Background
Malaria still remains a public health problem throughout tropical and sub-tropical regions of the world, with an estimated 214 million new cases and 438,000 deaths in 2015 [
1]. Four
Plasmodium species are recognised as causing human malaria;
Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale and
Plasmodium vivax, but recently a fifth species,
Plasmodium knowlesi, has been recognized as causing symptomatic malaria in humans [
2‐
4].
Plasmodium knowlesi, transmitted by the forest-dwelling
Anopheles from the Leucosphyrus group, is an emerging cause for zoonotic human malaria in Southeast Asia [
3,
5‐
7]. Malaysia has had a successful malaria control programme, aimed to eliminate malaria by 2020, with marked reductions in reported cases of
P. falciparum and
P. vivax, but there has been a recent increase in
P. knowlesi cases [
8‐
11].
Plasmodium knowlesi is now the most common cause of malaria in the Malaysian state of Sabah, accounting for 62 % of all malaria incidences in 2013 and presenting a threat to malaria elimination [
8].
It has been proposed that land use change, including deforestation, forest fragmentation and agricultural practices, has increased the incidence of
P. knowlesi by increasing the encroachment of humans into previously forested areas, allowing a higher interaction between vectors and human and macaque hosts [
9,
12]. The increase in
P. knowlesi cases may also be underestimated due to misdiagnosis during microscopic examination [
13,
14]. Microscopy of stained blood smears allows differentiation between species, but frequent misdiagnosis occurs in areas containing
P. falciparum,
P. vivax and
P. knowlesi [
14].
Monkeys, particularly the long-tailed macaque (
Macaca fascicularis) and the pig-tailed macaque (
Macaca nemestrina) found in Southeast Asia, are the two main natural hosts of
P. knowlesi [
15]. A study in Sabah showed nearly all patients with
P. knowlesi malaria had a recent history of forest or forest-edge exposure, and had seen a monkey in the preceding month [
16]. Most members of the Leucosphyrus group, from the genus
Anopheles, feed primarily on monkeys in the canopy and are capable of transmitting various
Plasmodium species [
17].
Anopheles balabacensis is the predominant vector of human malaria in Sabah [
18,
19], and has also been incriminated as a
P. knowlesi vector [
20,
21].
Most primates are arboreal. Although some species of chimpanzees, baboons and macaques rest and feed at ground level during the day, primates almost always sleep in the canopy during the night [
22]. As
Anopheles species generally bite between 6 p.m. and 6 a.m., primate roosting sites are potentially a key location for disease transmission between primate hosts. It is hypothesized that vectors are biting humans at ground level, but if given the opportunity, will bite at canopy level. This study expects that key vector species should be present in the disturbed forest habitats where people come into contact with monkeys, but it is unknown which species should expect to be present in primary forest.
Since transmission is increasing in Sabah, it is important to identify the P. knowlesi vectors present and understand their biting behaviour within forest habitats. While other vector assessments are ongoing in the Interior, West Coast, Kudat and Sandakan Divisions in Sabah, the Tawau Division has not been studied. This study was conducted to determine the vertical distribution of mosquitoes and their biting preference in Sabah, Malaysia.
Discussion
Studying the anthropophily of simian malaria vectors in the canopy of tropical forests is essential because the hosts are primarily arboreal. In order to develop, sustain or adapt a good control programme, it is important to monitor mosquito populations as well as their hosts and host-seeking preference, distribution and behaviour. Although previous studies in Southeast Asia have used monkey-baited traps at different canopy heights [
12,
40,
41], this is the first study to attempt human landing catches, using this method, in the canopy. This study found that there was a higher abundance and human landing rate of mosquitoes at ground level, where people tend to be, than in the canopy where the simian hosts reside. This trend was driven by
An. balabacensis, a key malaria vector in Sabah, and highlights the potential importance of this species in transmitting
Plasmodium species from simian to human hosts.
Anopheles balabacensis was the most abundant mosquito in all sampled areas, accounting for 70 % of all collected species.
Anopheles balabacensis is considered the most important vector of human malaria parasites on Banggi Island and mainland Sabah, Malaysia [
18,
19,
30,
42]. In Sabah,
An. balabacensis was found to be mainly exophagic, but could also be endophagic and exophilic [
30,
43]. These behaviours cause
An. balabacensis to be an effective vector of
P. knowlesi from human to primate hosts. There were also two distinct subpopulations, one more zoophilic and one more anthropophilic [
44‐
46].
Anopheles balabacensis occurs in forested areas, and readily bites human and monkey hosts, making it an ideal vector of simian malaria [
20,
47].
Currently insecticide-treated bed nets and indoor residual spraying are the two main control methods in Malaysia [
48]. This study showed
An. balabacensis bites as early as 18:00 h in the Tawau Division. Other studies have shown the species bites as early as 18:00 h in recent years in comparison to late night biters in previous decades [
44,
49‐
51]. Given that
An. balabacensis is early evening biting, highly anthropophilic, exophagic and exophilic, current control methods are not sufficient to break the transmission cycle of
P. knowlesi [
52]. In Vietnam and Cambodia, long-lasting insecticidal hammocks (LLIH) were shown to reduce malaria incidence and prevalence in forested areas, and may prove to be an additional effective tool in reduction of malaria in Malaysia [
52‐
54]. The use of repellents have been used for malaria control, but need to be tested in forest and plantations areas.
This study found a different community composition of mosquitoes in the canopy to that at ground level. Different mosquito species have particular flight distributions, with certain species flying and feeding close to ground, some species showing a preference for higher canopy heights, while others show a random distribution [
55,
56]. The percentage biting at different canopy heights can be affected by microclimate conditions, such as relative humidity, temperature, wind speed and rainfall [
57,
58], but may also change according to time of day [
59].
Moonlight appeared to have a significant impact on mosquito activity, with human landing rates increasing on bright nights. Although some studies have shown moonlight increases relative abundance of biting vectors [
60‐
64], others have shown a decrease [
65‐
68] or no effect at all [
69]. Collection bias was reduced in this study by collecting in each area under different phases of the moon.
This study also showed how forest disturbance affected mosquito abundance, species richness and human landing rates. Vector abundance was greater in the lightly modified virgin jungle reserve and heavily modified logged forest than in the unmodified primary forest. These results may be explained by the availability of larval breeding sites. Wheel tracks in logged areas due to logging activities can provide breeding sites for a range of mosquito species, whereas wheel tracks are not present within primary forests or virgin jungle reserves [
31]. Species richness, estimated by the Chao1 index and ACE, differed across forest disturbance and height, with logged forest and ground level having a higher species richness than primary forest and canopy.
Conclusions
This study has given an overview of mosquito species found in the Tawau Division, including the anthropophily of P. knowlesi vectors at canopy and ground levels. Anopheles
balabacensis was the predominant species found in primary forest, virgin jungle reserve and logged forest with a preference for landing on humans at ground level. As An. balabacensis is a vector of human and simian malaria, these findings will be useful for the planning of control strategies of malaria vectors.
Authors’ contributions
HLB conducted the fieldwork, analysis and wrote the manuscript. HLB, RME, IV, CD and JDM were involved in the design of the study. RME and JDM supervised the study. All authors read and approved the final manuscript.