Search

Menu
Search
in Ecology

First molecular confirmations of Anopheles dirus and Anopheles scanloni in Indonesia, with DNA of zoonotic, enzootic and human malarias detected in An. dirus

99 Views


Abstract

Despite Indonesia reporting high numbers of Plasmodium knowlesi malaria cases in North Sumatra Province, the Anopheles mosquito vectors remain unknown. This study identified the Leucosphyrus Group species present in Langkat Regency, North Sumatra, and the Plasmodium species DNA in their heads and thoraces. Mosquitoes collected by human landing catch were morphologically identified and their species identification subsequently confirmed using ITS2 sequencing as well as Dirus Complex (DiCSIP) and Anopheles scanloni-specific PCR. Reverse-transcription real-time and nested PCR assays targeting the 18 S rRNA gene were applied for Plasmodium species detection and identification. Of 597 morphologically identified Leucosphyrus Group mosquitoes, two species of the Dirus Complex were confirmed for the first time in Indonesia: 97.8% of specimens were Anopheles dirus with 2.2% being Anopheles scanloni. Seven An. dirus specimens were Plasmodium-positive, including mixed infections with P. inui, P. knowlesi, and/or P. vivax and one equivocal sample positive for P. coatneyi and P. knowlesi. BLAST analysis indicated possible cross-reactivity of P. fieldi primers with P. inui. This study provides the first molecular confirmation of An. dirus and confirms the presence of An. scanloni, two species of the Dirus Complex in North Sumatra. In addition, it demonstrates the presence of both macaque and human Plasmodium species DNA in An. dirus, suggesting the potential role of this species in zoonotic and human malaria transmission in this Indonesian region.

Data availability

The data sets used during this study are available in the JCU Research Data repository (https://doi.org/10.25903/rabx-g630). The DNA sequence data generated in this study are available through the GenBank database (https://www.ncbi.nlm.nih.gov/genbank/). The ITS2 sequences of An. dirus from North Sumatra have been deposited under accession numbers PQ589932 – PQ589940.

References

  1. World Health Organization. Despite continued impact of COVID-19, malaria cases and deaths remained stable in 2021, (2022). https://www.who.int/news/item/08-12-2022-despite-continued-impact-of-covid-19–malaria-cases-and-deaths-remained-stable-in-2021

  2. Huang, J. et al. Global burden of malaria before and after the COVID-19 pandemic based on the global burden of disease study 2021. Sci. Rep. 15, 9113. https://doi.org/10.1038/s41598-025-93487-3 (2025).

    Google Scholar 

  3. World Health Organization. Countries and territories certified malaria-free by WHO, (2025). https://www.who.int/teams/global-malaria-programme/elimination/countries-and-territories-certified-malaria-free-by-who

  4. World Health Organization. World malaria report 2024 (World Health Organization, 2025).

  5. Chin, W., Contacos, P. G., Coatney, G. R. & Kimball, H. R. A naturaly acquited quotidian-typemalaria in man transferable to monkeys. Science 149, 865. https://doi.org/10.1126/science.149.3686.865 (1965).

    Google Scholar 

  6. Barber, B. E., Rajahram, G. S., Grigg, M. J., William, T. & Anstey, N. M. World malaria report: time to acknowledge Plasmodium knowlesi malaria. Malar. J. 16, 135. https://doi.org/10.1186/s12936-017-1787-y (2017).

    Google Scholar 

  7. Lee, K. S. et al. Plasmodium knowlesi: reservoir hosts and tracking the emergence in humans and macaques. PLoS Pathog. 7, e1002015 (2011).

    Google Scholar 

  8. Figtree, M. et al. Plasmodium knowlesi in human, Indonesian Borneo. Emerg. Infect. Dis. 16, 672–674. https://doi.org/10.3201/eid1604.091624 (2010).

    Google Scholar 

  9. Fauziah, N. et al. Emerging malaria in Indonesia: An overview of Plasmodium knowlesi infections. Parasite Epidemiol. Control. 28, e00405. https://doi.org/10.1016/j.parepi.2024.e00405 (2025).

    Google Scholar 

  10. Sulistyaningsih, E., Fitri, L. E. & Löscher, T. Berens-Riha, N. Diagnostic difficulties with Plasmodium knowlesi infection in humans. Emerg. Infect. Dis. 16, 1033–1034. https://doi.org/10.3201/eid1606.100022 (2010).

    Google Scholar 

  11. Setiadi, W. et al. A zoonotic human infection with simian malaria, Plasmodium knowlesi, in Central Kalimantan, Indonesia. Malar. J. 15, 218. https://doi.org/10.1186/s12936-016-1272-z (2016).

    Google Scholar 

  12. Ompunsunggu, S. et al. Peneman baru Plasmodium knowlesi pada manusia di Kalimantan Tengah. Buletin Penelitian Kesehatan. 43, 63–75. https://doi.org/10.22435/bpk.v43i2.4140.63-76 (2014).

    Google Scholar 

  13. Herdiana, H. et al. Malaria risk factor assessment using active and passive surveillance data from Aceh Besar, Indonesia, a low endemic, malaria elimination setting with Plasmodium knowlesi, Plasmodium vivax, and Plasmodium falciparum. Malar J 15, 468, (2016). https://doi.org/10.1186/s12936-016-1523-z

  14. Coutrier, F. N. et al. Laboratory challenges of Plasmodium species identification in Aceh Province, Indonesia, a malaria elimination setting with newly discovered P. knowlesi. PLoS Negl. Trop. Dis. 12, e0006924. https://doi.org/10.1371/journal.pntd.0006924 (2018).

    Google Scholar 

  15. Lubis, I. N. D. et al. Contribution of Plasmodium knowlesi to multispecies human malaria infections in North Sumatera, Indonesia. J. Infect. Dis. 215, 1148–1155. https://doi.org/10.1093/infdis/jix091 (2017).

    Google Scholar 

  16. van de Straat, B. et al. Zoonotic malaria transmission and land use change in Southeast Asia: what is known about the vectors. Malar. J. 21, 109. https://doi.org/10.1186/s12936-022-04129-2 (2022).

    Google Scholar 

  17. Bin Said, I. et al. Systematic review of Plasmodium knowlesi in Indonesia: a risk of emergence in the context of capital relocation to Borneo? Parasit. Vectors. 15, 258. https://doi.org/10.1186/s13071-022-05375-8 (2022).

    Google Scholar 

  18. Maeno, Y. et al. Humans frequently exposed to a range of non-human primate malaria parasite species through the bites of Anopheles dirus mosquitoes in South-central Vietnam. Parasit. Vectors. 8 https://doi.org/10.1186/s13071-015-0995-y (2015).

  19. Vythilingam, I. et al. Plasmodium knowlesi malaria an emerging public health problem in Hulu Selangor, Selangor, Malaysia (2009–2013): epidemiologic and entomologic analysis. Parasit. Vectors. 7 https://doi.org/10.1186/1756-3305-7-436 (2014).

  20. Wong, M. L. 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. 9, e0004135. https://doi.org/10.1371/journal.pntd.0004135 (2015).

    Google Scholar 

  21. Vythilingam, I. et al. Natural transmission of Plasmodium knowlesi to humans by Anopheles latens in Sarawak, Malaysia. Trans. R Soc. Trop. Med. Hyg. 100, 1087–1088. https://doi.org/10.1016/j.trstmh.2006.02.006 (2006).

    Google Scholar 

  22. Wibowo, A. A., Umniyati, S. R., Hutagalung, J. & Rahayu, T. Confirmation of Anopheles balabacensis as natural vector of malaria caused by Plasmodium knowlesi inhabits forested areas in Kecamatan Balik Bukit, Western Lampung Regency. E3S Web Conf. 151, 01028, (2020). https://doi.org/10.1051/e3sconf/202015101028

  23. Ang, J. X. D. et al. New vectors in northern Sarawak, Malaysian Borneo, for the zoonotic malaria parasite, Plasmodium knowlesi. Parasit. Vectors. 13, 472. https://doi.org/10.1186/s13071-020-04345-2 (2020).

    Google Scholar 

  24. Hawkes, F. M. et al. Vector compositions change across forested to deforested ecotones in emerging areas of zoonotic malaria transmission in Malaysia. Sci. Rep. 9, 13312. https://doi.org/10.1038/s41598-019-49842-2 (2019).

    Google Scholar 

  25. De Ang, J. X., Yaman, K., Kadir, K. A., Matusop, A. & Singh, B. New vectors that are early feeders for Plasmodium knowlesi and other simian malaria parasites in Sarawak, Malaysian Borneo. Sci. Rep. 11 https://doi.org/10.1038/s41598-021-86107-3 (2021).

  26. Yanmanee, S. et al. Natural vectors of Plasmodium knowlesi and other primate, avian and ungulate malaria parasites in Narathiwat Province, Southern Thailand. Sci. Rep. 13, 8875. https://doi.org/10.1038/s41598-023-36017-3 (2023).

    Google Scholar 

  27. Wharton, R. H., Eyles, D. E., Warren, M. & Cheong, W. H. Studies to determine the vectors of monkey malaria Malaya. Ann. Trop. Med. Parasitol. 58, 56–77. https://doi.org/10.1080/00034983.1964.11686215 (1964).

    Google Scholar 

  28. Wharton, R. H. & Eyles, D. E. Anopheles hackeri, a vector of Plasmodium knowlesi in Malaya. Science 134, 279–280. https://doi.org/10.1126/science.134.3474.279 (1961).

    Google Scholar 

  29. Vidhya, P. T., Sunish, I. P., Maile, A. & Zahid, A. K. Anopheles sundaicus mosquitoes as vector for Plasmodium knowlesi, Andaman and Nicobar Islands, India. Emerg. Infect. Dis. 25, 817–820. https://doi.org/10.3201/eid2504.181668 (2019).

    Google Scholar 

  30. Jeslyn, W. P. et al. Molecular epidemiological investigation of Plasmodium knowlesi in humans and macaques in Singapore. Vector Borne Zoonotic Dis. 11, 131–135. https://doi.org/10.1089/vbz.2010.0024 (2011).

    Google Scholar 

  31. Herdiana, H. et al. Progress towards malaria elimination in Sabang municipality, Aceh, Indonesia. Malar. J. 12, 42. https://doi.org/10.1186/1475-2875-12-42 (2013).

    Google Scholar 

  32. Herdiana, H. et al. Two clusters of Plasmodium knowlesi cases in a malaria elimination area, Sabang Municipality, Aceh, Indonesia. Malar. J. 17, 186. https://doi.org/10.1186/s12936-018-2334-1 (2018).

    Google Scholar 

  33. Vythilingam, I., Chua, T. H., Liew, J. W. K., Manin, B. O. & Ferguson, H. M. The vectors of Plasmodium knowlesi and other simian malarias Southeast Asia: challenges in malaria elimination. Adv. Parasitol. 113, 131–189. https://doi.org/10.1016/bs.apar.2021.08.005 (2021).

    Google Scholar 

  34. van de Straat, B. Adapt or perish: defining malaria vector behaviours in a changing world PhD dissertation thesis, James Cook University, (2024).

  35. Kamau, E. et al. Development of a highly sensitive genus-specific quantitative reverse transcriptase real-time PCR assay for detection and quantitation of Plasmodium by amplifying RNA and DNA of the 18S rRNA genes. J. Clin. Microbiol. 49, 2946–2953. https://doi.org/10.1128/jcm.00276-11 (2011).

    Google Scholar 

  36. Snounou, G. et al. High sensitivity of detection of human malaria parasites by the use of nested polymerase chain reaction. Mol. Biochem. Parasitol. 61, 315–320. https://doi.org/10.1016/0166-6851(93)90077-B (1993).

    Google Scholar 

  37. Imwong, M. et al. Spurious amplification of a Plasmodium vivax small-subunit RNA gene by use of primers currently used to detect P. knowlesi. J. Clin. Microbiol. 47, 4173–4175. https://doi.org/10.1128/jcm.00811-09 (2009).

    Google Scholar 

  38. Badan Pusat Statistik Provinsi Sumatera Utara. Jumlah penduduk menurut jenis kelamin dan kabupaten/kota (jiwa) 2023, (2023). https://sumut.bps.go.id/id/statistics-table/2/NjUjMg==/nomor-penduduk-menurut-tipe-kelamin-dan-kabupaten-kota.html

  39. Vythilingam, I., Wong, M. L. & Wan-Yussof, W. S. Current status of Plasmodium knowlesi vectors: a public health concern? Parasitol 145, 32–40. https://doi.org/10.1017/S0031182016000901 (2018).

    Google Scholar 

  40. Tobin, R. J. et al. Updating estimates of Plasmodium knowlesi malaria risk in response to changing land use patterns across Southeast Asia. PLoS Negl. Trop. Dis. 18, e0011570. https://doi.org/10.1371/journal.pntd.0011570 (2024).

    Google Scholar 

  41. Sallum, M., Peyton, E., Harrison, B. & Wilkerson, R. Revision of the Leucosphyrus Group of Anopheles (Cellia) (Diptera, Culicidae). Rev. Bras. Entomol. 49 https://doi.org/10.1590/S0085-56262005000500001 (2005).

  42. Moyes, C. L. et al. Predicting the geographical distributions of the macaque hosts and mosquito vectors of Plasmodium knowlesi malaria in forested and non-forested areas. Parasit. Vectors. 9, 242. https://doi.org/10.1186/s13071-016-1527-0 (2016).

    Google Scholar 

  43. Shearer, F. M. et al. Estimating geographical variation in the risk of zoonotic Plasmodium knowlesi infection in countries eliminating malaria. PLoS Negl. Trop. Dis. 10, e0004915. https://doi.org/10.1371/journal.pntd.0004915 (2016).

    Google Scholar 

  44. Harbach, R. Composition and nature of the Culicidae (mosquitoes). (2024).

  45. Sallum, M. A. M., Foster, P. G., Li, C., Sithiprasasna, R. & Wilkerson, R. C. Phylogeny of the Leucosphyrus Group of Anopheles (Cellia) (Diptera: Culcidae) based on mitochondrial gene sequences. Ann. Entomol. 100, 27–35. https://doi.org/10.1603/0013-8746(2007)100[27:POTLGO]2.0.CO;2 (2007).

    Google Scholar 

  46. Harbach, R. E. Mosquito taxonomy inventory, (2024). https://mosquito-taxonomic-inventory.myspecies.info/node/11358

  47. Hii, J. & Rueda, L. M. Malaria vectors in the Greater Mekong Subregion: overview of malaria vectors and remaining challenges. Southeast. Asian J. Trop. Med. Public. Health. 44 (Suppl 1), 73–165 (2013). discussion 306 – 167.

    Google Scholar 

  48. Takano, K. T. et al. Partial mitochondrial DNA sequences suggest the existence of a cryptic species within the Leucosphyrus Group of the genus Anopheles (Diptera: Culicidae), forest malaria vectors, in northern Vietnam. Parasit. Vectors. 3, 41–41. https://doi.org/10.1186/1756-3305-3-41 (2010).

    Google Scholar 

  49. Tyagi, V. et al. Malaria vector Anopheles culicifacies sibling species differentiation using egg morphometry and morphology. Parasit. Vectors. 9, 202. https://doi.org/10.1186/s13071-016-1478-5 (2016).

    Google Scholar 

  50. Sharma, A. K. et al. Distribution of Anopheles culicifacies and detection of its sibling species E from Madhya Pradesh: Central India. J. Arthropod Borne Dis. 8, 186–196 (2014).

    Google Scholar 

  51. Tananchai, C. et al. Species diversity and biting activity of Anopheles dirus and Anopheles baimaii (Diptera: Culicidae) in a malaria prone area of western Thailand. Parasit. Vectors. 5, 211. https://doi.org/10.1186/1756-3305-5-211 (2012).

    Google Scholar 

  52. Obsomer, V., Defourny, P. & Coosemans, M. The Anopheles dirus complex: spatial distribution and environmental drivers. Malar. J. 6 https://doi.org/10.1186/1475-2875-6-26 (2007).

  53. Sinka, M. et al. The dominant Anopheles vectors of human malaria in the Asia-Pacific region: occurrence data, distribution maps and bionomic precis. Parasit. Vectors. 4, 89. https://doi.org/10.1186/1756-3305-4-89 (2011).

    Google Scholar 

  54. Manguin, S. et al. SCAR markers and multiplex PCR-based identification of isomorphic species in the Anopheles dirus complex in Southeast Asia. Med. Vet. Entomol. 16, 46–54. https://doi.org/10.1046/j.0269-283x.2002.00344.x (2002).

    Google Scholar 

  55. Morgan, K., Somboon, P. & Walton, C. in Anopheles mosquitoes – New insights into malaria vectors (ed S Manguin) Ch. 11, 327–355Intechopen, (2013).

  56. Permana, D. H. et al. The potential for zoonotic malaria transmission in five areas of Indonesia inhabited by non-human primates. Parasit. Vectors. 16, 267. https://doi.org/10.1186/s13071-023-05880-4 (2023).

    Google Scholar 

  57. Baimai, V., Kijchalao, U., Sawadwongporn, P. & Green, C. A. Geographic distribution and biting behaviour of four species of the Anopheles dirus complex (Diptera: Culicidae) in Thailand. Southeast. Asian J. Trop. Med. Public. Health. 19, 151–161 (1988).

    Google Scholar 

  58. Manguin, S., Garros, C., Dusfour, I., Harbach, R. E. & Coosemans, M. Bionomics, taxonomy, and distribution of the major malaria vector taxa of Anopheles subgenus Cellia in Southeast Asia: an updated review. Infect. Genet. Evol. 8, 489–503. https://doi.org/10.1016/j.meegid.2007.11.004 (2008).

    Google Scholar 

  59. Walton, C. et al. Identification of five species of the Anopheles dirus complex from Thailand, using allele-specific polymerase chain reaction. Med. Vet. Entomol. 13, 24–32. https://doi.org/10.1046/j.1365-2915.1999.00142.x (1999).

    Google Scholar 

  60. Saeung, M. et al. Dirus complex species identification PCR (DiCSIP) improves the identification of Anopheles dirus complex from the Greater Mekong Subregion. Parasit. Vectors. 17, 260. https://doi.org/10.1186/s13071-024-06321-6 (2024).

    Google Scholar 

  61. Rhosenberg, R., Andre, R. G. & Somchit, L. Highly efficient dry season transmission of malaria in Thailand. Trans. R Soc. Trop. Med. Hyg. 84, 22–28. https://doi.org/10.1016/0035-9203(90)90367-N (1990).

    Google Scholar 

  62. Rattanarithikul, R., Konishi, E. & Linthicum, K. J. Detection of Plasmodium vivax and Plasmodium falciparum circumsporozoite antigen in anopheline mosquitoes collected in southern Thailand. Am. J. Trop. Med. Hyg. 54, 114–121. https://doi.org/10.4269/ajtmh.1996.54.114 (1996).

    Google Scholar 

  63. Somboon, P., Aramrattana, A., Lines, J. & Webber, R. Entomological and epidemiological investigations of malaria transmission in relation to population movements in forest areas of north-west Thailand. Southeast. Asian J. Trop. Med. Public. Health. 29, 3–9 (1998).

    Google Scholar 

  64. Gingrich, J. B., Weatherhead, A., Sattabongkot, J., Pilakasiri, C. & Wirtz, R. A. Hyperendemic malaria in a Thai village: dependence of year-round transmission on focal and seasonally circumscribed mosquito (Diptera: Culicidae) habitats. J. Med. Entomol. 27, 1016–1026. https://doi.org/10.1093/jmedent/27.6.1016 (1990).

    Google Scholar 

  65. Rattaprasert, P., Chaksangchaichot, P., Wihokhoen, B., Suparach, N. & Sorosjinda-Nunthawarasilp, P. Detection of putative antimalarial-resistant Plasmodium vivax in Anopheles vectors at Thailand-Cambodia and Thailand-Myanmar borders. Southeast. Asian J. Trop. Med. Public. Health. 47, 182–193 (2016).

    Google Scholar 

  66. Poolphol, P. et al. Natural Plasmodium vivax infections in Anopheles mosquitoes in a malaria endemic area of northeastern Thailand. Parasitol. Res. 116, 3349–3359. https://doi.org/10.1007/s00436-017-5653-1 (2017).

    Google Scholar 

  67. Zhang, C. et al. Survey of malaria vectors on the Cambodia, Thailand and China-Laos Borders. Malar. J. 21, 399. https://doi.org/10.1186/s12936-022-04418-w (2022).

    Google Scholar 

  68. Toma, T. et al. Entomological surveys of malaria in Khammouane Province, Lao PDR, in 1999 and 2000. Southeast. Asian J. Trop. Med. Public. Health. 33, 532–546 (2002).

    Google Scholar 

  69. Vythilingam, I. et al. Epidemiology of malaria in Attapeu Province, Lao PDR in relation to entomological parameters. Trans. R Soc. Trop. Med. Hyg. 99, 833–839. https://doi.org/10.1016/j.trstmh.2005.06.012 (2005).

    Google Scholar 

  70. Van Dung, N. et al. Anopheles diversity, biting behaviour and transmission potential in forest and farm environments of Gia Lai province, Vietnam. Malar. J. 22, 204. https://doi.org/10.1186/s12936-023-04631-1 (2023).

    Google Scholar 

  71. Sun, D. et al. Surveillance and control of malaria vectors in Hainan Province, China from 1950 to 2021: a retrospective review. Trop. Med. Infect. Dis. 8 https://doi.org/10.3390/tropicalmed8030131 (2023).

  72. Htay, A. et al. Well-breeding Anopheles dirus and their role in malaria transmission in Myanmar. Southeast. Asian J. Trop. Med. Public. Health. 30, 447–453 (1999).

    Google Scholar 

  73. Oo, T. T., Storch, V. & Becker, N. Anopheles dirus and its role in malaria transmission in Myanmar. J. Vector Ecol. 28 2, 175–183 (2003).

    Google Scholar 

  74. Prakash, A., Bhattacharyya, D. R., Mohapatra, P. K. & Mahanta, J. Estimation of vectorial capacity of Anopheles dirus (Diptera: Culicidae) in a forest-fringed village of Assam (India). Vector Borne Zoonotic Dis. 1, 231–237. https://doi.org/10.1089/153036601753552594 (2001).

    Google Scholar 

  75. Nakazawa, S. et al. Anopheles dirus co-infection with human and monkey malaria parasites in Vietnam. Int. J. Parasitol. 39, 1533–1537. https://doi.org/10.1016/j.ijpara.2009.08.005 (2009).

    Google Scholar 

  76. Chinh, V. D. et al. Prevalence of human and non-human primate Plasmodium parasites in anopheline mosquitoes: a cross-sectional epidemiological study in Southern Vietnam. Trop. Med. Health. 47 https://doi.org/10.1186/s41182-019-0139-8 (2019).

  77. Marchand, R. P., Culleton, R., Maeno, Y., Quang, N. T. & Nakazawa, S. Co-infections of Plasmodium knowlesi, P. falciparum, and P. vivax among humans and Anopheles dirus mosquitoes, Southern Vietnam. Emerg. Infect. Dis. 17, 1232–1239. https://doi.org/10.3201/eid1707.101551 (2011).

    Google Scholar 

  78. Collins, W. E., Contacos, P. G., Guinn, E. G. & Held, J. R. Transmission of Plasmodium fieldi by Anopheles maculatus, A. stephensi, and A. balabacensis balabacensis. J Parasitol 54, 376–376, (1968). https://doi.org/10.2307/3276956

  79. Collins, W. E., Contacos, P. G., Skinner, J. C. & Guinn, E. G. Studies on the transmission of simian malaria V. infection and transmission of the B strain of Plasmodium cynomolgi. J. Parasitol. 58, 653–659. https://doi.org/10.2307/3278284 (1972).

    Google Scholar 

  80. Collins, W. E. et al. Transmission of the OS strain of Plasmodium inui to Saimiri sciureus boliviensis and Aotus azarae boliviensis monkeys by Anopheles dirus mosquitoes. J. Parasitol. 74, 502–503. https://doi.org/10.2307/3282066 (1988).

    Google Scholar 

  81. Collins, W. E., Warren, M., Sullivan, J. S. & Galland, G. G. Plasmodium coatneyi: observations on periodicity, mosquito infection, and transmission to Macaca mulatta monkeys. Am. J. Trop. Med. Hyg. 64, 101–110. https://doi.org/10.4269/ajtmh.2001.64.101 (2001).

    Google Scholar 

  82. Ta, T. H. et al. First case of a naturally acquired human infection with Plasmodium cynomolgi. Malar. J. 13, 68. https://doi.org/10.1186/1475-2875-13-68 (2014).

    Google Scholar 

  83. Davidson, G. et al. Forest Restoration and the Zoonotic Vector Anopheles balabacensis in Sabah. Malaysia EcoHealth. https://doi.org/10.1007/s10393-024-01675-w (2024).

    Google Scholar 

  84. Murdiyarso, L. S. et al. Plasmodium cynomolgi Infections not found in microscopy-diagnosed malaria cases across Sabah, Malaysia. Am. J. Trop. Med. Hyg. 112, 85–88. https://doi.org/10.4269/ajtmh.24-0264 (2024).

    Google Scholar 

  85. Braima, K. A. et al. Improved limit of detection for zoonotic Plasmodium knowlesi and P. cynomolgi surveillance using reverse transcription for total nucleic acid preserved samples or dried blood spots. PLoS Negl. Trop. Dis. 19, e0012129. https://doi.org/10.1371/journal.pntd.0012129 (2025).

    Google Scholar 

  86. Bustin, S. A. et al. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin. Chem. 55, 611–622. https://doi.org/10.1373/clinchem.2008.112797 (2009).

    Google Scholar 

  87. McCall, M. N., McMurray, H. R., Land, H. & Almudevar, A. On non-detects in qPCR data. Bioinformatics 30, 2310–2316. https://doi.org/10.1093/bioinformatics/btu239 (2014).

    Google Scholar 

  88. Kralik, P. & Ricchi, M. A basic guide to real time PCR in microbial diagnostics: definitions, parameters, and everything. Front. Microbiol. 8 https://doi.org/10.3389/fmicb.2017.00108 (2017).

  89. Grigg, M. J. et al. Plasmodium knowlesi detection methods for human infections-diagnosis and surveillance. Adv. Parasitol. 113, 77–130. https://doi.org/10.1016/bs.apar.2021.08.002 (2021).

    Google Scholar 

  90. Baimai, V. in Proceeding of the 3rd conference on malaria research, Thailand,. 146–162 (1989). 146–162 (1989). (1989).

  91. Harrison, L. E. et al. A multi-criteria framework for disease surveillance site selection: case study for Plasmodium knowlesi malaria in Indonesia. R Soc. Open. Sci. 11, 230641. https://doi.org/10.1098/rsos.230641 (2024).

    Google Scholar 

  92. Russell, T. L., Staunton, K. & Burkot, T. R. Standard operating procedure for performing human landing catch (HLC). (2022).

  93. World Health Organization. Malaria entomology and vector control. Guide for participants (World Health Organization, 2013).

  94. Silver, J. B. Mosquito ecology: field sampling methods. Third edition (Springer, 2008).

  95. O’Connor, C. & Soepanta, A. Illustrated key to female anophelines of Indonesia (Directorate of Communicable Disease, MoH and US Naval Medical Research, 1989).

  96. Beebe, N. W. & Saul, A. Discrimination of all members of the Anopheles punctulatus complex by polymerase chain reaction – restriction fragment length polymorphism analysis. Am. J. Trop. Med. Hyg. 53, 478–481. https://doi.org/10.4269/ajtmh.1995.53.478 (1995).

    Google Scholar 

Download references

Acknowledgements

We are grateful to the residents of Dusun II and V in Ujung Bandar Village, Salapian Sub-district Langkat Regency, North Sumatra, for their invaluable support in mosquito sampling and their hospitality during fieldwork. We thank Hidayatullah Nasution, Panusunan Hasibuan, and Ledy Afrida Sinaga (Balai Laboratorium Kesehatan Masyarakat Medan) for their contributions to the fieldwork and morphological identification of mosquitoes. We appreciate Mhd Ihza Fahrezi and Kania Haura Chalisaturahmi (Universitas Sumatra Utara) for their help with laboratory assays. We thank Theeraphap Chareonviriyaphap (Kasetsart University, Thailand) and Natapong Jupatanakul (Biotech, Thailand) for providing positive controls for the Dirus Complex species.

Funding

This study was funded by the ZOOMAL project (‘Evaluating zoonotic malaria and agricultural land use in Indonesia’; #LS-2019-116), Australian Centre for International Agricultural Research and the Indo-Pacific Centre for Health Security, Department of Foreign Affairs and Trade, Australian Government. BFS was supported by a James Cook University Postgraduate Research Scholarship. MJG is supported by a NHMRC EL2 Investigator grant.

Author information

Authors and Affiliations

  1. Australian Institute of Tropical Health and Medicine, James Cook University, Cairns, Australia

    Boni F. Sebayang, Bram van de Straat, Tanya L. Russell & Thomas R. Burkot

  2. Medan Public Health Laboratory Center, Ministry of Health of the Republic of Indonesia, Medan, Indonesia

    Ahadi Kurniawan

  3. Faculty of Medicine, Universitas Sumatera Utara, Medan, Indonesia

    Adzkia M. Haq & Inke N. D. Lubis

  4. Vector-borne and Zoonotic Research Group, Research Center of Public Health and Nutrition, Health Research Organization, BRIN, Salatiga, Indonesia

    Triwibowo A. Garjito

  5. Department of Medical Entomology, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand

    Manop Saeung

  6. HSM, Univ. Montpellier, CNRS, IRD, Montpellier, France

    Sylvie Manguin

  7. Menzies School of Health Research, Charles Darwin University, Darwin, Australia

    Matthew J. Grigg

  8. Institute for Health Research, University of Notre Dame Australia, Fremantle, Australia

    Matthew J. Grigg

Authors

  1. Boni F. Sebayang
    View author publications

    Search author on:PubMed Google Scholar

  2. Bram van de Straat
    View author publications

    Search author on:PubMed Google Scholar

  3. Ahadi Kurniawan
    View author publications

    Search author on:PubMed Google Scholar

  4. Adzkia M. Haq
    View author publications

    Search author on:PubMed Google Scholar

  5. Triwibowo A. Garjito
    View author publications

    Search author on:PubMed Google Scholar

  6. Manop Saeung
    View author publications

    Search author on:PubMed Google Scholar

  7. Sylvie Manguin
    View author publications

    Search author on:PubMed Google Scholar

  8. Inke N. D. Lubis
    View author publications

    Search author on:PubMed Google Scholar

  9. Matthew J. Grigg
    View author publications

    Search author on:PubMed Google Scholar

  10. Tanya L. Russell
    View author publications

    Search author on:PubMed Google Scholar

  11. Thomas R. Burkot
    View author publications

    Search author on:PubMed Google Scholar

Contributions

BFS, TRB, TLR and MJG conceived and designed the study. TRB, TLR, TAG, INDL and MJG advised on fieldwork. BvdS, AK, and BFS conducted field studies. BFS and AMH conducted the laboratory analyses with input from TAG, MS, SM, INDL, and MJG. BFS drafted the original draft of the manuscript, and MJG, TRB and TLR provided supervision. All authors reviewed and approved the final manuscript.

Corresponding author

Correspondence to
Boni F. Sebayang.

Ethics declarations

Competing interests

The authors declare no competing interests.

Ethical approval

This study was approved by the Universitas Sumatera Utara, Faculty of Medicine (application number 723/KEP/USU/2021) on 14 July 2021, and by James Cook University (application number H8583) on 29 September 2021. Procedures involving human participants followed the institutional and WHO guidelines and regulations for entomological vector surveillance.

Consent to participate

Human landing catch (HLC) activities were conducted strictly in accordance with the ethical protocols approved by Universitas Sumatera Utara and James Cook University. All mosquito collectors were adults (≥ 18 years old) and provided written informed consent prior to participation, in which the risks to the collector and the potential benefits to both the collector and the communities in which they worked were explained. Volunteers received basic training for collecting mosquitoes using an oral aspirator, including safety training, and measures were taken to minimize exposure to mosquito bites as specified in the ethical approvals.

Supplementary Information

The online version contains supplementary material available at the DOI link website.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary Material 1

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

Reprints and permissions

About this article

Cite this article

Sebayang, B.F., van de Straat, B., Kurniawan, A. et al. First molecular confirmations of Anopheles dirus and Anopheles scanloni in Indonesia, with DNA of zoonotic, enzootic and human malarias detected in An. dirus.
Sci Rep (2026). https://doi.org/10.1038/s41598-026-42478-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1038/s41598-026-42478-z

Keywords


  • Plasmodium knowlesi
  • Leucosphyrus Group

  • Anopheles dirus

  • Anopheles scanloni
  • North Sumatra
  • Enzootic malaria

Source: Ecology - nature.com

Continental-scale drivers of soil microbial extracellular polymeric substances

Sonophore enables autonomous observation of micronekton communities in the ocean twilight zone

This portal is not a newspaper as it is updated without periodicity. It cannot be considered an editorial product pursuant to law n. 62 of 7.03.2001. The author of the portal is not responsible for the content of comments to posts, the content of the linked sites. Some texts or images included in this portal are taken from the internet and, therefore, considered to be in the public domain; if their publication is violated, the copyright will be promptly communicated via e-mail. They will be immediately removed.

Back to Top
Close