Unexpected absence of genetic separation of a highly diverse population of hookworms from geographically isolated hosts
Graphical abstract
Introduction
Parasite population structure is dictated by their ability to disperse (Badets et al., 2011, Poulin et al., 2011, Rohde, 2002). The parasite populations of canine hookworm (Ancylostoma caninum) and human hookworm (Ancylostoma duodenale) are not associated with the site of parasite collection because of the dispersal of the host, with no geographical barriers for both canine and human hosts (Hawdon et al., 2001, Hu et al., 2002, Miranda et al., 2008, Moser et al., 2007). In contrast, we expect that hookworm, Uncinaria sanguinis, from the endangered Australian sea lion, Neophoca cinerea, whose population of fewer than 15,000 individuals (Shaughnessy et al., 2011) is distributed across highly fragmented island colonies along the South and Western Australian coast, would exhibit geographical segregation through the process of vicariance; extreme natal site fidelity of their female host (Campbell et al., 2008, Lowther et al., 2012) and transmammary mode of transmission, imply restricted parasite dispersal (Fig. 1). Mitochondrial DNA analysis of Australian sea lion females indicates little or no interchange of females between most breeding colonies, with marked genetic restriction of mtDNA occurring between the three largest colonies, Dangerous Reef, Seal Bay, and The Pages Islands (Campbell et al., 2008, Lowther et al., 2012). We therefore hypothesise that, as with maternally derived genes, the extreme natal site fidelity of females will result in geographical barriers to maternally transmitted pathogens, such as hookworm.
Hookworms are blood-feeding intestinal worms responsible for high levels of morbidity and mortality in a wide range of mammals (Hotez et al., 2005, Hotez et al., 2004). Hookworm females lay eggs which are passed into the environment in host faeces (Fig. 1). The free-living infective larvae hatch and then infect new hosts percutaneously (Hotez et al., 2004, Loukas and Prociv, 2001, Olsen and Lyons, 1965). However, unlike in other hosts, percutaneous infection with hookworm larvae is not recognised to result in patent infection or cause clinical disease in otariids (fur seals and sea lions) (Olsen and Lyons, 1965). Larvae remain dormant in the ventral abdominal blubber until parturition, at which time they migrate to the mammary tissue and infect pups via the colostrum. Neonatal otariids exhibit high parasite burdens soon after birth but eggs are not detected in pup faeces after 2–8 months of age (Lyons et al., 2011, Olsen and Lyons, 1965). Based on this life cycle, adult males are considered to be dead end hosts (Castinel et al., 2007, Olsen and Lyons, 1965).
The aim of this study was to investigate the existence of geographical barriers (=vicariance) for hookworms (U. sanguinis) among the three largest, genetically restricted, island colonies of the Australian sea lion, N. cinerea. We used the mitochondrial cytochrome c oxidase subunit I gene (cox1) locus of mtDNA to evaluate the genetic diversity and population structure of hookworms infecting N. cinerea pups at these colonies. The use of the cox1 locus was validated against mtDNA marker genes across two newly determined complete U. sanguinis mtDNA genomes. The results are compared directly to the genetic diversity of canine and human hookworms reported previously. Apparent absence of geographical barriers is discussed, together with its implications to the ecology of N. cinerea populations.
Section snippets
Specimens of hookworm (U. sanguinis) from Australian sea lion (N. cinerea) colonies in South Australia
Specimens of U. sanguinis were collected from Australian sea lion pups at three geographically isolated colonies in South Australia that show restriction of host mtDNA between colonies. All specimens were morphologically consistent with U. sanguinis (Marcus et al., 2014a). Samples were collected from Dangerous Reef, Spencer Gulf (34°48′54″S, 136°12′43″E), South Page Island, Backstairs Passage (35°46′37″S, 138°17′31″E) and Seal Bay, Kangaroo Island (35°59′40″S, 137°19′0″E) (Fig. 2). These
Results
Genetic diversity, based on cox1 from a single hookworm per host (n = 46, Dataset 1), revealed high haplotype diversity (h = 0.991) and high nucleotide diversity (π = 0.008) with 40 unique haplotypes identified (Table 1). Similarly, genetic diversity of hookworms within a single host was high, based on cox1 sequence of five worms from each of two individual pups, (Dataset 2 and 3); these demonstrated high haplotype and nucleotide diversity (Dataset 2: h = 1, π = 0.009, 5 haplotypes; Dataset 3: h = 0.900, π =
Discussion
The high diversity and low geographical differentiation of cox1 haplotypes of U. sanguinis from geographically and genetically distinct populations of N. cinerea rejects the vicariance hypothesis that female host natal site fidelity and the transmammary route of infection restricts hookworm gene flow between N. cinerea populations.
Lack of isolation by distance was previously documented for Nec. americanus and A. caninum with ΦST estimated as 0.12 and 0.28 respectively (Hawdon et al., 2001,
Acknowledgements
Funding for sample collection was provided by Australian Marine Mammal Centre, Australian Antarctic Division, Australian Federal Government, and the Winifred Violet Scott Foundation. Support was also provided by a Faculty of Veterinary Science Whitehead Bequest – Conservation. We thank Clarence Kennedy, Janet Simpson and the Staff of Seal Bay of the Department of Environment, Water and Natural Resources, South Australia and the many volunteers for their assistance in the field and sample
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