Identification of Plasmodium
Sporozoites of P. knowlesi and other simian malaria parasites were identified by nested PCR assays in An. balabacensis (n = 9) and An. barbirostris (s.l.) (n = 5) (subsequently identified following molecular characterisation as An. donaldi). The specimens of An. balabacensis and An. donaldi examined had sporozoite infection rates of 29.0 % and 27.8% respectively, which are exceedingly high compared to other vector studies in Malaysia. In the neighbouring Malaysian Borneo state of Sabah, An. balabacensis was identified as the vector for P. knowlesi with sporozoite rates ranging from 1.03 to 3.42% at three different sites [32]. Infection rates of < 2.00% were reported among vectors in central Sarawak and among An. cracens in Peninsular Malaysia [29, 31, 58]. The only other study known to have found a comparable sporozoite rate was a study done in Palawan, Philippines, where 29.4% of the examined An. balabacensis had sprorozoites [59]. The high sporozoite rates of the vectors could be attributed to the site of collection as well, which is consistent with the finding of the previous study involving An. latens in Kapit, Sarawak [58], where it was found that the sporozoite rate was highest in the forest, followed by the rates in the farm at the forest-fringe, and in the long house. Long Luping is an abandoned army camp which is far away from any human settlement. It is potentially a foraging site for the macaques due to the banana trees that grow around its perimeter. No vector control activities have been carried out since the army camp was abandoned and macaques were sighted at the site during the entomological surveys, suggesting that macaques and mosquitoes could forage and breed, respectively, undisturbed in this site.
The high infection rates could also be due to the way sampling was conducted compared to other reported studies [4, 30–32, 60, 61]. First, the present method recovers both sides of the salivary glands without rupturing any to check for sporozoites, increasing the yield of extracted Plasmodium DNA. Secondly, the present method does not retain the head of the specimens which might contain inhibitors which would disrupt PCR assays. PCR inhibitors were previously found to be present in the heads of Culex pipiens and An. punctipennis which caused false negative results in detection of Wolbachia pipientis in samples tested [62]. The efficiency of the PCR assay was later restored when specimens were decapitated prior to DNA extraction. Although we had high sporozoite infection rates by nested PCR assays, we failed to generate the longer (1500–2000 bp) Plasmodium SSU rDNA amplicons from five samples that were malaria-positive by nested PCR assays. This could be caused by the very low number of sporozoites present in the salivary glands of the mosquitoes and the low sensitivity of the PCR assay to amplify long fragments from a sample with low template concentration.
Diversity and density of Plasmodium infection in vectors
In line with the discovery of at least seven Plasmodium species infecting long-tailed macaques in Sarawak [63], it is unsurprising that multiple unidentified Plasmodium species were recovered from An. balabacensis in this study (Fig. 2). However, the low quantity of DNA extracted from the salivary glands prevented the sequencing of another gene, such as the mitochondrial genome, which would have been necessary to determine whether these are indeed novel species of Plasmodium. It is highly likely that unidentified Plasmodium species co-infected the vectors when they fed on macaques which are known to host a diverse range of Plasmodium [1, 4, 63 ,64].
Accurate identification of Plasmodium by PCR might also be impeded when uncharacterised Plasmodium, such as those found in sample LW45 (C10 and C13), could be detected by P. cynomolgi-specific primers (Fig. 3). This demonstrates the need for the sequencing of a considerable genomic locus length for proper identification of the species of Plasmodium. On the other hand, the unsuccessful attempts to sequence P. fieldi from sample LW67, and P. cynomolgi from LW45, could be due to the low density of P. fieldi and P. cynomolgi, respectively, among the other Plasmodium co-infecting each of these mosquitoes. As the PCR amplification prior to cloning amplifies the SSU rRNA genes of all Plasmodium species indiscriminately, the scarcity of any species of Plasmodium DNA in the sample reduces the chance of its amplicon being produced during PCR amplification. The difficulty in obtaining sequencing data of genes of Plasmodium derived from vectors is probably the main reason why previous studies on vectors of knowlesi malaria have only used nested PCR assays [15, 30, 32, 33, 61] and for the paucity of studies describing the diversity and density of Plasmodium infection in vectors [31, 60]. With an increasing number of zoonotic malaria infections worldwide [65–67], epidemiological studies of these inadequately studied species within human populations that come into close contact with macaques during activities in the forest and forest-fringe will also be required to monitor potential host-switch events.
Molecular characterisation of vectors and implications for vector control in Sarawak
Phylogenetic analyses confirmed that the Plasmodium-positive mosquitoes from Lawas, Sarawak, identified morphologically as An. balabacensis were An. balabacensis whereas those identified as An. barbirostris (s.l.) were An. donaldi. Anopheles balabacensis has been incriminated as a vector for P. knowlesi in Sabah, Malaysian Borneo and belongs to the Leucosphyrus Group which has been long thought to be the only species group capable of transmitting P. knowlesi in natural settings [4, 15, 30–32, 60, 61]. Anopheles kochi from the Kochi Group was suspected as a vector due to its high susceptibility to P. knowlesi infection under experimental conditions and its simiophilic biting behaviour but the parasite was never recovered from any An. kochi collected in the natural environment [4, 68, 69]. DNA of P. knowlesi was detected by PCR assays in 2019 from DNA extracted from the carcasses of An. donaldi in Sabah, Malaysian Borneo and from An. sundaicus in the Nicobar and Andaman Islands of India, [61, 70] whereas the Plasmodium DNA detected in the present study in Sarawak, Malaysian Borneo were recovered from the salivary glands of mosquitoes. In both Sabah and Sarawak, more detailed studies need to be conducted on the bionomics of An. donaldi and An. balabacensis to provide data for the implementation of appropriate vector control. Species-specific molecular assays should also be designed and utilised in future vector incrimination studies for this species range in order to correctly identify malaria-infective mosquitoes.
The incrimination of An. balabacensis and An. donaldi as novel vectors for P. knowlesi in northern Sarawak calls for re-evaluation of current and future vector control methods in the state. Detailed studies first need to be undertaken to determine the feeding behaviour and host preference of these vectors. The main vector control methods currently adopted by the Sarawak State Health Department for malaria control are the provision of insecticide treated bednets and the regular spraying of residual insecticide on houses in malarious areas. These would be of limited value against An. latens in central Sarawak which are mainly exophagic and acrodendrophilic [58]. Further studies need to be undertaken on An. donaldi and An. balabacensis in Lawas to determine the effectiveness of providing bednets to people. From the aspect of insecticide-based preventative measures, future insecticide resistance surveys should include both An. balabacensis and An. donaldi to ensure that the insecticide used would still be efficient in killing these vectors. As one of the main vector control methods currently adopted by the Sarawak State Health Department is the regular spraying of residual insecticide on houses in malarious area, spraying could also be considered for uninhabited buildings like the army camp in Long Luping, where human presence is intermittent. Apart from insecticides, clustered regularly interspaced short palindromic repeat (CRISPR)-based gene drive has been recently suggested as one possible method for the control of P. knowlesi vector(s) [71]. Gene drive is a biotechnology method used to increase the spread of a genetic trait (e.g. mosquito sterility/mosquito immunity against Plasmodium infection) into the wild population. As the system relies on the ability of the nuclease to recognise a specific nucleotide sequence and cause a double-stranded break on the chromosome [72], it is imperative that the genomes of the vectors of P. knowlesi are readily available for researchers in the field. Besides An. dirus which is a member of the Leucosphyrus Group, no other An. leucosphyrus (s.l.) and An. barbirostris (s.l.) genomes have been sequenced [73]. Genomic data of vectors of P. knowlesi incriminated so far in Peninsular Malaysia, Malaysian Borneo and Vietnam will be crucial in the development of a gene drive since it is an extremely species-specific vector control method [72]. Novel methods of vector control are clearly needed, and while waiting for these to be developed, personal protection and avoidance of being bitten by mosquitoes need to be advocated during public health promotion exercises as methods for the prevention and control of zoonotic malaria.