Background
The Bioko Island Malaria Control Project (BIMCP), funded by a consortium led by Marathon Oil Corporation and the Government of Equatorial Guinea, implemented a malaria control program on Bioko Island, Equatorial Guinea in 2004. The BIMCP employs vector control and malaria case management strategies to reduce and eventually eliminate malaria transmission on Bioko Island. Over the first 5 years of the malaria control programme there has been a 64 % reduction in malaria related death among under-5 year olds from 2004 to 2009 on Bioko Island [
1].
At its inception, the BIMCP’s anti-vector intervention was based on indoor residual spraying (IRS) of pyrethroid insecticides. This resulted in the elimination of
Anopheles gambiae S form and
Anopheles funestus populations from the island [
2]. While
Anopheles coluzzii (formerly
An. gambiae M form) and
Anopheles melas remained on the island, their population sizes were drastically reduced [
3,
4]. Though the BIMCP has been successful at reducing
Anopheles populations and malaria incidence [
1,
5], entomological inoculation rates (EIR) as high as 840 were observed on Bioko Island in 2009 [
6]. However, the EIR has been reduced dramatically since then (unpublished results), even though malaria remains a major public health burden on the island.
The efficacy of IRS and LLIN interventions are predicated on the feeding and resting behaviour of the vectors [
7].
Anopheles coluzzii and
An. melas are both largely endophagic and endophilic [
8]. However, a shift in mosquito host-seeking behaviours in response to anti-vector interventions has been observed in several malaria vectors in various parts of the world [
9]. In several parts of its range, including India, Thailand, China and Vietnam, the Asian vector
Anopheles minimus became more exophagic and exophilic, and in some cases more zoophilic and crepuscular, as a result of DDT spraying in the 1970s and 1980s [
10]. Following these changes, anti-malarial spraying was reportedly less effective at interrupting transmission [
7]. More recently, there have been several accounts of increased outdoor host-seeking in African
Anopheles populations following an increase in vector control campaigns [
10‐
15]. Some of these are probably phenotypic responses to the presence of excito-repellent insecticides, but some may be adaptive and due to evolved changes in genetically controlled behaviour—i.e., behavioural resistance (reviewed in [
9]).
A limited study on Bioko Island in 1993 reported that outdoor (human landing catch) HLC collections caught no anophelines, while parallel indoor HLC collections caught between 3.7 and 6.0 anophelines (
An. gambiae s.l. and
An. funestus) per person/night [
16]. However, these indoor/outdoor paired HLC collections were only conducted over two nights from three locations on Bioko Island [
16]. Though the indoor/outdoor sampling was marginal, these observations suggested that mosquito feeding was occurring primarily indoors and that IRS and/or LLIN interventions would be highly effective [
16,
17]. In 2009, the vector monitoring programme of the BIMCP was greatly expanded and paired indoor/outdoor HLC collections began in sentinel sites across the island. These HLC collections made clear that outdoor biting on Bioko Island was common in
An. coluzzii and
An. melas [
6,
18].
The current study examines the effects of vector control on An. coluzzii and An. melas abundance and feeding behaviour on Bioko Island from 2009 to 2014. Entomological surveys were conducted and analysed across four locations in Bioko Island containing varying combinations of the two Anopheles vectors. Mosquito collections were analysed for changes in mosquito abundance, species composition and the proportion of outdoor host-seeking An. gambiae s.l. at paired indoor/outdoor HLC site. There was a decrease in the number of mosquitoes caught per collector night and an increase in the proportion host-seeking outdoors for both An. coluzzii and An. melas in all four villages. The hypothesis that this shift was a phenotypic response due to insecticide repellency was tested, and concluded that this is probably not the primary underlying cause of the observed changes. This leaves the possibility that the observed shift in feeding behaviour may be due to adaptive changes in the malaria vectors of Bioko Island.
Discussion
Significant changes in
An. gambiae s.l. vector abundance and host-seeking behaviour occurred under the BIMCP between 2009 and 2014. During this time, the number of
An. gambiae s.l. collected per person night was reduced by 93 and 87 %, for indoor and outdoor collections respectively. The BIMCP began in 2004 and has reduced mosquito populations dramatically over its first 5 years [
2‐
4]. This data indicates that the BICMP has continued to reduce malaria mosquito populations, 6 years into the project. By 2014, all four villages had, on average, under ten
An. gambiae s.l. collected per person night. Although current vector control tools used on Bioko are only applied indoors, there was a decrease in the rate of both indoor and outdoor host-seeking mosquitoes. In comparing the decrease in mosquitos collected per person night between villages, Mongola had a larger reduction in host-seeking mosquitoes compared to Arena Blanca, Biabia and Balboa. This appears to be due to the large number of host-seeking
An. gambiae s.l. present at the beginning of the study in Mongola compared to the other villages (Fig.
2).
During this time, the proportion of host-seeking
An. gambiae s.l. caught outdoors, in paired indoor-outdoor human landing catches, has increased. On Bioko Island, the proportion caught outdoors in paired HLC catches has now reached over 80 % in some locations: this is higher than other recent reports of outdoor feeding proportions in response to IRS or LLINs [
10‐
15]. For example, in Benin, following large scale LLINs distribution, the proportion of
An. funestus caught outdoors in paired human-landing catches increased from 45 % in 2008 to 68.1 % in 2011 [
13]. In Tanzania, following two LLINs distributions, this proportion increased from 39.2 % in 1997 to 70.2 % in 2009 in
An. funestus, although
An. gambiae host-seeking behaviour did not change during this same period [
10].
High outdoor biting rates were reported for
An. coluzzii (then referred to as
An. gambiae M) on Bioko Island in 2009 [
6,
18]. This contrasted with reports from Bioko prior to 2004, in which outdoor biting was not observed [
16,
17]. This implies a behavioural shift favoring outdoor host-seeking, possibly as the result of intense selection pressure imposed by the indoor application of insecticides. This observation was consistent with other studies both in Africa and elsewhere reporting an increased proportion of outdoor host-seeking in response to IRS or LLINs [
10‐
15]. Here, we have shown that a shift in host-seeking behaviour has accompanied the IRS-based vector control programme under the BIMCP. Assuming that there was no systematic change over the study period in the methods used for human landing catches (including collector bias), or in the relative accessibility of the collectors stationed indoors and outdoors, this change in the proportion caught outdoors implies a change in the intrinsic behaviour of the local mosquitoes.
If this kind of behavioural shift is inherited, adapted and evolved, then it is worrisome. However, it is important to emphasize that so far, the substantial overall reduction in
Anopheles mosquito populations in Bioko has resulted in a decrease in the total number of outdoor blood meals (Fig.
2) [
26]. A recent analysis, that combined human night-time behaviour with mosquito host-seeking behaviour, showed that malaria transmission on Bioko Island is still primarily driven by indoor contact between vectors and humans [
26]. This is because humans are primarily indoors during the times when mosquitoes are searching for hosts. This is consistent with the malaria transmission studies conducted in Burkina Faso, Tanzania, Kenya, and Zambia (including those mentioned above) where almost all human exposure to
An. gambiae complex mosquitoes and
An. funestus occurred indoors, even though outdoor biting rates were high during collections [
27].
Nonetheless, although outdoor biting is not currently the primary site of malaria transmission, it is recognized as a considerable potential problem for the future. It is now widely accepted that outdoor transmission will need to be addressed in order to achieve the goal of eliminating malaria from many endemic areas [
9,
28,
29]. Indeed, it has been reported that among people protected by LLINs at night, about half of the exposure to malaria vectors occurs outdoors [
30,
31]. Furthermore, simulation studies have suggested that increased outdoor biting, if it is inherited and thus a form of behavioural resistance, may have as large an impact on our ability to control vectors as physiological forms of insecticide resistance [
32].
In some situations, a plausible explanation for an increase in outdoor host-seeking of malaria vectors during vector control programmes is the reduced availability of host indoors, e.g., because they are protected by bed nets. However, vector control on Bioko Island has consisted primarily of IRS, with average bed net coverage being low [
1]. Unless mosquitoes are greatly deterred or repelled by the insecticide, IRS does not make hosts unavailable to host-seeking mosquitoes. If the IRS insecticide exhibits deterrency/repellency effects on host-seeking
An. gambiae s.l., there would presumably be an increase in outdoor host-seeking in the month following an IRS spray round relative to the month prior, assuming that the houses used for sampling were sprayed. However, when the proportion of host-seeking mosquitoes caught outdoors in the months surrounding an IRS spray round was analysed, there was no increase in this proportion following spraying, regardless of the insecticide sprayed. Indeed, there was an increase in the proportion of indoor host-seeking mosquitoes in the month following a deltamethrin spray compared to the month prior. It is unlikely that this change in host-seeking behaviour is due to declining insecticide residues in the homes as previous reports suggest that IRS spray of bendiocarb remain on the interior walls of homes for 3 months and the long-lasting encapsulated deltamethrin is expected to last even longer [
33]. From 2009 to 2013, the houses which were sprayed were not individually recorded. This could undermine the power of this analyses. In 2014, the status of collection houses was recorded and all houses used in the collections that year were indeed sprayed. A large number of collections were included in this analyses (92 houses) and undoubtedly the vast majority of them would have been covered during the spray rounds. Therefore, it is unlikely there were a large number of unsprayed houses included in the collections which might confound the analysis. Currently, there is no clear explanation for why the proportion of indoor host-seeking mosquitoes would increase in the month following a deltamethrin spray compared to the month prior and future work will be required to uncover the basis for this behaviour.
If the behaviour of outdoor-host-seeking is heritable, and if outdoor hosts are available, then presumably the suppression of mosquito populations by heavy usage of IRS or LLINs could select for a mainly outdoor-feeding
Anopheles population. If such a population emerged, it would be fully adapted to avoid indoor-applied insecticide. This could result in a resurgence of malaria transmission and the failure of programmes based on current vector control tools [
9]. It cannot be assumed that the increased outdoor feeding has an underlying genetic basis, but this possibility is clearly of great concern.
Authors’ contributions
JIM data analysis and manuscript preparation. SP molecular analysis, data analyses and manuscript preparation. ZPH data analyses. MCM statistical analysis. GF, AM and HJO supervised and planned collections. VK and VPR molecular analyses. GG processed and analyzed IRS coverage data. IK and CS planned study design and collections, manuscript editing. JL manuscript editing, MAS supervised molecular analyses, planned study design and collections, manuscript preparation. All authors read and approved the final manuscript.