggs in the tretment n ontrol ups ounte fter 48 h. An oviposition inex ws etermine y: (numer of eggs li in tretment up numer of eggs li in ontrol up)/(totl numer of eggs within the experimentl ge). Eh relese rte of eh grss voltile extrt ws replite 5 times. Tent iossy Greenhouse ge tents (2 m 2 m 2 m; BioQuip, Rnho Domniguez, CA, USA) were use s multirry iossys to nlyse the oviposition preferene of An. riensis n An. oluzzii to the four wil grss voltile extrts n ontrol. The tents were kept in greenhouse t 27 ± C, 5 ± 5% RH, n t 2 h:2 h light:rk photoperio. As ove, 3 ml plsti ups fille with ml istille wter provie the oviposition sustrte. Tretment ups were onitione with the four wil grss voltile extrts in n mount orresponing to the voltiles relese uring.4 min from the iniviul grss speies, while the ontrol up ws onitione with the equivlent mount of n-hexne. Tretment n ontrol ups were set up in 5 5 mtrix (2 m etween ups). The mtrix ws hnge in etween replites (n = for eh Anopheles speies) to voi position effets of the tretments. Twenty femle mosquitoes, 48 h post-loo fe, were relese into the tents, n the numer of eggs ounte fter 24 h. The oviposition response ws sore y ounting the numer of eggs in the tretment n ontrol ups. Dt nlysis The ehviourl responses of grvi An. oluzzii n An. riensis in the win tunnel n two-hoie oviposition iossy were nlyse using nominl logisti fit moel, in whih hoie ws the epenent vrile, weighte y the numer of () mosquitoes in the ttrtion ssys n (2) eggs li in the oviposition ssys, with ose s the inepenent fixe effet n replite s rnom effet (JMP Pro 2... SAS Institute In., Cry, NC, USA). In the tent experiments (5-hoie oviposition ssys), the numer of eggs ws use s the weight, the hoie s the epenent vrile, the grsses n ontrol s the inepenent fixe effet, n the tent n replites s the rnom effets. The χ 2 n P vlue from the likelihoo rtio test re reporte here. Signifint ifferenes etween the iniviul oses (win tunnel n

4 Asmre et l. Mlr J (27) 6:65 Pge 4 of 9 two-hoie ssys) n grsses (five-hoie ssy) were etermine y os rtio pirwise omprisons. 3 Results Anopheles riensis lrvl ensity in nturl grss hitts No signifint effet of su-site lotion ws foun (F =.367, DF = 9, DF n = 3, P =.999). Hene, the lrvl unne t ollete from the reeing hitts ssoite with eh grss speies ws poole in susequent nlysis. Anopheles riensis ws the most unnt speies omprising more thn 4% of the speimens in the stuy re, n ws the only memer of the An. gmie sensu lto omplex to e ientifie following PCR nlyses of 48 mosquito lrve. A signifintly higher numer of An. riensis lrve were foun in E. pyrmilis ominte reeing hitts thn in ny of the other potentil reeing hitts (Fig. ). The numer of lrve in the remining hitts lso iffere signifintly, with reeing hitts ominte y E. stgnin ontining more lrve thn reeing hitts ominte y T. ltifoli (Fig. ). Interestingly, no lrve were foun in hitts ominte y C. ppyrus (Fig. ). Attrtion of An. riensis n An. oluzzii to grss voltiles Attrtion of An. riensis n An. oluzzii to the grss voltile extrts ws ose epenent n iffere mong the grss speies (An. riensis: Dose, χ 2 = 34.5, P <.; Grss, χ 2 = 3., P =.44; An. oluzzii: Dose, χ 2 = 46.25, P <.; Grss, χ 2 = 4.46, P <.) (Fig. 2). No signifint ifferene in the ttrtion of An. riensis to the voltile extrts of E. pyrmilis, E. stgnin n T. ltifoli ws foun, however, the ttrtion to the C. ppyrus voltile extrt ws signifintly lower thn eh of the other grss voltile extrts (Tle ). Attrtion of An. oluzzii to the voltile extrts of either the Poee (E. pyrmilis n E. stgnin), or the Typhee (T. ltifoli) n Cyperee (C. ppyrus), i not iffer. The ttrtion to the voltile extrt of the Typhee n Cyperee, however, ws signifintly lower thn tht of the Poee (Tle ). Oviposition response of An. riensis n An. oluzzii to grss voltiles The oviposition response of An. riensis n An. oluzzii to wter onitione with the grss voltile extrts ws oth ose epenent n iffere mong the grss speies (An. riensis: Dose, χ 2 = 94.67, P <.; Grss, χ 2 = 44.79, P <.; An. oluzzii: Dose, χ 2 = 47.35, P <.; Grss, χ 2 = 24.29, P <.) (Fig. 3). No signifint ifferenes in oviposition response were foun for An. oluzzii to wter Numer of lrve/ip Numer of lrve/ip E. pyrmilis T. ltifoli E. stgnin C. ppyrus Fig. Anopheles lrvl ensity in nturl reeing hitts ominte y four grss speies, Ehinohlo pyrmilis, Ehinohlo stgnin, Typh ltifoli n Cyperus ppyrus, ssye in erly (top) n lte (ottom) Septemer. The men lrvl ensities with ifferent letter esigntions re signifintly ifferent from one nother (univrite generl liner moel with Tukey s post ho nlysis; P <.5). Error rs represent the stnr error of the men onitione with voltile extrts of E. pyrmilis, E. stgnin n T. ltifoli, ut the oviposition response to these grss extrts were signifintly higher thn tht oserve for the C. ppyrus voltile extrt (Tle 2). Similrly, for the C. ppyrus voltile extrt, the oviposition response of An. riensis ws signifintly lower thn to tht of ll the other grss voltile extrts (Tle 2). In ontrst to An. oluzzii, the voltile extrt

5 Asmre et l. Mlr J (27) 6:65 Pge 5 of 9 An. riensis ttrtion (%) An. oluzzii ttrtion (%) 5 e 5 f.... min equivlent min equivlent min equivlent min equivlent Fig. 2 Attrtion of grvi Anopheles riensis n Anopheles oluzzii to grss voltile hespe extrts, from Ehinohlo pyrmilis (, e) Ehinohlo stgnin (, f), Typh ltifoli (, g) n Cyperus ppyrus (, h), respetively. Relese rte of the voltile hespe extrts is given in minute equivlents. The solvent ontrol ws hexne (). The men perent ttrtion vlues with the sme letters inite no signifint ifferene from one nother (nominl logisti fit moel, χ 2 n P <.5 from the likelihoo rtio test, signifint ifferenes were etermine y os rtio pirwise omprisons). Error rs represent the stnr error of the men g h Tle Cross-omprtive nlysis of the ttrtion of grvi mosquitoes to voltile extrts of grss speies Attrtion An. riensis An. oluzzii Level Level 2 Os rtio P Os rtio P E. pyrmilis E. stgnin E. pyrmilis T. ltifoli E. pyrmilis C. ppyrus <. E. stgnin T. ltifoli <. E. stgnin C. ppyrus <. T. ltifoli C. ppyrus The nlysis is one using nominl logisti regression moel fit α =.5 of T. ltifoli stimulte lower oviposition response in An. riensis thn tht of E. pyrmilis (Tle 2). Tent experiments response In the five-hoie oviposition ssy, the overll oviposition preferene hierrhy of oth Anopheles speies to the grss voltile extrts ws foun to e E. pyrmilis > E. stgnin T. ltifoli = C. ppyrus (Fig. 4). Wter onitione with voltile extrts of either E. pyrmilis or E. stgnin, eliite signifintly higher oviposition response thn the ontrol in oth An. riensis n An. oluzzii, while tht of T. ltifoli eliite signifintly higher response thn the ontrol in An. riensis lone (Fig. 4). In ontrst, the oviposition response of An. oluzzii to wter onitione with voltile extrts of T. ltifoli, s well s tht of C. ppyrus, ws signifintly lower thn tht of the ontrol. Disussion Fiel olletion of An. riensis lrve inite role for emergent vegettion in the oviposition site seletion n survivl of mlri mosquitoes. Lrvl ensities were highest in Poee-ssoite hitts, muh lower in Typh-ssoite sites n sent from Cyperus-ssoite sites. One potentil mehnism regulting the ifferentil istriution of lrve my e n oour-se oviposition site seletion preferene. Grvi femle An. riensis n An. oluzzii were, inee, foun to e ifferentilly ttrte to ll grss voltile extrts, yet were only stimulte to oviposit on wter onitione with Poee voltile extrts. This ws further supporte y multi-hoie oviposition ssys reveling tht oth speies emonstrte preferene hierrhy mong the grss voltiles, E. pyrmilis > E. stgnin > T. ltifoli C. ppyrus. These finings my lso reflet the unne of ville nutrients n toxins ssoite with these potentil lrvl hitts, whih my ffet

6 Asmre et l. Mlr J (27) 6:65 Pge 6 of 9 An. riensis oviposition preferene - e An. oluzzii oviposition preferene - f g.... min equivlent min equivlent min equivlent min equivlent h Fig. 3 Oviposition response of grvi Anopheles riensis n Anopheles oluzzii to grss voltile hespe extrts, from Ehinohlo pyrmilis (, e) Ehinohlo stgnin (, f), Typh ltifoli (, g) n Cyperus ppyrus (, h), respetively. Relese rte of the voltile hespe extrts is given in minute equivlents. The solvent ontrol ws hexne (). Men oviposition preferenes with the sme letters inite no signifint ifferene from one nother (nominl logisti fit moel, χ 2 n P <.5 from the likelihoo rtio test, signifint ifferenes were etermine y os rtio pirwise omprisons). Error rs represent the stnr error of the men Tle 2 Cross-omprtive nlysis of the oviposition response of grvi mosquitoes to voltile extrts of grss speies Oviposition An. riensis An. oluzzii Level Level 2 Os rtio P Os rtio P E. pyrmilis E. stgnin E. pyrmilis T. ltifoli E. pyrmilis C. ppyrus.5364 <..68 <. E. stgnin T. ltifoli E. stgnin C. ppyrus.6366 < <. T. ltifoli C. ppyrus.6894 < The nlysis is one using nominl logisti regression moel fit α =.5 lrvl survivl ifferentilly in the vrious grss-ssoite hitts. Emergent vegettion in quti hitts re ommonly ssoite with the presene or sene of An. gmie/ An. oluzzii n An. riensis lrve [, 7, 9, 9, 23, 36]. While the numer of Anopheles lrve ollete in this stuy were low, ue to the impt of El Niño in the stuy re in 24 25, the oserve ptterns of ssoition with vegettion re onsistent with previous reports [9, 9, 24]. In stuies in whih the vegettion hs een hrterise, hitts ssoite with Poee generlly hve higher An. gmie/an. oluzzii n An. riensis lrvl ensity ompre with tht of hitts ssoite with Typhee n Cyperee [9, 9, 24]. While vegettion in these hitts is known to influene hrteristis, suh s shing, temperture, wter flow, pretor unne n nutrients, they lso provie grvi mosquitoes with hemil ues importnt to hitt seletion. An inresing oy of eviene suggests tht nopheline mosquitoes mke use of olftory ues s positive initors for oviposition site seletion [8, 37 39]. While the fous of this stuy ws not to ientify the speifi slient voltiles in these ttrtive n versive grsses, the ehviourl results presente here inite strong n roust preferene for the hespe extrts of the Poee grsses. Interestingly, the mjority of the previously ientifie olftory ues tht rive the oviposition site seletion in An. gmie s.l. originte from wil n ultivte grsses of the Poee fmily [37 39]. These oours inlue α- n ß-pinene, 3-rene, ryophyllene, limonene n nonnl [8, 38 4], whih re not ffete y mehnil mge of the plnts, ut re thought to e onstitutively expresse [42 44]. Future work will e ime t ientifying the slient voltiles in

7 Asmre et l. Mlr J (27) 6:65 Pge 7 of 9 An. riensis; numer of eggs An. oluzzii; numer of eggs 2 2 erive from she mize pollen, whih re rih in wter solule proteins [49], hve een shown to enhne lrvl evelopment n growth, ult size n survivl [45, 46]. While pollen of other grss speies might lso onstitute supplementl nutriment for surfe feeing nophelines, the nutritive qulity of grss pollen vries ross speies [5, 5]. For exmple, typh pollen is known to e nutrient poor ompre with mize pollen [5, 5]. Femles tht selet lrvl hitts ssoite with grsses sheing nutrient rih pollen hve the potentil to inrese their own fitness y proviing their offspring with seletive vntges, s previously shown for mize pollen. Seletive pressures my lso e involve in the voine of C. ppyrus-ssoite hitts y grvi nophelines, s this grss seretes essentil oils tht rete thin film on the surfe of the wter, preventing mosquito lrve from rething [29, 3]. Conlusions The emonstrte hierrhil preferene of grvi An. oluzzii n An. riensis for grsses inites tht vegettion types ssoite with lrvl hitts re instrumentl in the oviposition hoie of the mlri mosquitoes. Current n future nlysis of Anopheles oviposition eology ssoite with grsses is likely to provie key lrvl hitt hrteristis tht my e integrte into urrent methos of lrvl ontrol. Also, ientifying voltile ues from grsses tht moulte grvi mlri mosquito ehviours hs istint potentil for the evelopment of tools to e use in future monitoring n ontrol methos. E. pyrmilis T. ltifoli E. stgnin C. ppyrus Fig. 4 Oviposition preferene hierrhy of Anopheles riensis (top) n Anopheles oluzzii (ottom) in multi-hoie ren, to wter onitione with voltile hespe extrts of the four grss speies, Ehinohlo pyrmilis, Ehinohlo stgnin, Typh ltifoli n Cyperus ppyrus. Men numers of eggs with the sme letters inite no signifint ifferene from one nother (nominl logisti fit moel, χ 2 n P <.5 from the likelihoo rtio test, signifint ifferenes were etermine y os rtio pirwise omprisons). Error rs represent the stnr error of the men the grsses tht eliite ttrtion in grvi nophelines in this stuy. Anopheles mosquitoes re likely ifferentilly ttrte to lrvl hitts tht re rih in nutrients, erive iretly from she pollen [45 47] n iniretly from umulte etritus n ssoite miro-orgnisms, n tht provie shelter from ioti n ioti threts [48]. Nutrients Arevitions DF: egrees of freeom; GLM: generlise liner moel; HSD: honest signifint ifferene; PCR: polymerse hin retion; RH: reltive humiity; χ 2 : Chi squre. Authors ontriutions RI, RH n SRH esigne the stuy. RI n SRH supervise n YA onute the experiments. SRH n YA onute the sttistil nlyses. YA proue the originl rft, while RI, SRH n RH ritilly revise the mnusript. All uthors re n pprove the finl mnusript. Author etils Deprtment of Zoologil Sienes, Ais A University, PO. Box 76, Ais A, Ethiopi. 2 Deprtment of Biologil Sienes, Dere Mrkos University, Dere Mrkos, Ethiopi. 3 Deprtment of Plnt Protetion Biology, Unit of Chemil Eology, Sweish University of Agriulturl Sienes, Alnrp, Sween. 4 Nturl Resoures Institute, University of Greenwih, Lonon, UK. Aknowlegements Ais A University n Dere Mrkos University re uly knowlege for filitting the stuy. The uthors re grteful to the lol ommunities in the shoreline villges of Lke Tn for their oopertion uring grss voltile olletion n lrvl smpling. The uthors wish to exten their hertfelt knowlegements to the lte Dr. Emiru Seyoum for his involvement in the erly stges of stuy esign n implementtion, n for his wisom. Competing interests The uthors elre tht they hve no ompeting interests.

8 Asmre et l. Mlr J (27) 6:65 Pge 8 of 9 Avilility of t n mterils The t sets supporting the onlusions of this rtile re provie in the mnusript. Funing This stuy ws finnilly supporte y grnt from the Sweish Interntionl Development Coopertion Ageny, Chemo-eologil mngement of Anopheles riensis (SWE-29-3) to RI. Reeive: 9 August 26 Aepte: 3 Jnury 27 Referenes. Neng BA, Simuni JA, Mugi JP, Githeko AK, Fillinger U. Proutivity of mlri vetors from ifferent hitt types in the western Keny highlns. PLoS ONE. 2;6:e White MT, Griffin JT, Churher TS, Ferguson NM, Bsáñez M-G, Ghni AC, et l. Moelling the impt of vetor ontrol interventions on Anopheles gmie popultion ynmis. Prsit Vetors. 2;4: Hmäk PA, Björkmn M, Rämert B, Hopkins RJ. Sle-epenent responses in ge herivores ffet ttk rtes in sptilly heterogeneous systems. Bsi Appl Eol. 29;: Rff KF, Anersson MN, Shlyter F. Host seletion y rk eetles: plying the os in high-stkes gme. Av Inset Physiol. 26;5: Wester B, Cré RT. Use of hitt oour y host-seeking insets. Biol Rev. 26. oi:./rv Gimnig JE, Omok M, Kmu L, Hwley WA. Chrteristis of lrvl nopheline (Dipter: Culiie) hitts in western Keny. J Me Entomol. 2;38: Minkw N, Sonye G, Mogi M, Yn G. Hitt hrteristis of Anopheles gmie s.s. lrve in Kenyn highln. Me Vet Entomol. 24;8: Vnwmeke SO, Somoon P, Hrh R, Isenstt M, Lmin EF, Wlton C, et l. Lnspe n ln over ftors influene the presene of Aees n Anopheles lrve. J Me Entomol. 27;44: Fillinger U, Somroek H, Mjmere S, vn Loon E, Tkken W, Linsy SW. Ientifying the most proutive reeing sites for mlri mosquitoes in The Gmi. Mlr J. 29;8:62.. Neng BA, Simuni JA, Mugi JP, Githeko AK. Physil, hemil n iologil hrteristis in hitts of high n low presene of nopheline lrve in western Keny highlns. PLoS ONE. 22;7:e Mushinzimn E, Mung S, Minkw N, Li L, Feng C-C, Bin L, et l. Lnspe eterminnts n remote sensing of nopheline mosquito lrvl hitts in the western Keny highlns. Mlr J. 26;5:3. 2. Dugss S, Linh JM, Torr SJ, Oyieke F, Linsy SW, Fillinger U. Eletri nets n stiky mterils for nlysing oviposition ehviour of grvi mlri vetors. Mlr J. 22;: Dugss S, Linh JM, Torr SJ, Linsy SW, Fillinger U. Evlution of the influene of eletri nets on the ehviour of oviposition site seeking Anopheles gmie s.s. Prsit Vetors. 24;7: Okl MN, Frnis B, Herrer-Vrel M, Fillinger U, Linsy SW. Wter vpor is pre-oviposition ttrtnt for the mlri vetor Anopheles gmie sensu strito. Mlr J. 23;2: Rejmánková E, Higshi R, Grieo J, Ahee N, Roerts D. Voltile sustnes from lrvl hitts meite speies-speifi oviposition in Anopheles mosquitoes. J Me Entomol. 25;42: Sum LA, Ogunugfor CB, Deng AL, Hssnli A. Regultion of oviposition in Anopheles gmie s.s.: role of inter- n intr-speifi signls. J Chem Eol. 28;34: Afify A, Glizi CG. Chemosensory ues for mosquito oviposition site seletion. J Me Entomol. 25;52: Linh JM, Okl MN, Herrer-Vrel M, Borg-Krlson AK, Torto B, Linsy SW, et l. Disovery of n oviposition ttrtnt for grvi mlri vetors of the Anopheles gmie speies omplex. Mlr J. 25;4:9. 9. Fillinger U, Sonye G, Killeen GF, Knols BG, Beher N. The prtil importne of permnent n semipermnent hitts for ontrolling quti stges of Anopheles gmie sensu lto mosquitoes: opertionl oservtion from rurl town in western Keny. Trop Me Int Helth. 24;9: Mjmere S, Fillinger U, Syer DR, Green C, Linsy SW. Sptil istriution of mosquito lrve n the potentil for trgete lrvl ontrol in The Gmi. Am J Trop Me Hyg. 28;79: Minkw N, Mung S, Atieli F, Mushinzimn E, Zhou G, Githeko AK, et l. Sptil istriution of nopheline lrvl hitts in western Kenyn highlns: effets of ln over types n topogrphy. Am J Trop Me Hyg. 25;73: Imhle SS, Pijmns KP, Mukn WR, vn Lmmeren R, Githeko AK, Tkken W. A longituinl stuy on Anopheles mosquito lrvl unne in istint geogrphil n environmentl settings in western Keny. Mlr J. 2;: Minkw N, Di GO, Sonye GO, Futmi K, Njeng SM. Mlri vetors in Lke Vitori n jent hitts in western Keny. PLoS ONE. 22;7:e Bøgh C, Clrke SE, Jwr M, Thoms CJ, Linsy SW. Lolize reeing of the Anopheles gmie omplex (Dipter: Culiie) long the River Gmi, West Afri. Bull Entomol Res. 23;93: Gougn LC, Rkotonrnry M, Boyer S, Lempérière G, Deheq J-S, Fontenille D. Aioti n ioti ftors ssoite with the presene of Anopheles riensis immtures n their unne in nturlly ourring n mn-me quti hitts. Prsit Vetors. 22;5: Khter EI, Sowilem MM, Sllm MF, Alhme AM. Eology n hitt hrteriztion of mosquitoes in Sui Ari. Trop Biome. 23;3: Gom LKH. Experimentl reeing of Anopheles gmie Giles in ppyrus swmps. Nture. 96;87: Yohnnes M, Hile M, Ghereyesus TA, Witten KH, Gethew A, Byss P, et l. Cn soure reution of mosquito lrvl hitt reue mlri trnsmission in Tigry, Ethiopi? Trop Me Int Helth. 25;: Mouhet G, Mnguin S, Siroulon G, Lventure S, Fye O, Onp AW, et l. Evolution of mlri in Afri for the pst 4 yers: impt of limti n humn ftors. 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9 Asmre et l. Mlr J (27) 6:65 Pge 9 of Yn Z-G, Wng C-Z. Similr ttrtiveness of mize voltiles inue y Helioverp rmiger n Pseuleti seprte to the generlist prsitoi Cmpoletis hloriee. Ent Exp Appl. 26;8: Ye-Eiyo Y, Pollk R, Spielmn A. Enhne evelopment in nture of lrvl Anopheles riensis mosquitoes feeing on mize pollen. Am J Trop Me Hyg. 2;63: Ye-Eiyo Y, Pollk RJ, Kiszewski A, Spielmn A. Enhnement of evelopment of lrvl Anopheles riensis y proximity to flowering mize (Ze mys) in turi wter n when rowe. Am J Trop Me Hyg. 23;68: Ye-Eiyo Y, Pollk RJ, Kiszewski A, Spielmn A. A omponent of mize pollen tht stimultes lrvl mosquitoes (Dipter: Culiie) to fee n inreses toxiity of miroil lrviies. J Me Entomol. 23;4: Merritt RW, D RH, Wlker ED. Feeing ehvior, nturl foo, n nutritionl reltionships of lrvl mosquitoes. Annu Rev Entomol. 992;37: Porter EK. Origins n geneti nonvriility of the proteins whih iffuse from mize pollen. Environ Helth Prospet. 98;37: Shmit JO, Thoenes SC, Levin MD. Survivl of honey ees Apis mellifer (Hymenopter: Apie), fe with vrious pollen soures. Ann Entomol So Am. 987;8: Roulston TH, Cne JH. Pollen nutritionl ontent n igestiility for nimls. Plnt Syst Evol. 2;222: Sumit your next mnusript to BioMe Centrl n we will help you t every step: We ept pre-sumission inquiries Our seletor tool helps you to fin the most relevnt journl We provie roun the lok ustomer support Convenient online sumission Thorough peer review Inlusion in PuMe n ll mjor inexing servies Mximum visiility for your reserh Sumit your mnusript t