Singapore's Anopheles sinensis Form A is susceptible to Plasmodium vivax isolates from the western Thailand-Myanmar border

Abstract

Background: Singapore has been certified 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 identified Anopheles sinensis as the most probable vector. As metaphase karyotype studies divided An. sinensis into two forms, A and B, with different vector competence: the investigation of vector competence of An. sinensis found in Singapore was thus pursued using Plasmodium vivax field isolates from the Thailand-Myanmar border.

Methods: Adults and larvae An. sinensis were collected from Singapore from 14 different locations, using various trapping and collection methods between September 2013 and January 2016. Molecular identification 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. Artificial 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-Myanmar P. vivax strain, suggesting a potential role as a malaria vector in Singapore.

Keywords: An. cracens; Anopheles sinensis Form A; Infection; Malaria vector; Sporozoites.

Fig. 1

Spatial distribution of Anopheles sinensis collected from 13 locations throughout Singapore for determining the taxonomic forms of Singapore’s An. sinensis. Additionally, sequences of samples from four locations labelled “x” and five 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. sinensis collected from a 2 years temporal study using Night-Catcher; Routine surveillance/Larvae reared to adult: Larvae collected through routine surveillance were sent into EHI lab for identification and were reared to adult stage; DNA barcode/larvae reared to adult stage: Anopheles sinensis larvae were collected for DNA barcode project (DNA sequences were retrieved from Genbank and were used as reference in our current taxonomic study [32]); Malaria outbreaks/Human Landing: Adult An. sinensis were collected during 2009 malaria outbreak via Human Landing (DNA sequences were retrieved from Genbank for our current study [8])

Fig. 2

Night Catcher is an in-house designed mosquito trap that enables trapping of mosquitoes at every hour. It was improvised from CDC light trap which uses incandescent light and dry ice (CO₂) as attractants

Fig. 3

Anopheles sinensis’ wing vein CuA (indicated by red arrow) showed a pale fringe spot and b dark fringe spot [35]

Fig. 4

Phylogenetic tree of the ITS2 genes of Anopheles hyrcanus group, constructed using the neighbor-joining algorithm. The values next to the nodes are bootstrap percentages based on 1000 replicates, and only bootstrap percentages above 70% are shown

Fig. 5

Phylogenetic tree of the CO1 genes of Anopheles hyrcanus group, constructed using the neighbor-joining algorithm. The values next to the nodes are bootstrap percentages based on 1000 replicates, and only bootstrap percentages above 70% are shown

Fig. 6

Photos of midguts of An. sinensis with growth of P. vivax oocysts (red globules). Visualizing midguts with oocysts growth at a ×4 magnification and b ×10 magnification

Fig. 7

Microscopy image of sporozoites released from salivary glands of infected An. sinensis Form A (black arrows). Visualizing sporozoites on KOVA glass slides at ×40 magnification