Field study
We conducted field studies in La
Lopé, Gabon in Central Africa from November to December 2016, and in
Rabai, Kenya in East Africa from April to May 2017. The period of field
study overlapped with the rainy season in each locality during which
mosquitoes had a large population size. La Lopé has an extensive
continuous tropical rainforest surrounding La Lopé village (Figure 1a).
The forest in Rabai, on the other hand, is more fragmented, with several
villages scattered around the forest patch (Figure 1b). In each
location, we searched for water-holding containers as potential mosquito
larval breeding sites in both the forests and nearby villages. A
potential larval breeding site was defined as a container holding at
least one mosquito larvae (not necessarily Ae. aegypti ) at the
time of sampling, which suggested that the site had been present long
enough for a mosquito to lay eggs. We categorized larval breeding sites
into three habitat groups: forest, peridomestic (outdoor containers in a
village area), and domestic (indoor containers) (Table 1), according to
their locations. We separated indoor and outdoor containers because
classical studies from the 1970s reported that, at least in Rabai,
Kenya, Ae. aegypti living indoor and outdoor showed distinct
behavioral and genetic differences (Leahy et al., 1978; McBride et al.,
2014; Petersen, 1977; Tabachnick et al., 1979; Trpis & Hausermann,
1975). Genetic analysis showed that these indoor mosquitoes in Rabai
were likely descendent of non-African Aaa (Brown et al., 2011;
Gloria‐Soria et al., 2016). However, this previously describedAaa -like indoor form was no longer found during our field
sampling (Rose et al., 2020; Xia et al., 2020).
In La Lopé, we visited 60 larval breeding sites in seven forest
locations and 38 sites in six village locations (Figure 1a, one village
location, Kazamabika village, is further away from the other village
locations). The sampling locations separate by 5-17 km. Forest larval
breeding sites were predominantly rock pools (n=49) around streams and
tree holes accumulating rainwater (n=11). Previous studies have
considered tree holes and rock pools as distinct mosquito larval habitat
groups (Soghigian et al., 2017). However, we only found a few tree holes
with complete data (n < 6 in all analyses), and comparing
between tree holes and rock pools is beyond the scope of this study.
Therefore, we grouped them as ‘natural containers.’ In the village,
mosquito larvae were found in a variety of artificial containers,
including construction bricks, tires, metal cans, and plastic
containers. Because residents in the village rarely store water indoors,
all village larval breeding sites were ‘peridomestic.’ In Rabai, Kenya,
we sampled 31 larval breeding sites consisting of mainly plastic
buckets, earthenware pots, and metal barrels in four villages. They were
mostly indoor (i.e., ‘domestic’) containers. The 37 larval breeding
sites in the Rabai forest were all tree holes holding rainwater (Figure
1b). We recorded the GPS coordinates of each sampling location (consist
of multiple larval breeding sites) in La Lopé, and of each larval
breeding site in Rabai, Kenya (Figure 1).
Upon identifying a potential larval breeding site in any habitat, we
measured 11-16 physical variables and collected water samples to analyze
bacterial and volatile profiles. Sample sizes for each category of
environmental variables were summarized in Table 1. Method details are
described in the following sections and the Appendix. We also collected
all mosquito larvae and pupae using pipets and reared them to adults in
field stations, keeping collections from different larval breeding sites
separate. Upon eclosion, adults were identified to genus and species
based on taxonomic keys using a dissection microscope in the field
(Rueda, 2004). We kept Ae. aegypti adults alive to establish lab
colonies for later behavioral experiments.
We categorized each larval breeding site (i.e., container) as ‘Ae.
aegypti present’ or ‘Ae. aegypti absent’ based on whether it
held any Ae. aegypti larvae or pupae (Table 1). It is worth
noting that the absence of Ae. aegypti did not necessarily
suggest an avoidance. Some sites may be suitable for oviposition and
larval development but not yet colonized by Ae. aegypti , and we
also could not observe unhatched eggs. Bearing this potential caveat, we
combined the three habitat categories and the two Ae. aegyptipresence status to generate six ‘larval breeding site groups’ (Table 1).
We focused on three comparisons for the analysis of environmental
conditions: 1) across larval breeding site groups, 2) across habitat
categories regardless of Ae. aegypti presence status, and 3)
between Ae. aegypti present and absent sites regardless of
habitats. In Rabai, almost all peridomestic and domestic larval breeding
sites sampled were present with Ae. aegypti . The only
peridomestic Ae. aegypti absent site was excluded from analyses
comparing between larval breeding site groups, but retained in
comparisons between habitats or between Ae. aegypti present vs.
absent sites.
The fieldwork in La Lopé was approved by the CENAREST with the
authorization AR0013/16/MESRS/CENAREST/CG/CST/CSAR, and by the La Lopé
National Parks with the authorization AE16008/PR/ANPN/SE/CS/AEPN. The
fieldwork in Rabai was approved by the Kenya Medical Research Institute
Scientific and Ethical Review Unit with the authorization
KEMRI/SERU/3433.