Background
Singapore was once rampant with malaria cases [
1]. Outbreaks in mainland Singapore and off-shore islands of Singapore involving the known malaria vectors, i.e.
Anopheles maculatus,
Anopheles epiroticus (previously known as
Anopheles sundaicus) and
Anopheles letifer, were reported from 1960s to 1970s [
2‐
4]. Singapore attained its malaria free status in November 1982 [
2]. The total malaria annual incidence rate fluctuated between 2.9 and 3.9 cases per 100,000 people from 1998 to 2007, and 0.5 to 2.6 per 100,000 people from 2008 to 2015 [
5]. The major causative parasite was
P. vivax, followed by
P. falciparum. While almost all cases were imported cases, there have been occasional sporadic malarial cases with no travel history (e.g. in 2010 and 2013) and 15 small sporadic localized transmissions with less than 50 cases in each outbreak [
5‐
9]. As a tourist and business hub, with high reliance on foreign personnel from malaria endemic countries, Singapore remains vulnerable to malaria unless the vector population is well understood and remains well controlled.
The last outbreaks occurred in the middle of 2009, when three clusters with a total of 29
vivax malaria patients, with no recent travel history, were identified by the Ministry of Health. Relapse cases in
vivax malaria amongst foreign workers from malaria endemic countries are common and defining if the cluster is due to local transmission is challenging. Therefore, molecular epidemiology was performed using the
msp3a and
msp1 genes of the parasite. It confirmed only two independent local transmissions in Mandai-Sungei Kadut and in Sembawang [
8]. The predominant
Anopheles found in the two areas was
Anopheles sinensis, a mosquito that was not previously recognized as a vector in Singapore. Transmission in Jurong could not be confirmed as the infecting parasite from the cases showed no genetic link among them. Correspondingly, no potential Anopheles vectors, including
An. sinensis, were found in the vicinity. Although
An. sinensis has been implicated as the malaria vector in some parts of Asia, including Korea, China, Japan and Vietnam, it has never been reported as a vector in Singapore [
10‐
23].
Anopheles sinensis is a member of the
Hyrcanus group. Due to morphological complexity and similarity among the members of the group, the members have often been misidentified and their respective vector status is confusing [
24,
25]. Furthermore, confirming
An. sinensis as vector has been made more complicated by the existence of two forms, i.e. Form A and B, both of which are morphologically identical [
26‐
28]. Yet, hybridization of these two forms showed they were genetically compatible, yielding viable progeny, complete synaptic polytene chromosomes and was said to exhibit cytological polymorphic races [
29,
30].
The vector competence of these two forms of
An. sinensis is not fully understood. To date, only a single study reported that
An. sinensis Form B was able to produce sporozoites in the salivary glands, while Form A could not [
31]. Based on the cytological polymorphism of
An. sinensis and on previous vector competence studies [
29‐
31], it was noteworthy that the two forms could have different vector abilities in malaria transmission depending on their geographic regions. This study aims to characterize Singapore’s strain of
An. sinensis, including its vector competence.
Discussion
In the 2009 malaria outbreaks in Singapore,
An. sinensis, was the predominant
Anopheles species found in local outbreak areas. Together with classical and molecular epidemiological data, it was suggested that
An. sinensis was the probable malaria vector [
8]. This study has now determined that
An. sinensis in Singapore belongs to Form A of the species and more importantly, provided evidence that it is a potential malaria vector in Singapore. Due to limitation in transferring of
Anopheles eggs and variation of rearing conditions, dissection of few mosquitoes in the initial four experiments could only be carried so as to confirm the successful development of
vivax oocysts in
An. sinensis Form A. Following that, minimal number of dissection for oocysts was needed, and infected
An. sinensis Form A mosquitoes could be reserved for salivary glands dissection on 16 DPI, which is essentially crucial to determine the vector status of
An. sinensis Form A.
Anopheles sinensis is classified under the
Hyrcanus group. Under this group, it comprises of several species having minute differences in their morphology. From eight species [
25], the total number of species within the
Hyrcanus group increased to 27 [
47,
48]. Using integrative taxonomy (the combination of morphological and molecular tools), Singapore’s
An. sinensis was, for the first time, confirmed to be Form A. All
An. sinensis collected from the field, including those collected from the 2009 local malaria outbreak [
8] formed a clade with Form A of Thailand. No Form B was found. Although
ITS2 showed homogeneity among the
An. sinensis in Singapore, the
COI analysis suggest some heterogeneity which probably could only be deciphered using techniques that provides better resolution e.g. Restriction-site Associated DNA sequencing (RADseq) [
49] or whole genome sequencing [
50].
Although there have been multiple reports of experimental infection that resulted in
An. sinensis producing sporozoites [
51‐
53], only two [
18,
54] natural infections of
An. sinensis have been recorded in the Southeast Asia. However, these findings were called into doubt [
25]. Thailand has never implicated
An. sinensis as an important malaria vector, with contrasting results in vector competencies being reported from two studies. One reported 61.5% of infected mosquitoes presenting with sporozoites; another showed only 5.88% in Form B and none in Form A [
31,
52]. On the contrary, we have shown that Singapore’s strain of
An. sinensis (Form A) is a potential vector of
P. vivax, with competency level nearly equivalent to
An. cracens. It could have been the vector of the 2009 local malaria outbreak. Together with the data from Korea, China and Thailand, the vector competencies of
An. sinensis appears to be highly dependent on the taxonomic forms [
31] and geographical areas of the mosquitoes, and the perhaps genetic diversity parasites [
51], Difference due to experimental design also cannot be excluded. More work is needed to understand
An. sinensis and its role in malaria transmission.
Although we are aware that experimental susceptibility tests do not necessarily reflect the role of malaria transmission in nature, such findings highlight the potential risk of
An. sinensis if its population is left uncurbed. The habitats of
An. sinensis in Singapore are not restricted to the rural, usually coastal, areas of Singapore, where typical malaria vectors were found. They appear to thrive well in urban freshwater bodies such as ponds and reservoirs that have become very integrated into the Singapore landscape. Being zoophilic, numerous reports classified
An. sinensis as an inefficient or an unimportant vector even though infections were detected naturally and experimentally [
13,
14,
51]. However, in an urbanized city like Singapore, where animals are scarce,
An. sinensis could readily bite human since human density is considerably high [
13,
15]. Thus, the risk of malaria transmission by
An. sinensis could not be disregarded, and warrants monitoring and surveillance. During the investigation and mitigation of
An. sinensis breeding, it was found that they can be controlled by removing algae that develop in these water bodies. More work is ongoing to determine the risk of
An. sinensis breeding in urban Singapore.
Authors’ contributions
BM, NLC, LR and FN conceived the study. PSC, CA and MABAR were responsible of colonization of mosquitoes. CA, PSC, and LPSG were responsible of infection, detection and quantification of oocysts and sporozoites in mosquitoes. PSC was responsible in the preparation of manuscript. PSC conducted the DNA extraction, PCR and sequence alignment and analysis. PC was responsible of parasite DNA extraction and qPCR. CCS, CC and BR worked on implementing the study on a field level and reviewed the manuscript; CCS and LDQ provide advice in the experimental designs of this study. All authors read and approved the final manuscript.