Pang et al. Malar J (2017) 16:465 DOI 10.1186/s12936-017-2114-3 Malaria Journal RESEARCH Open Access Singapore’s Anopheles sinensis Form A is susceptible to Plasmodium vivax isolates from the western Thailand–Myanmar border Sook‑Cheng Pang1,7†, Chiara Andolina2,3†, Benoit Malleret4,6, Peter R. Christensen3, Sai‑Gek Lam‑Phua1, Muhammad Alif Bin Abdul Razak1, Chee‑Seng Chong1, Daiqin Li7, Cindy S. Chu3, Bruce Russell5, Laurent Rénia4, Lee‑Ching Ng1* and Francois Nosten2,3 Abstract Background: Singapore has been certifed malaria-free by the World Health Organization since November 1982. However, sporadic autochthonous malaria outbreaks do occur. In one of the most recent outbreaks of vivax malaria, an entomological investigation identifed Anopheles sinensis as the most probable vector. As metaphase karyotype studies divided An. sinensis into two forms, A and B, with diferent vector competence: the investigation of vector competence of An. sinensis found in Singapore was thus pursued using Plasmodium vivax feld isolates from the Thai‑ land–Myanmar border. Methods: Adults and larvae An. sinensis were collected from Singapore from 14 diferent locations, using various trapping and collection methods between September 2013 and January 2016. Molecular identifcation of An. sinensis species were conducted by amplifying the ITS2 and CO1 region using PCR. Experimental infections of An. sinensis using blood from seven patients infected with P. vivax from the Thailand–Myanmar border were conducted with Anopheles cracens (An. dirus B) as control. Results: Phylogenetic analysis showed that An. sinensis ­(F22, ­F2 and collected from outbreak areas) found in Singapore was entirely Form A, and closely related to An. sinensis Form A from Thailand. Artifcial infection of these Singapore strain An. sinensis Form A resulted in the development of oocysts in four experiments, with the number of sporozoites produced by one An. sinensis ranging from 4301 to 14,538. Conclusions: Infection experiments showed that An. sinensis Form A from Singapore was susceptible to Thai–Myan‑ mar P. vivax strain, suggesting a potential role as a malaria vector in Singapore. Keywords: Malaria vector, Infection, Anopheles sinensis Form A, An. cracens, Sporozoites Background letifer, were reported from 1960s to 1970s [2–4]. Singa- Singapore was once rampant with malaria cases [1]. pore attained its malaria free status in November 1982 Outbreaks in mainland Singapore and of-shore islands [2]. Te total malaria annual incidence rate fuctuated of Singapore involving the known malaria vectors, i.e. between 2.9 and 3.9 cases per 100,000 people from 1998 Anopheles maculatus, Anopheles epiroticus (previ- to 2007, and 0.5 to 2.6 per 100,000 people from 2008 to ously known as Anopheles sundaicus) and Anopheles 2015 [5]. Te major causative parasite was P. vivax, fol- lowed by P. falciparum. While almost all cases were imported cases, there have been occasional sporadic *Correspondence: [email protected] malarial cases with no travel history (e.g. in 2010 and †Sook-Cheng Pang and Chiara Andolina contributed equally to this work 2013) and 15 small sporadic localized transmissions with 1 Environmental Health Institute, National Environment Agency, 11 Biopolis Way, Singapore 138667, Singapore less than 50 cases in each outbreak [5–9]. As a tourist and Full list of author information is available at the end of the article business hub, with high reliance on foreign personnel © The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Pang et al. Malar J (2017) 16:465 Page 2 of 12 from malaria endemic countries, Singapore remains vul- the laboratory. Following the frst collection, 103 adults nerable to malaria unless the vector population is well and larvae An. sinensis were collected from 13 diferent understood and remains well controlled. locations in Singapore between September 2013 and Te last outbreaks occurred in the middle of 2009, January 2016 (Fig. 1). Anopheles sinensis larvae were col- when three clusters with a total of 29 vivax malaria lected by the Environmental Health Ofcer (EHO) of patients, with no recent travel history, were identifed National Environment Agency (NEA) during the routine by the Ministry of Health. Relapse cases in vivax malaria malaria surveillance and were submitted to Environmen- amongst foreign workers from malaria endemic coun- tal Health Institute (EHI) for identifcation. Tey were tries are common and defning if the cluster is due to reared to adult for this study. local transmission is challenging. Terefore, molecular For the An. sinensis adults, some were collected using epidemiology was performed using the msp3a and msp1 modifed CDC-light traps during spatial distribution genes of the parasite. It confrmed only two independ- study and ad-hoc collection in response to public feed- ent local transmissions in Mandai-Sungei Kadut and in back on high mosquito population. A portion of adult Sembawang [8]. Te predominant Anopheles found in An. sinensis were collected through human landing catch the two areas was Anopheles sinensis, a mosquito that during 2009 malaria outbreak in malaria cluster areas was not previously recognized as a vector in Singapore. [8] and during surveillance by the Singapore Armed Transmission in Jurong could not be confrmed as the Forces in military training grounds [32]. Te remaining infecting parasite from the cases showed no genetic link adult An. sinensis were collected via Night-Catcher dur- among them. Correspondingly, no potential Anopheles ing temporal study and ad-hoc collection was conducted vectors, including An. sinensis, were found in the vicinity. due to high mosquito population. Night-Catcher, an in- Although An. sinensis has been implicated as the malaria house mosquito trap, which was improvised from CDC vector in some parts of Asia, including Korea, China, light trap, enables hourly collection of mosquitoes using Japan and Vietnam, it has never been reported as a vector incandescent light and dry ice (CO 2) as attractant (Fig. 2). in Singapore [10–23]. All collections were conducted from 7 p.m. to the 10 a.m. Anopheles sinensis is a member of the Hyrcanus group. the next morning. Due to morphological complexity and similarity among the members of the group, the members have often been Mosquito morphological identifcation misidentifed and their respective vector status is confus- Larvae and adult mosquitoes were identifed under com- ing [24, 25]. Furthermore, confrming An. sinensis as vec- pound microscope according to taxonomy keys [17, 33, tor has been made more complicated by the existence of 34]. Confrmed An. sinensis were reared in EHI’s insec- two forms, i.e. Form A and B, both of which are morpho- tary at 25 °C (± 2 °C) and 70% (± 10%) relative humidity. logically identical [26–28]. Yet, hybridization of these two Upon emergence, the adults were then reconfrmed mor- forms showed they were genetically compatible, yield- phologically to the species level according to taxonomy ing viable progeny, complete synaptic polytene chromo- keys [17, 33, 34]. Due to the absence of morphological somes and was said to exhibit cytological polymorphic trait diferences between the two forms of An. sinensis, races [29, 30]. these mosquitoes were further determined using molecu- Te vector competence of these two forms of An. sin- lar taxonomic tools to ensure the accurate form determi- ensis is not fully understood. To date, only a single study nation as well as purity of the colony. reported that An. sinensis Form B was able to produce sporozoites in the salivary glands, while Form A could Molecular taxonomy not [31]. Based on the cytological polymorphism of An. In order to identify the forms of An. sinensis in Singapore, sinensis and on previous vector competence studies [29– regions of both the COI and rDNA internal transcribed 31], it was noteworthy that the two forms could have dif- spacer (ITS2) genes were sequenced. All 103 An. sinen- ferent vector abilities in malaria transmission depending sis collected from 13 diferent locations were individually on their geographic regions. Tis study aims to charac- processed. Total DNA were extracted individually using terize Singapore’s strain of An. sinensis, including its vec- DNeasy blood and tissue kit following the manufactur- tor competence. er’s procedures (Qiagen, Hilden, Germany) and stored at − 20 °C until analysis. Two regions fanking the mito- Methods chondrial COI gene and ITS2 gene were amplifed by pol- Mosquito collection ymerase chain reaction (PCR) as described in previous In December 2013, larvae of the An. sinensis were col- studies [35–37]. Amplicons were then visualized on 2% lected from a grassy pool of a big feld at Changi Coast agarose gel stained with GelRed (Biotium Inc., USA), and Road, eastern Singapore and they were colonized in cleaned using Purelink PCR purifcation kit (Invitrogen Pang et al. Malar J (2017) 16:465 Page 3 of 12 Fig. 1 Spatial distribution of Anopheles sinensis collected from 13 locations throughout Singapore for determining the taxonomic forms of Singa‑ pore’s An. sinensis. Additionally, sequences of samples from four locations labelled “x” and fve locations labelled as “∆” were extracted from Genbank as references. Note: Spatial/CDC light trap: Spatial distribution study conducted in 2013 using CDC light trap; Singapore Armed Forces/Human Landing: Mosquitoes collected from Singapore Military training grounds via Human Landing method; Ad hoc/Night‑Catcher: Mosquitoes collected due to feedback on high Anopheles adult population using Night‑Catcher; Temporal/Night‑Catcher: An.