Journal of Eukaryotic Microbiology ISSN 1066-5234 ORIGINAL ARTICLE High Prevalence and Endemism of Trypanosomatids on a Small Caribbean Island Jan Votypka a,b, Petr Kmentc, Eva Kriegovaa, Mark J.A. Vermeijd,e, Patrick J. Keelingf, Vyacheslav Yurchenkoa,g,h & Julius Lukesa,i a Institute of Parasitology, Biology Centre, Czech Academy of Sciences, 370 05, Cesk e Budejovice (Budweis), Czech Republic b Department of Parasitology, Faculty of Science, Charles University, 128 44, Prague, Czech Republic c Department of Entomology, National Museum, 148 00, Prague, Czech Republic d Carmabi Foundation, Willemstad, Curacßao e Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, Amsterdam, The Netherlands f Department of Botany, University of British Columbia, Vancouver, BC, Canada g Faculty of Science, University of Ostrava, 710 00, Ostrava, Czech Republic h Martsinovsky Institute of Medical Parasitology, Tropical and Vector Borne Diseases, Sechenov University, Moscow, Russia i Faculty of Sciences, University of South Bohemia, 370 05, Cesk e Budejovice (Budweis), Czech Republic Keywords ABSTRACT Blastocrithidia; conservation; endemic species; evolution; Heteroptera; island We describe the monoxenous trypanosomatids parasitizing true bugs and flies ß biogeography theory; Leptomonas; on the island of Curacao. Out of 248 examined true bugs belonging to 17 spe- Phytomonas. cies, 93 individuals were found to be infected (overall 38% prevalence) by at least one trypanosomatid species (referred to as typing units; TUs). Out of 80 Correspondence flies, six were infected. All detected trypanosomatids were compared based J. Votypka, Department of Parasitology, on their 18S rRNA sequences with TUs parasitizing bugs and flies described Faculty of Science, Charles University, from mainland South America, allowing us to assess their diversity and distri- Vinicn a 7, 128 44 Prague, Czech Republic bution. Besides Leptomonas pyrrhocoris and Leptomonas seymouri, two Telephone number: +420-221-951-826; known species of the subfamily Leishmaniinae, our analysis revealed six new e-mail: [email protected] TUs falling into the groups ‘jaculum’, Blastocrithidia and Herpetomonas. More- and over, two new members of the genus Phytomonas and three new TUs J. Lukes, Institute of Parasitology, Biology belonging to the monophyletic group designated as ‘new clade II’ sensu Mol. Centre, Czech Academy of Sciences, Phylogenet. Evol, 69, 255 (2013) were isolated. The detected trypanosomatids Branisovsk a 31, 370 05, Ceske Budejovice were characterized by moderate diversity (13 TUs) species richness. Out of (Budweis), Czech Republic nine and four TUs from the heteropteran and dipteran hosts, respectively, 11 Telephone number: +420-387-775-416; TUs have not been encountered before. Although a sampling bias may partially e-mail: [email protected] affect the comparison between trypanosomatid communities on Curacßao and the mainland, the high proportion of unique TUs from the former location sug- Received: 22 October 2018; revised 13 gests that the prominent role of islands in increasing the global diversity of November 2018; accepted November 16, macroscopic organisms may also extend to their protistan parasites. 2018. Early View publication December 18, 2018 doi:10.1111/jeu.12704 SINCE the formulation of the theory of island biogeogra- lower species diversity than in areas of similar size on the phy (MacArthur and Wilson, 1967), islands have widely nearby mainland (Blondel 2000; Whittaker and Fernandez- been considered to be model systems for studying funda- Palacios 2007). It is generally assumed that islands har- mental principles in ecology, evolutionary biology and bio- bour endemic species and that the relative absence of geography because island communities are relatively predators creates (sub)species that would not survive simple in terms of biodiversity and ecological interactions elsewhere. Particularly very old and well isolated oceanic (Emerson 2002; Graham et al. 2017; Warren, et al. 2015). islands represent conditions resulting in higher levels of On islands, communities of macroscopic organisms show extant biodiversity and endemism of inhabiting organisms. © 2018 International Society of Protistologists 600 Journal of Eukaryotic Microbiology 2019, 66, 600–607 Votypka et al. Trypanosomatid Diversity on Curacßao Oceanic islands are therefore often considered as impor- for this Neotropical region (Borghesan et al. 2013; tant biodiversity hotspots (Myers et al. 2000). Kozminsky et al. 2015; Maslov et al. 2007, 2010; Teix- However, the situation is different on small islands that eira et al. 2011; Westenberger et al. 2004; Yurchenko are exposed to a strong influence from the mainland; due et al. 2006). to their proximity to the continent, such islands are consid- ered less conducive for the emergence of new and ende- MATERIALS AND METHODS mic species (Gray and Cavers 2014; Warren et al. 2015). Colonization from the mainland is very common, but the The Caribbean island of Curacßao (16N, 69W) is located number of biotopes is often smaller on the island than on approximately 66 km north of Venezuela, having a popula- the mainland. In addition, extinction rates on continental tion of 160,000 on an area of 444 km2. Although it is islands are high due to the relative scarcity of suitable home to several endemic animals, its fauna and flora are habitats and higher competition for limited resources highly similar to those of the nearby mainland. The island (Losos and Ricklefs 2010). These principles underlying is very dry, has a tropical savannah climate and xeric Island Biogeography Theory (IBT) were largely derived scrublands with various forms of cacti and thorny shrubs from studies of vertebrates, such as lizards, mammals and as the prevailing vegetation (http://www.meteo.cw/clima birds, and, to a lesser extent, from selected groups of te.php). insects and snails. However, very little is known about Insects, predominantly true bugs (Heteroptera) and islandic microorganisms including parasitic ones. smaller number of flies (Diptera: Brachycera), were col- Parasites can respond to the occurrence and distribution lected within two seasons in March and April 2015 and of their hosts. For example, human pathogens on oceanic April 2016 by sweep netting in the vicinity of the Carib- islands follow the basic predictions of IBT, increasing in bean Research and Management of Biodiversity (Carmabi) number with increasing size of oceanic islands when they research station in Piscadera Baai, and throughout a num- lie closer to the mainland (Jean et al. 2016). Understand- ber of biotopes of Curacßao. Collected true bugs were iden- ing the reasons why hosts lose, maintain or swap their tified to species using available keys and comparative parasites after colonizing new areas has long intrigued bio- collections, whereas flies were identified to the family geographers. IBT predicts that continental hosts will level only. Host specimens were dry-mounted and depos- harbour greater parasite richness than their island counter- ited in the collections of Department of Entomology, parts, retaining only a subset of the parasite species from National Museum, Prague, Czech Republic. the continental source pool (MacArthur and Wilson 1967). Within 24 h after capture, insects were killed and sur- Parasite richness increases with host density and diversity face-sterilized with 70% ethanol, washed and dissected in and is therefore expected to decline on small and remote 0.9% sterile saline solution and examined for parasites islands (Arneberg et al. 1998). Studies comparing differ- under a microscope as described previously (Votypka et al. ences in parasite diversity between the mainland and 2010, 2012a). To establish the primary cultures of try- nearby island communities are scarce. To the best of our panosomatids, contents of the insect intestines were culti- knowledge, pathogenic hemosporidians of birds are the vated in the Brain Heart Infusion as described elsewhere only protist parasites whose communities have been com- (Votypka et al. 2014; Yurchenko et al. 2016). Total geno- pared between the mainland and nearby islands (Illera mic DNA was isolated from the gut tissue, or in the case et al. 2015; Padilla et al. 2017; Perez-Rodr ıguez et al. of successful cultivations, from 1-ml of axenically grown 2013). cultures, using a protocol described previously (Votypka Monoxenous (= single-host) trypanosomatid flagellates et al. 2014). For amplifying the trypanosomatid 18S rRNA are confined to insects and represent the bulk of known gene, about 10 ng DNA was subjected to nested PCR diversity of the family Trypanosomatidae, while a smaller described by Seward et al. (2017), and obtained PCR prod- fraction of trypanosomatids comprises dixenous (= two- ucts were directly sequenced. The sequences were hosts) species that circulate between a vertebrate/plant aligned using Geneious software (version 10.0.6, https:// and an invertebrate host (Maslov et al. 2013; Podlipaev www.geneious.com), and phylogenetic trees were con- 2001). Phylogenetic studies strongly hint to the emer- structed using related sequences available in GenBank. gence of the dixenous flagellates that include members Alignments for phylogenetic analysis were generated in of the genera Trypanosoma and Leishmania and are Kalign, with ambiguously aligned positions in the trimmed responsible for numerous deadly diseases of humans alignment being removed manually in BioEdit. The final and other mammals, from their monoxenous