How often are male mosquitoes attracted to humans?

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.

Methods
Male attraction to humans – Aedes species comparison  

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 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.

Mosquito preferences for different human subjects

In our experiments we found that Ae. aegypti males exhibit preferences towards certain human subjects. While previous research has demonstrated differential attraction of female Ae. aegypti 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 Ae. aegypti and Ae. notoscriptus. 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). 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.