Indel-Informed Bayesian Analysis Suggests Cryptic Population

Infection, Genetics and Evolution 75 (2019) 103994 Contents lists available at ScienceDirect Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid Research Paper Indel-informed Bayesian analysis suggests cryptic population structure between Plasmodium knowlesi of humans and long-tailed macaques (Macaca T fascicularis) in Malaysian Borneo ⁎ JustinJ.S. Wilcox ,1, Abigail Kerschner, Hope Hollocher Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556-5688, USA ARTICLE INFO ABSTRACT Keywords: Plasmodium knowlesi is an important causative agent of malaria in humans of Southeast Asia. Macaques are Malaria natural hosts for this parasite, but little is conclusively known about its patterns of transmission within and Plasmodium knowlesi between these hosts. Here, we apply a comprehensive phylogenetic approach to test for patterns of cryptic Zoonosis population genetic structure between P. knowlesi isolated from humans and long-tailed macaques from the state Macaque of Sarawak in Malaysian Borneo. Our approach differs from previous investigations through our exhaustive use Host specificity of archival 18S Small Subunit rRNA (18S) gene sequences from Plasmodium and Hepatocystis species, our inclusion Phylogeny of insertion and deletion information during phylogenetic inference, and our application of Bayesian phylogenetic inference to this problem. We report distinct clades of P. knowlesi that predominantly contained sequences from either human or macaque hosts for paralogous A-type and S-type 18S gene loci. We report significant partitioning of sequence distances between host species across both types of loci, and confirmed that sequences of the same locus type showed significantly biased assortment into different clades depending on their host species. Our results support the zoonotic potential of Plasmodium knowlesi, but also suggest that humans may be preferentially infected with certain strains of this parasite. Broadly, such patterns could arise through pre- ferential zoonotic transmission of some parasite lineages or a disposition of parasites to transmit within, rather than between, human and macaque hosts. Available data are insufficient to address these hypotheses. Our results suggest that the epidemiology of P. knowlesi may be more complicated than previously assumed, and highlight the need for renewed and more vigorous explorations of transmission patterns in the fifth human malarial parasite. 1. Introduction malaria and the management of human-macaque interactions. The evidence to date unequivocally suggests that P. knowlesi has Plasmodium knowlesi has recently been acknowledged as an im- zoonotic origins in humans and is regularly transmitted between long- portant causative agent of human malaria in Southeast Asia (Singh tailed macaques (Macaca fascicularis) and humans. Several phylogenies et al., 2004; Cox-Singh et al., 2008; White, 2008). Although it is gen- have shown co-clustering of human and macaque P. knowlesi strains erally regarded as a zoonotic agent that humans incidentally acquire across multiple loci (Singh et al., 2004; Jeslyn et al., 2011; Lee et al., through sympatry with macaques, it can persist in human populations 2011; Jongwutiwes et al., 2011; Putaporntip et al., 2013), and experi- and accounts for a substantial portion of human malaria burden in some mental studies provide additional evidence for cross-transmission by regions (Cox-Singh and Singh, 2008; Cox-Singh et al., 2008; Barber showing that P. knowlesi from macaques will easily infect humans et al., 2012; William et al., 2013). The ubiquity of P. knowlesi in humans through direct inoculation (White et al., 2008; Singh and Daneshvar, of endemic regions provides the potential for transmission between 2013). Plasmodium knowlesi is also transmitted through many of the humans and obfuscates the relative roles of zoonotic, enzootic, and same vectors (most notably Anopheles species of the Leucosphyrus sub- (potentially) human-to-human transmission in driving human infec- group) as dedicated human malarias (Vythilingam et al., 2016) and has tions (Singh and Daneshvar, 2013; William et al., 2013). Resolving been reported to form gametocytes in humans (Singh et al., 2004; these relative roles is of paramount concern to the control of knowlesi- Maeno et al., 2017; Davidson et al., 2019), suggesting that human-to- ⁎ Corresponding author at: New York University Abu Dhabi, Abu Dhabi, United Arab Emirates. E-mail address: [email protected] (J.S. Wilcox). 1 Present Address: P.O. NYUAD 129188 New York University Abu Dhabi, Abu Dhabi, United Arab Emirates. https://doi.org/10.1016/j.meegid.2019.103994 Received 10 March 2019; Received in revised form 1 August 2019; Accepted 3 August 2019 Available online 14 August 2019 1567-1348/ © 2019 Published by Elsevier B.V. J.S. Wilcox, et al. Infection, Genetics and Evolution 75 (2019) 103994 human transmission of P. knowlesi could be possible as well (Marchand this consists of 413 sequences in total. Of these, we could attribute 47 et al., 2011), particularly as mosquito-vectored serial passage of Plas- sequences to P. knowlesi (see Supplementary Table 1). Sequences were modium knowlesi between humans has been achieved experimentally compiled from the Silva 128 database using an automated tcsh-shell (Chin et al., 1968). script; the Babesia divergens sequence AJ439713 was also included with Natural human-to-human transmission of P. knowlesi has not been these as an outgroup. This outgroup sequence was chosen as Babesia shown and is difficult to directly assess. Population substructuring be- spp. are among the closest relatives of Plasmodium spp. with available tween human and macaque hosts may, however, provide indirect evi- 18S sequence information. Sequences were aligned using the MafftQ- dence for human-to-human transmission. A study in Thailand has, for ins-i iterative alignment for structural RNAs (Katoh and Standley, example, reported clades of P. knowlesi that appear to occur more ty- 2013). Redundant sequences were removed using the CD-hit program pically in either humans or macaques, consistent with expectations (Li and Godzik, 2006; Fu et al., 2012), and remaining sequences human-to-human transmission (Putaporntip et al., 2013), despite the (N = 320, including the outgroup, of which 28 were attributable to P. low incidence of human knowlesi-malaria in this country (Jongwutiwes knowlesi) were realigned using the Q-INS-i iterative alignment. Pro- et al., 2011). While studies from Malaysia have, overwhelmingly shown cessed sequences were compiled into Nexus format in two partitions to phylogenetic cross-clustering of sequences from parasites of macaques produce an all-sequence indel-informed tree: one partition contained and humans (Singh et al., 2004; Jeslyn et al., 2011; Lee et al., 2011), full sequence data; the other contained only presence-absence data for they may provide some indirect evidence for cryptic partitioning be- each base in the alignment. Host and geographic information were re- tween hosts, by revealing: low haplotype overlap between parasites of corded for each remaining unique sequence using National Center for human and macaque hosts (Lee et al., 2011); tendencies for overlapping Bioinformation Technology (NCBI) Genbank; relevant publications haplotypes to occur at internal nodes of haplotype networks (Lee et al., were consulted when necessary. Plasmodium spp. possess multiple co- 2011; Yusof et al., 2016), which are reflective of more ancestral states pies of the 18S gene that are traditionally classified as A-type that are (Castelloe and Templeton, 1994; Templeton, 1998); and high levels of primarily transcribed during the asexual life stage of the parasite in its intraspecific genomic diversity of P. knowlesi relative to other human vertebrate host, and S-type that are primarily transcribed during the malarial agents with cryptic structuring of this diversity within humans sexual life stage of the parasite during its mosquito host (Waters, 1994; (Assefa et al., 2015; Divis et al., 2015; 2017). While these studies Gardner et al., 2002; Leclerc et al., 2004). As these two types of loci are provide a clear role for zoonosis, human-to-human transmission may the result of multiple duplication events, the true homologs of these loci occur and the primacy of zoonotic transmission as the only source of in other Plasmodium spp. are unknown, and as A-type and S-type loci human knowlesi-malaria infections remains an assumption (Assefa et al., are not definitively known to be homologs of one another across Plas- 2015). Under these circumstances of potentially incomplete assortment modium spp., all 18S paralog sequences were included in our analysis of P. knowlesi lineages infecting humans and macaques, more sensitive (Nishimoto et al., 2008; Jiang et al., 2010); ribosome type (i.e. A-type phylogenetic approaches may detect cryptic divisions between P. or S-type) was controlled for during down-stream data analysis (see knowlesi in these hosts. “2.3 Statistics”). In order to establish the role of indels in driving our Here, we utilize a customized, high-throughput pipeline to build a results, we also produced a tree using the full 18S dataset but excluding high-resolution indel-informed Bayesian tree of archived 18S gene se- the indel partition from the final Nexus file: an all-sequence no-indel quences from Plasmodium spp. and Hepatocystis spp., which are reported tree. to be contained within the genus Plasmodium (Galen et al., 2018). We In addition to producing

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