Sexually mature male mosquitoes leave their diurnal resting sites at species specific times to commence swarming (Figure ). The antennal fibrillae become erect and typically remain so for 1-2 h [14
], however in laboratory-reared An. stephensi
this period lasted up to six hours [37
]. Swarming commenced at various times for different species [25
]. As dusk nears, males begin to fly over the swarming arena [25
]. Gradually more males join forming large, loose aggregations. Eventually the males move from a distended, non-specific circling motion into a more rigid, condensed group [25
]. Swarms are comprised mainly of males (Table ) with numbers varying from approximately 5-5000 individuals.
The sexually mature male: A. diurnal indoor resting, B. 1 hour prior to swarming the antennal fibrillae become erect, C. males depart to commence swarming, D. After swarming ceases, male ingest a sugar meal prior to resuming indoor resting.
Captured anopheline males and females from various wild swarms
Both in the laboratory and in the field, females become active after the males. Hence copulations are not usually observed until 5-20 min after swarm initiation. In Malaysia, An. philippinensis
started mating 5 min after swarming commenced while An. hyrcanus
began mating 10 min later [33
]. An. gambiae
and An. funestus
males started mating approximately 10 min after swarming began [26
] and An. freeborni
commenced mating 5-10 min after swarming began. It is unknown why males appear at swarming arenas before females. Females may arrive later than males due to different phototactic stimuli. This may also be a behavioural response by males to ensure that they arrive at mating arenas before females to optimize their chances for mating.
Once formed, the swarm begins to move as a single unit (Figure ). Male An. gambiae
were observed to be unevenly distributed within the swarm with more males occupying the centre versus the periphery [38
]. This centralized positioning may increase a males' chance to acquire a mate by either occupying the space most frequented by females or being more likely to better detect approaching females. It may also be a function of males aligning themselves with a potential swarm marker.
Anopheles pharoensis Theobald swarming (Photo courtesy of J.D. Charlwood).
Swarming males have been reported to either mate with females as they penetrate the group [31
] or as they temporarily departed to secure a female flying nearby [23
]. Approaching females may be detected by their wing beat frequencies, which is typically lower than that of males. The range for detection is unknown, but in the laboratory An. gambiae
males could detect a female at distances of 50-75 mm [14
]. The ability of the male to detect conspecific female wing beats and reject heterospecific females has been proposed as another barrier to interspecific mating in An. gambiae s.l.
]. Males of some mosquito species attempt to harmonize their wing beats with nearby insects (sexual tuning) to determine if they are conspecific and of the opposite sex [41
] although this remains unknown for anophelines. Wild populations, however, display a greater variance in their tones resulting in widespread overlap and reduced discrimination of wing beat sound as a single way for males to identify conspecific females at a distance [42
The actual period during which mating occurs in the swarm is brief. Once mating commences, copulating pairs are seen usually for 10-30 min as darkness approaches [30
]. This shortened mating period may be related to the length of time spent in copula
. Copulation in An. gambiae
lasts 15-20 s [14
] and approximately 27 s in An. culicifacies
Detailed studies on copulation in Anopheles
have shown that as a male approaches the female he grasps her with his tarsal claw on his first pair of legs and then swings his abdomen up to clasp her genitalia. Once interlocked, the male releases his tarsal grasp of the female and they assume the venter-to-venter position and resume flying [14
]. The copulating pair then departs the swarm and flies off to complete the mating after which the male is assumed to rejoin the swarm. It is unknown if males will mate again on the same night, however observations exist of anopheline males returning to a swarm after mating (J.D. Charlwood, pers. comm.
Male courtship is considered to be absent in many swarming insects [45
]. Instead, when females are a limiting factor and the costs of mate finding are high, males may forgo "choosy" behaviours so as to not waste potential mating opportunities [46
]. This results in a "no-choice" scenario for both sexes as males cannot refuse a mate based on some phenotypic character and females will have limited choice, if any, in accepting a male's advances. There are no reported field observations of swarming males seeking out and rejecting a female. Possible indiscriminate mate choice by males was reported in wild An. funestus
. Wing lengths of females caught in copula
were not different from those of newly emerged females [32
]. Therefore, males are likely to mate with females of unknown quality as their chances of meeting and copulating are low. In contrast, tethered virgin females have been reported to dislodge males attempting to copulate in the laboratory [14
It remains unknown if males are capable of assessing female phenotypic quality and differentially allocate copulatory resources based on female quality during mating [46
]. Due to low female availability, the reported high risk of predation during swarming, and the energy budget required for doing so, males will have limited opportunities to mate [21
]. Therefore, it would benefit males to invest in quality inseminations thereby imposing monogamy on females via the insertion of a large volume of sperm and a mating plug. Natural behaviour corroborates this as polyandry is uncommon in wild An. gambiae
(2.5%) (reviewed in [21
]). However, males have been reported to resume swarming after mating. Therefore it may be possible for males to achieve a second mating on the same evening. This would mean that males might not be investing in one large ejaculate that depletes their capacity, but several smaller ones to ensure that they may mate with quality females. Indeed, laboratory reared An. gambiae
are capable of inseminating up to five females, of which two included sperm and mating plugs [48
Mating without swarming has been reported in several anophelines. Mating in alternate venues is often employed by individuals who are competitively disadvantaged [22
]. As in other insect groups, smaller males or males who are physiologically stressed and not capable of swarming for long periods of time may adopt alternate mating strategies to ensure they pass on their genes [22
]. Since the male antennal fibrillae become erect one hour before swarming commences, males should be sexually responsive to females in the vicinity even when not swarming ([23
] and reported in [34
]). Alternate venues may also favour sexual choice by allowing individuals a chance to select mates based on phenotypic qualities. Since females are considered to be monogamous, pre-copulatory choice would prevent mating with lesser quality males.
Once mating is completed, the female is assumed to become refractory to further mating due to the transfer of several male compounds. After ejaculation, males insert a mating plug composed of MAG substances [49
]. In other culicids, MAG substances are responsible for causing the switch of female behaviour from unmated to mated. This was originally reinforced by reported polyandry in An. gambiae s.l.
due to the inability of hybrid males with abnormal MAGs to prevent remating [50
]. However, in the laboratory it was determined that the presence of sperm in the spermatheca is responsible for the switchover state in anophelines [51
]. Though the function of MAG proteins in anophelines is unknown, several male-specific accessory gland proteins that are known to trigger post mating behavioural changes in Drosophila
] have recently been identified in An. gambiae
. Similarly, the insertion of the mating plug, and its associated substances, has been shown to turn off the mating machinery in An. gambiae
females possibly resulting in a physiological barrier to remating [53
]. Although superficially contradictory to earlier work [49
], the processing of the MAG substances within the female's atrium may be necessary to stimulate post-mating processes, which might have been inadvertently by-passed in the original experiments [51
The number of matings occurring in the swarm declines until darkness when most males disperse to rest and replenish energy reserves through nectar feeding. Males were found to expend up to 50% of their energy reserves during swarming and to feed shortly before dawn [54
]. This meal was used mainly to fuel flight during the period prior to scotophase [55
]. Nocturnal feeding, therefore, is required by males to replenish energy reserves utilized during mate seeking as well as for daily survival.
It has been argued that swarming is ritualistic and not important in mating (reviewed in [20
]). Swarming without mating [23
] or infrequent pairing [23
] has been observed for many species. However, a great number of copulating pairs have been observed in swarms of these same species [31
]. The crepuscular nature of swarming often hinders the researcher's ability to observe actual mating. Similarly, the number of sexually mature males far outnumbers the number of virgin females on any given evening [34
]. This would result in an underestimation of the actual number of matings. Swarming may still be the most efficient way for males and females to meet in highly dispersed populations.
It is, therefore, unknown what male or female phenotypic characters, if any, are associated with mating success. Similarly, of the males that do mate, it remains unknown whether they copulate with several females to increase their chance of securing a high quality female (i.e. cryptic male choice).