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Contents lists available at ScienceDirect

Acta Tropica

journa l h om epa ge: www.elsev ier .com/ locate /ac ta t ropica

ale reproductive biology of Aedes mosquitoes

lelia F. Olivaa,∗, David Damiensb, Mark Q. Benedictc

Polo d’Innovazione Genomica, Genetica e Biologia S.C.a.R.L., Edificio C, 3ˆ piano Polo Unico di Medicina ‘Santa Maria della Misericordia’, Loc. S. Andreaelle Fratte, 06132 Perugia, ItalyInsect Pest Control Laboratory, Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, FAO/IAEA Agriculture and Biotechnologyaboratories, A-2444 Seibersdorf, AustriaUniversità di Perugia, Dipartimento di Medicina Sperimentale e Scienze Biochimiche, Perugia, Italy

r t i c l e i n f o

rticle history:eceived 12 June 2013eceived in revised form9 November 2013ccepted 23 November 2013vailable online xxx

eywords:

a b s t r a c t

Among Aedes mosquitoes are species responsible for transmission of serious pathogens to humans. Tocope with the current threats to long-term effectiveness of the traditional vector control methods, non-conventional control strategies are being developed. These include autocidal control such as the releaseof sterile males (sterile insect technique) and the release of Wolbachia-infected males to induce sexualsterility (incompatible insect technique) and pathogen-refractory strain replacement variations usingWolbachia. Sterile male types of techniques particularly depend on released males’ ability to successfullymate with wild females. For that reason, a good understanding of male mating biology, including a thor-

edesale

eproductionate-selectionenetic controlterile insect technique

ough understanding of the reproductive system and mating capacity, increases the likelihood of successof such genetic vector control programmes. Here we review the literature concerning the reproduction ofAedes mosquitoes with an emphasis on the male biology. We consider sexual maturation, mate finding,insemination, male reproductive capacity, and the occurrence of multiple matings. We also discuss whichparameters are of greatest importance for the successful implementation of autocidal control methodsand propose questions for future research.

rnati

© 2013 Inte

. Background

Many Aedes species are vectors of viruses and nematodesesponsible for severe human diseases among which are dengue,ellow fever, chikungunya, and lymphatic filariasis (Lounibos,002; Gratz, 2004). The two species responsible for most diseases,edes aegypti and Aedes albopictus, have very effective invasiveapacities (Juliano and Lounibos, 2005). Their worldwide invasionas led to increasing disease risks (Reiter, 2001) and emergence ore-emergence of epidemics of dengue and other arboviral diseasesMackenzie et al., 2004).

Aedes mosquitoes are often found in high densities, breedingn rural as well as urban habitats. Larvae are able to develop inlean or polluted stagnant water and can colonize various artifi-ial habitats and peri-domestic containers, increasing their contactith humans and the potential for spreading diseases (Jansen

nd Beebe, 2010). Control of aedine populations usually relies on

Please cite this article in press as: Oliva, C.F., et al., Male rehttp://dx.doi.org/10.1016/j.actatropica.2013.11.021

arval control using insecticides and source removal, sometimesnvolving community participation. The location of larval sites inrivate properties makes the task of control by public institutions

∗ Corresponding author. Tel.: +39 0755858361.E-mail addresses: cleliaoliva@gmail.com (C.F. Oliva), D.Damiens@iaea.org

D. Damiens), mqbenedict@yahoo.com (M.Q. Benedict).

001-706X/$ – see front matter © 2013 International Atomic Energy Agency. Published bttp://dx.doi.org/10.1016/j.actatropica.2013.11.021

onal Atomic Energy Agency. Published by Elsevier B.V. All rights reserved.

extremely difficult and costly. Drastic control of larval sites and,after 1944, the use of DDT permitted the elimination of A. aegyptiin several countries of South America; however, the end of the con-trol measures were predictably followed by a rapid re-infestationfrom neighbouring areas (Soper, 1963). Over the last half-century,sustained control has rarely been achieved, and resurgence maypartly be ascribed to the high mobility and resilience of this speciesand the cessation of the control programmes (Reiter and Gubler,1997; Reiter, 2007).

The current vector control tools have failed to reduce mosquitopopulations in an effective and long-term way, therefore the addi-tional use of non-conventional methods (Alphey et al., 2010)relying on the principles of the sterile insect technique (SIT)are being considered in many regions. These methods rely onfrequent mass-releases of male mosquitoes following their radios-terilization (classical SIT, Robinson et al., 2009), alteration of theirprogeny’s survival (Phuc et al., 2007) or female flight capac-ity (Fu et al., 2010) or the reduction of the females’ fertility(Brelsfoard et al., 2008), or survival (McMeniman et al., 2009)via Wolbachia-mediated cytoplasmic incompatibility (Brelsfoardand Dobson, 2009). Alternative strategies consist of replacing

productive biology of Aedes mosquitoes. Acta Trop. (2013),

the wild population with mosquitoes refractory to the pathogenso that disease transmission to humans cannot occur (Speranc aand Capurro, 2007; Moreira et al., 2009; Hoffmann et al.,2011).

y Elsevier B.V. All rights reserved.

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Releases of sterile individuals have permitted the success-ul regional elimination of important pests, such as the New

orld screwworm (Cochliomyia hominivorax Coquerel), theediterranean fruit fly (Ceratitis capitata Wiedemann), and theexican fruit fly (Anastrepha ludens Loew) (Krafsur, 1998; Klassen

nd Curtis, 2005). SIT has been tested for mosquito control in the970s using various sterilizing approaches (chemosterilization,

onizing radiation, cytoplasmic incompatibility, or chromosomeranslocations) (Benedict and Robinson, 2003; Dame et al., 2009),nd research is currently being performed to improve the differentechnical steps (Robinson et al., 2009). Some field trials havehown encouraging potential of the releases of sterile mosquitoesn reducing wild population of Anopheles albimanus (Lofgren et al.,974; Weidhaas et al., 1974), A. albopictus (Bellini et al., 2007,013), A. aegypti (Harris et al., 2012; Lacroix et al., 2012), and Aedesolynesiensis (O’Connor et al., 2012).

Control methods that interfere with the reproduction of annsect can be improved with a sound knowledge of its mating biol-gy. Our understanding of the reproductive physiology of Aedespecies is still incomplete. Most studies cover the female aspects ofeproduction of the main disease vector, A. aegypti, and date fromhe 70s. However the recent development of transgenic mosquitoesnd application of molecular biology techniques have permitteddvances in the understanding of the mating system. We reviewhe existing literature on this topic concerning the Aedes genus andaise some crucial research questions that would help to improvehe efficiency and effectiveness of genetic control approaches.

. Male sexual maturation

A successful mating involves sequences of events that per-it mate interception, engagement of the genitalia, transfer of

emen, and storage of sperm by the female. Mating is precededy mate location and interaction, which usually requires appropri-te epigamic behaviour. The sperm stored by the female mosquitoill be used for egg fertilization shortly before the egg is laid. The

bility of released males to participate in and perform each stepnd the viability and competitiveness of their sperm will impacthe population control programmes.

Mosquito males must complete two easily observable physicalhanges prior to becoming sexually active after eclosion. Duringhe first day after emergence of Aedes males, the fibrillar hairs ofhe antennae become fully erect and usually remain so through-ut the lifetime, except in some old males (Roth, 1948; Fay, 1964;oster and Lea, 1975a; Nijhout, 1977). The erection of the fibrillaere important for female location as they allow males to respondo female flight sounds (Roth, 1948).

In addition, males undergo a permanent 180◦ rotation of theerminalia part of the genitalia; as a result, the claspers become ori-nted ventrally and the aedaegus lies above the anus. This rotations common among the Diptera and was suggested to result from andjustment of the copulation position (Lamb, 1922). Mosquitoesssume a venter-to-venter position; pairs of Anopheles usuallyace opposite directions (Roth, 1948; Charlwood and Jones, 1979),hereas Aedes more frequently face the same direction, and Culex

pecies were observed to equally use both attitudes (Roth, 1948).he different mating attitude was correlated with differences inhe structure of the male claspers between those species (Roth,948). Aedes males usually first grasp the female dorsally and thenapidly change to a ventral position (Roth, 1948). The 180◦ rotationf mosquito genitalia allows the claspers to be ventrally oriented, in

Please cite this article in press as: Oliva, C.F., et al., Male rehttp://dx.doi.org/10.1016/j.actatropica.2013.11.021

hich position they can be used to grasp the females’ cerci (Fig. 1).oreover, the genital pore of the male lies on the antero-dorsal sur-

ace of the aedeagus, which must then be rotated prior to copulationSpielman, 1964, 1966).

the sperm transfer during copulation (represented by the arrows). Adapted fromClements (1999) and Jones (1968).

The time required to complete terminalia rotation variesbetween species from 18 to 24 h for A. aegypti (Roth, 1948), 11 to25 h for A. albopictus (Miyagi and Toma, 1982; Oliva et al., 2012),around 12 and 40 h, respectively, for Aedes iriomotensis and Aedesatriisimilis (Miyagi and Toma, 1982), 30 h for Aedes taeniorhynchus(Provost et al., 1961), and nearly 4 days for Aedes provocans (Smithand Gadawski, 1994). Before completion of sexual maturation,males may attempt to make contact with females (Roth, 1948;Cabrera and Jaffe, 2007) but no copulation or swarm activity isobserved in A. aegypti males less than 24 h old (Cabrera and Jaffe,2007). Given the importance of terminalia rotation for mating abil-ity, it is notable that the radiosterilization of A. albopictus did notaffect the speed of rotation of the male terminalia (Oliva et al.,2012); however, closer observations of the antennal fibrillae insterilized or genetically modified males would be of interest.

3. Finding a mate

3.1. Male mate location behaviour and encounter sites

Contrary to the situation in anophelines in which males formstation-keeping swarms into which females fly and mate – usuallyin relation to some feature of the environment (Howell and Knols,2009) – Aedes males do not require the formation of such swarmsto attract females and mate. Aedes demonstrate wide behaviouralplasticity that allows them to mate in single pairs or in aggregations.This same flexibility permits mating throughout the day thoughcrepuscular peaks have been observed. An individual male Aedescan fly singly to intercept a female to mate (Downes, 1969) and,contrary to anophelines, matings where only one pair is presentin a small arena can be very easily performed. However, swarm-ing behaviour has been described, as small groups of males can beobserved to fly in a figure-eight pattern similarly to small swarmsnear a blood-meal source (Wright et al., 1966; Loor and DeFoliart,1970; Hartberg, 1971; Gubler and Bhattacharya, 1972), at the feet ofa walking blood meal host (Ali and Rozeboom, 1971), over the cor-ners of release cages (Edman et al., 1972), at the feet of large trees(Tuten et al., 2013), or over larval sites (unpublished observations).

productive biology of Aedes mosquitoes. Acta Trop. (2013),

Aedes swarming activity is a mate-locating behaviour; accordingto Cabrera and Jaffe (2007) it indeed enhances mating opportu-nities and increases copulation frequency but it is not a requiredbehaviour.

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Cabrera and Jaffe (2007) have reported the participation of anverage of 23 A. aegypti males in swarms formed in laboratoryonditions, in the presence of a blood–meal source. In the field,–40 male A. albopictus swarming around the observer (Gubler andhattacharya, 1972) and an average of only five male A. polyne-iensis swarming at the bases of trees (Tuten et al., 2013) wereeported. Swarms lasted from 5 to 16 min under laboratory condi-ions (Cabrera and Jaffe, 2007) and a high swarming activity during–10 min was also reported in the field (Gubler and Bhattacharya,972). The mating activity peaked between 5 and 10 min afterhe beginning of the swarm (Gubler and Bhattacharya, 1972), theuration of which was positively correlated with the likelihood ofopulation taking place (Cabrera and Jaffe, 2007). The main stim-lus for mate-seeking flying behaviour is likely to be the presencef females (Dyar, 1920, 1928; Nijhout and Craig, 1971; Gubler andhattacharya, 1972; Jaenson, 1985; Cabrera and Jaffe, 2007; Catort al., 2011) which is increased by the presence of a blood-mealource and by the onset of photophase, as females’ biting activ-ty peaks immediately after sunrise and before sunset (Hartberg,971; Edman et al., 1972; Foster and Lea, 1975a; Cabrera and Jaffe,007; Tuten et al., 2013). Quantitative and descriptive reports of thewarming and mating sites of Aedes species in the field are, how-ver, lacking, but due to the relatively limited dispersal of maleedes in urban areas (Muir and Kay, 1998; Bellini et al., 2010;ergero et al., 2013) these would be very useful to identify theest release areas for autocidal and population replacement pro-rammes.

.2. The interacting roles of olfaction and hearing in mateecognition

Cabrera and Jaffe (2007) reported the production of a volatileaggregation pheromone” by swarming males that stimulates bothemale and male flight and attracts both sexes to the swarm; theemale olfactory response to swarming males’ odour prevailed over

host odour. In response to this olfactory stimulus, females show characteristic very rapid agitated flight (Cabrera and Jaffe, 2007).emale wing beat also attracts males, as the females’ frequencyas been shown to stimulate male Aedes to pursue and seize the

emale (Roth, 1948). A. aegypti males would not attempt to cop-late with non-flying females (Spielman, 1964) nor with newlymerged females, which have a different wing beat frequency thanature ones (Roth, 1948; Brogdon, 1994). Studies on Anopheles

Pennetier et al., 2010), Culex (Warren et al., 2009), ToxorhynchitesGibson and Russell, 2006; Cator et al., 2009; Pennetier et al., 2010),nd Aedes (Cator et al., 2009) species have shown that a pair ofemale and male mosquitoes can alter their flight-tone frequencynd synchronize them before copulating. Interestingly, a femaleill not attempt or succeed to harmonize with every male, sug-

esting a certain degree of mate choice from either the male oremale according to the partner’s wing beat frequency (Gibson et al.,010). In anophelines, flight tone frequency has been shown to varyith body size, which supports the hypothesis of mate assessment

Cator et al., 2010). In this genus, it is also involved in species recog-ition (Pennetier et al., 2010). However, this does not appear to behe case for aedines (Roth, 1948; Nijhout and Craig, 1971), whereecognition would occur after the first contact through a species-pecific pheromone stimulus that triggers the male to shift to theoitus position (Nijhout and Craig, 1971). However, several experi-ental cross-matings between different species of Aedes have been

eported, though they always resulted in infertile progeny (Roth,948; Ali and Rozeboom, 1971; Ali et al., 1971; Lee et al., 2009). This

Please cite this article in press as: Oliva, C.F., et al., Male rehttp://dx.doi.org/10.1016/j.actatropica.2013.11.021

esult illustrates that barriers to normally rare interspecific matingehaviours can sometimes easily be overcome in the laboratory.t would be worth investigating further whether the olfactory cuemitted by females and the one emitted by swarming males are also

PRESSa xxx (2013) xxx– xxx 3

involved in mate choice. Both the ability to produce and recognizepheromones and wing beat frequency are crucial parameters forAedes mating; ensuring that they are not impaired in males that arereleased as part of a genetic control programme should be a priority.

4. Transfer of sperm and male AG secretions to the femalereproductive tract

In aedines, copulation is brief and usually lasts less than 1 min(Roth, 1948; Jones, 1974; Oliva et al., 2013). In A. aegypti, 6 s ofcontact was sufficient for insemination (Roth, 1948; Jones, 1974).Most of the copulations of A. albopictus observed in the field had amedian duration of 8 s (Gubler and Bhattacharya, 1972), against 45 sfor a long-colonized laboratory strain of A. albopictus (Oliva et al.,2013). The absence of predation risk under laboratory conditionsmight have led to the loss of the propensity for rapid copulation.

Following abdominal contact, engagement of the female cerci bythe male claspers, and probably a stimulus from the female cercion the sensillae of the male claspers, the male aedaegus becomeseverted and extended, the female vagina is widened to enlarge theopening into the bursa inseminalis (BI, Fig. 1) (Spielman, 1964;Spielman et al., 1974).

Males transfer a mixture of sperm and accessory glands (AG)secretions. The two seminal vesicles containing the sperm mergeinto the ejaculatory duct, and are joined by the paired AG near theanterior end of the duct (Fig. 1) (Spielman, 1964). The semen flowsthrough the ejaculatory duct, which leads directly to the genitalpore located near the base of the aedaegus, and contractions of themale AG provide pressure for the sperm transfer (Spielman, 1964).Semen transfer itself requires only a few seconds (Lum, 1961a). TheBI is the first, but temporary, sperm storage organ in Aedes females(Barr, 1974). Within 30 s after termination of coitus, the femaleatrium regains its precopulatory appearance, and sperm begin toconcentrate close to the spermathecal eminence with their headsoriented towards the spermathecal vestibule (Spielman, 1964). Theabundant AG secretion provides a liquid medium for sperm move-ment and plays a role in their migration to the spermathecae (Barr,1974). This migration occurred from 2 to 5 min after the act of copu-lation began for A. aegypti (Spielman, 1964) and A. albopictus (Olivaet al., 2013).

Most of the Culicinae females have three spermathecae whilefemales of the Anophelinae subfamily have only one. This presentsquestions regarding how the various spermathecae are filled andtheir sperm used during fertilization. In the case of Aedes species,females possess one large central spermatheca and two smallerlateral ones. Following mating with a sexually mature male thathas not mated previously, the majority of females have two sper-mathecae (the larger one and one of the small) filled with sperm:96% of females in A. polynesiensis (Chambers et al., 2011), and 91%in A. albopictus (Oliva et al., 2013). However, Jones and Wheeler(1965a) reported the surprisingly high rate of 42% and 92% offemale A. aegypti with three spermathecae filled with sperm afterforced-mating and cage-mating respectively, where 10 males and10 females were kept in small cages for several hours. These datacontrast with most of the studies reporting that one of the threespermathecae usually remains empty in female A. aegypti (Roth,1948; Lum, 1961b; Jones and Wheeler, 1965b). This highlights thediscrepancies that can result from different test protocols. Forcedmating may allow the male to transfer more sperm as the femaleis not able to reject the male and to end the mating prematurely;indeed Jones and Wheeler (1965a) reported that forced-matings

productive biology of Aedes mosquitoes. Acta Trop. (2013),

lasted twice as long as free mating. On the other hand, mating insmall cages with several males may favour multiple inseminationof the female thus contributing to more sperm being transferredto the spermathecae. As a consequence the resulting insemination

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nformation will not reflect the actual insemination capacity ofales neither the insemination rate of females occurring in a

atural setting. According to Jones and Wheeler (1965a), one thirdf the sperm cells transferred to the BI did not reach the spermath-cae. The male AG secretion transferred to the female contains aranular material that remains in the BI where it gradually clustersnd solidifies (Spielman et al., 1969; Oliva et al., 2013). The spermhat failed to reach the spermathecae become compressed intohe posterior end of the BI, lose their motility by the second dayfter copulation, and are then digested and absorbed in the BISpielman, 1964). It is still unclear what the consequences of thearge amount of sperm that is not used for fertilization could bend why the third sperm reservoir usually remains empty.

. Male reproductive capacity

Mosquito testes are filled with undifferentiated cells, sperma-ocytes and spermatozoa in various stages of development (Barr,974). Spermatogenesis in the testes begins in the larval stage andontinues throughout the adult life of the mosquito until spermato-ysts are exhausted of spermatogonia (undifferentiated germ cells)Lum, 1961a; Barr, 1974; Clements, 2000). The spermatozoa maturen the testes and are then stored in the seminal vesicles duringexual maturation, shortly after emergence until the second dayf adult life (Jones, 1967; Foster and Lea, 1975a). The two largeG of male mosquitoes accumulate secretions (Jones and Wheeler,965a) during the first two days after emergence (Hodapp andones, 1961; Lum, 1961b; Foster and Lea, 1975b). The maximumuantity of sperm in the seminal vesicles as well as of secretoryaterial in the AG was reached in 2-day-old male A. aegypti (Foster

nd Lea, 1975a).From 6000 to 6500 spermatozoa were counted in each testis

f late stage A. aegypti pupae (Jones, 1967), and about 5000 inhe seminal vesicles of 3–7 day-old unmated adults (Jones and

heeler, 1965a). Sperm production is positively correlated withge and body size (Ponlawat and Harrington, 2007), and probablyo a greater extent with temperature (Bader and Williams, 2012).

ale A. aegypti transferred about 1000–2000 sperm to the femaleI, two-thirds of which would later reach the spermathecae (Jonesnd Wheeler, 1965a). Jones and Wheeler (1965a) determined thatut of an average of 1847 sperm transferred by 3- to 7-day-old male. aegypti, on average 660 and 486 sperm cells would reach the

arge and one of the small spermathecae, respectively. The quan-ity of sperm transferred increases with male body size and agePonlawat and Harrington, 2009). Interestingly, wild male A. aegyptiransferred more sperm than laboratory-reared males (Ponlawatnd Harrington, 2009). Although this might be due to infraspecificariations between strains, one interpretation is that wild malesttempt to transfer a maximum quantity of semen to the encoun-ered female compare to a laboratory setting where probabilities to

eet females are higher and selection favours males that are morearsimonious. This difference highlights once again the discrepan-ies observed between laboratory and field studies and emphasizeshe importance of conducting experiments with young strains.

As shown by several studies (Roth, 1948), Aedes males attempt asany copulations as possible even though they might be unable to

ransfer any more sperm after the initial matings. Laboratory stud-es showed that male A. albopictus could fertilize up to 20 femalesver their lifetime when offered new females daily (Boyer et al.,011) and male A. aegypti could transfer accessory gland secretionso up to 22 females (Foster and Lea, 1975b). After repeated suc-essive matings the stock of spermatozoa in the seminal vesicles

Please cite this article in press as: Oliva, C.F., et al., Male rehttp://dx.doi.org/10.1016/j.actatropica.2013.11.021

nd the secretion in the AG become depleted; in male A. aegyptioth are nearly empty following six inseminations in rapid suc-ession, but they are replenished after three days without sexualctivity (Jones, 1967; Dapples et al., 1974). As expected, the speed

PRESSa xxx (2013) xxx– xxx

of seminal depletion was shown to be slower for larger males in A.aegypti (Helinski and Harrington, 2011). It is likely that some com-petitive males mate several females while others do not mate at allbut the distribution of males contributing few versus many matingsin the field remains unknown.

The induction of sterility by irradiation damages the early stagesof spermatozoa (Proverbs, 1969) and therefore lessens the possibil-ity to replenish the seminal vesicles with mature sperm. Therefore,it is of interest in the context of genetic control programmes toknow whether this treatment also affects male mating capacity tothe extent that it reduces programme effectiveness. Reductions inmating capacity are expected to reduce effectiveness if it resultsin remating or is correlated with lower mating competitiveness.Radiosterilized male A. albopictus can inseminate a maximum of7 females, after which their capacity to replenish testes with newsperm seems impossible; over the same period of mating opportu-nities, untreated males could inseminate a maximum of 11 females(Oliva et al., 2013). Genetically modified male A. aegypti insemi-nated an average of 6.6 females over their lifetime, which was sig-nificantly less than the average of 11.5 females inseminated by theiruntransformed counterparts (Bargielowski et al., 2011); howeverthe ability of these transgenic mosquitoes to replenish the semi-nal vesicles with sperm has not been reported, and as noted above,depending on the frequency of remating and male mating success,these reductions may or may not affect programme success.

6. Multiple insemination and sperm use

6.1. Male behaviour to promote female monogamy

Similarly to the majority of male insects, male Aedes are polygy-nous, and this might be favoured by their vast supply of sperm, rapidcopulation, and the likelihood of encountering numerous femalesin relatively dense concentrations at feeding, resting, or oviposi-tion sites (Thornhill and Alcock, 2001). On the other hand, malestend to induce monogamy in female by transferring (1) a sufficientquantity of sperm to fertilize all the eggs the female will lay dur-ing her lifetime (Jones, 1968) and (2) AG secretions that act as ashort-term physical barrier (George, 1967; Spielman et al., 1967,1969; Oliva et al., 2013) and a long-term chemical barrier to furtherinsemination (Helinski et al., 2012b). The AG secretions contain agranular material that gradually clusters and solidifies within the BIafter about 40 min post-insemination (Spielman et al., 1967, 1969;Oliva et al., 2013) and could temporarily play a similar role to themating plug in Anopheles. This content is dissolved 24–48 h afterthe mating (Lum, 1961b; Oliva et al., 2013), but the prevention ofreinsemination is maintained (Helinski et al., 2012b; Oliva et al.,2013). The pheromone responsible for inducing this monogamy inAedes has been shown to be matrone (Fuchs et al., 1968) which actsdirectly on the female nervous system (Gwadz, 1972). The transferof AG secretions in mosquitoes is thought to be a remnant of nuptialgifts ensuring female monogamy, although the nuptial gift mighthave become superfluous with the development of the ability tostore enough energy with sugar and blood feeding (Yuval, 2006).Nutritional benefits of the transfer of male AG secretions in insectshave been proposed to result in a higher fecundity (Thornhill andAlcock, 2001), but no significant increase was observed betweenonce- and multiply-mated female A. aegypti (Lang, 1956; Helinskiand Harrington, 2012). Yuval (2006) suggested that the male trans-fer of AG secretions has lost its nutritive function and solely evolvedto prevent subsequent female insemination.

productive biology of Aedes mosquitoes. Acta Trop. (2013),

6.2. What are the odds for multiple insemination of females?

However, female Aedes possess physiological and behaviouralfeatures which could classify them as polyandric. The presence of

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ultiple spermathecae are usually associated with a polygamicehaviour and cryptic choice for sperm use (Eberhard, 1996;immons, 2001), though the later has not yet been demonstrated inosquitoes. The likelihood of polyandry in insect females is shaped

y their pattern of sexual receptivity over time. Unlike many insectpecies, the behavioural receptivity (i.e. acceptance of copulation)f Aedes females does not decrease with age (Gwadz and Craig,968) nor after the first mating (Oliva et al., 2013). They can beubjected to frequent copulation attempts in experimental situ-tions (Roth, 1948; Jones, 1974). A single female A. aegypti leftogether with 11 males was recorded to be subjected to 50 cop-lation attempts by different males in 1 h, however the number ofctual successful insemination was not observed (Roth, 1948).

On the other hand, the physiological receptivity to insemina-ion ceases 40 min after females’ first insemination (Craig, 1967;liva et al., 2013). Williams and Berger (1980) reported the trans-

er of semen to the BI of 22% of A. aegypti females after the 5thonotrophic cycle, although no observations of the use of the newperm for egg fertilization were made. Given that the physical andhemical barriers to female reinsemination are not active immedi-tely after her first mating, it seems intuitively likely that a virginemale subjected to such a high frequency of copulations could benseminated multiple times. Recently, proof of the occurrence of

ultiple-insemination in the field has been established, as 14%f female A. aegypti were inseminated by at least two males inemi-field enclosures (Helinski et al., 2012a) and 26% of female A.lbopictus caught in the field laid multi-fathered progeny (Boyert al., 2012).

As multiple insemination of female Aedes exists in low propor-ion in the field, it is possible that male avoid this kind of mating.

minority of 33% of female A. aegypti caught in copula in the fieldy Gubler and Bhattacharya (1972) were parous, which led themo suggest a preference of wild males for nulliparous females foropulation. Mated females of this species were much less likely tocoustically interact with a male by matching her flight tone, sug-esting a reduced sensitivity to male stimuli (Cator et al., 2009),hich might explain Gubler and Bhattacharya’s observation. The

xistence of a male choice for the female partner in A. aegypti wasut forward (Gwadz et al., 1971; Jones, 1974), although there iso evidence yet that they are able to detect female mating state.olerstock et al. (2002) reported a change in the proportion of someuticular hydrocarbons (CHC) in Aedes females after mating, anduggested that this could act as a mating history indicator, simi-arly to some dipterans including Drosophila melanogaster (Meigen)Zawitowski and Richmond, 1986). This has however not yet beenemonstrated in Aedes but would be worth studying. In a laboratoryetting, male A. albopictus did not refuse to copulate with non-virginemales, but a shorter copulation time was observed compared toopulation with virgin or freshly-mated (less than 40 min before)emales, and those copulations did not lead to sperm transfer (Olivat al., 2013). It therefore appears that male Aedes would attempto copulate with any females but refuse or fail to ejaculate whenhe females have already been inseminated for a period longerhan 40 min. The actual attractiveness of a mated female for maleseserves further investigations.

The pattern of sperm usage in mosquito females inseminatedultiple times is not yet understood and there has been no report

o far of sperm competition in any mosquito species. Female A.lbopictus inseminated multiple times have oviposited eggs fer-ilized by the different males during the first gonotrophic cycleBoyer et al., 2012; Oliva et al., 2013). The occurrence of spermisplacement therefore seems to be absent in Aedes species. Sperm

Please cite this article in press as: Oliva, C.F., et al., Male rehttp://dx.doi.org/10.1016/j.actatropica.2013.11.021

recedence (i.e. non-random use of sperm) has been reported forome Drosophila species. Non-equal participation of sperm fromwo male A. albopictus in egg fertilization was observed over succes-ive gonotrophic cycles (Oliva et al., 2013); however this could be

PRESSa xxx (2013) xxx– xxx 5

ascribed to a different quantity of sperm being transferred by eachmale rather than to sperm precedence. Precise studies of the storageand use of sperm from two or more males in Aedes spermathecaewould allow a greater understanding of their mating biology.

6.3. Consequences of polyandry for genetic control programmes

In the context of genetic control programmes, the consequencesof female polygamy have been questioned. In monogamous species,the main issue consists in ensuring that the released males areable to copulate, inseminate, and inhibit female receptivity simi-larly to wild males. On the other hand, if polyandry occurs, spermcompetitiveness in fertilizing eggs, sperm numbers, and a male’sability to perform subsequent matings become of higher concern(Barclay, 2005; Whitten and Mahon, 2005). Models have shownthat polyandry is not an adverse factor to autocidal programmes,as long as the sperm of a previous mating is not replaced ateach successive mating and sperm from both males are simi-larly competitive (Knipling, 1964; Whitten, 1971; Barclay, 2005;Perez-Staples et al., 2013). Populations of polygamous insects havebeen successfully eradicated using the SIT (melon fly: Steineret al., 1965). In some species an increased incidence of remat-ing in females mated with sterile relative to fertile ones has beenreported, and has been ascribed to a smaller quantity of spermtransferred by the sterile males (boll weevils: Haynes and Mitchell,1977), or to a reduced efficacy of sterile males’ sperm-associatedaccessory gland products to inhibit female receptivity (South Amer-ican fruit fly: Abraham et al., 2012). However, in A. albopictus (Olivaet al., 2013) and Anopheles arabiensis (Helinski and Knols, 2008;Helinski et al., 2008) similar proportions of multiple-inseminationswere reported when females were mated first with radio-sterilizedthen untreated males or vice versa, suggesting a similar capacityto inseminate and that the female post-mating responses wereinduced in the same way by untreated or sterile males. In sucha situation polyandry should not alter the genetic control pro-grammes’ efficiency but in fact might be a benefit as released malescould, under some circumstances, mate with immigrant insemi-nated females (Pates and Curtis, 2005). The important questions toconsider for genetic control programmes are the possible differen-tial ability of sterile and wild males to prevent female remating ordifferential use of sterile and fertile sperm for eggs fertilization.

7. Future directions for Aedes population control

Genetic control approaches to suppress or replace disease-transmitting vectors act directly on the reproduction of a wildpopulation, and therefore it is fundamental to have a good under-standing of the reproductive biology of the target species. Thecurrent knowledge of Aedes mating biology is still lacking, in partic-ular regarding mating and swarming activity in the field, the fateof sperm in the female reproductive tract and factors that affectmate choice. Recent studies have permitted important advances inunderstanding the role of the male AG secretion on mated femalebehaviour but a more thorough understanding of the consequencesof multiple inseminations is needed.

The outdoor mating habits of Aedes mosquitoes are relativelyunknown compared with the wealth of information we haveabout mating under laboratory conditions. When mosquitoesare confined in small laboratory cages under un-natural setting,results of mating studies can hardly reflect what is happeningin the field. A better understanding of the behaviour of wild

productive biology of Aedes mosquitoes. Acta Trop. (2013),

males is essential to be able to anticipate and optimise the fateof released individuals. Some key research questions that wouldbring important information for the general knowledge and for theimplementation of Aedes population control programmes are listed

ARTICLE IN PRESSG Model

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6 C.F. Oliva et al. / Acta Tropica xxx (2013) xxx– xxx

Table 1Male reproductive biology of Aedes mosquitoes: key research questions for release programmes (see references in the text).

What do we already know? What do we still want to know?

Finding a mate: Released males have to be able to locate and copulate with wild females.General• Males swarm in small groups near blood-meal source, at the bases of trees,or over larval sites.• A volatile “aggregation” pheromone stimulating females is produced byswarming males.• Change in the proportion of some cuticular hydrocarbons (CHC) aftermating in females.• Importance of flight and wing beat frequency in pair forming.

General• Spatial, qualitative, and quantitative description of Aedes swarms in the field.• Proportion of mating occurring with or without group swarming activity inthe field.• Chemical nature of the aggregation pheromone.• Mechanism and cause of CHC changes in females.• Effect of CHC proportion changes on the copulatory behaviour of males.Released male• Effect of the induction of sterility or genetic modification on the ability toproduce swarming pheromones, on the flight activity (wing beat frequency),or on the swarming behaviour.

Insemination and reproductive success: released males must be capable of transferring semen and inducing the post-mating response in females.General• A mixture of sperm and AG secretions transferred by the male to thefemale BI.• AG secretions involved in preventing further insemination of the female.• Depletion of male reproductive system after a series of inseminations andreplenishment after a few days.Released male• Ability to mature new sperm cells after depletion impaired byradiosterilization.• Reduced number of females inseminated by radiosterilized and GM males.

General• Distribution of number of females inseminated in the field by males.• Variation in the reproductive success between geographically distant strains.Released male• Effect of any genetic modification on male’s ability to mature new spermcells after depletion.• Effect of the induction of sterility or genetic modification on male’s ability toproduce new AG secretion after depletion.• Effect of size and culture on male mating competitiveness.

Female multiple insemination: released males must have the same probabilities as wild males in inseminating a non-virgin female or preventing reinsemination.The sperm of released males must equally compete with the sperm of wild males for the egg fertilization.General• Evidence of multiple inseminations occurring in the field.• Sperm from more than one male can be stored and used for the eggfertilization only if all matings take place within a short period (40 min afterthe first mating).Released male

General• Ability of males to detect female mating state and preferentially choosenulliparous females.• Benefits of multiple inseminations for female (increased fecundity, survival,genetic advantage).• Mechanisms of sperm transfer and storage in once- and twice-mated females.

ie

A

Gao

R

A

A

A

A

B

B

B

B

• Ability to prevent female reinsemination once the physical or chemicalbarriers are in place, and to inseminate previously mated females notaffected by the radiosterilization.

n Table 1. We summarized the current knowledge associated withach question and the directions that deserve further research.

cknowledgements

This work was supported by the IAEA research under the CRP43002. We are grateful to R. Lees for useful comments and writingssistance and to D. Chadee, J. Gilles, and R. Lees for proof readingf the article.

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