Recent studies on windborne migration of African malaria vectors raise new questions about this previously unrecognized behaviour, including questions regarding the fraction of windborne migrants in the population, as well as how this fraction might change across species, seasons, or ecological zones, among other factors. Identification of migrants in field experiments could help address such questions. In this study, wild mosquitoes were subjected to a novel, field-adapted tethered-flight assay, in order to separate them into mosquitoes with high flight activity (HFA) or low flight activity (LFA), employing flight aptitude indices reflecting flight persistence (i.e., longest flight duration, and total flight) and restlessness (i.e., flight bouts). Albeit not without exceptions, based on previous flight-mill studies, HFA is likely to be more common in long-distance migrants than LFA [31,37,65,66]. Accordingly, we evaluated variation in HFA mosquitoes, as putative migrants in Sahelian populations of An. coluzzii, An. gambiae s.s., and An. arabiensis, over various seasons and gonotrophic states. Although differences between groups were moderate, consistent with our predictions, we found elevated HFA mosquitoes in the wet season and among gravid females. However, predictions regarding species variation during the wet season were less certain. Based on Dao et al. [41], migrants were initially predicted only in An. gambiae s.s. and An. arabiensis; however, based on the aerial sampling of Huestis et al. [30], the presence of HFAs across all three species was predicted, with higher HFAs in An. coluzzii, followed by An. gambiae s.s. Consistent with Huestis et al. [30], results showed the highest proportion of HFA in An. coluzzii, with the lowest being in in An. arabiensis. Moreover, during the wet-season, An. coluzzii HFAs exhibited larger wings than conspecific LFAs.
Additional analysis indicated that wings of wet-season, An. coluzzii HFAs exhibited allometric change. Overall, these results agree with recent literature, which has found the dominance of gravid An. coluzzii flying during the wet season at altitudes of 40-290 m above ground [30].
Although an ultimate ‘comprehensive flight index,’ as well as cutoff values to distinguish between long-distance migrants and appetitive flyers are yet to be found, ad hoc indices and values have been successfully used, e.g., [33,34,36]. This study followed flight-mill based studies seeking to identify long-distance migrant insects that often relied on 1) total flight; 2) longest flight; and 3) the number of flight bouts [31,37-39]. In this study’s findings, the low absolute value of the correlation coefficient between the longest flight bout and flight bouts (r = -0.43, Fig. S3, bottom-right panel) highlights both high degree of independence of these indices and a degree of distinction between exhibiting flight persistence vs. restlessness. Only 10.5% of HFA mosquitoes based on longest flight were classified as such by flight bouts (unlike longest and total flight sharing 90.3% of HFAs), reaffirming that these modalities of flights are distinct. Overall, out of six comparisons (Table S1), HFA mosquito comparisons based on total flight revealed significant differences in five tests, whereas the same comparisons based on longest flight and flight bouts revealed significant differences in three tests and one test, respectively. This suggests that persistence of flight is a more relevant modality for long-distance migration, similar to most other studies [37]. Notably, the variable longest flight bout showed consistent trends with total flight in five (of six) comparisons, whereas the variable flight bouts showed consistent trends in only two comparisons (Table S1).
Flight aptitude variation over seasons, species and gonotrophic states
Previous work in the Sahel has shown that An. coluzzii populations build up rapidly after the first rains (i.e. May-June), and decline towards the late wet season (i.e., October), presumably entering aestivation [41]. In contrast, both An. gambiae s.s. and An. arabiensis populations absent during the dry season build up around 6 weeks after the emergence of An. coluzzii, quickly vanishing with the drying-up of surface water. The population dynamics of both An. gambiae s.s. and An. arabiensis suggest immigration (using reliable wind systems) from southerly sources, where breeding sites are perennial. Based on these findings, minimal HFAs in An. coluzzii but elevated HFAs in both An. gambiae s.s. and An. arabiensis, were predicted to occur mostly during the wet season. Additionally, because long-distance migration in most insect species occurs before reproduction [2,36,58,66], elevated HFAs in non-blood-fed females were expected, compared to gravid females. Unlike freshly blood-fed females which are burdened by the largest weight due to high water content of the bloodmeal, gravid females’ weight is intermediate and closer to the weight of the unfed female than to her weight when fully engorged [63]. Combined with the additional energy reserves that the bloodmeal offers, gravid females may be well suited to embark on long flights [45,63]. There may be additional benefits to additional mass dependent on the flight modality while propelled by the wind such as gliding, soaring, which additional studies may uncover [67]. Finally, adding nutritional analysis with future tethered flight assays may shed light on the energetic content before and after the flight and possibly the allocation of nutritional reserves between reproduction and flight.
Overall, the results agree with the predictions based on the aerial sampling results, specifically regarding 1) elevated HFA during the wet season; 2) elevated HFAs among gravid mosquitoes; and 3) the presence of HFAs among all species, with highest flight aptitude in An. coluzzii and lowest flight aptitude in An. arabiensis. However, uncertainty remains, due to partial consistencies concerning the different flight aptitude indices, as well as coarse discrimination as a result of low statistical power among groups (Table S1). For example, this uncertainty is reflected in the statistically non-significant differences in An. coluzzii between the early dry season (December-February) and the late dry season (March-April, Fig. 5), as well as the statistically non-significant difference between An. gambiae s.s. and the other two species (Fig. 6).
Wing morphometry and flight aptitude
Morphological differences between wings of HFAs and LFAs can provide strong evidence in support of migrators classification while on the ground and reveal distinct developmental program(s) for long-distance migrants. Our findings show that during the wet season, An. coluzzii HFAs had a larger wing area, attributable to an increase in both wing width and length, when compared with the LFAs. These results were not confounded by variation in body size between seasons as the analysis was confined to the wet season, when no seasonal change in wing length was detected (Fig. S3, in agreement with previous results [63,64]). The larger wings of HFAs may reflect isometric increases in all aspects of body size; alternatively, it may indicate an allometric change (e.g., an increase in wing area independent of body size). This is difficult to resolve with wild, mostly gravid mosquitoes due to the fact that variations in dry weight may confound bloodmeal size (and number) with body size. However, the allometric increase in wing width (over that expected by wing length) of An. coluzzii HFAs during the wet season further supports the validity of the classification and suggests that migrants undergo a distinct developmental plan prior to adult eclosion/emergence.
Interpretation of flight behaviour
Our prediction of higher flight activity during the wet season may sound counter-intuitive at first but fits with other empirical results and with theoretical expectations; No evidence to-date has shown high altitude flight in the early wet seasons (May-June). Huestis et al., [30] have shown that An. coluzzii and An. gambiae s.s. were collected in altitude from late July through November, similarly to flight aptitude results presented here. As discussed in Huestis et al., [30] the migration during the mid-wet season (July-September) probably follows the changing resources generated by the patchwork of precipitation that falls along the ITCZ as it sweeps through the Sahel northwards and then southwards. Unlike the early part of the season, in the later part of the wet season, i.e. October-November, the increase in elevated flight activity may represent individuals embarking on southerly, return flights before the dry season onset. Accordingly, results presented, both from altitude and the ground, support migration ‘within the Sahel’ and possibly emigration from the Sahel in October-November, when winds carrying insect southwards are more common. Additionally, these results indicate a similar capacity of these species for long-distance migration. The absence of evidence for high altitude migration during May-June may be due to the fact that during this period long-distance flight is suppressed, as conditions in Thierola are optimal, (i.e., minimal crowding, predation, competition) until local density increases (in late July when the ITCZ may well be some distance away), forming new optimal resources elsewhere. According to this hypothesis, elevated emigration will be detected in populations south of Thierola, maybe even south of Bamako, where the ITCZ (and the rains) arrives a month or so earlier.
In some species windborne migration appears to be a mandatory phase of the young adult [68], however, based on the low fraction of HFA (based on total flight), the members of the An. gambiae complex are considered here as an example of ‘partial migrators’ [38,57], in which the majority of individuals in the population do not engage in long-distance migration, even during times when migration peaks. Moreover, after arrival, immigrants will exhibit LFA; therefore, these results may identify only emigrants prior to their journey. Since the migratory phase may last only 1-3 days, we would expect to have only 1-2 days, at most, to capture a migrant before they embark on their journey. Thus, a large sample size is required to represent migrants among the more numerous ‘appetitive flyers’, which adds noise to the data and limits the statistical power of detecting differences or trends.
To date, no information is available concerning mosquito flight behaviour at high altitudes. Even if tethered flight assays accurately identify long-distance migrants, the flight data generated in the assay is unlikely to mirror free flight behaviour of mosquitoes at altitude. For example, the total flight duration may greatly underestimate actual flight in altitude – simulations based on aerial sampling data [30] suggests night-long migratory flights in some cases. Likewise, flight-mill results may fail to exhibit flight patterns matching expectations based on migration due to technical, as well as biological reasons [31,35,37,39]. Fair examples might include a lack of lift generation and a lack of tarsal contact, both of which may lead to unrealistically extended flight. On the other hand, the lack of sensory cues from either air movement, temperature and humidity gradients, or apparent ground movement, may curtail flights. In the present study, the flight aptitude assay relied on fixed-tethered mosquitoes placed in 50 mL Falcon tubes to partially isolate them from surrounding environmental cues. As a result, they may express intrinsically driven flight, as previously suggested in studies regarding locusts and moths [69,70]. Finally, flight assays for migrant identification will be more informative when additional information is gathered on each mosquito to assess agreement with other aspects of the migration syndrome, pertaining to optimal locomotor drive in young, pre-reproductive adults, with metabolism switching between flight characteristics and ovary development [36,54,58,65,71,72]. Examples of additional information might include combining data on nutritional reserves (typically elevated before migration) necessary to fuel extended flight, responses to host or oviposition site cues (typically inhibited prior and during migration) [73], levels of cuticular hydrocarbons (presumably elevated prior to migration to enhance desiccation tolerance) [64], and transcriptome analyses along with morphometrical analyses of size/shape of wings, thorax, and spiracles.