Species composition of collected mosquitoes
A total of 192 mosquitoes were collected from Long Tengoa (n=86) and Long Luping (n=106) (Table 1). Based on morphological characteristics, mosquitoes from the genus Anopheles were the most abundant (n=65; 33%), followed by Culex (n=41; 21%), Aedes (n=40; 21%), Armigeres (n=31; 16%), Mansonia (n=7; 4%), Malaya (n=6; 3%), and Uranotaenia (n=2,1%).
A higher proportion of culicines were found in Long Tengoa (n=72; 84%), while anophelines predominated half of the total collection in Long Luping (n=61, 48%). Most An. balabacensis (n=31) and An. barbirostris (n=18) mosquitoes were caught at Long Luping while members of the Anopheles Umbrosus (An. letifer, An. roperi, and An. umbrosus) were collected in Long Tengoa, and these were absent in Long Luping (Table 1).
Table 1. Anopheles mosquitoes collected from two sites in Lawas.
Genera/Species
|
Number of mosquitoes
|
Long Tengoa
|
Long Luping
|
Total
|
An. balabacensis
|
0
|
31
|
31
|
An. latens
|
1
|
1
|
2
|
An. barbirostris
|
0
|
18
|
18
|
An. donaldi
|
3
|
0
|
3
|
An. letifer
|
5
|
0
|
5
|
An. roperi
|
1
|
0
|
1
|
An. umbrosus
|
4
|
0
|
4
|
An. tessellatus
|
0
|
1
|
1
|
Aedes
|
9
|
31
|
40
|
Armigeres
|
31
|
0
|
31
|
Culex
|
31
|
10
|
41
|
Malaya
|
0
|
6
|
6
|
Mansonia
|
1
|
6
|
7
|
Uranotaenia
|
0
|
2
|
2
|
Total
|
86
|
106
|
192
|
Plasmodium species in Anopheles mosquitoes
DNA extracted from the salivary glands of 65 anopheline mosquitoes were subjected to nested PCR assays and Plasmodium DNA was only detected in samples derived from 10 An. balabacensis and 6 An. barbirostris collected in Long Luping. Of 31 An. balabacensis, 9 (29.0 %) were found to carry P. knowlesi and other simian malaria parasites while 5 (27.8 %) An. barbirostris were found to be infected with only P. knowlesi (Table 2). The identity of the species of Plasmodium could not be determined for 2 of the Plasmodium-positive samples by using the species-specific PCR primers for P. coatneyi, P. cynomolgi, P. inui, P. fieldi, and P. knowlesi.
Table 2. Summary of results of PCR assays for Plasmodium-positive samples from Long Luping.
Species
|
Number analysed by PCR assays
|
Number of positive samples
|
Sample ID (LW)
|
Genus-specific
PCR assay
|
Species-specific
PCR assays*
|
An. balabacensis
|
30
|
10
|
1: No species identified
6: Pk
1: Pcy + Pk
1: Pcy + Pfld + Pin
1: Pin
|
101
31, 32, 49, 50, 51, 59
45
67
74
|
An. barbirostris
|
18
|
6
|
1: No species identified
5: Pk
|
38
44, 47, 48, 57, 58
|
*Pct = P. coatneyi, Pcy = P. cynomolgi; Pfld = P. fieldi; Pin = P. inui; Pk = P. knowlesi.
All 14 samples which were tested positive for at least one simian malaria parasite were subjected to another round of semi-nested PCR to obtain a longer fragment of the SSUrRNA for sequencing. Amplicons could not be generated for 5 (3 An. balabacensis and 2 An. barbirostris) of the samples infected with only P. knowlesi. As a result, a total of 38 SSUrRNA fragments were cloned and sequenced from 9 anophelines (6 An. balabacensis and 3 An. barbirostris) which were Plasmodium-positive by nested PCR assays. The phylogenetic tree (Figure 2) constructed using the Plasmodium A- and S-type SSUrRNA sequences showed that An. balabacensis were harbouring sporozoites of P. cynomolgi (LW67), P. inui (LW67 and LW74), and P. knowlesi (LW45, LW59, LW31, LW49) in their salivary glands while An. barbirostris carried only P. knowlesi (LW47, LW57, LW58). Three An. balabacensis (LW45, LW67, LW74) were shown to carry more than 1 species of Plasmodium while the other 2 An. balabacensis (LW31, LW49) were infected with only P. knowlesi. Multiple A-type SSUrRNA sequences derived from the salivary glands of mosquitoes LW45, LW67, and LW74 formed several distinct clades instead of grouping with any of the clades formed by the reference sequences (Figure 2). This indicates that An. balabacensis is not only harbouring sporozoites of P. cynomolgi, P. inui, and P. knowlesi, but also sporozoites of unknown species of Plasmodium.
Molecular characterisation of Anopheles mosquitoes
The ITS2 region and CO1 gene [see Additional Files 3 & 4 for multiple sequence alignment of CO1] of anophelines carrying simian malaria parasites were further sequenced to confirm their identities. Phylogenetic analyses of the ITS2 region and CO1 gene of An. barbirostris s. l. from Thailand, West Sumatra, West Java, and South Kalimantan showed that samples from each localities have formed distinct clades (Figures 3 & 4). The phylogenetic tree constructed using the ML method with the ITS2 sequences showed that An. barbirostris collected in this study from the Lawas district clustered together with those from Selangor, Peninsular Malaysia (Accession no.: KJ462243, KJ462248, and KJ462247). In addition, the ITS2 sequences from Selangor are also the only ones with identical length to the ITS2 of An. barbirostris characterised in the current study (Table 4). These An. barbirostris will be referred to as An. barbirostris Clade VI. On the other hand, the ML tree of the CO1 gene of An. barbirostris Clade VI collected in Lawas, Malaysian Borneo appeared to be distinct from the other An. barbirostris s. l. (Figure 4). An. barbirostris Clade VI and An. barbirostris s. l. however, are shown to more closely related to each other than to the outgroup, An. coustani.
Table 4. Lengths of 5.8S-ITS2-28S region of members of the Barbirostris Subgroup amplified by primers 5.8SF and 28SR.
Species
|
Reference
|
Locality
|
Amplicon size
(bp)
|
An. barbirostris s.s
|
[33]
|
South Kalimantan, Thailand
|
1674-1677
|
An. vanderwulpi
|
Sumatra
|
1858
|
An. barbirostris Clade III
|
Thailand
|
1860-1862
|
An. barbirostris Clade IV
|
Sumatra, Thailand
|
1713-1718
|
An. campestris
|
Thailand
|
1652
|
An. barbirostris VI
|
[45]
|
Selangor, Peninsular Malaysia
|
1576
|
An. barbirostris VI
|
Current study
|
Sarawak, Malaysian Borneo
|
The CO1 fragment of An. balabacensis obtained in this study showed close phylogenetic relationship (Figure 5) with the other An. balabacensis sequences. Those collected from this study in Sarawak, Malaysian Borneo clustered together with the An. balabacensis in Eastern Sabah, Malaysian Borneo (Accession no.: DQ897940), forming a sister clade to the An. balabacensis from South Kalimantan, Indonesian Borneo (Accession no.: DQ897941).