CA

BRTa

b

c

d

e

U

a

ARRAA

KASM

1

hmilw2cttth

0h

Acta Tropica 132S (2014) S53–S63

Contents lists available at ScienceDirect

Acta Tropica

jo ur nal home p age: www.elsev ier .com/ locate /ac ta t ropica

haracterization of swarming and mating behaviour betweennopheles coluzzii and Anopheles melas in a sympatry area of Benin

enoît S. Assogbaa, Luc Djogbénoua,∗, Jacques Saizonoua, Abdoulaye Diabatéc,och K. Dabiréc, Nicolas Moirouxe, Jérémie R.L. Gillesd, Michel Makoutodéa,hierry Baldetb,e

Institut Régional de Santé Publique, Université d’Abomey Calavi, 01BP918 Cotonou, BeninCentre de Recherche Entomologique de Cotonou, Institut de Recherches pour le Développement, UMR224, 06BP2604 Cotonou, BeninInstitut de Recherche en Science de la Santé/Centre Muraz, BP 545 Bobo-Dioulasso, Burkina FasoInsect Pest Control Laboratory, Joint FAO/IAEA Division of Nuclear Techniques in Food, International Atomic Energy Agency, Vienna, AustriaInstitut de Recherche pour le Développement (IRD), Maladies Infectieuses et Vecteurs: Ecologie, Génétique, Evolution et Contrôle (MIVEGEC),MR UM1-UM2 – CNRS 5290 – IRD 224, Montpellier, France

r t i c l e i n f o

rticle history:eceived 11 June 2013eceived in revised form 4 September 2013ccepted 7 September 2013vailable online 7 October 2013

eywords:nopheles gambiaewarmalaria vector control

a b s t r a c t

The swarm structure of two sibling species, Anopheles gambiae coluzzii and Anopheles melas, was charac-terize to explore the ecological and environmental parameters associated with the formation of swarmsand their spatial distribution. Swarms and breeding sites were searched and sampled between Januaryand December 2010, and larval and adult samples were identified by PCR. During the dry season, 456swarms of An. gambiae s.l. were sampled from 38 swarm sites yielding a total of 23,274 males and 76females. Of these 38 swarming sites, 18 were composed exclusively of An. gambiae coluzzii and 20 exclu-sively of An. melas, presenting clear evidence of reproductive swarm segregation. The species makeup ofcouples sampled from swarms also demonstrated assortative mating. The swarm site localization wasclose to human dwellings in the case of the An. gambiae coluzzii and on salt production sites for An. melas.At the peak of the rainy season, swarms of An. melas were absent. These findings offer evidence that the

ecological speciation of these two sibling species of mosquitoes is associated with spatial swarm seg-regation and assortative mating, providing strong support for the hypothesis that mate recognition iscurrently maintaining adaptive differentiation and promoting ecological speciation. Further studies onthe swarming and mating systems of An. gambiae, with the prospect of producing a predictive model ofswarm distribution, are needed to inform any future efforts to implement strategies based on the use of

nal A

GMM or SIT.

Copyright © Internatio

. Introduction

Vector-borne diseases cause a considerable burden on humanealth in tropical and subtropical regions. Malaria alone, trans-itted exclusively by Anopheles mosquitoes (Diptera: Culicidae)

nfected with Plasmodium protozoan parasites, causes 243 mil-ion cases each year, most of which occur in sub-Saharan Africa

ith 863,000 deaths in 2009, mainly children under five (WHO,010). Despite efforts to prevent and control the disease, theonsequences of malaria persist. The development of drug resis-ance within populations of Plasmodium and the inability, so far,

o formulate effective vaccines have limited the direct means ofackling malaria (Greenwood and Alonso, 2002). Vector controlas been historically the cheapest and most successful approach

∗ Corresponding author. Tel.: +229 95428543; fax: +229 21341672.E-mail address: ldjogbenou22002@yahoo.fr (L. Djogbénou).

001-706X/$ – see front matter. Copyright © International Atomic Energy Agency 2013. Pttp://dx.doi.org/10.1016/j.actatropica.2013.09.006

tomic Energy Agency 2013. Published by Elsevier B.V. All rights reserved.

(Pampana, 1969) and evolutionary vector biology is now offeringfresh perspectives on it (Michalakis and Renaud, 2009b). Anotherreason for revisiting vector control is one might think that peopletransmit the parasite as a vector, but do not show the symp-toms of malaria and are not treated (Macdonald, 1957; Maxwellet al., 1999; McKenzie, 2000). Thus, vector control should be thepriority for the fight against malaria (Michalakis and Renaud,2009a).

Traditional strategies aimed at tackling malaria have oftenfocused on reducing human–mosquito contact with insecticide-treated bed nets and indoor residual spraying, principally throughthe use of insecticides (Gu and Novak, 2009; Takken and Knols,2009; Zhou et al., 2010). However, the rapid appearance ofinsecticide resistance in vector species is hampering the eradica-

tion of this devastating disease (Baleta, 2009; Djogbénou, 2009;Djogbenou et al., 2010), and efforts aimed at developing novel andmore effective malaria control strategies are intensifying (Takkenand Knols, 2009). New approaches aim to render wild vector

ublished by Elsevier B.V. All rights reserved.

S Tropic

penanmpmrtfmatsettfliiso

vpmsAAsbfpwsegi2lgiLWetwtsitiaatofhet(Ctwi

54 B.S. Assogba et al. / Acta

opulations refractory to malaria transmission (Ito et al., 2002; Kimt al., 2004) or to release sterile males to progressively suppressatural mosquito populations (Alphey et al., 2002, 2008; Benedictnd Robinson, 2003; Knipling, 1959; Thomas et al., 2000). Theseovel approaches require a thorough understanding of mosquitoating (Howell and Knols, 2009), a facet of mosquito biology

oorly understood so far and this study aims to help investigate. Aajor concern is the presence of reproductive barriers that would

educe the spread of the refractoriness genes between subpopula-ions, or prevent released sterile males from mating with all targetemales. A second concern is the possibility that laboratory adapted

osquitoes would not be able to compete for mates in the wildnd therefore the genes of interest would not be integrated intohe natural population’s gene pool. Success will depend on under-tanding patterns of mosquito reproduction that are relevant toffective transgene spread. In this paper, we seek to investigatehe parameters related to the swarming event which involved inhe mechanisms of reproductive barriers that have restricted geneow between two species of Anopheles gambiae sensu lato, these

nformation’s could be further used in explaining the reproductivesolation between An. coluzzii (form M) and An. gambiae s.s. (form S)ince the issue of ecological speciation within this important vectorf malaria is not yet resolved.

The An. gambiae complex, the major and most efficient malariaector in Africa, mates in swarm (Downes, 1968). It is a com-lex of at least seven morphologically indistinguishable species ofosquitoes in the genus Anopheles (Hunt et al., 1998). Three of these

pecies are encountered in West Africa and in the south of Benin:n. gambiae s.s. GILES (1902), An arabiensis PATTON (1905) andnopheles melas THEOBALD (1903). An. gambiae s.s. is undergoingpeciation (Coluzzi et al., 2002; della Torre et al., 2002). Early studiesased on chromosomal inversions of An. gambiae s.s. in West Africaound five partially isolated populations based on combinations ofaracentric inversions on the right arm of chromosome 2. Theseere named Forest, Savanna, Bamako, Mopti and Bissau chromo-

omal forms (Bryan et al., 1982; Coluzzi et al., 1985, 1979; Touret al., 1998). The chromosomal forms exhibit different degrees ofene flow between them, and their spatial and seasonal distributionndicates that they are adapted to different niches (Manoukis et al.,008; Sogoba et al., 2008; Toure et al., 1998). It is now well estab-

ished, through molecular and population genetics studies, that An.ambiae s.s. is currently in the process of speciation for putativencipient species, M and S molecular forms (Cassone et al., 2008;ehmann et al., 2003; Stump et al., 2005a,b; Turner et al., 2005;

hite et al., 2010; Wondji et al., 2002). Fixed nucleotide differ-nces in X-linked ribosomal DNA genes led to the designation ofwo “molecular forms”, named M and S (Favia et al., 2001), amonghich gene flow appears highly restricted (Tripet et al., 2001), to

he extent that both forms are currently recognized as incipientpecies (della Torre et al., 2005). Recently, An. gambiae s.s. “M form”s named Anopheles coluzzii while the “S form” retains the nomino-ypical name An. gambiae s.s. (Coetzee et al., 2013). No post-matingsolation mechanism is known between the M and S forms: viablend fertile hybrids can be readily obtained in the laboratory, at nopparent fitness cost (Diabaté et al., 2007). However, strong assor-ative mating is observed in nature as evidenced by the low ratef heterogamous inseminations detected in wild females of bothorms (Tripet et al., 2001), and the rarity or complete absence ofybrid genotypes in areas where both forms co-exist (della Torret al., 2001, 2005; Wondji et al., 2005). The S form is widespreadhroughout tropical Africa and is presumed to be the ancestral formAyala and Coluzzi, 2005), while the M form occurs only in West and

entral Africa (Masendu et al., 2004). Typically, the S form peaks inhe rainy season, exploiting rain-dependent puddles as larval sites,hereas the M form predominates in more arid conditions and

n association with irrigated sites such as rice fields (della Torre

a 132S (2014) S53–S63

et al., 2005; Diabaté et al., 2002, 2003b, 2004). The rate of naturalhybridization between the molecular forms is usually thought to bebelow 1% (della Torre et al., 2001; Wondji et al., 2005), although M/Shybrids have been recorded at much higher rates in western-mostWest Africa, in restricted locations, up to 7% in Gambia and 19–24%in Guinea-Bissau (Caputo et al., 2008; Oliveira et al., 2008) suggest-ing different mating behaviour of the forms in these environments.Whether this deficit of hybrids reflects hybrid inferiority in the fieldis not known, but laboratory studies have found no evidence forreduced fitness in hybrids (Diabaté et al., 2007). Strong assortativemating between the molecular forms in the field has been described(Tripet et al., 2001), but its underlying mechanisms are not knownyet. Ecological and biological factors that break down the reproduc-tive isolation between the forms in these environments have notbeen investigated yet and await discovery.

An. gambiae s.l. mates in flight at specific mating stations,and very often over specific landmarks known as swarm mark-ers (Charlwood et al., 2002; Downes, 1969; Yuval, 2006). Theswarms are composed of males; females typically approach aswarm, acquire a mate and leave in copula. The way which thesexes are attracted to each other may contribute to the specificmate recognition systems, which facilitate species identificationand prevent hybridization (Clements, 1999). Insects use a variety ofstimuli to bring males and females together for mating, includingpheromones, visual signals and sound signals, which can oper-ate over long and short ranges (Clements, 1999). However, thehypothesis that flight-tone is used for differential mate recogni-tion was not supported by experiments in the laboratory (Pennetieret al., 2010; Tripet et al., 2005). Additionally, a recent study usinga mark–release-recapture experiment of M and S forms in naturalhouses (with an absence of swarm markers) found no evidence forassortative mating indoors (Dao et al., 2008), suggesting that chem-ical and sound cues are not involved, at least under these conditions.Moreover, very few studies were focused on An. melas geographicaldistribution in salt-water breeder and its swarm’s characterizationin West African (Charlwood, 1980; Coetzee et al., 2000). Given thegoal of understanding pre-reproductive isolation between the Mand S forms of An. gambiae s.s. (in the process of speciation) forprediction of the spatial distribution of swarms, we proposed toperform a comparative study between the reproductive behavioursof An. gambiae s.s. and An. melas. These species have evolved strongreproductive isolation, although the species barrier is still perme-able (Akogbeto, 1995; Powell et al., 1999). They were found to besympatric in our study site (Akogbeto, 1995). Our hypothesis wasthat swarms of these two species should differ in their general char-acteristics (physical marker on the ground and other parametersrelated to the swarming process in each species) and that shouldaccount for their reproductive isolation. The current research thatwas conducted in an area of the Guinean forest belt in SouthernBenin aims to characterize the swarm structure and location of bothspecies and study the ecological and environmental parametersassociated with their formation.

2. Materials and methods

2.1. Study area

This study was carried out in Djègbadji village (6◦12′36′′ N –2◦20′00′′ E), in the town of Ouidah, 40 km from the Cotonou coastallagoon area of Benin (West Africa) from January to December2010. The area is characterized by degraded mangrove swamp,

covers an area of 2 km2 and contains six clusters of habitationssurrounded by mangrove swamp. Mean annual rainfall is about1230 mm with four seasons: two rainy seasons (April–August andOctober–November) and two dry seasons (December–March and

Tropic

Ausasmtvba

2

tesMwafb1nscecd

tsufcPkfc

owmoogewA

2

etW(atsrftc

B.S. Assogba et al. / Acta

ugust–September). Because of the irrigation system, the fieldssed to grow vegetables contain permanent mosquito breedingites which are preferentially colonized by An. gambiae s.s. withn abundant vector population at the end of the long rainy sea-on (Akogbeto, 1995). Previous studies on the bio-ecology of An.elas in coastal lagoons in Benin have shown that this malaria vec-

or is abundant from February to September (Akogbeto, 1995). Theillage has more than 1500 inhabitants and fishing, salt makingy craftsmen, market gardening and food farming are the mainctivities. Sheep, goats, pigs and cows are also reared in the village.

.2. Swarm events and breeding sites survey

Swarms were actively surveyed across the village by a fewrained observers, starting at sunset and looking towards the light-st part of the sky from ground to about 4 m above. Once located,warms were sampled using an insect net (Diabaté et al., 2003a).osquitoes were transferred into cups; individual mosquitoesere killed with chloroform, identified morphologically (Gillies

nd Coetzee, 1987), counted and kept on silica gel in 1.5 ml tubesor molecular analysis to identify the members of the An. gam-iae complex present in the swarm (Favia et al., 2001; Scott et al.,993). The location of the swarm, start and end of swarming time,umber of mating couples observed from swarm, estimated swarmize and height above the ground were recorded. Samples wereollected from swarms that occurred at the same location over sev-ral evenings. Mating couples (mating event) were also identified,ounted and collected as often as possible as they fell or flew outuring the swarm events.

Mosquito breeding sites were also actively searched for acrosshe village during the same study period. Once located, breedingites were identified by a specific code and salinity was measuredsing a conduct-meter (HAN-HI98130). Larvae samples were per-ormed using a calibrated dipper method (twice per month). Afterollections, larvae were transferred to the Institut Régional de Santéublique (IRSP) insectary and reared to the adult stage. Adults wereilled with chloroform, labelled and kept on silica gel in 1.5 ml tubesor molecular analysis to identify the members of the An. gambiaeomplex present in the breeding site.

A set of 50 mosquitoes was randomly selected from each groupf collected specimens (swarms and breeding sites). Genomic DNAas extracted from individual females using a protocol slightlyodified (using cetyltrimethylammonium bromide (CTAB) instead

f sodium dodecyl sulfate (SDS)) from Collins et al. (1987). Speciesf the An. gambiae complex and M and S molecular forms of An.ambiae s.s. were determined using PCR (Favia et al., 2001; Scottt al., 1993). Furthermore, males and females from mating pairsere also subsequently identified as species and molecular form ofn. gambiae s.s.

.3. Swarm markers (landmarks)

To understand the role of physical elements around the swarmvent sites, with the aim of using this information in the predic-ion of the spatial distribution of swarms of An. gambiae s.l. in

est Africa for vector control purposes, we recorded all landmarksswarm markers) under the swarm sites in the village identifiedbove. These markers were subsequently monitored once a montho identify and record those which were eventually selected by eachpecies of An. gambiae s.l. for swarming. All swarm markers were

ecorded and photographed and each marker found was monitoredor three consecutive nights each month to confirm their use byhe males for swarming. An estimation of the number of males thatonstituted each swarm and the number of couples were recorded.

a 132S (2014) S53–S63 S55

2.4. Dynamic indoor resting collection: species composition

Indoor resting mosquitoes were also collected using an insec-ticide spray catch method to estimate population dynamics ofmembers of the An. gambiae complex present throughout the vil-lage with special interest paid to temporal variation in the relativefrequencies of An. gambiae s.l. species which occurred in the vil-lage. The collection was done the day after the last swarm surveyto avoid affecting swarm compositions with the insecticide spray,from July 2010 to April 2011 early in the morning (5:00 to 7:00 am,GMT) and once per month. To ascertain that the pattern of swarmdistribution across the village was not a by-product of spatial distri-bution of the species within the village, 4–5 houses located withineach of 7 island agglomerations and near or far from each swarmingsite, were selected for indoor collections. All specimens were mor-phologically identified (Gillies and Coetzee, 1987). Genomic DNAwas extracted from single mosquitoes and polymerase chain reac-tion (PCR) was used to amplify the intergenic spacer of the rDNAto identify the member of the An. gambiae complex (An. melas orAn. gambiae s.s. M form) as described previously (Favia et al., 2001;Scott et al., 1993). Specimens of An. gambiae s.l. were individuallystored in cups and given a unique identification number and packedaccording to their point of collection. In the lab, they were preservedand subsequently analyzed for species/molecular form as describedabove.

2.5. Mapping of the study area

We used the satellite image provided by CENS© (2003)/Distri-bution Spot Image S.A/ISIS as background data to map mosquitobreeding sites, human dwellings, swarm locations and theanimal round up places using a global positioning system (GPS-GARMIN®) with measurements of latitude and longitude accurateto within 2 m during the dry season correspond to stable climaticconditions.

2.6. Data analysis

Analysis of variance (ANOVA) was performed to compareswarming marker with swarm size and mating events betweendifferent swarms (Kruskal–Wallis test), and swarming character-istics (swarm size, swarming height, swarming period and matingevents) between An. gambiae s.s. M form and An. melas swarms(Mann–Whitney test), using GraphPad Prism 5 software. Linearregression analysis was used to test the interaction between mat-ing events and swarm size using the same software. The analysis ofspatial distribution of swarms was carried out with Esri ArcGis®

software. This tool was used to compare the independent vari-able distribution (human dwelling and breeding sites position)to random distribution of swarms. The dependent variable wasthe presence of an An. gambiae s.l. swarm. Independent ecolog-ical variables included number of houses, and major breedingsites.

3. Results

3.1. Swarm events and breeding site characteristics

Swarms usually appeared in the same location every evening,and were sampled when its size was near the peak. During thestudy period, a total of 456 swarm events were recorded, sam-pled and mapped from 38 swarming sites in the village yielding

23,274 males and 76 females which were identified morphologi-cally as An. gambiae s.l. (Table 1; Fig. 1). The molecular analyseson pools of 50 mosquitoes collected from swarms indicated thatall swarms were species-specific. Eighteen of the 38 swarms were

S56 B.S. Assogba et al. / Acta Tropica 132S (2014) S53–S63

Table 1Specific composition of An. gambiae swarms in Djègbadji village.

Swarmingsites code

An. gambiaesampled fromswarm

Other speciesfrom swarm

Molecularidentification(PCR)

Swarm types Mating eventfrom swarm

Species ofmating couplesfemales)

Mating couplesstatus

Males Females % of An.gambiae M

% of An.melas

Observed Sampled

SWCo 01 54 00 00 50 100 00 14 06 An. gambiae M AssortativeSWCo 02 59 00 00 50 100 00 15 08 An. gambiae M AssortativeSWCo 03 77 02 00 50 100 00 18 02 An. gambiae M AssortativeSWCo 04 298 00 00 50 100 00 21 13 An. gambiae M AssortativeSWCo 05 134 05 00 50 100 00 12 01 An. gambiae M AssortativeSWDo 01 339 03 00 50 100 00 29 03 An. gambiae M AssortativeSWDo 02 446 00 00 50 100 00 68 07 An. gambiae M AssortativeSWDo 03 212 00 00 50 100 00 54 09 An. gambiae M AssortativeSWAg 01 1290 06 00 50 100 00 76 13 An. gambiae M AssortativeSWAg 02 1172 02 00 50 100 00 11 03 An. gambiae M AssortativeSWAg 03 1467 00 00 50 100 00 46 14 An. gambiae M AssortativeSWAn 02 357 05 00 50 100 00 17 06 An. gambiae M AssortativeSWAn 03 126 00 00 50 100 00 32 08 An. gambiae M AssortativeSWAn 04 76 00 00 50 100 00 47 12 An. gambiae M AssortativeSWKo 01 82 04 00 50 100 00 14 05 An. gambiae M AssortativeSWKo 02 69 08 00 50 100 00 17 01 An. gambiae M AssortativeSWDe 01 310 00 00 50 100 00 34 06 An. gambiae M AssortativeSWDe 01 127 00 00 50 100 00 29 11 An. gambiae M AssortativeSWDj 01 836 00 00 50 00 100 34 10 An. melas AssortativeSWDj 02 328 04 00 50 00 100 29 06 An. melas AssortativeSWDj 03 1128 06 00 50 00 100 47 17 An. melas AssortativeSWDj 04 1058 02 02 Culex 50 00 100 56 02 An. melas AssortativeSWHo 01 1299 00 04 Culex 50 00 100 39 14 An. melas AssortativeSWHo 02 989 00 00 50 00 100 59 11 An. melas AssortativeSWHo 03 447 00 00 50 00 100 25 08 An. melas AssortativeSWHo 04 789 05 02 Culex 50 00 100 74 17 An. melas AssortativeSWHo 05 573 00 00 50 00 100 44 16 An. melas AssortativeSWHo 06 278 07 00 50 00 100 48 05 An. melas AssortativeSWHo 07 989 03 00 50 00 100 36 06 An. melas AssortativeSWHo 08 1121 00 05 Culex 50 00 100 62 11 An. melas AssortativeSWHo 09 895 04 00 50 00 100 31 11 An. melas AssortativeSWHo 10 26 01 00 50 00 100 14 03 An. melas AssortativeSWHo 11 734 00 00 50 00 100 16 04 An. melas AssortativeSWHo 12 1299 03 00 50 00 100 62 08 An. melas AssortativeSWFo 01 1672 00 00 50 00 100 43 11 An. melas AssortativeSWFo 02 1512 05 00 50 00 100 57 14 An. melas AssortativeSWFo 03 278 00 00 50 00 100 21 06 An. melas Assortative

SWFo 04 328 01 00 50 00

38 23,274 76 13 1900 –

Fig. 1. Distribution map of swarms, breeding sites, human habit

100 51 13 An. melas Assortative– 1402 321 – –

ations, and rest sites of cattle and pigs in Djègbadji village.

B.S. Assogba et al. / Acta Tropica 132S (2014) S53–S63 S57

Fig. 2. (a) Comparison of swarming height between species: species on the X axis and swarming height (distance between ground and the middle of the swarm in metres)on the Y axis. (b) Comparison of swarming duration between species: species on X axis and swarming duration (in min) on Y axis. (a and b) Grey points indicate swarmingh betws and Ac ating

cesAe(sbfccptosdisvmeic

vsbba

eight and bars indicate the mean with SEM for each species. Statistical differencesize correlation with mating events correlation, respectively, for An. gambiae s.s. Mollected from swarm) on the Y axis. Statistically significant correlations between m

omposed exclusively of An. gambiae s.s. M form and the 20 oth-rs were composed exclusively of An. melas (Table 1). No mixedwarm of both species was recorded. Also no An. Arabiensis andn. gambiae s.s. (S form) were identified from these swarms. How-ver mixed swarms were recorded with another mosquito’s genusCulex quinquefasciatus) for four An. melas swarming sites. A total 76ingle females collected from different swarms showed that theyelonged to the same specie as the males which were collectedrom the same swarm. 321 mating couples were collected, 128ouples collected were An. gambiae s.s. M form swarms and 193ouples were An. melas swarms. All couples were homogeneouslyaired (male and female being of the same species). We comparedhe characteristics of several swarms made up of each species. Webserved a slight variation of swarming duration between the twopecies (p = 0.0148; Fig. 1b). In contrast, we observed significantifference in the swarming height (p < 0.0001; Fig. 2a). The swarm-

ng site localization was close to human dwellings for An. gambiae.s. M from and on salt production sites for An. melas within theillage study area (Fig. 1). Also the comparison highlighted thatating events between the two species were significantly differ-

nt (p < 0.0001), with a higher number of mating events occurringn swarms of An. melas. It was not possible to determine with anyertainty the swarm size for either species in our study.

Overall 33 breeding sites (where the presence of mosquito lar-ae was recorded) were identified. As we identified for swarm

pecific composition, 19 breeding sites were specific for An. gam-iae s.s. M form with an average salinity of around 0.47 g/l and 14reeding sites were specific for An. melas with an average salinityround 23.9 g/l.

een means are indicated (non-parametric Mann–Whitney U test). (c and d) Swarmn. melas: number of couples on the X axis and swarm size (number of individuals

event and swarming size are indicated (linear regression).

3.2. Distribution of the species among indoor resting sites

Collections of indoor resting mosquitoes were carried outmainly in human dwellings from July 2010 to early April 2011. Intotal 320 mosquito collections in 33 rooms were conducted, whichgenerated 907 An. gambiae s.l. specimens. After molecular analysis74.20% were found to be An. gambiae s.s. M form while 25.80% com-prised An. melas. An. gambiae s.s. M form and An. melas were bothfound in all collections of indoor resting mosquitoes. There wasno spatial difference in the relative frequencies of species or formsin the study area (Fig. 3). An. gambiae s.s. M form and An. melaswere spread out through the village, found co-inhabiting housesand found in the vicinity of swarms of different species (Fig. 3).More interestingly, An. gambiae s.s. M form males and An. melasfemales were found in the same houses, in contrary to the patternobserved with the swarm events where we observed uncon-fused spatial segregation of the swarming sites between the twospecies.

3.3. Swarm markers (landmarks)

To understand the role of physical markers in swarm site selec-tion by each species, all swarm sites in the village were recorded.309 potential swarming sites were identified in the study village (byactively examining the swarm markers we observed from others

swarm sites) and allotted to five groups (Fig. 4). Of the 309 potentialswarming sites, 18 sites were seen to be used by An. gambiae s.s. Mform for swarming with 44% of these being patches of bare ground,7% were wells, 5% were pig feeder sites and 9% were woodpiles

S58 B.S. Assogba et al. / Acta Tropica 132S (2014) S53–S63

Fig. 3. Indoor resting site locations and species composition of An. gambiae s.s. M and An. melas in Djègbadji village. Each pie chart represents an individual swarm, the sizeof which indicates the number of individuals. Circle indicated in the legend corresponds.

Fig. 4. Picture of representative swarm markers; the arrows indicate the exact placement of the swarm in each site.

05101520253035404550

0

50

100

150

200

250

Bareground

Basketfilter

Pig feederWellplace

Woodpile

Re

lativ

e fr

eq

ue

nc

y (%

)

Co

un

t M

ark

er

Swarm Marker of An. gambiae ss M form

Nombre Positive Relative freqency

0

10

20

30

40

50

60

0

50

100

150

200

250

Bareground

Basketfilter

Pig feederWellplace

Woodpile Re

lativ

e fr

eq

ue

nc

y (%

)

Co

un

t M

ark

ers

Swarm Marker of An. melas

Nombre Positive Relative frequency

Fig. 5. Different types of physical markers in study area and their relative frequency of occupation by swarms. X axis: swarming marker types. Left Y axis: (black) numberof swarming markers of this type present in the study site, (white) number of swarming markers positive for swarms. Right Y axis: (grey) relative frequency of positiveswarming markers.

B.S. Assogba et al. / Acta Tropica 132S (2014) S53–S63 S59

F biae s.o rmingY rence

(ppbaeufTeF

Fhib

ig. 6. Relationship between swarm markers and size and mating events of An. gamf individual mosquitoes that represent swarm size on Y axis. (c and d) Various swa

axis. The bars indicate mean (grey) with SEM (black). Statistically significant diffe

Fig. 5). 20 sites were used by An. melas for swarming with 48% beingatches of bare ground and 2% being basket filters (Fig. 5). Althoughatches of bare ground were the most common swarm marker usedy the two species (Fig. 5), many swarms of each species were notssociated to a specific swarm marker (p > 0.05 when comparingach swarm marker between the two species). An. melas swarmssed the bare ground of salt making sites and An. gambiae s.s. M

orm used the bare ground located within human dwellings (Fig. 7).he comparison of mating events between different swarm mark-rs of the two species showed a significant difference (p < 0.0001;ig. 6a–d) suggesting swarming site markers affect mating events.

ig. 7. Relationship between swarm site and distribution of mosquito breeding sites anabitations (Dist Sw Habitations) and breeding sites (Dist Sw breeding sites). Y axis: di

ndicate the distance from swarming site to habitations and breeding sites. The bars inetween means are indicated (non-parametric Mann–Whitney U test).

s. M form and An. melas. (a and b) Various swarming markers on X axis and number markers on X axis and number of mating events (couples) formed from swarm on

between mean is indicated (non-parametric Kruskal–Wallis test).

3.4. Spatial and temporal distribution of swarms

A swarming distribution map was constructed, integratinghuman dwellings, animal round up places, and breeding and swarmsites position (Fig. 1). This map showed a strong spatial segrega-tion of swarming sites between An. gambiae s.s. M form and An.melas. The swarming sites of An. gambiae M form were concen-

trated within the island agglomerations whereas those of An. melasoccurred preferentially in the salt making areas (apart from thevillage), hence swarms of An. gambiae s.s. M form were closer tohuman habitations than swarms of An. melas (Fig. 7). Likewise,

d human houses in the study area. X axis: distance from swarming site to humanstance from swarming site to human habitations and breeding sites. Grey pointsdicate the mean with SEM for each group and statistically significant differences

S Tropic

Aasst

ctsssifdm1sTtwsntdOstwia

4

bsdotbshtIwsestiso(Ymq

pDtsmTee

60 B.S. Assogba et al. / Acta

n. melas breeding sites were found within salt making areas rel-tively far from the village near the swarming sites (where thealinity was high). Concerning An. gambiae s.s. M form, the breedingites were concentrated in an area distant from the village charac-erized by low salinity (Fig. 1).

We attempted to determine the species composition of swarmsollected throughout the season using the data from the geno-yping analyses in order to assess the dynamics of the number ofwarm events and the presence or absence of breeding sites of eachpecies. During the study period, 11,780 specimens collected fromwarms were identified by PCR (30 males from each swarm dur-ng the dry season and 20 males in the rainy season). Only the Morm of An. gambiae s.s. and An. melas were reported. During thery season (from May to November), the arrival time of the firstale at the swarming sites was 17:45 pm (GMT) for each species,

0 min after sunset occurred at 17:35 pm, whereas during the rainyeason swarming began around 18:10 pm (GMT) for both species.his difference was associated to the sunset being at a differentime in different seasons. Furthermore, during the rainy season,e observed the flooding of the mangrove lead to the decrease in

alinity to an average of 0.27 g/l. At the peak of the rainy season,o An. melas breeding sites were recorded. At this moment, whenhe salt making sites were all inundated, we clearly observed theecrease in An. melas swarm size until their total disappearance inctober (Fig. 8). During the rainy season, additional An. gambiae

.s. M form breeding sites were recorded within the village due tohe rainfall, and their swarm size increased (Fig. 8) in accordanceith the indoor resting dynamics (Fig. 9). There was no variation

n swarming height or duration for either species between the drynd the rainy season.

. Discussion

Based on field observations, the breeding and swarmingehaviour of two, closely related, mosquito species, An. gambiae.s. M form and An. melas, is described and compared. We foundifferent characteristics in both breeding and swarming behaviourf these species, which are certainly involved in their reproduc-ive isolation (Powell et al., 1999). For the two species, swarmingegan at the same time (at sunset). Males always started swarmingome minutes before the first copulation was observed – it per-aps being “better for a male to arrive a couple of minutes earlyo the swarm rather than two seconds too late” (Williams, 1992).n most mosquitoes, mating is initiated in flight and is associated

ith swarming behaviour of the males (Downes, 1969). In thistudy the same observation was made. There is a lack of knowl-dge about partner choice in the otherwise extensively studiedpecies, An. gambiae complex, and this can partly be attributed tohe elusiveness of swarming in the field and the difficulty in find-ng swarms to study, let alone to investigate partner choice withinwarms. Several authors have attempted to increase understandingf the specific role of this male behaviour in the swarming eventCharlwood et al., 2002; Diabaté et al., 2011; Ng’habi et al., 2008;uval et al., 1993). Further studies are needed to clarify the deter-inants of mating success of individual males with respect to their

uality.During the swarming event, females fly into swarms and depart

aired with a male (Charlwood et al., 2002; Charlwood, 1980;iabaté et al., 2006; Marchand, 1984) this behaviour charac-

erized mating events in the observed mosquito species in thistudy. A strong correlation between swarm size and number of

ating events was observed in the two species from our data.

his correlation was also observed in a previous study (Diabatét al., 2011), and if true this adds support to the female prefer-nce choice model, since increasing swarm size offers a greater

a 132S (2014) S53–S63

possibility for a female to find their preferential mate according totheir specific criteria. According to evolutionary theory, this is likelybecause these females would pay the highest cost of mating withan incompetent male, because they mate only once in their life-time (Goma, 1963), whereas males can mate several times (Bryan,1968; Charlwood, 1980). It has also been demonstrated that femalemanakins, a lek-breeding bird species, choose males according totheir genetic quality traits (Ryder et al., 2009, 2010). The criteria forchoice of sexual partner among An. gambiae complex species arestill poorly known. Furthermore, we found a correlation betweenhigh An. gambiae s.s. M form swarm size and the occurrence of therainy season and a greater An. melas swarm size in the dry sea-son. This result suggests that a correlation exists between swarmbehaviour and a species’ bio-ecology (Akogbeto et al., 1995; Huntet al., 1998). Briefly, swarm size and the number of mating eventswere associated with population size. This information can be usedfor decreasing vector populations, informing the implementationand surveillance of any vector control programme based on Geneti-cally Modified Mosquitoes (GMM) in the context of malaria control.

A strong pattern of spatial segregation between An. gambiaes.s. M form and An. melas swarms was observed, showing thatthe two species share distinct species-specific mating units insharp contrast to the mixed composition observed for the twospecies through indoor collections. This observation suggests thatspatial swarm segregation probably contributes strongly to assor-tative mating between An. gambiae s.s. M form and An. melas, witheach species having a specific swarming behaviour. This mecha-nism of reproductive isolation implies that females (which werefound indoors) have to choose the spatial location where males oftheir own species aggregate over locations for other swarm events.Some evidence to reinforce this assumption was shown by dataobtained from the species analysis of mosquito samples collectedfrom swarms. All specimens from each collection (representing anindividual swarm) belonged to the same species. What is intriguingis that we do not know how females locate male swarms. How-ever, in the context of GMM or SIT male releases, our data are veryhelpful in identifying the release sites which would maximize con-tact with wild females. Overall, further study is needed with othersspecies of the Anopheles complex to confirm that spatial segrega-tion between the two species investigated in this study is relevantin the explanation of reproductive isolation mechanisms.

Studies on the swarming and mating behaviour in the fieldsuggests that An. gambiae males avoid contact with interspecificpartners mainly by swarming at different heights above the ground(Charlwood et al., 2002). Here, we observed a significant differencein swarming height above the ground between An. gambiae s.s. Mform and An. melas. However, no swarms were collected at a heighthigher than 4 m above the ground. The swarming heights of An.gambiae s.s. M form in our data in Benin was lower than that pre-viously observed for the same species and molecular form of An.gambiae s.s. from Burkina Faso (2–3 m above the ground) (Diabatéet al., 2003a). This suggests that the swarming height could varyeither according to adaptation to the environmental conditions(temperature, humidity, sunset time) or to the vector biology (An.gambiae s.s. M form from Benin corresponds to the Forest chro-mosomal form in degraded forest environments, and An. gambiaes.s. M form from Burkina Faso corresponds to the Mopti chromo-somal form in a wet savannah environment (della Torre et al., 2001).Swarms have been reported to form at varying heights as well, pos-sibly determined by species-appropriate swarm markers or relatedto orientation with swarm markers (Charlwood et al., 2002, 2003;Charlwood, 1980; Marchand, 1984).

An. gambiae s.s., the major malaria vector in Africa, swarms overspecific landmarks known as swarm markers presumably guided bya visual cues (Charlwood et al., 2002; Diabaté et al., 2003a, 2009;Downes, 1969; Yuval, 2006). It is unknown how males aggregate

B.S. Assogba et al. / Acta Tropica 132S (2014) S53–S63 S61

0

100

200

300

400

500

600

700

800

0

200

400

600

800

1000

1200

1400

1600

1800

2000

OctoberJulyAprilJanuary

Mo

nth

ly a

ccu

mu

late

d r

ain

fall

(m

m)

Mon

thly

sw

arm

siz

e m

ean

(In

div

idu

al)

Month

An. gambiae ss M form An. melas Rainfall

F h moI he NA(

odmioAlpmaspTssior(

itw

Fo

ig. 8. Dynamic evolution of swarm size and rainfall. Rainfall is presented for eacnteractive Online Visualization and analysis Infrastructure (Giovanni) as part of thttp://disc2.nascom.nasa.gov/Giovanni/tovas/TRMM).

r what factors influence the sustenance of swarms. Our resultsemonstrate that patches of bare ground were observed to be theost popular swarming marker used by the two species stud-

ed (Fig. 5). However, An. melas swarms were mainly recordedver bare ground close to or within the salt production sites, andn. gambiae s.s. M form swarms were observed over bare ground

ocated outdoor and within human property. Several studies haveointed out that An. gambiae s.s. use a relatively wide range ofarker types (Charlwood et al., 2002; Diabaté et al., 2003a, 2011),

nd a similar pattern was observed here with the swarms of thispecies being observed above other marker types in addition toatches of bare ground which was common to the two species.he results here indicate there is no likelihood of markers beingpecific to each species, since there is no specific marker for eachpecies which dominates. This has previously been demonstratedn other swarming behaviour studies in which the authors did notbserve an obvious association between markers and the occur-ence of swarming events of specific Anopheles complex membersCharlwood et al., 2002; Diabaté et al., 2003a, 2011).

We tried to investigate the spatial distribution of the breed-

ng sites of each species. We cannot categorically state that thewo species exploit different location for laying eggs, becausee observed some breeding sites for each species which were

ig. 9. Dynamic evolution of An. gambiae s.s. M form and An. melas indoor resting at studyf individual mosquitoes collected from human habitations in the study area for each spe

nth from January 2010 to December 2010 and were acquired using the GES-DISCSA’s Goddard Earth Sciences (GES), Data and Information Services Center (DISC)

relatively close despite most of the others being separated by longerdistances. The significant difference we found was that the level ofsalinity of breeding sites of An. melas was at least 50 times higherthan that for An. gambiae s.s. M form. From these results, the geneticisolation between sympatric populations of An. gambiae s.s. M formand An. melas could also be partly explained by their breedingbehaviour through the recognition of breeding sites with a highlevel of salinity by An. melas females. The breeding sites of the twospecies were mostly located far from each other apart from 3 An.melas breeding sites, which were close to those of An. gambiae s.s. Mform. This observation allows us to speculate on the possibility thatAn. melas (which was described to breed in brackish water) may alsobreed in fresh water. Already, authors have collected An. melas (inNigeria) and An. merus (in Tanzania) from breeding sites containingfresh water where An. gambiae was expected to be found (Bushrod,1981; Ebenezer et al., 2012). Our speculation on this statementcould be reinforced by these studies, but further studies need tobe done both in the laboratory and in the field to confirm this the-ory. Two very recent studies investigated the blood feeding andhost seeking behaviour of the complex of An. gambiae from Ghana

and Equatorial Guinea, respectively. Both studies showed that An.melas could feed on humans to an equal level indoors as outdoors,as with An. gambiae s.s. (Reddy et al., 2011). In relation to these

area from July 2010 to April 2011. The bars indicate monthly totals for the numbercies.

S Tropic

fig

5

iSbmtspscTtvilgmirammva

A

aGt

R

A

A

A

A

B

B

B

B

B

B

C

C

62 B.S. Assogba et al. / Acta

ndings, the collection through indoor resting of An. melas and An.ambiae together in most houses is not surprising.

. Conclusion

Mating is one aspect of behaviour that has been much ignoredn mosquito biology. Yet, the success of a transgenic release orIT strategy depends on normal, competitive mating occurringetween introduced and wild individuals. A research require-ent in order to improve the new vector control strategies is

o investigate the correlation between the mating frequency ofterile males with wild females and the dynamics of the vectoropulation. The work in this paper showing the involvement ofwarming behaviour in assortative mating within the An. gambiaeomplex in Benin is upstream knowledge to that which is needed.hus, bio-ecological knowledge is provided which will contributeo further investigation into mating system behaviour to informector control programmes through the release of genetically mod-fied or sterile males. However, the required establishment ofaboratory cultures and subsequent genetic transformation of tar-et mosquito species may result in insects with widely differentating behaviours compared to their wild siblings. Unless compet-

tive ability and mating behaviour are adequately understood, theelease of transgenic or sterilized mosquitoes may result in failureskin to those observed in several former SIT studies. Furthermore,alaria vectors currently viewed as “secondary” such as An. melasay expand to dominate residual transmission and act as primary

ectors following the successful implementation of interventionsimed at current priority vector species (Bayoh et al., 2010).

cknowledgements

This work was supported by a grant from TDR/WHO ID: A80690nd by IAEA Research Contract No. 16412/R0 under the CRP43002. Authors are grateful to L. Djossou for his technical assis-

ance during laboratory assays and mosquito collections.

eferences

kogbeto, M., 1995. Entomological study on the malaria transmission in coastal andlagoon areas: the case of a village built on a brackish lake. Ann. Soc. Belg. Med.Trop. 75, 219–227.

lphey, L., Beard, C.B., Billingsley, P., Coetzee, M., Crisanti, A., Curtis, C., Eggleston, P.,Godfray, C., Hemingway, J., Jacobs Lorena, M., James, A.A., Kafatos, F.C., Mukwaya,L.G., Paton, M., Powell, J.R., Schneider, W., Scott, T.W., Sina, B., Sinden, R., Sinkins,S., Spielman, A., Toure, Y., Collins, F.H., 2002. Malaria control with geneticallymanipulated insect vectors. Science (Washington) 298, 119–121.

lphey, L., Nimmo, D., O’Connell, S., Alphey, N., 2008. Insect population suppressionusing engineered insects. In: Transgenesis and the Management of Vector-BorneDisease., pp. 93–103.

yala, F.J., Coluzzi, M., 2005. Chromosome speciation: humans, Drosophila, andmosquitoes. Proc. Natl. Acad. Sci. U.S.A. 102 (Suppl. 1), 6535–6542.

aleta, A., 2009. Insecticide resistance threatens malaria control in Africa. Lancet374, 1581–1582.

ayoh, M.N., Mathias, D.K., Odiere, M.R., Mutuku, F.M., Kamau, L., Gimnig, J.E., Vulule,J.M., Hawley, W.A., Hamel, M.J., Walker, E.D., 2010. Anopheles gambiae: historicalpopulation decline associated with regional distribution of insecticide-treatedbed nets in western Nyanza Province, Kenya. Malar. J. 9, 62.

enedict, M.Q., Robinson, A.S., 2003. The first releases of transgenic mosquitoes: anargument for the sterile insect technique. Trends Parasitol. 19, 349–355.

ryan, J., Deco, M., Petrarca, V., Coluzzi, M., 1982. Inversion polymorphism and incip-ient speciation in Anopheles gambiae s. str. in the Gambia, West Africa. Genetica59, 167–176.

ryan, J.H., 1968. Results of consecutive matings of female Anopheles gambiae sp. Bwith fertile and sterile males. Nature 218, 489.

ushrod, F., 1981. The Anopheles gambiae Giles complex and Bancroftian filariasistransmission in a Tanzanian coastal village. Ann. Trop. Med. Parasitol. 75, 93.

aputo, B., Nwakanma, D., Jawara, M., Adiamoh, M., Dia, I., Konate, L., Petrarca, V.,Conway, D.J., della Torre, A., 2008. Anopheles gambiae complex along the Gambia

river, with particular reference to the molecular forms of An. gambiae s.s. Malar.J. 7, 182.

assone, B.J., Mouline, K., Hahn, M.W., White, B.J., Pombi, M., Simard, F., Costan-tini, C., Besansky, N.J., 2008. Differential gene expression in incipient species ofAnopheles gambiae. Mol. Ecol. 17, 2491–2504.

a 132S (2014) S53–S63

Charlwood, J., Pinto, J., Sousa, C., Madsen, H., Ferreira, C., Do Rosario, V., 2002. Theswarming and mating behaviour of Anopheles gambiae ss (Diptera: Culicidae)from Sao Tome Island. J. Vector Ecol. 27, 178–183.

Charlwood, J.D., Pinto, J., Sousa, C.A., Ferreira, C., Petrarca, V., Rosario Vdo, E., 2003.‘A mate or a meal’ – pre-gravid behaviour of female Anopheles gambiae from theislands of Sao Tome and Principe, West Africa. Malar. J. 2, 9.

Charlwood, J.D.J., Jones, M.D.R., 1980. Mating in the mosquito, Anopheles gambiae.11. Swarming behaviour. Physiol. Entomol. 5, 315–320.

Clements, A., 1999. The Biology of Mosquitoes, vol. 2: Sensory Reception andBehaviour, CAB International, Wallingford, United Kingdom.

Coetzee, M., Craig, M., Le Sueur, D., 2000. Distribution of African malariamosquitoes belonging to the Anopheles gambiae complex. Parasitol. Today 16,74–77.

Coetzee, M., Hunt, R.H., Wilkerson, R., Torre, A.D., Coulibaly, M.B., Besansky, N.J.,2013. Anopheles coluzzii and Anopheles amharicus, new members of the Anophelesgambiae complex. Zootaxa 3619, 246–274.

Collins, F.H., Mendez, M.A., Rasmussen, M.O., Mehaffey, P.C., Besansky, N.J., Finnerty,V., 1987. A ribosomal RNA gene probe differentiates member species of theAnopheles gambiae complex. Am. J. Trop. Med. Hyg. 37, 37–41.

Coluzzi, M., Petrarca, V., di Deco, M.A., 1985. Chromosomal inversion intergra-dation and incipient speciation in Anopheles gambiae. Ital. J. Zool. 52,45–63.

Coluzzi, M., Sabatini, A., della Torre, A., Di Deco, M.A., Petrarca, V., 2002. A polytenechromosome analysis of the Anopheles gambiae species complex. Science 298,1415–1418.

Coluzzi, M., Sabatini, A., Petrarca, V., Di Deco, M., 1979. Chromosomal differentia-tion and adaptation to human environments in the Anopheles gambiae complex.Trans. R. Soc. Trop. Med. Hyg. 73, 483–497.

Dao, A., Adamou, A., Yaro, A.S., Maiga, H.M., Kassogue, Y., Traore, S.F., Lehmann, T.,2008. Assessment of alternative mating strategies in Anopheles gambiae: doesmating occur indoors. J. Med. Entomol. 45, 643–652.

della Torre, A., Costantini, C., Besansky, N.J., Caccone, A., Petrarca, V., Powell, J.R.,Coluzzi, M., 2002. Speciation within Anopheles gambiae – the glass is half full.Science 298, 115–117.

della Torre, A., Fanello, C., Akogbeto, M., Dossou-yovo, J., Favia, G., Petrarca, V.,Coluzzi, M., 2001. Molecular evidence of incipient speciation within Anophelesgambiae s.s. in West Africa. Insect Mol. Biol. 10, 9–18.

della Torre, A., Tu, Z., Petrarca, V., 2005. On the distribution and genetic differenti-ation of Anopheles gambiae s.s. molecular forms. Insect Biochem. Mol. Biol. 35,755–769.

Diabaté, A., Dabire, R.K., Millogo, N., Lehmann, T., 2007. Evaluating the effect ofpostmating isolation between molecular forms of Anopheles gambiae (Diptera:Culicidae). J. Med. Entomol. 44, 60–64.

Diabaté, A., Baldet, T., Brengues, C., Kengne, P., Dabire, K.R., Simard, F., Chandre,F., Hougard, J.M., Hemingway, J., Ouedraogo, J.B., Fontenille, D., 2003a. Naturalswarming behaviour of the molecular M form of Anopheles gambiae. Trans. R.Soc. Trop. Med. Hyg. 97, 713–716.

Diabaté, A., Baldet, T., Chandre, F., Akoobeto, M., Guiguemde, T.R., Darriet, F.,Brengues, C., Guillet, P., Hemingway, J., Small, G.J., Hougard, J.M., 2002. The role ofagricultural use of insecticides in resistance to pyrethroids in Anopheles gambiaes.l. in Burkina Faso. Am. J. Trop. Med. Hyg. 67, 617–622.

Diabaté, A., Baldet, T., Chandre, F., Dabire, K.R., Kengne, P., Guiguemde, T.R., Simard,F., Guillet, P., Hemingway, J., Hougard, J.M., 2003b. KDR mutation, a geneticmarker to assess events of introgression between the molecular M and S formsof Anopheles gambiae (Diptera: Culicidae) in the tropical savannah area of WestAfrica. J. Med. Entomol. 40, 195–198.

Diabaté, A., Brengues, C., Baldet, T., Dabire, K.R., Hougard, J.M., Akogbeto, M.,Kengne, P., Simard, F., Guillet, P., Hemingway, J., Chandre, F., 2004. The spreadof the Leu-Phe kdr mutation through Anopheles gambiae complex in BurkinaFaso: genetic introgression and de novo phenomena. Trop. Med. Int. Health 9,1267–1273.

Diabaté, A., Dabire, R.K., Kengne, P., Brengues, C., Baldet, T., Ouari, A., Simard, F.,Lehmann, T., 2006. Mixed swarms of the molecular M and S forms of Anophe-les gambiae (Diptera: Culicidae) in sympatric area from Burkina Faso. J. Med.Entomol. 43, 480–483.

Diabaté, A., Dao, A., Adamou, A., Gonzalez, R., Manoukis, N.C., Traoré, S.F., Gwadz,R.W., Lehmann, T., 2009. Spatial swarm segregation and reproductive isolationbetween the molecular forms of Anopheles gambiae. Proc. R. Soc. B: Biol. Sci. 276,4215–4422.

Diabaté, A., Dao, A., Diallo, M., Huestis, D.L., Lehmann, T., 2011. Spatial distributionand male mating success of Anopheles gambiae swarms. BMC Evol. Biol. 11, 184.

Djogbénou, L., 2009. Lutte antivectorielle contre le paludisme et résistance desvecteurs aux insecticides en Afrique. Med. Trop. 69, 160–164.

Djogbenou, L., Noel, V., Agnew, P., 2010. Costs of insensitive acetylcholinesteraseinsecticide resistance for the malaria vector Anopheles gambiae homozygous forthe G119S mutation. Malar. J. 9, 12.

Downes, J., 1969. The swarming and mating flight of Diptera. Annu. Rev. Entomol.14, 271–298.

Downes, J.A., 1968. The swarming and mating flight of Diptera. Annu. Rev. Entomol.14, 271–298.

Ebenezer, A., Okiwelu, S., Agi, P., Noutcha, M., Awolola, T., Oduola, A., 2012. Species

Composition of the Anopheles gambiae Complex Across Eco-Vegetational Zonesin Bayelsa State, Niger Delta Region, Nigeria.

Favia, G., Lanfrancotti, A., Spanos, L., Siden Kiamos, I., Louis, C., 2001. Molecularcharacterization of ribosomal DNA polymorphisms discriminating among chro-mosomal forms of Anopheles gambiae s.s. Insect Mol. Biol. 10, 19–23.

Tropic

G

G

GG

HH

I

K

K

L

MM

M

M

M

MMM

N

O

PP

P

R

B.S. Assogba et al. / Acta

illies, M., Coetzee, M., 1987. A Supplement to the Anophelinae of Africa South of theSahara (Afrotropical region). The South African Institute for Medical Research,Johannesburg.

oma, L.K.H., 1963. Tests for multiple insemination in Anopheles gambiae Giles.Nature (Lond.) 197, 99–100.

reenwood, B., Alonso, P., 2002. Malaria Vaccine Trials.u, W., Novak, R.J., 2009. Predicting the impact of insecticide-treated bed nets on

malaria transmission: the devil is in the detail. Malar. J. 8, 256.owell, P.I., Knols, B.G.J., 2009. Male mating biology. Malar. J. 8, S8.unt, R.H., Coetzee, M., Fettene, M., 1998. The Anopheles gambiae complex: a new

species from Ethiopia. Trans. R. Soc. Trop. Med. Hyg. 92, 231–235.to, J., Ghosh, A., Moreira, L.A., Wimmer, E.A., Jacobs Lorena, M., 2002. Transgenic

anopheline mosquitoes impaired in transmission of a malaria parasite. Nature(London) 417, 452–455.

im, W., Koo, H., Richman, A.M., Seeley, D., Vizioli, J., Klocko, A.D., O’brochta, D.A.,2004. Ectopic expression of a cecropin transgene in the human malaria vectormosquito Anopheles gambiae (Diptera: Culicidae): effects on susceptibility toPlasmodium. J. Med. Entomol. 41, 447–455.

nipling, E.F., 1959. Sterile-male method of population control. Science (New York,NY) 130, 902.

ehmann, T., Licht, M., Elissa, N., Maega, B.T., Chimumbwa, J.M., Watsenga, F.T.,Wondji, C.S., Simard, F., Hawley, W.A., 2003. Population structure of Anophelesgambiae in Africa. J. Hered. 94, 133–147.

acdonald, G., 1957. The Epidemiology and Control of Malaria.anoukis, N.C., Powell, J.R., Toure, M.B., Sacko, A., Edillo, F.E., Coulibaly, M.B.,

Traore, S.F., Taylor, C.E., Besansky, N.J., 2008. A test of the chromosomal the-ory of ecotypic speciation in Anopheles gambiae. Proc. Natl. Acad. Sci. U.S.A. 105,2940–2945.

archand, R., 1984. Field observations on swarming and mating in Anopheles gam-biae mosquitoes in Tanzania. Neth. J. Zool. 34, 367–387.

asendu, H.T., Hunt, R.H., Govere, J., Brooke, B.D., Awolola, T.S., Coetzee, M., 2004.The sympatric occurrence of two molecular forms of the malaria vector Anophe-les gambiae Giles sensu stricto in Kanyemba, in the Zambezi Valley, Zimbabwe.Trans. R. Soc. Trop. Med. Hyg. 98, 393–396.

axwell, C., Myamba, J., Njunwa, K., Greenwood, B., Curtis, C., 1999. Comparison ofbednets impregnated with different pyrethroids for their impact on mosquitoesand on re-infection with malaria after clearance of pre-existing infections withchlorproguanil-dapsone. Trans. R. Soc. Trop. Med. Hyg. 93, 4–11.

cKenzie, F., 2000. Why model malaria? Parasitol. Today 16, 511–516.ichalakis, Y., Renaud, F., 2009a. Evolution in vector control. Nature 462, 298–300.ichalakis, Y., Renaud, F., 2009b. Malaria: evolution in vector control. Nature 462,

298–300.g’habi, K.R., Huho, B.J., Nkwengulila, G., Killeen, G.F., Knols, B.G.J., Ferguson, H.M.,

2008. Sexual selection in mosquito swarms: may the best man lose? Anim.Behav. 76, 105–112.

liveira, E., Salgueiro, P., Palsson, K., Vicente, J.L., Arez, A.P., Jaenson, T.G., Caccone, A.,Pinto, J., 2008. High levels of hybridization between molecular forms of Anophe-les gambiae from Guinea Bissau. J. Med. Entomol. 45, 1057–1063.

ampana, E., 1969. A Textbook of Malaria Eradication, 2nd edition.ennetier, C., Warren, B., Dabiré, K.R., Russell, I.J., Gibson, G., 2010. Singing on the

wing as a mechanism for species recognition in the malarial mosquito Anophelesgambiae. Curr. Biol. 20, 131–136.

owell, J., Petrarca, V., della Torre, A., Caccone, A., Coluzzi, M., 1999. Populationstructure, speciation, and introgression in the Anopheles gambiae complex.Parassitologia 41, 101–113.

eddy, M.R., Overgaard, H.J., Abaga, S., Reddy, V.P., Caccone, A., Kiszewski, A.E.,Slotman, M.A., 2011. Outdoor host seeking behaviour of Anopheles gambiae

a 132S (2014) S53–S63 S63

mosquitoes following initiation of malaria vector control on Bioko Island, Equa-torial Guinea. Malar. J. 10, 184.

Ryder, T., Tori, W., Blake, J., Loiselle, B., Parker, P., 2010. Mate choice for genetic qual-ity: a test of the heterozygosity and compatibility hypotheses in a lek-breedingbird. Behav. Ecol. 21, 203–210.

Ryder, T.B., Parker, P.G., Blake, J.G., Loiselle, B.A., 2009. It takes two to tango: repro-ductive skew and social correlates of male mating success in a lek-breeding bird.Proc. R. Soc. B: Biol. Sci. 276, 2377–3238.

Scott, J.A., Brogdon, W.G., Collins, F.H., 1993. Identification of single specimens ofthe Anopheles gambiae complex by the polymerase chain reaction. Am. J. Trop.Med. Hyg. 49, 520.

Sogoba, N., Vounatsou, P., Bagayoko, M., Doumbia, S., Dolo, G., Gosoniu, L., Traore,S., Smith, T., Toure, Y., 2008. Spatial distribution of the chromosomal forms ofAnopheles gambiae in Mali. Malar. J. 7, 205.

Stump, A.D., Fitzpatrick, M.C., Lobo, N.F., Traore, S., Sagnon, N., Costantini, C., Collins,F.H., Besansky, N.J., 2005a. Centromere-proximal differentiation and speciationin Anopheles gambiae. Proc. Natl. Acad. Sci. U.S.A. 102, 15930–15935.

Stump, A.D., Shoener, J.A., Costantini, C., Sagnon, N.F., Besansky, N.J., 2005b. Sex-linked differentiation between incipient species of Anopheles gambiae. Genetics169, 1509–1519.

Takken, W., Knols, B.G.J., 2009. Malaria vector control: current and future strategies.Trends Parasitol. 25, 101–104.

Thomas, D.D., Donnelly, C.A., Wood, R.J., Alphey, L.S., 2000. Insect population controlusing a dominant, repressible, lethal genetic system. Science 287, 2474–2476.

Toure, Y., Petrarca, V., Traore, S., Coulibaly, A., Maiga, H., Sankare, O., Sow, M., DiDeco, M., Coluzzi, M., 1998. The distribution and inversion polymorphism ofchromosomally recognized taxa of the Anopheles gambiae complex in Mali, WestAfrica. Parassitologia 40, 477–511.

Tripet, F., Thiemann, T., Lanzaro, G.C., 2005. Effect of seminal fluids in mat-ing between M and S forms of Anopheles gambiae. J. Med. Entomol. 42,596–603.

Tripet, F., Toure, Y.T., Taylor, C.E., Norris, D.E., Dolo, G., Lanzaro, G.C., 2001. DNAanalysis of transferred sperm reveals significant levels of gene flow betweenmolecular forms of Anopheles gambiae. Mol. Ecol. 10, 1725–1732.

Turner, T.L., Hahn, M.W., Nuzhdin, S.V., 2005. Genomic islands of speciation inAnopheles gambiae. PLoS Biol. 3, e285.

White, B.J., Cheng, C., Simard, F., Costantini, C., Besansky, N.J., 2010. Genetic associ-ation of physically unlinked islands of genomic divergence in incipient speciesof Anopheles gambiae. Mol. Ecol. 19, 925–939.

WHO, 2010. World Malaria Report 2010. World Health Organization, Geneva.Williams, G.C., 1992. Natural Selection: Domains, Levels and Challenges. Oxford

University Press, USA.Wondji, C., Frederic, S., Petrarca, V., Etang, J., Santolamazza, F., della Torre, A., Fonte-

nille, D., 2005. Species and populations of the Anopheles gambiae complex inCameroon with special emphasis on chromosomal and molecular forms ofAnopheles gambiae s.s. J. Med. Entomol. 42, 998–1005.

Wondji, C., Simard, F., Fontenille, D., 2002. Evidence for genetic differentiationbetween the molecular forms M and S within the forest chromosomal form ofAnopheles gambiae in an area of sympatry. Insect Mol. Biol. 11, 11–19.

Yuval, B., 2006. Mating systems of blood-feeding flies. Annu. Rev. Entomol. 51,413–440.

Yuval, B., Wekesa, J., Washino, R., 1993. Effect of body size on swarming behavior and

mating success of male Anopheles freeborni (Diptera: Culicidae). J. Insect Behav.6, 333–342.

Zhou, G., Githeko, A.K., Minakawa, N., Yan, G., 2010. Community-wide benefits of tar-geted indoor residual spray for malaria control in the Western Kenya Highland.Malar. J. 9, 67.