Assessment of Alternative Mating Strategies in Anopheles gambiae: Does Mating Occur Indoors?

Assessment of Alternative Mating Strategies in Anopheles gambiae: Does Mating Occur Indoors?
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  B EHAVIOR , C HEMICAL  E COLOGY Assessment of Alternative Mating Strategies in  Anopheles gambiae :Does Mating Occur Indoors? ADAMA DAO, 1 ABDOULAYE ADAMOU, 1 ALPHA SEYDOU YARO, 1 HAMIDOU MOUSSA MAI ¨ GA, 2 YAYA KASSOGUE, 1 SE´ KOU FANTAMADY TRAORE´ , 1 AND  TOVI LEHMANN 3 J. Med. Entomol. 45(4): 643Ð652 (2008) ABSTRACT  Mating in  Anopheles gambiae  has been observed only in outdoor swarms. Here weevaluate whether mating also occurs indoors. Mark-release-recapture of virgin males and females innatural houses showed that mating occurred over a single day even when mosquitoes can leave thehouse through exit traps and without adaptation to laboratory conditions. In these experiments,insemination rate in the M molecular form of   An. gambiae  (and  An. arabiensis ) was higher than thatof the S form (15 versus 6%). Under these conditions, smaller females of the M form mated morefrequentlythanlargerfemalesofthatform.Samplingmosquitoesthroughoutthedayshowedthatbothsexes enter houses around sunrise and leave around sunset, staying indoors together from dawn todusk. In an area dominated by the M form, the daily rate of insemination in samples from exit trapswas  5% higher than in those from entry traps, implying that mating occurred indoors. Importantly,frequency of cross mating between the molecular forms was as high as that between members of thesame form, indicating that, indoors, assortative mating breaks down. Altogether, these results suggestthat indoor mating is an alternative mating strategy of the M molecular form of   An. gambiae . Becausenaturally occurring mating couples have not yet been observed indoors, this conclusion awaitsvalidation. KEY WORDS  Anopheles gambiae , alternative mating strategies, molecular formsMosquitomatingbehaviorisapoorlyunderstoodfacetof their life, partly because direct observations arehampered by their small size, speed, and crepuscularor nocturnal activity. Hence, only a few studies ad-dressmatingbehavioroftheprincipalAfricanmalaria vector,  Anopheles gambiae  under natural conditions(Charlwood and Jones 1980, Marchand 1984, Yuval etal. 1992, Charlwood et al. 2002b, Diabate et al. 2003,2006). The  Anopheles gambiae  complex consists of seven sibling species (Coluzzi et al. 1979, 1985, Huntet al. 1998, Toure et al. 1998). The M and S molecularforms of   An. gambiae  s.s. represent incipient species(Coluzzi et al. 1985, Toure et al. 1998, della Torre etal. 2001, Favia et al. 2001, Gentile et al. 2001, Muk-abayire et al. 2001, Lehmann et al. 2003, Turner et al.2005, but see Lanzaro et al. 1998, Yawson et al. 2007).Many mosquito species initiate mating in swarmsthat typically consist of a group of males ßying inzigzag pattern 1Ð3 m above ground (often over a distinct marker) and forming a distinct  “ cloud, ”  intowhichfemalesßyanddepartpairedwithamale.How-ever, swarms are not involved in mating of many cu-licine and anopheline species (Nielsen and Haeger1960, McIver et al. 1980, Lounibos et al. 1998) wheremating occurs near the vertebrate host or near larvalsites (Yuval 2006). As noted by Downes (1969),swarmingindifferent “ arenas ” canfacilitateprematingisolation.Thematingbehaviorof   An.gambiae iskeytounderstand the mechanisms of reproductive isolationof the partly sympatric molecular forms and siblingspecies. Observing only Þve couples in 34 swarms(Marchand 1984) casts doubt if mating occurs only inswarms.Althoughmorecoupleswereobservedin  An.gambiae  swarms elsewhere (Charlwood et al. 2002,Diabate et al. 2003, 2006), this issue remains relevantbecause mating can be initiated in different ways bydifferentindividualswithinspecies(EmlenandOring1977, Thornhill and Alcock 1983, Yuval 2006). Alter-native mating strategies are often used by males thathave low prospects of mating under  “ standard ”  con-ditions because of competitive disadvantage, such assmallerbodysize(ThornhillandAlcock1983,Yuvaletal. 1993, Charlwood et al. 2002a, Okanda et al. 2002).For example,  An. labranchiae atroparvus  swarms out-doors and indoors, yet most females are mated insheltered  “ resting ”  sites (Cambounac and Hill 1940).Although mating in  An. gambiae  is initiated in out-door swarms, the possibility that mating also occursindependent of swarms has not been addressed. Toevaluate if   An. gambiae  mates indoors, we (1) con-ducted experiments using progeny of wild females in 1 Malaria Research and Training Center, 1805, Point G, Bamako,Mali. 2 Faculty of Sciences and Techniques, University of Bamako, Ba-mako, Mali. 3 Corresponding author: Laboratory of Malaria and Vector Re-search,NIAID,NIH,12735TwinbrookParkway,Rockville,MD20852(e-mail:  natural settings; (2) determined whether males andfemales naturally co-occur in houses, (3) assessedwhether insemination rate is higher in females sam-pledfromexitversusentrytrapsasexpectedifmatingindoors occurred naturally; and (4) evaluated thespeciÞcity in indoor mating between the molecularforms. Materials and MethodsStudy Sites.  Indoor mark, release, and recapturematingexperimentsandassessmentofthepresenceof males with virgin females indoors were conducted inthe villages of Banambani (8  3   W, 12  48   N) andDone ´  gue ´  bougou(7  59  5  W,12  48  38  N),located3Ð4km apart and  15 km north of Bamako, Mali. Thesefarming communities are situated among gently roll-inghillscoveredwithvegetationcharacteristicsofwetsavanna.Mosthouseshaveasingleroom,builtofmud,shaped like a cylinder, and covered by a conic thatchroof.Experiments comparing natural insemination ratesin mosquitoes entering and exiting houses were car-riedoutinthevillageSokouraninearNiono(14  17  N,8  5   W). It is a rice cultivation area located 350 kmnortheastofBamakoinahabitatofdrysavannawhere,away from the river and the irrigation canals, vegeta-tion is sparse. The inhabitants live in mud-walledhouses, typically designed as a rectangular prism(box), with one door and two windows. Unlike invillages near Bamako, in Niono, many inhabitants usebednets regularly, but most bednets are not impreg-nated with insecticides. Mosquitoes Used in Release Experiments.  Femalesof   An. gambiae  s.l. were collected from houses in Ba-nambaniandDone ´  gue ´  bougouusingaspiratorsduringOctoberÐNovember2004,JulyÐAugust2005,andJulyÐAugust 2007. They were transported to the insectaryat the Malaria Research and Training Center in theUniversity of Bamako and maintained at 27  C and75Ð85% RH. Blood-fed half-gravid and gravid femaleswereplacedin1-galcagesandkeptfor2dbeforetheywere placed individually in a 50-ml Falcon tube con-taining 15 ml deionized water for oviposition. A stripofÞlterpaper(2cmwide)surroundedthewateredge,providing a wet surface to collect the eggs. FemaleswholaideggswereidentiÞedtospeciesandmolecularform using molecular assays (Fanello et al. 2002).First-instar larvae were pooled by species and molec-ular form in groups of 200 per pan. Pans (25 cm widthby 30 cm length by 6 cm depth) were Þlled with 400ml of deionized water, and 0.1 g ground Þsh food(TetraminTetra,Inc.,Melle,Germany)wasprovideddaily. When most larvae reached the third instar, 400mlofwaterwasadded.Pupaewerecollecteddailyandtransferred into emergence cages. Emerging malesand females were separated within 24 h to preventmating. Virginity of females was conÞrmed by dissec-tion and examination of spermatheca (below) from 8to 20 females from each cage. All veriÞcations ( n  339) were negative. Indoor Release Experiments.  Several one-roomhouses in Banambani and Done ´  gue ´  bougou were se-lected for the experiments. Before release, all open-ingsofthehouseinthevillageweresealedbynets,andexittrapsweremountedinthescreenthatcoveredthedooropening(Fig.1A).Fivetraps(30by30by30cm)weremountedonthedoor,spacedmoreorlessevenlyfrombottomtotop.Onetrap(60by60by60cm)wasmounted on the window. To increase survival andrecapture rate, spiders and cobwebs were removed,three to Þve wet towels were hung to increase airhumidity, and three pads of cottons soaked in 5%sucrose solution were suspended by strings from theceiling.Afewhoursbeforerelease,virginfemales(2Ð5d old) were dusted with a ßuorescent dye (DayGlo;DayGloColor,Cleveland,OH).Eachspeciesandmo-lecular form was marked with a different color. Maleswere not marked. Males and females were releasedinto the house together, 4Ð1 h before sunset. Mosqui-toes were removed from exit traps continuouslythroughout the experiment to prevent mating in theexit traps, and males and females were placed in sep-arate cages. The next morning, mosquitoes were col-lected indoors using a different aspirator and a differ-ent cage for each mosquito color. After the livecollection,pyrethrumspraycollection(PSC)wasper-formed to collect all remaining mosquitoes.Inseminationstatuswasdeterminedbyexamination(  100 magniÞcation or higher) of dissected sper-matheca, pressed under a microscope coverslip, forthe presence of sperm. All recaptured mosquitoeswere preserved in 85% ethanol, and their species andmolecular form were determined as described above.To determine whether smaller body size is a deter-minant of indoor mating, one wing of randomly se-lected females ( n  190) was removed, mounted on a slide, and measured to the nearest 0.1 mm using a dissecting scope equipped with micrometer ruler un-der  10 magniÞcation. Abundance of Males and Females in Houses.  Maleand female mosquitoes were collected from Þvehouses in Done ´  gue ´  bougou or Banambani by PSC ateach of the following times: 0600, 0700, 1700, 1800,1830, 1900, 2000, 2200, and 2400 hours. Weekly col-lectionswereperformedfromJulytoSeptember2005alternately in the each village, allowing 2 wk beforerepeated sampling of the same house. Natural Indoor Mating Assessment: Using Entryand Exit Traps.  To ascertain if indoor mating occursnaturally, experiments were undertaken to comparefemale insemination rates in entry and exit trapsmounted on houses during AprilÐMay 2006 in Sokou-rani (Niono), when mosquito density was high. If indoor mating occurs naturally, insemination of fe-males from exit traps was expected to be higher thanin females from entry traps as long as mosquitoes canalsoenterandexitthehousethroughanotherwindowordoorthatwasnotÞttedwithtraps.Weassumedthatindoormortalitywasnothigherinvirginfemalesthaninmatedfemales.IntheÞrstexperiment,funnel-basedentryandexittrapswereused.Eachtrapmeasured30by 30 by 30 cm. Two exit and two entry traps were644 J OURNAL OF  M EDICAL  E NTOMOLOGY  Vol. 45, no. 4  mounted on three different houses every other nightforfournights.Thetrapsweremountedinpairsonthedoor and on one window similar to those depicted inFig. 1A. Mosquitoes freely entered and exited thehousethroughtheotherwindow,whichwasleftopen.In the second experiment, larger traps (50 by 50 cmwide by 200 cm high) were used instead. One entrytrap was mounted inside the house, and one exit trapwasmountedoutside(Fig.1B).Funnelswerenotusedin these traps, and mosquitoes ßew into them ingreater numbers. Mosquitoes entered and exited thehouse through the door opening, which was looselycovered by a cloth curtain, leaving space for ßyingmosquitoes. Such curtains were seen on many housesin that area (Fig. 1B). As customary in this region,three to four people slept in each house, and often a few people slept outside. The experiment did notchangeanyoftheseconditions,butweconÞrmedthatnoinsecticide-impregnatedbednetshavebeenusedin experimental houses. Traps were installed before1400 hours for 24 h (Þrst series) or 3Ð4 consecutived (second series). To prevent mating inside traps,mosquitoes were continuously collected, and malesand females were immediately separated in differ-ent cups and pooled based on the following timeintervals: 1500  1700; 1700Ð1900; 1900Ð2000; 2000Ð2400; 2400Ð0300; 0300Ð0600; 0600Ð0900; 0900Ð1200;and1200  1500hours.Duringtheexperiments,sunsetand sunrise times were close to 1900 and 0600 hours,respectively. The gonotrophic stage of females wasrecorded, and their insemination status was deter-mined. Specificity of Indoor Mating Between the Molecu-lar Forms.  The mating speciÞcity of the molecularforms was assessed using indoor mark-release-recap-ture(MRR)experimentsinBanambaniandDone ´  gue ´  - Fig. 1.  Mounting exit traps in MRR experiments in Doneguebougou (top) and in experiments to assess natural insem-ination in Niono (bottom) using entry and exit traps (see text for details). July 2008 D AO ET AL .: I NDOOR  M ATING OF  An. gambiae  645  bougou from July to August 2007. The same proce-dures described above were used to produce virginoffspring of wild females, conduct the MRR experi-ments, and process the adults, but no exit traps wereused. Two experimental designs were used, both withmales of one molecular form. In the single femaledesign,thefemaleswereallofasingleform,eitherthesame or the other form of the males (i.e.,   M  M ,  M  S ,  S  S , and  S  M ). In mix female design, fe-males from the two molecular forms were releasedtogether in equal numbers (i.e.,  M  M&S ,  S  M&S ).Every combination in the single female design wasreplicated three times, and those of the mix femaledesign were replicated four times. The Þrst tworeplicationswerecarriedoutwith200mosquitoesof each form and sex. In subsequent experiments, 150males and a total of 150 females were used in bothexperimental designs (i.e., 75:75 M:S females in themix female design). The ßuorescent dyes were usedalternately; females of each form were marked withthe same color combinations across replicates andcolor was not confounded in molecular form.Inadditiontoanalysisoffemaleinseminationstatususing logistic regression, we calculated a speciÞcityindex for each form in each experimental design inevery replication as the difference between insemi-nationratesofthepureversuscrossformstandardizedby the average insemination rate across forms: [Ip  Ic]/[(Ip    Ic)/2], where Ip and Ic represent theinsemination rate of the females of the same and theotherformasthatofthemales,respectively.Theindexvariedfromtwo(highestspeciÞcity)to  2(completepreference of the other form), and 0 indicated nospeciÞcity. ResultsInsemination Rate in Indoor Mark, Release, andRecaptured Mosquitoes.  To test whether  An. gambiae might mate in natural houses, virgin ßuorescentlymarked females and unmarked males (2Ð5 d old, F 1 offspring of wild caught females) were released intohouses, and insemination status was determined thenextmorning.Exittraps(Fig.1A)allowedmosquitoesto leave the house. Overall across species and molec-ular forms, the insemination rate was 13% (range,1Ð27%; Table 1). The fraction of females and malescollected in exit traps varied considerably betweenexperiments (7Ð83% for females and 31Ð99% formales), possibly because of the relative humidity in-doororoutdoor,moonlightintensity,etc.Overall,62%of the females and 80% of the males were collectedfromexittraps.Inseminationratesinfemalescollectedin houses (14%) and in the exit traps (13%) did notdiffer( P   0.28,Gtest;df   1).Mosquitoescouldnotmateinexittrapsbecausetheywereremovedassoonas they entered.Tocomparetheinseminationrateofthespeciesandforms, we used logistic regression accommodatingvariation among experiments (excluding the experi-ment without the S form; Table 1). Differences be-tween species and molecular forms were found ( P   0.0001; Table 2), although insemination rates variedamong experiments ( P   0.0001; Table 2). The inter-actionbetweenexperimentandspecies/formwasnotsigniÞcant ( P   0.56; Table 2) and was removed. In-semination rate of the S form (6%) was lower ( P   0.001) than that of the M form (15%) and  An. ara-biensis  (18%), whereas the difference between the M Table 1. Assessment of indoor mating using mark, release, recapture experiments Release dateFemale recaptured/released Insemination rate (%)M S Ar M S Ar Mean19 Nov. 2004 32/60 44/100 32/100 3.1 0 0 1.013 Jul. 2005 28/100 0/0 32/100 3.6 Ñ 12.5 8.129 Jul. 2005 60/100 59/100 62/100 8.3 5.1 4.8 6.121 Aug. 2005 67/100 70/100 62/100 35.8 8.6 38.7 27.731 Aug. 2005 43/100 32/100 25/100 16.3 3.1 24 14.5Overall 230/460 206/400 213/500 16.5 4.9 17.4 12.9M, S, and Ar represent M form, S form, and  An. Arabiensis,  respectively. The no. of females and males released per species/forms was equal.The insemination rates are based on dissection of all recaptured females. Table 2. Body size (wing length, mm) difference between inseminated and uninseminated females SourceANOVA results Means (mm) and signiÞcance a of differencedf F/MS  P   SpForm Inseminated Unnseminated  P  Model 8 15.0/0.293 0.0001Error 219 ND/0.020 ND Ar 3.20 3.18 0.6Experiment 3 15.9/0.311 0.0001 M 2.97 3.08 0.0012Species/form 2 29.6/0.581 0.0009 S 3.15 3.20 0.3Insemination 1 3.3/0.064 0.073SpFo  insemination b 2 4.1/0.080 0.018 All 3.11 3.16 0.073 a Statistical signiÞcance of the contrast between inseminated and uninseminated females of   An. arabiensis  (Ar) and the M and S molecularforms of   An. gambiae. b Interaction between species/form and insemination.ND, not determined. 646 J OURNAL OF  M EDICAL  E NTOMOLOGY  Vol. 45, no. 4  form and  An. arabiensis  were not signiÞcant ( P   0.8;Table 2). These results show that both species andmolecularformscanmateindoorswithoutadaptationto laboratory conditions even if they can leave thehouse through exit traps. The results suggest that in-door conditions are more suitable for  An. arabiensis and the M form of   An. gambiae  than for the S form.Notably, nearly 90% of the females remained virginsundertheseconditionsdespiteaccesstomalesandtheadvantage of early reproduction, suggesting that theypreferred mating in outdoor swarms. Nevertheless,these experimental results did not show that this oc-curs naturally.In experimental houses, ßight activity peaked  5Ð10 min after sunset and remained so for up to anhour. During this time, a humming sound, producedby ßying mosquitoes was heard. Swarms, deÞned asgroups of males ßying together and forming a station-ary “ cloud, ” werenotobservedindoors.Instead,manymalesandfemalesconcentratedonthebottomhalfof thescreencoveringthedoorandontheadjacentwallsand engaged in short (  20 cm) ßights. Small clustersof mosquitoes (6Ð20) were also observed near theeaves in houses with thatch roofs. Couples matingwere observed ßying and falling to ground near thebetter illuminated door area by twilight (this wasespecially noticeable if white sheets were spread onthe ßoor). However, the events leading to coupleformation were not observed.Toassessiffemalebodysizeaffectedherprobabilityof mating indoors, we compared wing length of allinseminated females and that of a random sample of uninseminated females from each replicate experi-ment.Overall,inseminatedfemalestendtobesmallerthanuniseminatedfemales( P   0.073),butonlyintheM form was this difference signiÞcant ( P     0.0012;Table 2). Abundance of Males and Females in Houses.  Col-lections of males and females in houses were con-ducted throughout the day in Banambani andDone ´  gue ´  bougou by PSC. Male density indoors in-creased rapidly at sunrise (0615 hours) and remainedstable (average  4 males per house, max  36) untilsunset (1845 hours, sunset time varied between 1830and 1900 hours during the experiment). Male densitydeclined sharply to near zero by 1900 hours (Fig. 2),precluding indoor mating during the rest of the nightwhen male density was effectively zero. Female den-sity indoors followed this pattern, but female densityremained above zero throughout the night (Fig. 2). Fig. 2.  Mosquito density in houses estimated using pyrethrum spray catch in Doneguebougou and Banambani (pooled)over time showing variation among houses. In box-whisker plots, the box extends between the 25th and the 75th percentile,i.e., across one interquartile range (IQR), and the whiskers extend up to the most extreme value but not beyond 1.5 timesthe IQR. o, outlier values located 1.5Ð3 IQR from the median;  * extreme outliers located over three IQR. July 2008 D AO ET AL .: I NDOOR  M ATING OF  An. gambiae  647
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