Molecular characterisation of blood meal sources in Anopheles maculatus from a Plasmodium knowlesi transmission area in Seremban, Negeri Sembilan, Malaysia
DOI:
https://doi.org/10.63719/imrj.2026.01(SE).06Keywords:
Zoonotic malaria, Anopheles maculatus, Plasmodium knowlesi, host preference, COI geneAbstract
Zoonotic malaria, particularly caused by Plasmodium knowlesi, has emerged as a major public health concern in Southeast Asia. Although Malaysia successfully eliminated indigenous human malaria transmission in 2018, the continued rise in zoonotic malaria cases remains a significant challenge to achieving the WHO malaria elimination certification. Anopheles maculatus has been experimentally shown to support the complete sporogonic development of P. knowlesi under laboratory conditions. As the primary vector of human malaria in Peninsular Malaysia, An. maculatus is also considered a potential vector of P. knowlesi in natural settings, given its high abundance in areas where human cases of knowlesi malaria have been reported. Therefore, this study aimed to determine the blood meal sources of An. maculatus collected using the human landing catch (HLC) method between March and June 2024. A total of 150 visibly non-engorged mosquitoes were subjected to nested PCR targeting vertebrate DNA, followed by species-specific PCR assays for the detection of human and macaque DNA. Of the 150 samples analysed, 70 (46.7%) were positive for vertebrate DNA. All 70 positive samples contained human DNA, while no macaque DNA was detected. These findings indicate a strong association with human blood meals among An. maculatus collected in the study area and provide insights into the host-feeding behaviour of this species in a zoonotic malaria-endemic setting. The findings contribute to a better understanding of local malaria transmission ecology and may support future vector surveillance and control strategies. Further studies across broader geographical and ecological settings are recommended to better characterise the blood-feeding sources of An. maculatus and their epidemiological significance in zoonotic malaria transmission.
References
Singh B, Kim Sung L, Matusop A, Radhakrishnan A, Shamsul SS, Cox-Singh J, et al. A large focus of naturally acquired Plasmodium knowlesi infections in human beings. Lancet. 2004;363(9414):1017–24. doi:10.1016/S0140-6736(04)15836-4.
World Health Organization. World malaria report 2024. Geneva: World Health Organization; 2024.
Ministry of Health Malaysia. Malaysia malaria surveillance report 2024. Putrajaya: Ministry of Health Malaysia; 2024.
Vythilingam I, NoorAzian YM, Huat TC, Jiram AI, Yusri YM, Azahari AH, et al. Plasmodium knowlesi in humans, macaques and mosquitoes in Peninsular Malaysia. Parasit Vectors. 2008;1:26. doi:10.1186/1756-3305-1-26
Fornace KM, Brock PM, Abidin TR, Grignard L, Herman LS, Chua TH, et al. Environmental risk factors and exposure to the zoonotic malaria parasite Plasmodium knowlesi across northern Sabah, Malaysia: a population-based cross-sectional survey. Lancet Planet Health. 2016;1:e179–86. doi:10.1016/s2542-5196(19)30045-2
Tan CH, Vythilingam I, Matusop A, Chan ST, Singh B. Bionomics of Anopheles latens in Kapit, Sarawak, Malaysian Borneo in relation to the transmission of zoonotic simian malaria parasite Plasmodium knowlesi. Malar J. 2008;7:52. doi:10.1186/1475-2875-7-52
Jiram AI, Vythilingam I, NoorAzian YM, Yusof YM, Azahari AH, Fong MY. Entomologic investigation of Plasmodium knowlesi vectors in Kuala Lipis, Pahang, Malaysia. Malar J. 2012 Jun 22;11:213. doi:10.1186/1475-2875-11-213
Wong ML, Chua TH, Leong CS, Khaw LT, Fornace K, Wan-Sulaiman WY, et al. Seasonal and spatial dynamics of the primary vector of Plasmodium knowlesi within a major transmission focus in Sabah, Malaysia. PLoS Negl Trop Dis. 2015;9(10):e0004135. doi:10.1371/journal.pntd.0004135
Manin BO, Ferguson HM, Vythilingam I, Fornace K, Drakeley CJ, Chua TH, et al. Investigating the contribution of peri-domestic transmission to risk of zoonotic malaria infection in humans. PLoS Negl Trop Dis. 2016;10(10):e0005064. doi:10.1371/journal.pntd.0005064
Wharton RH. The biology of Anopheles hackeri and Anopheles maculatus in relation to malaria transmission in Malaya. Kuala Lumpur: Institute for Medical Research; 1962.
Rahman WA, Adanan CR. Malaria and Anopheles mosquitoes in Malaysia. Southeast Asian J Trop Med Public Health. 1997;28(3):599–605. doi:10.1016/j.actatropica.2005.06.022
Collins WE, Warren M, Skinner JC, Richardson BB, Kearse TS. Infection of Anopheles maculatus with Plasmodium knowlesi. J Parasitol. 1967;53(4):778–82.
Sebayang BF, van de Straat B, Kurniawan A, et al. Evidence incriminating Anopheles maculatus as a potential vector of Plasmodium knowlesi and Plasmodium inui. Sci Rep. 2026;16:6515. doi:10.1038/s41598-026-35946-z
Sinka ME, Bangs MJ, Manguin S, Chareonviriyaphap T, Patil AP, Temperley WH, Gething PW, Elyazar IR, Kabaria CW, Harbach RE, Hay SI. The dominant Anopheles vectors of human malaria in the Asia-Pacific region: occurrence data, distribution maps and bionomic précis. Parasit Vectors. 2011 May 25;4:89. doi:10.1186/1756-3305-4-89.
Kent RJ. Molecular methods for arthropod bloodmeal identification and applications to ecological and vector-borne disease studies. Mol Ecol Resour. 2009;9(1):4–18. doi:10.1111/j.1755-0998.2008.02469.x
Fornace KM, Abidin TR, Alexander N, Brock P, Grigg MJ, Murphy A, William T, Menon J, Drakeley CJ, Cox J. Association between Landscape Factors and Spatial Patterns of Plasmodium knowlesi Infections in Sabah, Malaysia. Emerg Infect Dis. 2016 Feb;22(2):201-8. doi:10.3201/eid2202.150656.
Kent RJ, Norris DE. Identification of mammalian blood meals in mosquitoes by a multiplexed polymerase chain reaction targeting cytochrome b. Am J Trop Med Hyg. 2005;73(2):336–42.
Logue K, Keven JB, Cannon MV, Reimer L, Siba P, Walker ED, et al. Unbiased characterization of Anopheles mosquito blood meals by targeted high-throughput sequencing. PLoS Negl Trop Dis. 2016;10(3):e0004512. doi:10.1371/journal.pntd.0004512
Technical Meeting for the Malaria Elimination Strategic Plan, Vector-Borne Disease Control Sector, Disease Control Division, Ministry of Health Malaysia
Reid JA. 1968. Anopheline Mosquitoes of Malaya and Borneo. Studies from the Institute for Medical Research Malaysia No. 31.
Alcaide M, Rico C, Ruiz S, Soriguer R, Muñoz J, Figuerola J. Disentangling vector-borne transmission networks: a universal DNA barcoding method to identify vertebrate hosts from arthropod bloodmeals. PLoS One. 2009;4(9):e7092. doi:10.1371/journal.pone.0007092.
Gunathilaka N, Denipitiya T, Hapugoda M, Abeyewickreme W, Wickremasinghe R. Determination of the foraging behaviour and blood meal source of malaria vector mosquitoes in Trincomalee District of Sri Lanka using a multiplex real time polymerase chain reaction assay. Malar J. 2016;15:242. doi:10.1186/s12936-016-1279-5.
Muenworn V, Sungvornyothin S, Kongmee M, Polsomboon S, Bangs MJ, Akrathanakul P, et al. Biting activity and host preference of the malaria vectors Anopheles maculatus and Anopheles sawadwongporni (Diptera: Culicidae) in Thailand. J Vector Ecol. 2009;34(1):62-9. doi:10.1111/j.1948-7134.2009.00008.x.
Rahman WA, Adanan CR, Abu Hassan A. Species composition of adult Anopheles populations and their breeding habitats in Hulu Perak district, Peninsular Malaysia. Southeast Asian J Trop Med Public Health. 2002;33(3):547-50.
Jeyaprakasam NK, Low VL, Liew JWK, Pramasivan S, Wan-Sulaiman WY, Saeung A, et al. Blood meal analysis of Anopheles vectors of simian malaria based on laboratory and field studies.Sci Rep. 2022;12(1):354. doi:10.1038/s41598-021-04106-w.
