The Seasonal Dynamics and Biting Behavior of Potential Anopheles Vectors of Plasmodium Knowlesi in Palawan, Philippines Richard Paul Malijan Research Institute for Tropical Medicine Frank Mechan Liverpool School of Tropical Medicine Jessie C Braganza Jr Research Institute for Tropical Medicine Kristelle Mae R. Valle Research Institute for Tropical Medicine Ferdinand V. Salazar Research Institute for Tropical Medicine Majhalia M Torno Research Institute for Tropical Medicine Wilfredo E Aure Research Institute for Tropical Medicine Brian A Bacay Research Institute for Tropical Medicine Fe Esperanza Espino Research Institute of Tropical Medicine Stephen J Torr Liverpool School of Tropical Medicine Kimberly M Fornace London School of Hygiene & Tropical Medicine Chris Drakeley London School of Hygiene & Tropical Medicine Heather M Ferguson ( [email protected] ) University of Glasgow Centre for Virus Research https://orcid.org/0000-0002-9625-5176 Research Article Keywords: Anopheles balabacensis, Anopheles avirostris, Plasmodium knowlesi, Vector behavior, Philippines Posted Date: June 3rd, 2021 DOI: https://doi.org/10.21203/rs.3.rs-561091/v1 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at Parasites & Vectors on July 7th, 2021. See the published version at https://doi.org/10.1186/s13071-021-04853-9. 1 The seasonal dynamics and biting behavior of potential Anopheles vectors of 2 Plasmodium knowlesi in Palawan, Philippines 3 4 Richard Paul B Malijan1, Frank Mechan2, Jessie C Braganza Jr1,, Kristelle Mae R Valle1, 5 Ferdinand V Salazar1, Majhalia M Torno1,3, Wilfredo E Aure1, Brian A Bacay1, Fe Esperanza 6 Espino4, Stephen J Torr2, Kimberly M. Fornace5, Chris Drakeley5, , and Heather M Ferguson6* 7 8 Affiliations: 9 10 1 Department of Medical Entomology, Research Institute for Tropical Medicine, Alabang, 11 Muntinlupa City, Metro Manila 1781, Philippines 12 2 Department of Vector Biology, Liverpool School of Tropical Medicine, Liverpool, UK, L3 13 5Q4 14 3 –Current Address, Taxonomy & Pesticide Efficacy Branch, Vector Biology & Control 15 Division, Environment Health Institute, National Environment Agency, Ministry of 16 Sustainability and the Environment, 11 Biopolis Way, Singapore 138667 17 4 Department of Parasitology, Research Institute for Tropical Medicine, Alabang, 18 Muntinlupa City, Metro Manila 1781, Philippines 19 5 Faculty of Infectious and Tropical Diseases, London School of Tropical Medicine and 20 Hygiene, London, UK, WC1E 7HT 21 6 – Institute of Biodiversity, Animal Health and Comparative Medicine, University of 22 Glasgow, Glasgow, UK, G12 8QQ 23 24 *Corresponding Author 1 25 26 Abstract 27 Background: A small number of human cases of the zoonotic malaria Plasmodium knowlesi 28 have been reported in Palawan Island, the Philippines. Identification of potential vector species 29 and their bionomics is crucial for understanding of human exposure risk in this setting. Here, 30 we combined longitudinal surveillance with a trap-evaluation study to address knowledge gaps 31 about the ecology and potential for zoonotic spillover of this macaque malaria in Palawan 32 Island. 33 Methods: The abundance, diversity and biting behavior of human-biting Anopheles 34 mosquitoes were assessed through monthly outdoor Human Landing Catches (HLC) in three 35 ecotypes representing different land use (forest-edge, forest and agricultural area) across 8 36 months. Additionally, the host preference and biting activity of potential Anopheles vectors 37 was assessed through comparison of their abundance and capture time in traps baited with 38 humans (HLC, human-baited electrocuting net – HEN) or macaques (monkey-baited trap – 39 MBT, monkey-baited electrocuting net – HEN). All female Anopheles mosquitoes were tested 40 for presence of Plasmodium parasite by PCR. 41 Results: Previously incriminated vectors Anopheles balabacensis and An. flavirostris 42 accounted for >95% of Anophelines caught in longitudinal surveillance. However human 43 biting densities were relatively low (An. balabacensis: 0.34 -1.20 per night, An. flavirostris: 0- 44 2 bites per night). Biting densities of An. balabacensis were highest in the forest-edge while 45 An. flavirostris was most abundant in the agricultural area. The abundance of An. balabacensis 46 and An. flavirostris was significantly higher in HLC than MBT. None of the 357 female 47 Anopheles mosquitoes tested for Plasmodium infection were positive. 48 Conclusions: The relatively low density and lack of malaria infection in Anopheles mosquitoes 49 sampled here indicates that exposure to P. knowlesi in this setting is considerably lower than 2 50 neighbouring countries (Malaysia) where it is now the primary cause of malaria in humans. 51 Although Anophelines had lower abundance in MBT than HLC, An. balabacensis and An. 52 flavirostris were caught by both methods suggesting they could act as bridge vectors between 53 humans and macaques. These species bite primarily outdoors and in early evening; confirming 54 that Insecticide Treated Nets are unlikely to provide protection against P. knowlesi vectors. 55 56 Keywords: Anopheles balabacensis, Anopheles flavirostris, Plasmodium knowlesi, Vector 57 behavior, Philippines 58 3 59 60 Background 61 The Philippines has established a sub national/territorial malaria elimination strategy, 62 through which zero indigenous cases were reported in 78 out of 81 provinces in 2019(1-3). The 63 primary malaria species of public health importance in the Philippines are Plasmodium 64 falciparum and P. vivax which respectively comprise ~88% and 9% of the total indigenous 65 malaria cases(1). Malaria transmission in the country is now confined to a few provinces 66 including Palawan(1-3). Concern has been raised that the emergence of the zoonotic malaria 67 parasite P. knowlesi as a public health problem in several Southeast Asian countries may 68 threaten regional elimination(4, 5). Human cases of P. knowlesi infection in Palawan, Philippines 69 were first confirmed in 2008, based on molecular detection from blood slides that had been 70 previously diagnosed by microscopy as P. malariae(6). Recent serological work indicates P. 71 knowlesi transmission in Palawan is ongoing, with community sampling reporting that 1.1% 72 of individuals tested positive for the P. knowlesi specific PkSERA3 ag1 antibody response(7). 73 In response to the emerging threat of P. knowlesi, an international collaboration was established 74 in 2012 to investigate the risk factors of human infections and identify populations at risk. The 75 ‘MONKEYBAR’ project focused its investigation in two known areas of transmission: Sabah 76 in Malaysian Borneo and Palawan Island in the Philippines(7, 8). Although human infections of 77 P. knowlesi have been reported in both setting(5, 7, 9), cases have been sporadic in Palawan(6, 10, 78 11) whilst P. knowlesi is now the leading cause of human malaria in Sabah(5, 12, 13). 79 80 The primary reservoirs of P. knowlesi are the long-tailed (Macaca fascicularis) and pig-tailed 81 (M. nemestrina) macaques that are widely distributed throughout Southeast Asia(14, 15). Long- 82 tailed macaques are the only monkey species in the Philippines, and are widely distributed 83 throughout the country including Palawan(16). While Long-tailed macaques have been 4 84 confirmed as reservoirs of P. knowlesi in Palawan(17), there is limited understanding of the 85 ecology of P. knowlesi transmission and potential for human spillover in this setting. Of 86 particular concern is whether human P. knowlesi cases will continue to be sporadic and rare in 87 Palawan, or transition into substantial spillover into human populations as has occurred in the 88 neighbouring area of Sabah, Malaysian Borneo; which is <100km across the sea from Palawan. 89 Variation in epidemiological potential may be related to differences in vector species and their 90 interactions with human and macaque host species. Understanding the local ecology of 91 transmission is vital to identify both spillover potential and control strategies(14). 92 Competent Anopheles species that feed on both human and monkey hosts could act of 93 P. knowlesi bridge vectors(15, 18) . Mosquitoes in the Anopheles Leucosphyrus group have been 94 implicated as P. knowlesi vectors and capable of cross-species transfer between macaques to 95 humans(12, 18-21). Primary vector species vary geographically(22-25); with An. balabacensis and 96 An. donaldi being the most important in Sabah(26, 27). In the Philippines, there has been 97 relatively limited investigations of Anopheles vectors of simian malaria. Early work (1970s) 98 indicated that An. balabacensis is the most likely vector of simian malaria on Palawan(28) ; 99 however there been no recent confirmation of the role of this vector within the period of P. 100 knowlesi emergence in humans in Palawan. 101 Investigating the ecology and behavior of potential vectors of P. knowlesi and 102 incrimination of the vector species responsible for cross-species transmission are essential in 103 identifying human populations at risk and developing appropriate control strategies(29). The 104 gold standard for directly measuring human exposure and risk of malaria infection is the 105 Human Landing Catch (HLC)(30, 31). However, it raises ethical concerns by exposing people to 106 mosquitoes that might be infected with mosquito-borne diseases; many of which have no