METHODS: Individuals of An. balabacensis were collected in the field in Ranau district, Sabah to establish a laboratory colony. Induced mating was used, and the life history parameters of the progeny were recorded. The age-stage, two-sex life table approach was used in the analysis. The culture conditions in the laboratory were 9 h light:15 h dark, mean temperature 25.7 °C ± 0.05 and relative humidity 75.8% ± 0.31.
RESULTS: The eggs hatched within 2 days, and the larval stage lasted for 10.5 days in total, with duration of instar stages I, II, III and IV of 2.3, 3.7, 2.3, 2.2 days, respectively. The maximum total fecundity was 729 for one particular female, while the maximum female age-specific mean fecundity (mx) was 142 at age 59 days. The gross reproductive rate or number of offspring per individual was about 102. On average, each female laid 1.81 ± 0.19 (range 1-7) batches of eggs, with 63% of the females producing only one batch; only one female laid six batches, while one other laid seven. Each batch comprised 159 ± 17.1 eggs (range 5-224) and the female ratio of offspring was 0.28 ± 0.06. The intrinsic rate of increase, finite rate of increase, net reproductive rate, mean generation time and doubling time were, respectively, 0.12 ± 0.01 day-1, 1.12 ± 0.01 day-1, 46.2 ± 14.97, 33.02 ± 1.85 and 5.97 days.
CONCLUSIONS: Both the net reproductive rate and intrinsic rate of increase of An. balabacensis are lower than those of other species in published studies. Our results can be used to improve models of P. knowlesi transmission and to set a baseline for assessing the impacts of environmental change on malaria dynamics. Furthermore, incorporating these population parameters of An. balabacensis into spatial and temporal models on the transmission of P. knowlesi would provide better insight and increase the accuracy of epidemiological forecasting.
METHODOLOGY/PRINCIPAL FINDINGS: Anopheles spp. were sampled using human landing catch (HLC) method at Paradason village in Kudat district of Sabah. The collected Anopheles were identified morphologically and then subjected to total DNA extraction and polymerase chain reaction (PCR) to detect Plasmodium parasites in the mosquitoes. Identification of Plasmodium spp. was confirmed by sequencing the SSU rRNA gene with species specific primers. MEGA4 software was then used to analyse the SSU rRNA sequences and bulid the phylogenetic tree for inferring the relationship between simian malaria parasites in Sabah. PCR results showed that only 1.61% (23/1,425) of the screened An. balabacensis were infected with one or two of the five simian Plasmodium spp. found in Sabah, viz. Plasmodium coatneyi, P. inui, P. fieldi, P. cynomolgi and P. knowlesi. Sequence analysis of SSU rRNA of Plasmodium isolates showed high percentage of identity within the same Plasmodium sp. group. The phylogenetic tree based on the consensus sequences of P. knowlesi showed 99.7%-100.0% nucleotide identity among the isolates from An. balabacensis, human patients and a long-tailed macaque from the same locality.
CONCLUSIONS/SIGNIFICANCE: This is the first study showing high molecular identity between the P. knowlesi isolates from An. balabacensis, human patients and a long-tailed macaque in Sabah. The other common simian Plasmodium spp. found in long-tailed macaques and also detected in An. balabacensis were P. coatneyi, P. inui, P. fieldi and P. cynomolgi. The high percentage identity of nucleotide sequences between the P. knowlesi isolates from the long-tailed macaque, An. balabacensis and human patients suggests a close genetic relationship between the parasites from these hosts.
RESULTS: A total of 1599 Anopheles specimens were collected in the village, of which about 90% were An. balabacensis. Anopheles balabacensis was present throughout the year and was the dominant Anopheles species in all habitat types. The shrub bushes habitat had the highest Anopheles species diversity while forest edge had the greatest number of Anopheles individuals caught. GLMM analysis indicated that An. balabacensis abundance was not affected by the type of habitats, and it was more active during the early and late night compared to predawn and dawn. PCR assay showed that 1.61% of the tested An. balabacensis were positive for malaria parasites, most of which were caught in oil palm estates and infected with one to two Plasmodium species.
CONCLUSIONS: The identification of infected vectors in a range of habitats, including agricultural and farming areas, illustrates the potential for humans to be exposed to P. knowlesi outside forested areas. This finding contributes to a growing body of evidence implicating environmental changes due to deforestation, expansion of agricultural and farming areas, and development of human settlements near to forest fringes in the emergence of P. knowlesi in Sabah.
RESULTS: HLC caught more An. balabacensis than any other method (3.6 per night). In contrast, no An. balabacensis were collected in MBT collections, which generally performed poorly for all mosquito taxa. Anopheles vector species including An. balabacensis were sampled in both HENET and MENET collections, but at a mean abundance of less than 1 per night. There was no difference between HENET and MENET in the overall abundance (P = 0.05) or proportion (P = 0.7) of An. balabacensis. The estimated diversity of Anopheles species was marginally higher in electrocuting net than HLC collections, and similar in collections made with humans or monkey hosts.
CONCLUSIONS: Host-baited electrocuting nets had moderate success for sampling known zoonotic malaria vectors. The primary vector An. balabacensis was collected with electrocuting nets baited both with humans and macaques, but at a considerably lower density than the HLC standard. However, electrocuting nets were considerably more successful than monkey-baited traps and representatively characterised anopheline species diversity. Consequently, their use allows inferences about relative mosquito attraction to be meaningfully interpreted while eliminating confounding factors due to trapping method. On this basis, electrocuting net traps should be considered as a useful standardised method for investigating vector contact with humans and wildlife reservoirs.