See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/259204876

Male reproductive biology of Aedes mosquitoes

Article  in  Acta Tropica · December 2013

DOI: 10.1016/j.actatropica.2013.11.021 · Source: PubMed

CITATIONS READS

75 2,089

3 authors:

Clelia Felice Oliva Damiens David

CTIFL unemployed
22 PUBLICATIONS   588 CITATIONS    66 PUBLICATIONS   1,489 CITATIONS   

SEE PROFILE SEE PROFILE

Mark Quentin Benedict

Centers for Disease Control and Prevention
149 PUBLICATIONS   6,181 CITATIONS   

SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Revolutionizing Insect Control (Revolinc) View project

Sterile Insect Technique (SIT) View project

All content following this page was uploaded by Mark Quentin Benedict on 01 October 2019.

The user has requested enhancement of the downloaded file.

 ARTICLE IN PRESS
G Model
ACTROP-3239; No. of Pages 8

Acta Tropica xxx (2013) xxx–xxx

Contents lists available at ScienceDirect

Acta Tropica
journal homepage: www.elsevier.com/locate/actatropica

Male reproductive biology of Aedes mosquitoes

Clelia F. Oliva a,∗ , David Damiens b , Mark Q. Benedict c
a
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. Andrea
delle Fratte, 06132 Perugia, Italy
b
Insect Pest Control Laboratory, Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, FAO/IAEA Agriculture and Biotechnology
Laboratories, A-2444 Seibersdorf, Austria
c
Università di Perugia, Dipartimento di Medicina Sperimentale e Scienze Biochimiche, Perugia, Italy

a r t i c l e i n f o a b s t r a c t

Article history: Among Aedes mosquitoes are species responsible for transmission of serious pathogens to humans. To
Received 12 June 2013 cope with the current threats to long-term effectiveness of the traditional vector control methods, non-
Received in revised form conventional control strategies are being developed. These include autocidal control such as the release
19 November 2013
of sterile males (sterile insect technique) and the release of Wolbachia-infected males to induce sexual
Accepted 23 November 2013
sterility (incompatible insect technique) and pathogen-refractory strain replacement variations using
Available online xxx
Wolbachia. Sterile male types of techniques particularly depend on released males’ ability to successfully
mate with wild females. For that reason, a good understanding of male mating biology, including a thor-
Keywords:
Aedes
ough understanding of the reproductive system and mating capacity, increases the likelihood of success
Male of such genetic vector control programmes. Here we review the literature concerning the reproduction of
Reproduction Aedes mosquitoes with an emphasis on the male biology. We consider sexual maturation, mate finding,
Mate-selection insemination, male reproductive capacity, and the occurrence of multiple matings. We also discuss which
Genetic control parameters are of greatest importance for the successful implementation of autocidal control methods
Sterile insect technique and propose questions for future research.
© 2013 International Atomic Energy Agency. Published by Elsevier B.V. All rights reserved.

1. Background
Many Aedes species are vectors of viruses and nematodes
responsible for severe human diseases among which are dengue,
yellow fever, chikungunya, and lymphatic filariasis (Lounibos,
2002; Gratz, 2004). The two species responsible for most diseases,
Aedes aegypti and Aedes albopictus, have very effective invasive
capacities (Juliano and Lounibos, 2005). Their worldwide invasion
has led to increasing disease risks (Reiter, 2001) and emergence or
re-emergence of epidemics of dengue and other arboviral diseases
(Mackenzie et al., 2004).
Aedes mosquitoes are often found in high densities, breeding
in rural as well as urban habitats. Larvae are able to develop in
clean or polluted stagnant water and can colonize various artifi-
cial habitats and peri-domestic containers, increasing their contact
with humans and the potential for spreading diseases (Jansen
and Beebe, 2010). Control of aedine populations usually relies on
larval control using insecticides and source removal, sometimes
involving community participation. The location of larval sites in
private 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).
extremely difficult and costly. Drastic control of larval sites and,
after 1944, the use of DDT permitted the elimination of A. aegypti
in several countries of South America; however, the end of the con-
trol measures were predictably followed by a rapid re-infestation
from neighbouring areas (Soper, 1963). Over the last half-century,
sustained control has rarely been achieved, and resurgence may
partly be ascribed to the high mobility and resilience of this species
and the cessation of the control programmes (Reiter and Gubler,
1997; Reiter, 2007).
The current vector control tools have failed to reduce mosquito
populations 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 on
frequent mass-releases of male mosquitoes following their radios-
terilization (classical SIT, Robinson et al., 2009), alteration of their
progeny’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 (Brelsfoard
and Dobson, 2009). Alternative strategies consist of replacing
the wild population with mosquitoes refractory to the pathogen
so that disease transmission to humans cannot occur (Sperança
and Capurro, 2007; Moreira et al., 2009; Hoffmann et al.,
2011).

0001-706X/$ – see front matter © 2013 International Atomic Energy Agency. Published by Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.actatropica.2013.11.021

Please cite this article in press as: Oliva, C.F., et al., Male reproductive biology of Aedes mosquitoes. Acta Trop. (2013),
http://dx.doi.org/10.1016/j.actatropica.2013.11.021
 ARTICLE IN PRESS
G Model
ACTROP-3239; No. of Pages 8

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

Releases of sterile individuals have permitted the success-

ful regional elimination of important pests, such as the New
World screwworm (Cochliomyia hominivorax Coquerel), the
Mediterranean fruit fly (Ceratitis capitata Wiedemann), and the
Mexican fruit fly (Anastrepha ludens Loew) (Krafsur, 1998; Klassen
and Curtis, 2005). SIT has been tested for mosquito control in the
1970s using various sterilizing approaches (chemosterilization,
ionizing radiation, cytoplasmic incompatibility, or chromosome
translocations) (Benedict and Robinson, 2003; Dame et al., 2009),
and research is currently being performed to improve the different
technical steps (Robinson et al., 2009). Some field trials have
shown encouraging potential of the releases of sterile mosquitoes
in reducing wild population of Anopheles albimanus (Lofgren et al.,
1974; Weidhaas et al., 1974), A. albopictus (Bellini et al., 2007,
2013), A. aegypti (Harris et al., 2012; Lacroix et al., 2012), and Aedes
polynesiensis (O’Connor et al., 2012).
Control methods that interfere with the reproduction of an
insect can be improved with a sound knowledge of its mating biol-
ogy. Our understanding of the reproductive physiology of Aedes
species is still incomplete. Most studies cover the female aspects of Fig. 1. The reproductive system of female (in red) and male (in blue) Aedes and
reproduction of the main disease vector, A. aegypti, and date from the sperm transfer during copulation (represented by the arrows). Adapted from
the 70s. However the recent development of transgenic mosquitoes Clements (1999) and Jones (1968).
and application of molecular biology techniques have permitted
advances in the understanding of the mating system. We review
The time required to complete terminalia rotation varies
the existing literature on this topic concerning the Aedes genus and
between species from 18 to 24 h for A. aegypti (Roth, 1948), 11 to
raise some crucial research questions that would help to improve
25 h for A. albopictus (Miyagi and Toma, 1982; Oliva et al., 2012),
the efficiency and effectiveness of genetic control approaches.
around 12 and 40 h, respectively, for Aedes iriomotensis and Aedes
atriisimilis (Miyagi and Toma, 1982), 30 h for Aedes taeniorhynchus
(Provost et al., 1961), and nearly 4 days for Aedes provocans (Smith
2. Male sexual maturation
and Gadawski, 1994). Before completion of sexual maturation,
males may attempt to make contact with females (Roth, 1948;
A successful mating involves sequences of events that per-
Cabrera and Jaffe, 2007) but no copulation or swarm activity is
mit mate interception, engagement of the genitalia, transfer of
observed in A. aegypti males less than 24 h old (Cabrera and Jaffe,
semen, and storage of sperm by the female. Mating is preceded
2007). Given the importance of terminalia rotation for mating abil-
by mate location and interaction, which usually requires appropri-
ity, it is notable that the radiosterilization of A. albopictus did not
ate epigamic behaviour. The sperm stored by the female mosquito
affect the speed of rotation of the male terminalia (Oliva et al.,
will be used for egg fertilization shortly before the egg is laid. The
2012); however, closer observations of the antennal fibrillae in
ability of released males to participate in and perform each step
sterilized or genetically modified males would be of interest.
and the viability and competitiveness of their sperm will impact
the population control programmes.
Mosquito males must complete two easily observable physical 3. Finding a mate
changes prior to becoming sexually active after eclosion. During
the first day after emergence of Aedes males, the fibrillar hairs of 3.1. Male mate location behaviour and encounter sites
the antennae become fully erect and usually remain so through-
out the lifetime, except in some old males (Roth, 1948; Fay, 1964; Contrary to the situation in anophelines in which males form
Foster and Lea, 1975a; Nijhout, 1977). The erection of the fibrillae station-keeping swarms into which females fly and mate – usually
are important for female location as they allow males to respond in relation to some feature of the environment (Howell and Knols,
to female flight sounds (Roth, 1948). 2009) – Aedes males do not require the formation of such swarms
In addition, males undergo a permanent 180◦ rotation of the to attract females and mate. Aedes demonstrate wide behavioural
terminalia part of the genitalia; as a result, the claspers become ori- plasticity that allows them to mate in single pairs or in aggregations.
ented ventrally and the aedaegus lies above the anus. This rotation This same flexibility permits mating throughout the day though
is common among the Diptera and was suggested to result from an crepuscular peaks have been observed. An individual male Aedes
adjustment of the copulation position (Lamb, 1922). Mosquitoes can fly singly to intercept a female to mate (Downes, 1969) and,
assume a venter-to-venter position; pairs of Anopheles usually contrary to anophelines, matings where only one pair is present
face opposite directions (Roth, 1948; Charlwood and Jones, 1979), in a small arena can be very easily performed. However, swarm-
whereas Aedes more frequently face the same direction, and Culex ing behaviour has been described, as small groups of males can be
species were observed to equally use both attitudes (Roth, 1948). observed to fly in a figure-eight pattern similarly to small swarms
The different mating attitude was correlated with differences in near a blood-meal source (Wright et al., 1966; Loor and DeFoliart,
the structure of the male claspers between those species (Roth, 1970; Hartberg, 1971; Gubler and Bhattacharya, 1972), at the feet of
1948). Aedes males usually first grasp the female dorsally and then a walking blood meal host (Ali and Rozeboom, 1971), over the cor-
rapidly change to a ventral position (Roth, 1948). The 180◦ rotation ners of release cages (Edman et al., 1972), at the feet of large trees
of mosquito genitalia allows the claspers to be ventrally oriented, in (Tuten et al., 2013), or over larval sites (unpublished observations).
which position they can be used to grasp the females’ cerci (Fig. 1). Aedes swarming activity is a mate-locating behaviour; according
Moreover, the genital pore of the male lies on the antero-dorsal sur- to Cabrera and Jaffe (2007) it indeed enhances mating opportu-
face of the aedeagus, which must then be rotated prior to copulation nities and increases copulation frequency but it is not a required
(Spielman, 1964, 1966). behaviour.

Please cite this article in press as: Oliva, C.F., et al., Male reproductive biology of Aedes mosquitoes. Acta Trop. (2013),
http://dx.doi.org/10.1016/j.actatropica.2013.11.021
 ARTICLE IN PRESS
G Model
ACTROP-3239; No. of Pages 8
C.F. Oliva et al. / Acta Tropica xxx (2013) xxx–xxx
Cabrera and Jaffe (2007) have reported the participation of an
average of 23 A. aegypti males in swarms formed in laboratory
conditions, in the presence of a blood–meal source. In the field,
3–40 male A. albopictus swarming around the observer (Gubler and
Bhattacharya, 1972) and an average of only five male A. polyne-
siensis swarming at the bases of trees (Tuten et al., 2013) were
reported. Swarms lasted from 5 to 16 min under laboratory condi-
tions (Cabrera and Jaffe, 2007) and a high swarming activity during
5–10 min was also reported in the field (Gubler and Bhattacharya,
1972). The mating activity peaked between 5 and 10 min after
the beginning of the swarm (Gubler and Bhattacharya, 1972), the
duration of which was positively correlated with the likelihood of
copulation taking place (Cabrera and Jaffe, 2007). The main stim-
ulus for mate-seeking flying behaviour is likely to be the presence
of females (Dyar, 1920, 1928; Nijhout and Craig, 1971; Gubler and
Bhattacharya, 1972; Jaenson, 1985; Cabrera and Jaffe, 2007; Cator
et al., 2011) which is increased by the presence of a blood-meal
source and by the onset of photophase, as females’ biting activ-
ity peaks immediately after sunrise and before sunset (Hartberg,
1971; Edman et al., 1972; Foster and Lea, 1975a; Cabrera and Jaffe,
2007; Tuten et al., 2013). Quantitative and descriptive reports of the
swarming and mating sites of Aedes species in the field are, how-
ever, lacking, but due to the relatively limited dispersal of male
Aedes in urban areas (Muir and Kay, 1998; Bellini et al., 2010;
Bergero et al., 2013) these would be very useful to identify the
best release areas for autocidal and population replacement pro-
grammes.
3.2. The interacting roles of olfaction and hearing in mate
recognition
Cabrera and Jaffe (2007) reported the production of a volatile
“aggregation pheromone” by swarming males that stimulates both
female and male flight and attracts both sexes to the swarm; the
female olfactory response to swarming males’ odour prevailed over
a host odour. In response to this olfactory stimulus, females show
a characteristic very rapid agitated flight (Cabrera and Jaffe, 2007).
Female wing beat also attracts males, as the females’ frequency
has been shown to stimulate male Aedes to pursue and seize the
female (Roth, 1948). A. aegypti males would not attempt to cop-
ulate with non-flying females (Spielman, 1964) nor with newly
emerged females, which have a different wing beat frequency than
mature ones (Roth, 1948; Brogdon, 1994). Studies on Anopheles
(Pennetier et al., 2010), Culex (Warren et al., 2009), Toxorhynchites
(Gibson and Russell, 2006; Cator et al., 2009; Pennetier et al., 2010),
and Aedes (Cator et al., 2009) species have shown that a pair of
female and male mosquitoes can alter their flight-tone frequency
and synchronize them before copulating. Interestingly, a female
will not attempt or succeed to harmonize with every male, sug-
gesting a certain degree of mate choice from either the male or
female according to the partner’s wing beat frequency (Gibson et al.,
2010). In anophelines, flight tone frequency has been shown to vary
with body size, which supports the hypothesis of mate assessment
(Cator et al., 2010). In this genus, it is also involved in species recog-
nition (Pennetier et al., 2010). However, this does not appear to be
the case for aedines (Roth, 1948; Nijhout and Craig, 1971), where
recognition would occur after the first contact through a species-
specific pheromone stimulus that triggers the male to shift to the
coitus position (Nijhout and Craig, 1971). However, several experi-
mental cross-matings between different species of Aedes have been
reported, though they always resulted in infertile progeny (Roth,
1948; Ali and Rozeboom, 1971; Ali et al., 1971; Lee et al., 2009). This
result illustrates that barriers to normally rare interspecific mating
behaviours can sometimes easily be overcome in the laboratory.
It would be worth investigating further whether the olfactory cue
emitted by females and the one emitted by swarming males are also
Please cite this article in press as: Oliva, C.F., et al., Male reproductive biology of Aedes mosquitoes. Acta Trop. (2013),
http://dx.doi.org/10.1016/j.actatropica.2013.11.021
3
involved in mate choice. Both the ability to produce and recognize
pheromones and wing beat frequency are crucial parameters for
Aedes mating; ensuring that they are not impaired in males that are
released as part of a genetic control programme should be a priority.
4. Transfer of sperm and male AG secretions to the female
reproductive 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 of
contact was sufficient for insemination (Roth, 1948; Jones, 1974).
Most of the copulations of A. albopictus observed in the field had a
median duration of 8 s (Gubler and Bhattacharya, 1972), against 45 s
for a long-colonized laboratory strain of A. albopictus (Oliva et al.,
2013). The absence of predation risk under laboratory conditions
might have led to the loss of the propensity for rapid copulation.
Following abdominal contact, engagement of the female cerci by
the male claspers, and probably a stimulus from the female cerci
on the sensillae of the male claspers, the male aedaegus becomes
everted and extended, the female vagina is widened to enlarge the
opening 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 merge
into the ejaculatory duct, and are joined by the paired AG near the
anterior end of the duct (Fig. 1) (Spielman, 1964). The semen flows
through the ejaculatory duct, which leads directly to the genital
pore located near the base of the aedaegus, and contractions of the
male AG provide pressure for the sperm transfer (Spielman, 1964).
Semen transfer itself requires only a few seconds (Lum, 1961a). The
BI is the first, but temporary, sperm storage organ in Aedes females
(Barr, 1974). Within 30 s after termination of coitus, the female
atrium regains its precopulatory appearance, and sperm begin to
concentrate close to the spermathecal eminence with their heads
oriented towards the spermathecal vestibule (Spielman, 1964). The
abundant 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 (Oliva
et al., 2013).
Most of the Culicinae females have three spermathecae while
females of the Anophelinae subfamily have only one. This presents
questions regarding how the various spermathecae are filled and
their sperm used during fertilization. In the case of Aedes species,
females possess one large central spermatheca and two smaller
lateral ones. Following mating with a sexually mature male that
has 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% of
female A. aegypti with three spermathecae filled with sperm after
forced-mating and cage-mating respectively, where 10 males and
10 females were kept in small cages for several hours. These data
contrast with most of the studies reporting that one of the three
spermathecae usually remains empty in female A. aegypti (Roth,
1948; Lum, 1961b; Jones and Wheeler, 1965b). This highlights the
discrepancies that can result from different test protocols. Forced
mating may allow the male to transfer more sperm as the female
is not able to reject the male and to end the mating prematurely;
indeed Jones and Wheeler (1965a) reported that forced-matings
lasted twice as long as free mating. On the other hand, mating in
small cages with several males may favour multiple insemination
of the female thus contributing to more sperm being transferred
to the spermathecae. As a consequence the resulting insemination
 ARTICLE IN PRESS
G Model
ACTROP-3239; No. of Pages 8
4 C.F. Oliva et al. / Acta Tropica xxx (2013) xxx–xxx
information will not reflect the actual insemination capacity of
males neither the insemination rate of females occurring in a
natural setting. According to Jones and Wheeler (1965a), one third
of the sperm cells transferred to the BI did not reach the spermath-
ecae. The male AG secretion transferred to the female contains a
granular material that remains in the BI where it gradually clusters
and solidifies (Spielman et al., 1969; Oliva et al., 2013). The sperm
that failed to reach the spermathecae become compressed into
the posterior end of the BI, lose their motility by the second day
after copulation, and are then digested and absorbed in the BI
(Spielman, 1964). It is still unclear what the consequences of the
large amount of sperm that is not used for fertilization could be
and why the third sperm reservoir usually remains empty.
5. Male reproductive capacity
Mosquito testes are filled with undifferentiated cells, sperma-
tocytes and spermatozoa in various stages of development (Barr,
1974). Spermatogenesis in the testes begins in the larval stage and
continues throughout the adult life of the mosquito until spermato-
cysts are exhausted of spermatogonia (undifferentiated germ cells)
(Lum, 1961a; Barr, 1974; Clements, 2000). The spermatozoa mature
in the testes and are then stored in the seminal vesicles during
sexual maturation, shortly after emergence until the second day
of adult life (Jones, 1967; Foster and Lea, 1975a). The two large
AG of male mosquitoes accumulate secretions (Jones and Wheeler,
1965a) during the first two days after emergence (Hodapp and
Jones, 1961; Lum, 1961b; Foster and Lea, 1975b). The maximum
quantity of sperm in the seminal vesicles as well as of secretory
material in the AG was reached in 2-day-old male A. aegypti (Foster
and Lea, 1975a).
From 6000 to 6500 spermatozoa were counted in each testis
of late stage A. aegypti pupae (Jones, 1967), and about 5000 in
the seminal vesicles of 3–7 day-old unmated adults (Jones and
Wheeler, 1965a). Sperm production is positively correlated with
age and body size (Ponlawat and Harrington, 2007), and probably
to a greater extent with temperature (Bader and Williams, 2012).
Male A. aegypti transferred about 1000–2000 sperm to the female
BI, two-thirds of which would later reach the spermathecae (Jones
and Wheeler, 1965a). Jones and Wheeler (1965a) determined that
out of an average of 1847 sperm transferred by 3- to 7-day-old male
A. aegypti, on average 660 and 486 sperm cells would reach the
large and one of the small spermathecae, respectively. The quan-
tity of sperm transferred increases with male body size and age
(Ponlawat and Harrington, 2009). Interestingly, wild male A. aegypti
transferred more sperm than laboratory-reared males (Ponlawat
and Harrington, 2009). Although this might be due to infraspecific
variations between strains, one interpretation is that wild males
attempt to transfer a maximum quantity of semen to the encoun-
tered female compare to a laboratory setting where probabilities to
meet females are higher and selection favours males that are more
parsimonious. This difference highlights once again the discrepan-
cies observed between laboratory and field studies and emphasizes
the importance of conducting experiments with young strains.
As shown by several studies (Roth, 1948), Aedes males attempt as
many copulations as possible even though they might be unable to
transfer any more sperm after the initial matings. Laboratory stud-
ies showed that male A. albopictus could fertilize up to 20 females
over their lifetime when offered new females daily (Boyer et al.,
2011) and male A. aegypti could transfer accessory gland secretions
to up to 22 females (Foster and Lea, 1975b). After repeated suc-
cessive matings the stock of spermatozoa in the seminal vesicles
and the secretion in the AG become depleted; in male A. aegypti
both are nearly empty following six inseminations in rapid suc-
cession, but they are replenished after three days without sexual
activity (Jones, 1967; Dapples et al., 1974). As expected, the speed
Please cite this article in press as: Oliva, C.F., et al., Male reproductive biology of Aedes mosquitoes. Acta Trop. (2013),
http://dx.doi.org/10.1016/j.actatropica.2013.11.021
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 all
but the distribution of males contributing few versus many matings
in the field remains unknown.
The induction of sterility by irradiation damages the early stages
of 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 to
know whether this treatment also affects male mating capacity to
the extent that it reduces programme effectiveness. Reductions in
mating capacity are expected to reduce effectiveness if it results
in remating or is correlated with lower mating competitiveness.
Radiosterilized male A. albopictus can inseminate a maximum of
7 females, after which their capacity to replenish testes with new
sperm 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 their
untransformed counterparts (Bargielowski et al., 2011); however
the 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, rapid
copulation, and the likelihood of encountering numerous females
in relatively dense concentrations at feeding, resting, or oviposi-
tion sites (Thornhill and Alcock, 2001). On the other hand, males
tend to induce monogamy in female by transferring (1) a sufficient
quantity of sperm to fertilize all the eggs the female will lay dur-
ing her lifetime (Jones, 1968) and (2) AG secretions that act as a
short-term physical barrier (George, 1967; Spielman et al., 1967,
1969; Oliva et al., 2013) and a long-term chemical barrier to further
insemination (Helinski et al., 2012b). The AG secretions contain a
granular material that gradually clusters and solidifies within the BI
after about 40 min post-insemination (Spielman et al., 1967, 1969;
Oliva et al., 2013) and could temporarily play a similar role to the
mating plug in Anopheles. This content is dissolved 24–48 h after
the mating (Lum, 1961b; Oliva et al., 2013), but the prevention of
reinsemination is maintained (Helinski et al., 2012b; Oliva et al.,
2013). The pheromone responsible for inducing this monogamy in
Aedes has been shown to be matrone (Fuchs et al., 1968) which acts
directly on the female nervous system (Gwadz, 1972). The transfer
of AG secretions in mosquitoes is thought to be a remnant of nuptial
gifts ensuring female monogamy, although the nuptial gift might
have become superfluous with the development of the ability to
store enough energy with sugar and blood feeding (Yuval, 2006).
Nutritional benefits of the transfer of male AG secretions in insects
have been proposed to result in a higher fecundity (Thornhill and
Alcock, 2001), but no significant increase was observed between
once- and multiply-mated female A. aegypti (Lang, 1956; Helinski
and Harrington, 2012). Yuval (2006) suggested that the male trans-
fer of AG secretions has lost its nutritive function and solely evolved
to prevent subsequent female insemination.
6.2. What are the odds for multiple insemination of females?
However, female Aedes possess physiological and behavioural
features which could classify them as polyandric. The presence of
 ARTICLE IN PRESS
G Model
ACTROP-3239; No. of Pages 8

C.F. Oliva et al. / Acta Tropica xxx (2013) xxx–xxx 5

multiple spermathecae are usually associated with a polygamic
behaviour and cryptic choice for sperm use (Eberhard, 1996;
Simmons, 2001), though the later has not yet been demonstrated in
mosquitoes. The likelihood of polyandry in insect females is shaped
by their pattern of sexual receptivity over time. Unlike many insect
species, the behavioural receptivity (i.e. acceptance of copulation)
of Aedes females does not decrease with age (Gwadz and Craig,
1968) nor after the first mating (Oliva et al., 2013). They can be
subjected to frequent copulation attempts in experimental situ-
ations (Roth, 1948; Jones, 1974). A single female A. aegypti left
together with 11 males was recorded to be subjected to 50 cop-
ulation attempts by different males in 1 h, however the number of
actual successful insemination was not observed (Roth, 1948).
On the other hand, the physiological receptivity to insemina-
tion ceases 40 min after females’ first insemination (Craig, 1967;
Oliva et al., 2013). Williams and Berger (1980) reported the trans-
fer of semen to the BI of 22% of A. aegypti females after the 5th
gonotrophic cycle, although no observations of the use of the new
sperm for egg fertilization were made. Given that the physical and
chemical barriers to female reinsemination are not active immedi-
ately after her first mating, it seems intuitively likely that a virgin
female subjected to such a high frequency of copulations could be
inseminated multiple times. Recently, proof of the occurrence of
multiple-insemination in the field has been established, as 14%
of female A. aegypti were inseminated by at least two males in
semi-field enclosures (Helinski et al., 2012a) and 26% of female A.
albopictus caught in the field laid multi-fathered progeny (Boyer
et al., 2012).
As multiple insemination of female Aedes exists in low propor-
tion in the field, it is possible that male avoid this kind of mating.
A minority of 33% of female A. aegypti caught in copula in the field
by Gubler and Bhattacharya (1972) were parous, which led them
to suggest a preference of wild males for nulliparous females for
copulation. Mated females of this species were much less likely to
acoustically interact with a male by matching her flight tone, sug-
gesting a reduced sensitivity to male stimuli (Cator et al., 2009),
which might explain Gubler and Bhattacharya’s observation. The
existence of a male choice for the female partner in A. aegypti was
put forward (Gwadz et al., 1971; Jones, 1974), although there is
no evidence yet that they are able to detect female mating state.
Polerstock et al. (2002) reported a change in the proportion of some
cuticular hydrocarbons (CHC) in Aedes females after mating, and
suggested that this could act as a mating history indicator, simi-
larly to some dipterans including Drosophila melanogaster (Meigen)
(Zawitowski and Richmond, 1986). This has however not yet been
demonstrated in Aedes but would be worth studying. In a laboratory
setting, male A. albopictus did not refuse to copulate with non-virgin
females, but a shorter copulation time was observed compared to
copulation with virgin or freshly-mated (less than 40 min before)
females, and those copulations did not lead to sperm transfer (Oliva
et al., 2013). It therefore appears that male Aedes would attempt
to copulate with any females but refuse or fail to ejaculate when
the females have already been inseminated for a period longer
than 40 min. The actual attractiveness of a mated female for males
deserves further investigations.
The pattern of sperm usage in mosquito females inseminated
multiple times is not yet understood and there has been no report
so far of sperm competition in any mosquito species. Female A.
albopictus inseminated multiple times have oviposited eggs fer-
tilized by the different males during the first gonotrophic cycle
(Boyer et al., 2012; Oliva et al., 2013). The occurrence of sperm
displacement therefore seems to be absent in Aedes species. Sperm
precedence (i.e. non-random use of sperm) has been reported for
some Drosophila species. Non-equal participation of sperm from
two male A. albopictus in egg fertilization was observed over succes-
sive gonotrophic cycles (Oliva et al., 2013); however this could be
ascribed to a different quantity of sperm being transferred by each
male rather than to sperm precedence. Precise studies of the storage
and use of sperm from two or more males in Aedes spermathecae
would 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 consequences
of female polygamy have been questioned. In monogamous species,
the main issue consists in ensuring that the released males are
able to copulate, inseminate, and inhibit female receptivity simi-
larly to wild males. On the other hand, if polyandry occurs, sperm
competitiveness in fertilizing eggs, sperm numbers, and a male’s
ability to perform subsequent matings become of higher concern
(Barclay, 2005; Whitten and Mahon, 2005). Models have shown
that polyandry is not an adverse factor to autocidal programmes,
as long as the sperm of a previous mating is not replaced at
each 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 have
been successfully eradicated using the SIT (melon fly: Steiner
et al., 1965). In some species an increased incidence of remat-
ing in females mated with sterile relative to fertile ones has been
reported, and has been ascribed to a smaller quantity of sperm
transferred by the sterile males (boll weevils: Haynes and Mitchell,
1977), or to a reduced efficacy of sterile males’ sperm-associated
accessory gland products to inhibit female receptivity (South Amer-
ican fruit fly: Abraham et al., 2012). However, in A. albopictus (Oliva
et al., 2013) and Anopheles arabiensis (Helinski and Knols, 2008;
Helinski et al., 2008) similar proportions of multiple-inseminations
were reported when females were mated first with radio-sterilized
then untreated males or vice versa, suggesting a similar capacity
to inseminate and that the female post-mating responses were
induced in the same way by untreated or sterile males. In such
a situation polyandry should not alter the genetic control pro-
grammes’ efficiency but in fact might be a benefit as released males
could, under some circumstances, mate with immigrant insemi-
nated females (Pates and Curtis, 2005). The important questions to
consider for genetic control programmes are the possible differen-
tial ability of sterile and wild males to prevent female remating or
differential 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 wild
population, and therefore it is fundamental to have a good under-
standing of the reproductive biology of the target species. The
current knowledge of Aedes mating biology is still lacking, in partic-
ular regarding mating and swarming activity in the field, the fate
of sperm in the female reproductive tract and factors that affect
mate choice. Recent studies have permitted important advances in
understanding the role of the male AG secretion on mated female
behaviour but a more thorough understanding of the consequences
of multiple inseminations is needed.
The outdoor mating habits of Aedes mosquitoes are relatively
unknown compared with the wealth of information we have
about mating under laboratory conditions. When mosquitoes
are confined in small laboratory cages under un-natural setting,
results of mating studies can hardly reflect what is happening
in the field. A better understanding of the behaviour of wild
males is essential to be able to anticipate and optimise the fate
of released individuals. Some key research questions that would
bring important information for the general knowledge and for the
implementation of Aedes population control programmes are listed

Please cite this article in press as: Oliva, C.F., et al., Male reproductive biology of Aedes mosquitoes. Acta Trop. (2013),
http://dx.doi.org/10.1016/j.actatropica.2013.11.021
 ARTICLE IN PRESS
G Model
ACTROP-3239; No. of Pages 8
6 C.F. Oliva et al. / Acta Tropica xxx (2013) xxx–xxx
Table 1
Male reproductive biology of Aedes mosquitoes: key research questions for release programmes (see references in the text).
What do we already 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 by
swarming males.
• Change in the proportion of some cuticular hydrocarbons (CHC) after
mating in females.
• Importance of flight and wing beat frequency in pair forming.
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 the
female BI.
• AG secretions involved in preventing further insemination of the female.
• Depletion of male reproductive system after a series of inseminations and
replenishment after a few days.
Released male
• Ability to mature new sperm cells after depletion impaired by
radiosterilization.
• Reduced number of females inseminated by radiosterilized and GM males.
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 egg
fertilization only if all matings take place within a short period (40 min after
the first mating).
Released male
• Ability to prevent female reinsemination once the physical or chemical
barriers are in place, and to inseminate previously mated females not
affected by the radiosterilization.
in Table 1. We summarized the current knowledge associated with
each question and the directions that deserve further research.
Acknowledgements
This work was supported by the IAEA research under the CRP
G43002. We are grateful to R. Lees for useful comments and writing
assistance and to D. Chadee, J. Gilles, and R. Lees for proof reading
of the article.
References
Abraham, S., Cladera, J., Goane, L., Teresa Vera, M., 2012. Factors affecting Anastrepha
fraterculus female receptivity modulation by accessory gland products. J. Insect
Physiol. 58, 1–6.
Ali, S.R., Rozeboom, L.E., 1971. Cross-mating between Aedes (S.) polynesiensis marks
and Aedes (S.) albopictus Skuse in a large cage. Mosq. News 31, 80–84.
Ali, S.R., Rozeboom, L.E., Lloyd, E., 1971. Cross-insemination frequencies between
strains of Aedes albopictus and members of the Aedes scutellaris group. J. Med.
Entomol. 8, 263–265.
Alphey, L., Benedict, M., Bellini, R., Clark, G.G., Dame, D.A., Service, M.W., Dobson,
S.L., 2010. Sterile-insect methods for control of mosquito-borne diseases: an
analysis. Vector Borne Zoonotic Dis. 10, 295–311.
Bader, C.A., Williams, C.R., 2012. Mating, ovariole number and sperm production
of the dengue vector mosquito Aedes aegypti (L.) in Australia: broad thermal
optima provide the capacity for survival in a changing climate. Physiol. Entomol.
37, 136–144.
Barclay, H.J., 2005. Mathematical models for the use of sterile insects. In: Dyck,
V.A., Hendrichs, J., Robinson, A.S. (Eds.), Sterile Insect Technique. Principles and
Practice in Area-wide Integrated Pest Management. Springer Netherlands, Dor-
drecht, The Netherlands, pp. 147–174.
Bargielowski, I., Alphey, L., Koella, J.C., 2011. Cost of mating and insemination capac-
ity of a genetically modified mosquito Aedes aegypti OX513A compared to its
wild type counterpart. PLoS ONE 6, e26086.
Barr, A.R., 1974. Symposium on reproduction of arthropods of medical and veteri-
nary importance. V. Reproduction in Diptera of medical importance with special
reference to mosquitoes. J. Med. Entomol. 11, 35–40.
Please cite this article in press as: Oliva, C.F., et al., Male reproductive biology of Aedes mosquitoes. Acta Trop. (2013),
http://dx.doi.org/10.1016/j.actatropica.2013.11.021
What do we still want to know?
General
• Spatial, qualitative, and quantitative description of Aedes swarms in the field.
• Proportion of mating occurring with or without group swarming activity in
the 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 to
produce swarming pheromones, on the flight activity (wing beat frequency),
or on the swarming behaviour.
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 sperm
cells after depletion.
• Effect of the induction of sterility or genetic modification on male’s ability to
produce new AG secretion after depletion.
• Effect of size and culture on male mating competitiveness.
General
• Ability of males to detect female mating state and preferentially choose
nulliparous females.
• Benefits of multiple inseminations for female (increased fecundity, survival,
genetic advantage).
• Mechanisms of sperm transfer and storage in once- and twice-mated females.
Released male
• Pattern of use of multiple-origin sperm for egg fertilization.
Bellini, R., Calvitti, M., Medici, A., Carrieri, M., Celli, G., Maini, S., 2007. Use of the
sterile insect technique against Aedes albopictus in Italy: first results of a pilot
trial. In: Vreysen, M.J.B., Robinson, A.S., Hendrichs, J. (Eds.), Area-wide Control
of Insect Pests. From Research to Field Implementation. Springer, Dordrecht,
Netherlands, pp. 505–515.
Bellini, R., Albieri, A., Balestrino, F., Carrieri, M., Porretta, D., Urbanelli, S., Calvitti, M.,
Moretti, R., Maini, S., 2010. Dispersal and survival of Aedes albopictus (Diptera:
Culicidae) in Italian urban areas and significance for sterile insect technique
application. J. Med. Entomol. 47, 1082–1091.
Bellini, R., Medici, A., Puggioli, A., Balestrino, F., Carrieri, M., 2013. Pilot field tri-
als with Aedes albopictus irradiated sterile males in Italian urban areas. J. Med.
Entomol. 50, 317–325.
Benedict, M., Robinson, A.S., 2003. The first releases of transgenic mosquitoes: an
argument for the sterile insect technique. Trends Parasitol. 19, 349–355.
Bergero, P.E., Ruggiero, C.A., Lombardo, R., Schweigmann, N.J., Solari, H.G., 2013.
Dispersal of Aedes aegypti: field study in temperate areas using a novel method.
J. Vector Borne Dis. 50, 163–170.
Boyer, S., Gilles, J., Merancienne, D., Lemperière, G., Fontenille, D., 2011. Sexual per-
formance of male mosquito Aedes albopictus. Med. Vet. Entomol. 25, 454–459.
Boyer, S., Toty, C., Jacquet, M., Lempérière, G., Fontenille, D., 2012. Evidence of mul-
tiple inseminations in the field in Aedes albopictus. PLoS ONE 7, e42040.
Brelsfoard, C.L., Séchan, Y., Dobson, S.L., 2008. Interspecific hybridization yields
strategy for South Pacific filariasis vector elimination. PLoS Negl. Trop. Dis. 2,
e129.
Brelsfoard, C.L., Dobson, S.L., 2009. Wolbachia-based strategies to control insect
pests and disease vectors. Asia-Pac. J. Mol. Biol. 17, 55–63.
Brogdon, W.G., 1994. Measurement of flight tone differences between female Aedes
aegypti and A. albopictus (Diptera: Culicidae). J. Med. Entomol. 31, 700–703.
Cabrera, M., Jaffe, K., 2007. An aggregation pheromone modulates lekking behavior
in the vector mosquito Aedes aegypti (Diptera: Culicidae). J. Am. Mosq. Control
Assoc. 23, 1–10.
Cator, L.J., Arthur, B.J., Harrington, L.C., Hoy, R.R., 2009. Harmonic convergence in the
love songs of the dengue vector mosquito. Science 323, 1077–1079.
Cator, L.J., Ng’habi, K.R., Hoy, R.R., Harrington, L.C., 2010. Sizing up a mate: variation
in production and response to acoustic signals in Anopheles gambiae. Behav. Ecol.
21, 1033–1039.
Cator, L.J., Arthur, B.J., Ponlawat, A., Harrington, L.C., 2011. Behavioral observations
and sound recordings of free-flight mating swarms of A. aegypti (Diptera: Culi-
cidae) in Thailand. J. Med. Entomol. 48, 941–946.
Chambers, E.W., Hapairai, L., Peel, B.A., Bossin, H., Dobson, S.L., 2011. Male mating
competitiveness of a Wolbachia-introgressed Aedes polynesiensis strain under
semi-field conditions. PLoS Negl. Trop. Dis. 5, e1271.
 ARTICLE IN PRESS
G Model
ACTROP-3239; No. of Pages 8
C.F. Oliva et al. / Acta Tropica xxx (2013) xxx–xxx
Charlwood, J.D., Jones, M., 1979. Mating behaviour in the mosquito, Anophe-
les gambiae s.l. I. Close range and contact behaviour. Physiol. Entomol. 4,
111–120.
Clements, A.N., 1999. The Biology of Mosquitoes. Sensory, Reception, and Behaviour,
vol. 2. CABI Publishing, New York, NY.
Clements, A.N., 2000. The Biology of Mosquitoes. Development, Nutrition and Repro-
duction, vol. 1. CABI Publishing, New York, NY.
Craig Jr., G.B., 1967. Mosquitoes: female monogamy induced by male accessory gland
substance. Science 156, 1499–1501.
Dame, D.A., Curtis, C.F., Benedict, M.Q., Robinson, A.S., Knols, B.G., 2009. Historical
applications of induced sterilisation in field populations of mosquitoes. Malaria
J. 8, S2.
Dapples, C.C., Foster, W.A., Lea, A.O., 1974. Ultrastructure of the accessory gland of
the male mosquito, Aedes aegypti (L.) (Diptera: Culicidae). Int. J. Insect Morphol.
Embryol. 3, 279–291.
Downes, J.A., 1969. The swarming and mating flight of diptera. Annu. Rev. Entomol.
14, 271–298.
Dyar, H.G., 1920. The mosquitoes of British Columbia and the Yukon territory, Canada
(Diptera, Culicidae). Insecutor Inscitiae Menstruus 8, 1–27.
Dyar, H.G., 1928. The Mosquitoes of the Americas. Carnegie Institution of Washing-
ton, Washington, DC, No. 387.
Eberhard, W., 1996. Female Control: Sexual Selection by Cryptic Choice. Princeton
University Press, Princeton, NJ.
Edman, J.D., Haeger, J.S., Bidlingmayer, W.L., Dow, R.P., Nayar, J.K., Provost, M.W.,
1972. Sexual behavior of mosquitoes. 4. Field observations on mating and insem-
ination of marked broods of Aedes taeniorhynchus. Ann. Entomol. Soc. Am. 65,
848–852.
Fay, R., 1964. The biology and bionomics of Aedes aegypti in the laboratory. Mosq.
News 24, 300–308.
Foster, W.A., Lea, A.O., 1975a. Sexual behavior maturation in male Aedes triseriatus
(Diptera: Culicidae): a re-examination. J. Med. Entomol. 12, 459–463.
Foster, W.A., Lea, A.O., 1975b. Renewable fecundity of male Aedes aegypti following
replenishment of seminal vesicles and accessory glands. J. Insect Physiol. 21,
1085–1090.
Fu, G., Lees, R.S., Nimmo, D., Aw, D., Jin, L., Gray, P., Berendonk, T.U., White-Cooper, H.,
Scaife, S., Kim Phuc, H., Marinotti, O., Jasinskiene, N., James, A.A., Alphey, L., 2010.
Female-specific flightless phenotype for mosquito control. Proc. Natl. Acad. Sci.
107, 4550–4554.
Fuchs, M.S., Craig, G.B., Hiss, E.A., 1968. The biochemical basis of female monogamy
in mosquitoes. I. Extraction of the active principle from Aedes aegypti. Life Sci. 7,
835–839.
George, J.A., 1967. Effect of mating sequence on egg-hatch from female Aedes aegypti
(L.) mated with irradiated and normal males. Mosq. News 27, 82–86.
Gibson, G., Russell, I., 2006. Flying in tune: sexual recognition in mosquitoes. Curr.
Biol. 16, 1311–1316.
Gibson, G., Warren, B., Russell, I.J., 2010. Humming in tune: sex and species recog-
nition by mosquitoes on the wing. J. Assoc. Res. Otolaryngol. 11, 527–540.
Gratz, N.G., 2004. Critical review of the vector status of Aedes albopictus. Med. Vet.
Entomol. 18, 215–227.
Gubler, D., Bhattacharya, N., 1972. Swarming and mating of Aedes (S.) albopictus in
nature. Mosq. News 32, 219–223.
Gwadz, R.W., 1972. Neuro-hormonal regulation of sexual receptivity in female Aedes
aegypti. J. Insect Physiol. 18, 259–266.
Gwadz, R., Craig Jr., G., 1968. Sexual receptivity in female Aedes aegypti. Mosq. News
28, 586–594.
Gwadz, R.W., Craig, G.B., Hickey, W.A., 1971. Female sexual behavior as the mech-
anism rendering Aedes aegypti refractory to insemination. Biol. Bull. 140,
201–214.
Harris, A.F., McKemey, A.R., Nimmo, D., Curtis, Z., Black, I., Morgan, S.A., Oviedo,
M.N., Lacroix, R., Naish, N., Morrison, N.I., Collado, A., Stevenson, J., Scaife, S.,
Dafa’alla, T., Fu, G., Phillips, C., Miles, A., Raduan, N., Kelly, N., Beech, C., Donnelly,
C.A., Petrie, W.D., Alphey, L., 2012. Successful suppression of a field mosquito
population by sustained release of engineered male mosquitoes. Nature 30,
828–830.
Hartberg, W.K., 1971. Observations on the mating behaviour of Aedes aegypti in
nature. Bull. World Health Organ. 45, 847–850.
Haynes, J.W., Mitchell, E.B., 1977. Fractionated irradiation of boll weevils during
pupal development: effect of sperm depletion and transfer as measured by
female responsiveness. J. Econ. Entomol. 70, 411–412.
Helinski, M.E.H., Knols, B.G.J., 2008. Mating competitiveness of male Anopheles ara-
biensis mosquitoes irradiated with a partially or fully sterilizing dose in small
and large laboratory cages. J. Med. Entomol. 45, 698–705.
Helinski, M.E.H., Harrington, L.C., 2011. Male mating history and body size influ-
ence female fecundity and longevity of the dengue vector Aedes aegypti. J. Med.
Entomol. 48, 202–211.
Helinski, M.E.H., Harrington, L.C., 2012. The role of male harassment on female fit-
ness for the dengue vector mosquito Aedes aegypti. Behav. Ecol. Sociobiol. 66,
1131–1140.
Helinski, M.E., Hood, R.C., Knols, B.G., 2008. A stable isotope dual-labelling approach
to detect multiple insemination in un-irradiated and irradiated Anopheles ara-
biensis mosquitoes. Parasit. Vectors 1, 9.
Helinski, M.E.H., Valerio, L., Facchinelli, L., Scott, T.W., Ramsey, J., Harrington, L.C.,
2012a. Evidence of polyandry for Aedes aegypti in semifield enclosures. Am. J.
Trop. Med. Hyg. 86, 635–641.
Helinski, M.E.H., Deewatthanawong, P., Sirot, L.K., Wolfner, M.F., Harrington, L.C.,
2012b. Duration and dose-dependency of female sexual receptivity responses
Please cite this article in press as: Oliva, C.F., et al., Male reproductive biology of Aedes mosquitoes. Acta Trop. (2013),
http://dx.doi.org/10.1016/j.actatropica.2013.11.021
7
to seminal fluid proteins in Aedes albopictus and Ae. aegypti mosquitoes. J. Insect
Physiol. 58, 1307–1313.
Hodapp, C.J., Jones, J.C., 1961. The anatomy of the adult male reproduction sys-
tem of Aedes aegypti (Linnaeus) (Diptera, Culicidae). Ann. Entomol. Soc. Am. 54,
832–844.
Hoffmann, A.A., Montgomery, B.L., Popovici, J., Iturbe-Ormaetxe, I., Johnson, P.H.,
Muzzi, F., Greenfield, M., Durkan, M., Leong, Y.S., Dong, Y., Cook, H., Axford,
J., Callahan, A.G., Kenny, N., Omodei, C., McGraw, E.A., Ryan, P.A., Ritchie, S.A.,
Turelli, M., O’Neill, S.L., 2011. Successful establishment of Wolbachia in Aedes
populations to suppress dengue transmission. Nature 476, 454–457.
Howell, P.I., Knols, B.G., 2009. Male mating biology. Malaria J. 8, S8.
Jaenson, T.G., 1985. Attraction to mammals of male mosquitoes with special
reference to Aedes diantaeus in Sweden. J. Am. Mosq. Control Assoc. 1,
195–198.
Jansen, C.C., Beebe, N.W., 2010. The dengue vector Aedes aegypti: what comes next.
Microbes Infect. 12, 272–279.
Jones, J.C., 1967. Spermatocysts in Aedes aegypti (Linnaeus). Biol. Bull. 132, 23–33.
Jones, J.C., 1968. The sexual life of a mosquito. Sci. Am. 218, 108–115.
Jones, J.C., 1974. Sexual activities during single and multiple cohabitations in Aedes
aegypti mosquitoes. Physiol. Entomol. 48, 185–191.
Jones, J.C., Wheeler, R.E., 1965a. Studies on spermathecal filling in Aedes aegypti
(Linnaeus). I. Description. Biol. Bull. 129, 134–150.
Jones, J.C., Wheeler, R.E., 1965b. Studies on spermathecal filling in Aedes aegypti
(Linnaeus). II. Experimental. Biol. Bull. 129, 532–545.
Juliano, S.A., Philip Lounibos, L., 2005. Ecology of invasive mosquitoes: effects on
resident species and on human health. Ecol. Lett. 8, 558–574.
Klassen, W., Curtis, C.F., 2005. History of the sterile insect technique. In: dyck,
V.A., Hendrichs, J., Robinson, A.S. (Eds.), Sterile Insect Technique. Principles and
Practice in Area-wide Integrated Pest Management. Springer Netherlands, Dor-
drecht, The Netherlands, pp. 3–36.
Knipling, E.F., 1964. The Potential Role of the Sterility Method for Insect Population
Control with Special Reference to Combining this Method with Conventional
Methods. Agricultural Research Service (USDA), Washington, DC.
Krafsur, E., 1998. Sterile insect technique for suppressing and eradicating insect
populations: 55 years and counting. J. Agric. Entomol. 15, 303–317.
Lacroix, R., McKemey, A.R., Raduan, N., Kwee Wee, L., Hong Ming, W., Guat Ney,
T., Rahidah, A.A., Salman, S., Subramaniam, S., Nordin, S., Hanum, O.A.T., Anga-
muthu, N., Marlina Mansor, C., Lees, S., Naish, R.S., Scaife, N., Gray, S., Labbé, P.,
Beech, G., Nimmo, C., Alphey, D., Vasan, L., Han Lim, S.S., Wasi, L.A., Murad, N.S.,
2012. Open field release of genetically engineered sterile male Aedes aegypti in
Malaysia. PLoS ONE 7, e42771.
Lamb, C.G., 1922. The geometry of insect pairing. Proc. Biol. Sci. 94, 1–11.
Lang, C.A., 1956. The influence of mating on egg production by Aedes aegypti. Am. J.
Trop. Med. Hyg. 5, 909–914.
Lee, H.L., Aramu, M., Nazni, W.A., Selvi, S., Vasan, S., 2009. No evidence for successful
interspecific cross-mating of transgenic Aedes aegypti (L.) and wild type Aedes
albopictus Skuse. Trop. Biomed. 26, 312–319.
Lofgren, C.S., Dame, D.A., Breeland, S.G., Weidhaas, D.E., Jeffery, G., Kaiser, R., Ford,
H.R., Boston, M.D., Baldwin, K.F., 1974. Release of chemosterilized males for the
control of Anopheles albimanus in El Salvador. III. Field methods and population
control. Am. J. Trop. Med. Hyg. 23, 288–297.
Loor, K.A., DeFoliart, G.R., 1970. Field observations on the biology of Aedes triseriatus.
Mosq. News 30, 60–64.
Lounibos, L.P., 2002. Invasions by insect vectors of human disease. Annu. Rev. Ento-
mol. 47, 233–266.
Lum, P.T.M., 1961a. The reproductive system of some Florida mosquitoes. I. The male
reproductive tract. Ann. Entomol. Soc. Am. 54, 397–401.
Lum, P.T.M., 1961b. The reproductive system of some Florida mosquitoes. II. The
male accessory glands and their role. Ann. Entomol. Soc. Am. 54, 430–433.
Mackenzie, J.S., Gubler, D.J., Petersen, L.R., 2004. Emerging flaviviruses: the spread
and resurgence of Japanese encephalitis, west Nile and dengue viruses. Nat. Med.
10, S98–S109.
McMeniman, C.J., Lane, R.V., Cass, B.N., Fong, A.W.C., Sidhu, M., Wang, Y.F., O’Neill,
S.L., 2009. Stable introduction of a life-shortening Wolbachia infection into the
Mosquito Aedes aegypti. Science 323, 141–144.
Miyagi, I., Toma, T., 1982. Studies on the mosquitoes in the Yaeyama islands, Japan
6. Colonization and bionomics of Aedes (Verralina) iriomotensis and Aedes (Ver-
rallina) atriisimiles. Mosq. News 42, 28–33.
Moreira, L.A., Iturbe-Ormaetxe, I., Jeffery, J.A., Lu, G., Pyke, A.T., Hedges, L.M., Rocha,
B.C., Hall-Mendelin, S., Day, A., Riegler, M., Hugo, L.E., Johnson, K.N., Kay, B.H.,
McGraw, E.A., van den Hurk, A.F., Ryan, P.A., O’Neill, S.L., 2009. A Wolbachia
symbiont in Aedes aegypti limits infection with dengue, chikungunya, and Plas-
modium. Cell 139, 1268–1278.
Muir, L.E., Kay, B.H., 1998. Aedes aegypti survival and dispersal estimated by mark-
release-recapture in northern Australia. Am. J. Trop. Med. Hyg. 58, 277–282.
Nijhout, H.F., 1977. Control of antennal hair erection in male mosquitoes. Biol. Bull.
153, 591–603.
Nijhout, H.F., Craig Jr., G.B., 1971. Reproductive isolation in Stegomyia mosquitoes.
III Evidence for a sexual pheromone. Entomol. Exp. Appl. 14, 399–412.
O’Connor, L., Plichart, C., Sang, A.C., Brelsfoard, C.L., Bossin, H.C., Dobson, S.L., 2012.
Open release of male mosquitoes infected with a Wolbachia biopesticide: field
performance and infection containment. PLoS Negl. Trop. Dis. 6, e1797.
Oliva, C.F., Jacquet, M., Gilles, J., Lempérière, G., Maquart, P.-O., Quilici, S., Schoone-
man, F., Vreysen, M.J.B., Boyer, S., 2012. The sterile insect technique for
controlling populations of Aedes albopictus (Diptera: Culicidae) on Reunion
Island: mating vigour of sterilized males. PLoS ONE 7, e49414.
 ARTICLE IN PRESS
G Model
ACTROP-3239; No. of Pages 8
8 C.F. Oliva et al. / Acta Tropica xxx (2013) xxx–xxx
Oliva, C.F., Damiens, D., Vreysen, M.J.B., Lempérière, G., Gilles, J., 2013. Reproductive
strategies of Aedes albopictus (Diptera: Culicidae) and implications for the sterile
insect technique. PLoS ONE, 8, e78884.
Pates, H., Curtis, C., 2005. Mosquito behavior and vector control. Annu. Rev. Entomol.
50, 53–70.
Pennetier, 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 Anopheles
gambiae. Curr. Biol. 20, 131–136.
Perez-Staples, D., Shelly, T.E., Yuval, B., 2013. Female mating failure and the failure
of mating in sterile insect programs. Entomol. Exp. Appl. 146, 66–78.
Phuc, H., Andreasen, M.H., Burton, R.S., Vass, C., Epton, M.J., Pape, G., Fu, G., Condon,
K.C., Scaife, S., Donnelly, C.A., Coleman, P.G., White-Cooper, H., Alphey, L., 2007.
Late-acting dominant lethal genetic systems and mosquito control. BMC Biol. 5,
11.
Polerstock, A.R., Eigenbrode, S.R., Klowden, M.J., 2002. Mating alters the cuticu-
lar hydrocarbons of female Anopheles gambiae sensu stricto and Aedes aegypti
(Diptera: Culicidae). J. Med. Entomol. 39, 545–552.
Ponlawat, A., Harrington, L.C., 2007. Age and body size influence male sperm capacity
of the dengue vector Aedes aegypti (Diptera: Culicidae). J. Med. Entomol. 44,
422–426.
Ponlawat, A., Harrington, L.C., 2009. Factors associated with male mating success of
the dengue vector mosquito, Aedes aegypti. Am. J. Trop. Med. Hyg. 80, 395–400.
Proverbs, M.D., 1969. Induced sterilization and control of insects. Annu. Rev. Ento-
mol. 14, 81–102.
Provost, M.W., Lum, P.T.M., Branch, N., 1961. Rotation of male terminalia in Aedes
taeniorhynchus (Diptera: Culicidae) as affected by temperature. Ann. Entomol.
Soc. Am. 54, 896–900.
Reiter, P., 2001. Climate change and mosquito-borne disease. Environ. Health Per-
spect. 109 (Suppl. 1), 141–161.
Reiter, P., 2007. Oviposition, dispersal, and survival in Aedes aegypti: implications
for the efficacy of control strategies. Vector Borne Zoonotic Dis. 7, 261–273.
Reiter, P., Gubler, D.J., 1997. Surveillance and control of urban dengue vectors. In:
Gubler, D.J., Kuno, G. (Eds.), Dengue and Dengue Hemorrhagic Fever. CAB Inter-
national, London, pp. 425–462.
Robinson, A.S., Knols, B.G., Voigt, G., Hendrichs, J., 2009. Conceptual framework and
rationale. Malaria J. 8, S1.
Roth, L.M., 1948. A study of mosquito behavior. An experimental laboratory study
of the sexual behavior of Aedes aegypti (Linnaeus). Am. Midl. Nat. 40, 265–352.
Simmons, L.W., 2001. Sperm Competition and its Evolutionary Consequences in the
Insects. Princeton University Press, Princeton, NJ.
Smith, S.M., Gadawski, R.M., 1994. Swarming and mating in Aedes provocans
(Diptera: Culicidae). Gt. Lakes Entomol. 27, 175–184.
Soper, F.L., 1963. The elimination of urban yellow fever in the Americas through the
eradication of Aedes aegypti. Am. J. Public Health Nations Health 53, 7–16.
Please cite this article in press as: Oliva, C.F., et al., Male reproductive biology of Aedes mosquitoes. Acta Trop. (2013),
http://dx.doi.org/10.1016/j.actatropica.2013.11.021
View publication stats
Sperança, M.A., Capurro, M.L., 2007. Perspectives in the control of infectious dis-
eases by transgenic mosquitoes in the post-genomic era – a review. Mem. Inst.
Oswaldo Cruz 102, 425–433.
Spielman, A., 1964. The mechanics of copulation in Aedes aegypti. Biol. Bull. 127,
324–344.
Spielman, A., 1966. The functional anatomy of the copulatory apparatus of male
Culex pipiens (Diptera: Culicidae). Ann. Entomol. Soc. Am. 59, 309–314.
Spielman, A., Leahy, M.G., Skaff, V., 1967. Seminal loss in repeatedly mated female
Aedes aegypti. Biol. Bull. 132, 404–412.
Spielman, A., Leahy, M.G., Skaff, V., 1969. Failure of effective insemination of young
female Aedes aegypti mosquitoes. J. Insect Physiol. 15, 1471–1479.
Spielman, A., Waterman, R.E., Meller, S.M., 1974. Diversity in patterns of coital con-
tact of mosquitoes: a scanning electron microscopic study. J. Morphol. 142,
187–203.
Steiner, L.F., Harris, E.J., Mitchell, W.C., Fujimoto, M.S., Christenson, L.D., 1965. Melon
fly eradication by overflooding with sterile flies. J. Econ. Entomol. 58, 519–522.
Thornhill, R., Alcock, J., 2001. The Evolution of Insect Mating Systems. iUniverse.com,
Inc., Lincoln, USA.
Tuten, H.C., Stone, C.M., Dobson, S.L., 2013. Swarming behavior of Aedes polynesiensis
(Diptera: Culicidae) and characterization of swarm markers in American Samoa.
J. Med. Entomol. 50, 740–747.
Yuval, B., 2006. Mating systems of blood-feeding flies. Annu. Rev. Entomol. 51,
413–440.
Warren, B., Gibson, G., Russell, I.J., 2009. Sex recognition through midflight mat-
ing duets in Culex mosquitoes is mediated by acoustic distortion. Curr. Biol. 19,
485–491.
Weidhaas, D.E., Breeland, S.G., Lofgren, C.S., Dame, D.A., Kaiser, R., 1974. Release
of chemosterilized males for the control of Anopheles albimanus in El Sal-
vador. III. Field methods and population control. Am. J. Trop. Med. Hyg. 23,
298–308.
Whitten, M.J., 1971. Selection for polygamy and the sterile-insect technique. J. Econ.
Entomol. 64, 1310–1311.
Whitten, M., Mahon, R., 2005. Misconceptions and constraints. In: Dyck, V.A., Hen-
drichs, J., Robinson, A.S. (Eds.), Sterile Insect Technique. Principles and Practice in
Area-wide Integrated Pest Management. Springer Netherlands, Dordrecht, The
Netherlands, pp. 601–626.
Williams, R.W., Berger, A., 1980. The relation of female polygamy to gonotrophic
activity in the ROCK strain of Aedes aegypti. Mosq. News 40, 597–604.
Wright, J.E., Kappus, K.D., Venard, C.E., 1966. Swarming and mating behavior in lab-
oratory colonies of Aedes triseriatus (Diptera: Culicidae). Ann. Entomol. Soc. Am.
59, 1110–1112.
Zawitowski, S., Richmond, R., 1986. Inhibition of courtship and mating of Drosophila
melanogaster by the male-produced lipid, cis-vaccenyl acetate. J. Insect Physiol.
32, 189–192.