DOI: 10.5061/dryad.tb2rbp04x
title: How often are male mosquitoes attracted to humans?

description: Many mosquito species live close to humans where females feed on human
 blood. While male mosquitoes do not feed on blood, it has long been
 recognized that males of some species can be attracted to human hosts. To
 investigate how commonly male mosquitoes are attracted to humans, we
 review existing literature and performed human-baited field trials. We
 then undertook further laboratory and tent experiments to examine
 attraction to humans in males and females of three
 common Aedes species. We find that male attraction to
 humans is restricted to a handful of species including Aedes
 aegypti and Ae. albopictus. The presence of male
 host-seeking behaviour in some species may promote mating success and
 contribute to these species being globally invasive. Male and
 female Ae. aegypti show similar patterns in
 preferential attraction between different human subjects. We also
 demonstrate that mosquito repellents applied to human skin repel male
 mosquitoes. Our findings provide insights into mosquito evolution and
 behaviour and have implications for mosquito control programs,
 particularly those that involve monitoring of the male mosquito
 population.
description: <em><strong>Male
 attraction to humans
 – </strong></em><strong>Aedes</strong><em><strong> species comparison  </strong></em> We conducted experiments on mosquito attraction to humans using three species: <em>Ae. aegypti, Ae. notoscriptus</em>, and <em>Ae. vigilax</em>. The experiments were conducted in a 3 x 3 m tent under constant light levels and at room temperature. Each trial involved releasing 100 males, aged between 1 and 2 weeks, that had previously been allowed to mate, into the tent. The males were given 30 minutes to acclimate before the experiment began. The experiments were filmed using GoPro Hero 10 cameras placed at either end of the tent, with white panels (84.1 x 118.9 cm) as a background. In each trial, one side was baited with a human subject, while the other side was left unbaited as a control. Subjects stood facing the camera with their bare feet and shins in the field of view, with this position remaining consistent across trials. Subjects did not wear any perfume. The side of the baited and unbaited treatment was alternated for each trial. The number of trials, human subjects, and number of days of the experiments are summarized in Table S1 in the associated manuscript. The same batch of males was used for multiple trials on the same day but replaced daily. Treatments were recorded for 30 minutes using the time-lapse function immediately after the human subject assumed their position inside the tent. The number of mosquitoes in view of the camera was scored every 20 s, distinguishing between males that were in flight and males that landed on the human subject. For data analysis, we calculated the average number of male mosquitoes in each category over the entire trial period. <em><strong>Mosquito preferences for different human subjects</strong></em> In our experiments we found that<em> Ae. aegypti</em> males exhibit preferences towards certain human subjects. While previous research has demonstrated differential attraction of female <em>Ae. aegypti</em> mosquitoes to different human hosts, this has not yet been quantitatively reported in males. We conducted additional experiments in which we used a consistent set of five human subjects (coded A-E) who stood in pairs in opposite positions in the tent setup described in 2.3.2. The subjects were filmed for 5 minutes on each side before the sides were swapped and the procedure was repeated. This was done for each pairwise combination of the five subjects (20 combinations in total), with a fresh batch of males being used for each day of four separate days. For data analysis, we calculated the average number of male mosquitoes in view (combining flight and landed) over the 5 minutes of each trial for each human subject. We then tested all pairwise combinations of the same five human subjects for their attraction to female <em>Ae. aegypti</em> and <em>Ae. notoscriptus</em>. We used a two-port olfactometer (30 x 30 x 30 cm) similar to those used in previous studies by Ross et al (37) and Amos et al (32). The mosquitoes used in this experiment were 6-7 days post-emergence and had been allowed to mate prior to the experiment. We released approximately 50 females into the set-up and allowed them to acclimatise for approximately one minute. A box fan placed at the opposite end of the cage drew air through two traps into the cage. Pairs of subjects placed one hand each in front of one of the traps. After 5 minutes, we closed the entrance to the traps and counted the number of females in each trap and individuals remaining in the cage. The combinations of subjects and sides were randomized until all 20 pairwise comparisons between subjects and sides were completed. We repeated the experiment using the same 5 subjects for another four days using a fresh batch of females each day for a total of 10 replicates (5 per side). <em>Aedes vigilax</em> females were not assessed in this experiment due to relatively low rates of attraction to humans observed in a pilot trial using this olfactometer design. Prior to data analysis, we calculated a preference index for each person to reflect the relative attraction of each subject by dividing the number of mosquitoes attracted to one human subject by the number of mosquitoes attracted to both subjects. We determined the average preference index for all replicates of each human subject. Statistical analyses were performed using the preference index averaged across replicates.  
description: # Male attraction to humans: *Aedes species* comparison ## Datesets
 corresponding to the below methods: Landing\_tent\_*Ae. aegypti*,
 Landing\_tent\_*Ae.notoscriptus*, Landing\_tent\_*Ae.vigilax* #### Data
 was collected as follows: We conducted experiments on mosquito attraction
 to humans using three species: *Ae. aegypti*, *Ae. notoscriptus* and *Ae.
 vigilax*. The experiments were conducted in a 3 Å\~ 3 m tent under
 constant light levels and at room temperature. Each trial involved
 releasing 100 males, aged between one and two weeks, that had previously
 been allowed to mate, into the tent. The males were given 30 min to
 acclimatize before the experiment began. The experiments were filmed using
 GoPro Hero 10 cameras placed at either end of the tent, with white panels
 (84.1 Å\~ 118.9 cm) as a background. In each trial, one side was baited
 with a human subject, while the other side was left unbaited as a control.
 Subjects stood facing the camera with their bare feet and shins in the
 field of view, with this position remaining consistent across trials.
 Subjects did not wear any perfume. The side of the baited and unbaited
 treatment was alternated for each trial. The number of trials, human
 subjects and number of days of the experiments are summarized in
 electronic supplementary material, table S1. The same batch of males was
 used for multiple trials on the same day but replaced daily. Treatments
 were recorded for 30 min using the time-lapse function immediately after
 the human subject assumed their position inside the tent. The number of
 mosquitoes in view of the camera was scored every 20 s, distinguishing
 between males that were in flight and males that landed on the human
 subject. For data analysis, we calculated the average number of male
 mosquitoes in each category over the entire trial period. ####
 Interpretation of the datasets: Columns: * Species: the species tested *
 Person: which human subject was tested at the time * Treatment: either
 human or control. Control here means blank; human means subject was tested
 * Observation: 1- 90 counts (every second frame was counted) * Landed:
 number of mosquitoes that were landed on the human subject at the given
 observation * Flying: number of mosquitoes that were flying in camera view
 at the given observation * Total: the number of landed and flying
 mosquitoes combined at the given observation ## Datesets corresponding to
 the below methods: Female\_preferences\_Ae.aegypti,
 Male\_preferences\_Ae.aegypti, Female\_preferences\_Ae.notoscriptus ####
 Mosquito preferences for different human subjects In our experiments, we
 found that *Ae. aegypti* males exhibit preferences towards certain human
 subjects (see electronic supplementary material, figure S3). While
 previous research has demonstrated differential attraction of female *Ae.
 aegypti* mosquitoes to different human hosts [16,37,38], this has not yet
 been quantitatively reported in males. We conducted additional experiments
 in which we used a consistent set of five human subjects (coded A–E) who
 stood in pairs in opposite positions in the tent set-up described in
 §2.3.2. The subjects were filmed for 5 min on each side before the sides
 were swapped and the procedure was repeated. This was done for each
 pairwise combination of the five subjects (20 combinations in total), with
 a fresh batch of males being used for each day of four separate days. The
 footage was scored as described in §2.3.2. For data analysis, we
 calculated the average number of male mosquitoes in view (combining flight
 and landed) over the 5 min of each trial for each human subject. We then
 tested all pairwise combinations of the same five human subjects for their
 attraction to female *Ae. aegypti* and *Ae. notoscriptus*. We used a
 two-port olfactometer (30 Å\~ 30 Å\~ 30 cm) similar to those used in
 previous studies by Ross et al. [39] and Amos et al. [34]. The mosquitoes
 used in this experiment were 6–7 days post-emergence and had been allowed
 to mate prior to the experiment. We released approximately 50 females into
 the set-up and allowed them to acclimatize for approximately 1 min. A box
 fan placed at the opposite end of the cage drew air through two traps into
 the cage. Pairs of subjects placed one hand each in front of one of the
 traps. After 5 min, we closed the entrance to the traps and counted the
 number of females in each trap and individuals remaining in the cage. The
 combinations of subjects and sides were randomized until all 20 pairwise
 comparisons between subjects and sides were completed. We repeated the
 experiment using the same five subjects for another 4 days using a fresh
 batch of females each day for a total of 10 replicates (five per side).
 *Aedes vigilax* females were not assessed in this experiment due to
 relatively low rates of attraction to humans observed in a pilot trial
 using this olfactometer design. Prior to data analysis, we calculated a
 preference index for each person to reflect the relative attraction of
 each subject by dividing the number of mosquitoes attracted to one human
 subject by the number of mosquitoes attracted to both subjects. We
 determined the average preference index for all replicates of each human
 subject. Statistical analyses were performed using the preference index
 averaged across replicates. Effect of mosquito repellent on male
 mosquitoes that show attraction to humans After we confirmed that male
 *Ae. aegypti* show attraction to humans in our tent experiments, we tested
 whether they are repelled by a commercial mosquito repellent (Aerogard
 tropical strength insect repellent, Reckitt Benckiser, NSW, Australia)
 containing 191 g kg−1 diethyltoluamide and 40 g kg−1 N-octyl
 bicycloheptene dicarboximide. We used the same tent set-up as described in
 §2.3.2. The repellent was applied to the knees downwards to one of the two
 human subjects positioned on either site of the tent within 5 min before
 the trial began. The number of males in view was recorded every 20 s for
 10 min. The person wearing the repellent and the sides of the treatment
 and control were randomized. We ran 20 trials over 5 days with a rotation
 of nine human subjects, with the batch of 100 *Ae. aegypti* males replaced
 each day. The footage was scored as described in §2.3.2. For data
 analysis, we calculated the average number of male mosquitoes in view
 (combining flight and landed) over the 5 min of each trial. ####
 Interpretation of the columns of the tables: * Species: which species was
 tested * Left: human subject that was tested on the left hand site of the
 tent * Right: human subject that was tested on the right hand site of the
 tent * Observation: 1- 15 counts (every second frame was counted) *
 Landed: number of mosquitoes that were landed on the human subject at the
 given observation * Flying: number of mosquitoes that were flying in
 camera view at the given observation * Total: the number of landed and
 flying mosquitoes combined at the given observation * Percent: the precent
 of attracted mosqutioes for the human subject compared to the human
 subject they were tested against. This was then used to calculate a
 preference index for each subject to test whether certain human subjects
 were overall more attractive than others. *** If you have any questions
 about the data presented here, please email me at:
 veronique\_paris@hotmail.de I am very happy to provide more detail or
 explain any analysis.