The flight activity patterns of two sympatric and cryptic species of the An. farauti complex, An. farauti (s.s.) and An. hinesorum, in northern Queensland, Australia were characterized. We investigated the potential temporal differences in behaviour between the species, including total daily activity, accumulation dynamics, and dusk/early night-related flight activity, and their relationship to reproductive state. Both An. farauti and An. hinesorum exhibited daily nocturnal rhythms of flight activity with a pronounced peak at the onset of night at the end of dusk, and a smaller but obvious peak towards the end of night at dawn. During the remainder of the scotophase, intermittent and often lower level activity was observed. This general profile of 24 h activity is consistent with observations described previously in adult anopheline mosquitoes, including An. farauti [35, 37, 47, 55]. Such ‘bimodal’ activity patterns are preserved under constant dark conditions, indicating the underlying circadian clock contributes to the regulation of the behaviour. Furthermore, the bout of intense activity observed occurring at dusk/early night (ZT12–13) is relatively short-lived, being less than 45 min, and matches the length of a similar dusk bout of activity documented in the anopheline mosquitoes in the An. gambiae complex and in An. stephensi [8, 35, 37, 40, 49, 56].
Diel analysis of flight activity behaviour in the An. farauti complex has thus far been limited to a single study of adult female An. farauti Laveran [presumed An. farauti (s.s.)], examined under differing photoperiods . The study by Taylor  differs considerably from the present analysis, as it used acoustic recordings to assay flight activity, exposed mosquitoes to abrupt transitions from light to dark and dark to light, and the temporal resolution of analysis was relatively large (1 hour bins only) . Furthermore, it is unclear whether the subjects studied were captured as adults or reared from larvae. In the present study, we attempted to tease apart potential species- and reproductive stage/age-specific differences for two species in the An. farauti complex. Wild-caught female mosquitoes were subjected to a LD cycle with dawn and dusk transitions, recorded beam-break activity by minute, and analysed data by hourly and minute-to-minute resolution, thereby allowing for a precise temporal evaluation of behaviour .
When the accumulation of flight activity is measured from the start of daytime (ZT0), activity accumulates more rapidly in An. hinesorum than An. farauti. This separation becomes obvious shortly after the onset of night at ZT12, and when analyzed specifically at 75% accumulation, we see a clear significant difference. This difference is mostly due to the higher level of relative activity expressed by An. hinesorum compared to An. farauti in the dusk/early night-related bout of intense activity (~ZT12–13). The raw counts within this 1-h time bin show An. hinesorum exhibiting levels of activity twice as large as those by An. farauti. Interestingly, when we compare dusk/early night flight activity, the higher level of activity occurred for a duration of ~20 min between ZT11:55 and ZT12:15. The lower level of activity, as measured in raw counts or following Z-scored normalization, that occurs later and intermittently throughout the remainder of the scotophase/night is comparable between the two species. This focuses our attention on the dusk/early night related activity and that may relate to one or a combination of natural behaviours observed in An. farauti complex mosquitoes, such as host-seeking, and behaviours reported in other anophelines, such as migration, sugar-feeding, egg laying in gravids, and in virgins, swarming and mating. This differential activity pattern may represent an aspect of behaviour(s) that is elevated in An. hinesorum, e.g. increased feeding activity and/or longer distance/duration of flight.
Anopheles hinesorum began its nocturnal activity 5 min earlier on average than An. farauti, and the maximum peak of activity in An. hinesorum was observed to occur 4 min earlier than for An. farauti. This apparent ‘shift’ in the activity profile is of particular interest because the differences occur close to ZT12, the onset of night. The intense level and subsequent peak of activity occurring during the early night is the most dominant and consistent feature of the diel profile. This distinct profile may represent a combination of behavioural drives, many of which have been studied in anophelines and in particular members of the An. gambiae complex. These include migration, sugar-feeding, host-seeking and biting, all of which commence during twilight and the onset of night [21, 56,57,58,59].
As the majority of captured mosquitoes in the study were identified as either nulliparous or parous females, and few were scored as virgin and none scored as gravid, this makes swarming/mating and oviposition unlikely behaviours to be represented in the underlying flight activity drives of mosquitoes observed here. However, swarming and mating in other anophelines occurs during a small temporal window during dusk [60,61,62], and peak ovipositioning occurs during the first hour of the night [37, 39, 63], both of which are coincident with the peak in flight activity of An. farauti complex mosquitoes in the present study.
Due to the reproductive states of the majority of mosquitoes in the study, it is more likely that the intense early nocturnal activity in the flight profile in part reflects other factors, such as movement from resting sites, i.e. daily migration. In An. gambiae, dusk/early night is the time when females depart from their daytime resting sites [57,58,59]. Furthermore, the flight range for An. farauti has been estimated to be as much as 1.6 km .
Sugar-feeding activity in An. gambiae occurs almost exclusively at night and during the dusk/dawn transitions . However, the expression of this diel pattern is dependent upon the reproductive state of the female . Under laboratory conditions, in inseminated An. gambiae females that were either prevented from blood-feeding or blood-fed but prevented from ovipositioning, a high level of sugar-feeding activity was observed during the scotophase and with a noticeable peak during late dusk and early scotophase. Therefore, in the present study, it is possible that the nocturnal flight activity profiles of An. farauti complex females, and especially the dusk/early night peak, reflects this characteristic sugar-feeding behaviour.
Finally, host-seeking behaviour and the propensity to bite are also likely contributing drives underlying the diel flight activity profiles of An. farauti and An. hinesorum. Studies of An. farauti and to a lesser degree An. hinesorum host-seeking behaviour have been undertaken in the Solomon Islands and PNG, primarily using human landing catch (HLC) assays. In many locales, the nocturnal biting profiles for An. farauti consistently show a majority of feeding early in the night. For example, in the Solomon Islands (Haleta, Central Province), 82% of mosquitoes were captured between 18:00 and 21:00 h, and only 18% between midnight and 06:00 h . A major peak occurred at 19:00-20:00 h, declining thereafter, and then a small increase in capture rate occurred at the end of the night/dawn between 05:00-06:00 h . In Honiara, Solomon Islands, as much as 84% of An. farauti mosquitoes captured by HLCs was during the first hour following sunset . However, in comparison in PNG (Mirap, Madang Province) the shape of the profile is different: an early night peak remains, although a secondary peak occurred between 23:00 h and midnight, i.e. during the middle of the night .
A single study reports on the outdoor biting cycle of An. hinesorum as compared to An. farauti in PNG (Madang and East Sepik provinces) . While An. hinesorum exhibited an early biting profile, with 66% activity occurring between 18:00 and 22:00 h, An. farauti (s.s.) exhibited a uniform biting profile throughout the entire night and with no obvious peaks. Clearly this study reveals differences in the biting cycle between the two species, as well as An. hinesorum in this locale being an early night biter.
There are three major factors thought to contribute to the shape of the An. farauti host-seeking biting profile, namely long-term exposure to insecticide, outdoor or indoor location of the host and the lunar cycle. In areas where An. farauti (s.s.) populations have been exposed to residual spraying of DDT and/or introduction of insecticidal treated bednets, mosquitoes show significant shifts in their biting profiles, developing an exaggerated peak in biting during the onset of night, as well as increasing the number of outdoor versus indoor biting encounters [1, 8, 18,19,20,21,22,23]. For example, in the Solomon Islands (Baunasughu, Manibwenta and Maniparegho) prior to exposure to insecticide, indoor biting profiles showed a more uniform pattern during the first half of the night or show peak biting at midnight . Outdoor biting prior to insecticide exposure still showed a peak close to the onset of the night and highest levels of biting occurred during the first half of the night. Following exposure, both indoor and outdoor patterns became similar, with as much as 45% of nocturnal biting events occurring at dusk and the onset of night (18:30–19:30 h) . Finally, moonlight can impact the nocturnal biting cycle of An. farauti . In PNG (Maraga, Madang Province), the biting cycle of An. farauti showed an initial peak at the onset of night at all times of the lunar cycle. However, on moonless nights, this initial peak was most prominent, followed by a secondary peak occurring before midnight. Under a waxing moon, activity was concentrated in the first half of the night; under a waning moon activity was also highest during the first half of the night, but activity during the second half of the night remained relatively high compared to other phases of the moon. During a full moon, peak biting occurred in the middle of the night.
Relating the natural biting cycles of An. farauti and An. hinesorum is obviously complex due to these differing environmental factors. However, the location in Queensland, Australia where the mosquitoes were captured is not an area with known exposure to insecticides, mosquitoes were captured outdoors, and the conditions in the experimental chamber when assayed for flight activity were 0 lux at night (i.e. the equivalent to a moonless night). Based on the studies from the Solomon Islands and PNG, and under these environmental conditions in Australia, we might predict that the An. farauti (s.s.) mosquitoes would express a diel biting profile with a major peak occurring at the end of dusk into the early night, followed by continued lower level host-seeking activity during the first half of the night. In fact, a recent study in northern Queensland reveals An. farauti (s.s.) during the dry season as exhibiting early night peak biting. Biting was greatest at the onset of night, and while rates declined throughout the scotophase, a secondary peak occurred at 21:00-22:00 h followed by a small increase occurring near to dawn (W. K. Chow, R. D. Cooper, L. Rigby, P. Pickering, M. Lokhorst and N. Beebe, personal communication). Clearly, there is a correlative relationship between the diel cycles of flight activity and host-seeking activity for An. farauti (s.s.) in Queensland. This includes the intermittent flight activity observed during the scotophase, but especially so at the onset of night when peak outdoor biting occurs, and towards the end of night/dawn when there is a small but distinct rise in both activities. Therefore, it is likely that the diel flight activity profile reflects components of the drive to host seek and blood-feed.
In An. farauti there were differences in the flight activity profiles between nulliparous and parous mosquitoes: activity at the onset of night (ZT12–13) was higher in parous group, while conversely, activity was greater during the middle of the night (ZT16–17) in the nulliparous population. It is unclear what this finding means in terms of behavioural drives that may be different between these groups, except that the parous population is predicted to be older. There are known differences in the biting profiles of anophelines, where parous females tend to bite later than nulliparous females [65,66,67,68]. However, this phenomenon observed in the field may be associated with gravids ovipositing early in the night, thereby delaying host-seeking activity during the same night. However, it is unclear whether the differences observed in the flight activity profiles between the reproductive states/ages represent intrinsic differences in biting activity.
Thirty minutes after lights off at ~ZT12.5, An. farauti exhibited a small but significant additional elevation of activity compared to An. hinesorum for 8 min. While the meaning of this differential species activity in terms of natural behaviour is unknown, a similar differential pattern of behaviour was documented for An. gambiae and An. coluzzii males . Here, the two An. gambiae complex species show a separation in their pattern of activity in both the rise in activity after activity onset occurs during dusk, and on the decline of activity following its peak shorty after the onset of night. In that study the authors speculate that this differential pattern of activity represents either earlier swarm-assembly behaviour, or some aspect of pre/post-swarming behaviour(s), e.g. increased sugar-feeding activity and/or longer distance/duration of flight. This hypothesis may equally apply to An. hinesorum and An. farauti.
Another bout of differential activity was observed approximately an hour after lights off at ~ZT13 and for a longer duration of time (for ≤ 30 min). As this occurs in the scotophase well after the dusk/onset of night-related high intensity bout of activity, this ‘event’ may underlie a natural behaviour distinct from those associated with dusk and the onset of night.
When reproductive status/age is considered, the enhanced level of flight activity at dusk/onset of night in An. hinesorum versus An. farauti was conserved in both groups, namely inseminated nulliparous and inseminated parous. Furthermore, a distinct species difference was observed in the inseminated nulliparous group during the middle of the scotophase/night (ZT15–16), in which An. hinesorum mosquitoes had a pronounced elevated bout of activity. This revealed a conspicuous spike amongst the relatively low level activity recorded during the remainder of the scotophase. In An. hinesorum, flight activity during this hour was not only considerably higher compared to An. farauti but was relatively high compared to almost all times of the night excluding the dusk peak. Interestingly, this elevation of flight activity among inseminated nulliparous An. hinesorum at ZT15–16 is consistent with a previous study of HLAs of An. farauti complex mosquitoes in PNG (Madang and East Sepik provinces) . In this study, biting-landing catches in An. hinesorum were higher during the one hour in the middle of the night at midnight as compared to the preceding two hours and proceeding four hours. This variable pattern of landing catches was not observed for An. farauti, which was more uniformly distributed during the night. It is unclear whether this is simply a coincidence or whether the flight activity pattern observed here is associated with host-seeking behaviour and subsequent human encounters.
These behavioural data provide novel insights into the temporal regulation of flight activity that are important for ongoing efforts to understand An. farauti complex mosquitoes in Australia, the Solomon Islands, PNG, Indonesia and Vanuatu. Historically, northern Australia was malaria-endemic and, while now eliminated, there remains a risk of re-introduction and local transmission due to presence of An. farauti, and possibly An. hinesorum. As recent as 2002 there was an outbreak of P. vivax transmission in the Cairns area of Northern Queensland, illustrating the potential for local malaria transmission following introduction of the malaria parasite by infected people. Thus, the An. farauti complex poses a plausible risk for malaria transmission as Australia is surrounded by countries where malaria is endemic [1, 7]. These data also contribute to our understanding of An. farauti complex mosquitoes in malaria endemic areas, including the Solomon Islands and Papua New Guinea, where as much as 6% of the human population is infected with P. falciparum, P. vivax, or P. malariae [14, 15]. Furthermore, as the Queensland populations of An. farauti and An. hinesorum are not routinely exposed to insecticides, these behavioural data can serve as a baseline for comparison with mosquitoes from the Solomon Islands, where the timing of nocturnal biting activity has been shaped by continuing insecticide selective pressure (from insecticide treated bednets and/or residual spraying) [11, 18, 20]. Although An. farauti and An. hinesorum are morphologically indistinguishable species and are known to reside in slightly different habitats, differences in the timing of their flight behaviour, as proposed in this paper, and in host-seeking behaviour [10, 11], make vector control complicated in the areas in which the two species coexist. Understanding the causative factors underlying differences in flight activity may have real implications for controlling these two species, particularly as the number of recommended interventions expands.
The shifts in An. farauti complex biting activity from the middle of the night to earlier in the evening following insecticidal pressure (from insecticide treated bednets and/or indoor residual spraying of insecticides) [20,21,22], is a function of changes in the flight activity patterns of the mosquitoes, especially for host-seeking females. Further analysis of the flight activity of wild populations of An. farauti and An. hinesorum that have been exposed to long-term insecticide pressure would allow for this hypothesis to be tested. Such populations exist in regions of the Solomon Islands in particular. Therefore, the present analysis may provide a baseline for comparing populations of species in the An. farauti complex.
There are certain limitations to the interpretation of these data as the experimental conditions do not represent true unconstrained flight conditions in the wild. The absence of males and insufficient numbers of virgin females in this study limit our understanding of potential species differences in the timing of mating and swarming activity. In other anophelines, it is the males that first assemble the swarm around dusk into which virgin females then enter and mate [37, 69], and it has been proposed that differential timing of swarming might contribute in part to species-specific segregation of mating in sympatric An. gambiae complex mosquitoes [35, 70, 71]. Therefore, the timing of male activity may be more relevant in the context of temporal control of intraspecific mating . However, these data do provide some insight into the precise timing of aspects of flight behaviour in wild populations of predominately inseminated host-seeking mosquitoes (versus laboratory-raised colonies). It is therefore possible that additional but subtle differences exist between the two species and reproductive/age categories.