New vectors in northern Sarawak, Malaysian Borneo, for the zoonotic malaria parasite, Plasmodium knowlesi

Abstract

Background: The vectors for Plasmodium knowlesi, a significant cause of human malaria in Southeast Asia, identified previously in nature all belong to the Anopheles Leucosphyrus Group. Only one study has been previously undertaken in Sarawak, Malaysian Borneo, to identify vectors of P. knowlesi , where Anopheles latens was incriminated as the vector in Kapit, central Sarawak. A study was therefore undertaken to identify malaria vectors in a different location in Sarawak. Methods: Mosquitoes found landing on humans and resting on leaves over a 5-day period at two sites in the Lawas District of northern Sarawak were collected and identified. DNA samples extracted from salivary glands of Anopheles mosquitoes were subjected to nested PCR malaria-detection assays. The small sub-unit ribosomal RNA (SSUrRNA) genes of Plasmodium , and the internal transcribed spacer 2 (ITSII) and mitochondrial cytochrome c oxidase subunit 1 (COI) sequences of the mosquitoes were derived from the Plasmodium -positive samples for phylogenetic analyses. Results: A total of 65 anophelines and 127 culicines were collected. By PCR, six An. balabacensis and five An. barbirostris Clade VI were found to have single P. knowlesi infecions while three other An. balabacensis had either single, double or triple infections with P. inui, P. fieldi, P. cynomolgi and P. knowlesi . Phylogenetic analyses of the Plasmodium SSUrRNA genes confirmed 3 An. barbirostris Clade VI and 3 An. balabacensis with single P. knowlesi infections, while 3 other An. balabacensis had two or more Plasmodium species of P. inui, P. knowlesi, P. cynomolgi and possibly novel species of Plasmodium . Phylogenies inferred from the ITSII and COI sequences of An. balabacensis and An. barbirostris Clade VI indicate that the former is genetically indistinguishable from An. balabacensis in Borneo while the latter is a novel sibling species belonging to the Anopheles Barbirostris Subgroup. Conclusions: New vectors for P. knowlesi in Sarawak were identified, including An. barbirostris Clade VI , which is a species that does not belong to the Anopheles Leucosphyrus Group.