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Lineage-Specific Molecular Probing Reveals Novel Diversity and Ecological Partitioning of Haplosporidians

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Lineage-Specific Molecular Probing Reveals Novel Diversity and Ecological Partitioning of Haplosporidians The ISME Journal (2014) 8, 177–186 & 2014 International Society for Microbial Ecology All rights reserved 1751-7362/14 www.nature.com/ismej ORIGINAL ARTICLE Lineage-specific molecular probing reveals novel diversity and ecological partitioning of haplosporidians Hanna Hartikainen1, Oliver S Ashford1,Ce´dric Berney1, Beth Okamura1, Stephen W Feist2, Craig Baker-Austin2, Grant D Stentiford2,3 and David Bass1 1Department of Life Sciences, The Natural History Museum, London, UK; 2Centre for Environment, Fisheries and Aquaculture Science (Cefas), The Nothe, UK and 3European Union Reference Laboratory for Crustacean Diseases, Centre for Environment, Fisheries and Aquaculture Science (Cefas), The Nothe, UK Haplosporidians are rhizarian parasites of mostly marine invertebrates. They include the causative agents of diseases of commercially important molluscs, including MSX disease in oysters. Despite their importance for food security, their diversity and distributions are poorly known. We used a combination of group-specific PCR primers to probe environmental DNA samples from planktonic and benthic environments in Europe, South Africa and Panama. This revealed several highly distinct novel clades, novel lineages within known clades and seasonal (spring vs autumn) and habitat- related (brackish vs littoral) variation in assemblage composition. High frequencies of haplospor- idian lineages in the water column provide the first evidence for life cycles involving planktonic hosts, host-free stages or both. The general absence of haplosporidian lineages from all large online sequence data sets emphasises the importance of lineage-specific approaches for studying these highly divergent and diverse lineages. Combined with host-based field surveys, environmental sampling for pathogens will enhance future detection of known and novel pathogens and the assessment of disease risk. The ISME Journal (2014) 8, 177–186; doi:10.1038/ismej.2013.136; published online 22 August 2013 Subject Category: Microbial ecology and functional diversity of natural habitats Keywords: Ascetosporea; Haplosporidia; Haplosporidium; Minchinia; Bonamia; Urosporidium Introduction The notable lack of information on haplosporidian diversity revealed by environmental sampling to Characterising the diversity and distribution of date may reflect absence or low representation parasites remains a challenge due to their patchy in environmental sequencing databases (due to, for spatial and temporal distributions, host-restricted example, short-lived zoospores), artefacts of sampling occurrence and poorly known life cycles. This is strategy and/or the use of broadly targeted PCR particularly true for microbial endoparasites, which primers that are unsuitable for the highly divergent are typically undersampled relative to macropara- genes that may characterise many parasitic taxa (for sites (for example, helminths). However, many para- example, Canning and Okamura, 2004). sites disperse and complete their life cycles via Haplosporidia include the causative agents of release of infectious stages into the environment or the commercially significant oyster diseases MSX they infect small hosts that are incorporated in such (caused by Haplosporidium nelsoni (Burreson and samples. Environmental DNA detection techniques Ford, 2004)), bonamiosis (caused by various Bonamia may therefore offer an untapped and powerful means spp. (Pichot et al., 1980; Carnegie et al., 2006)) of characterising parasite assemblages, particularly in and agents of several decapod crustacean diseases view of how such approaches have revolutionised (Stentiford et al., 2004; Utari et al., 2012). our understanding of microbial diversity in a wide The reticulate amoebae, Filoreta and Gromia,are range of habitat types (Moreira and Lo´pez-Garcıa, the closest known free-living relatives; phytomyxids, 2002; Richards and Bass, 2005; Bik et al., 2012). vampyrellids and other Cercozoa are more distantly related (Cavalier-Smith and Chao, 2003; Bass et al., 2009). There are currently four recognised haplospor- Correspondence: H Hartikainen, Department of Life Sciences, idian genera: (1) Bonamia: four described species, all The Natural History Museum, London SW7 5BD, UK. E-mail: [email protected] economically important oyster parasites in temperate Received 15 March 2013; revised 12 June 2013; accepted 9 July waters; (2) Minchinia: a sparsely recorded genus of 2013; published online 22 August 2013 five species with sequence data from molluscs from Haplosporidian diversity H Hartikainen et al 178 the US Atlantic coast, Western Australia and Europe; the basis for prediction and mitigation of diseases (3) Urosporidium: one described species with caused by haplosporidians and other economically sequence data (U. crescens;(Floreset al., 1996) and important pathogens. another sequenced specimen (Reece et al., 2004), both occurring as hyperparasites of trematode worms; Materials and methods and (4) Haplosporidium: the most intensively studied and diverse genus (approximately 40 species Sampling protocol to date) and perhaps paraphyletic (Burreson and Environmental samples were collected from two Ford, 2004). Most known members of the genus infect localities in Weymouth (WEY), SW England: an molluscs, but some infect crustaceans (Burreson and intertidal rock pool in the Newton’s Cove (35 p.p.t. Ford, 2004; Stentiford et al., 2004) and annelids salinity; 50134N, 2122W) and a muddy, tidal channel (Siddall and Aguado, 2006). A minority of lineages in the Fleet Estuary (o10–30 p.p.t. salinity; 50135’N, infect freshwater molluscs (Molloy et al., 2012). 2128’W). Both sites were sampled during low tide in Additionally, some ‘haplosporidians’ that have not July 2011, October 2011 and April 2012. Approxi- been taxonomically characterised and/or do not mately 150 l of water was passed serially through branch robustly with characterised taxa on molecular 100-, 50- and 20-mm meshes, the material collected phylogenies have been sequenced (Reece et al., 2004). from the meshes using spatulas, kept cool and To date, methods of haplosporidian discovery frozen at À 80 1C on return. Samples were thawed have been largely based on direct histological and as part of the lysis step of the DNA extraction, molecular characterisation of infected host tissues. thus avoiding degradation caused by enzymes Although this approach is appropriate for many released from freeze–thaw-lysed cells. The filtered studies, screening a wide range of potential hosts is water was further processed using pressure filtration not generally feasible and is therefore not a good (Sartorius, Stedim, Go¨ttingen, Germany) onto way to estimate the diversity or size of the group. 142-mm diameter, 0.45-mm polycarbonate filter We hypothesised that there are many undetected papers. The papers were scraped using razor blades, haplosporidian lineages, as has been demonstrated scrapings frozen in liquid nitrogen and stored at in free-living protists (Bass and Cavalier-Smith, À 80 1C. Sixteen B2 g sediment samples were 2004; Bra˚te et al., 2010; Howe et al., 2011; Jones collected from each site in July and October. et al., 2011). Because haplosporidians are important To provide a wider geographic and ecological pathogens with global impacts on aquaculture, it is representation, five further sample sets were ana- important to appreciate their diversity, particularly lysed: (1) South Africa (SA): 120 water (B2 l) and as changing climates and intensifying farming sediment (B1–2 g) samples collected in December activities may provoke previously non-problematic 2011 from freshwater and marine sites in South parasites to emerge, causing outbreaks, high Africa, (2) BioMarKs (BM): matched DNA and cDNA mortalities and threats to aquaculture (Okamura from water column and sediment samples from and Feist, 2011). Knowledge of spatial and temporal eight coastal marine sites from the North Sea, the abundances of infectious stages in the environment Mediterranean, the Baltic and the Black Sea (col- may contribute to food security and aquaculture lected as part of the BioMarKs consortium, http:// sustainability, for instance, by providing early www.biomarks.eu) (Bass et al., 2012; Logares et al., warning systems of pathogen presence and planning 2012; Pawlowski et al., 2013), (3) Exeter (EXE): 118 tools, for example, for the siting of mollusc culture DNA extractions enriched in small metazoans (by in low disease-risk regions. filter fractionation, picking of specimens and so on) Our aims were: (1) to characterise the spatial and from freshwater and marine water samples from temporal patterns of occurrence of known and novel southern England and the Mediterranean (K Hamil- haplosporidian lineages in a range of planktonic and ton and B Williams, personal communication), (4) sediment samples, (2) to examine haplosporidian Estuarine gradient (EST): DNA from 32 marine-to- diversity in a variety of marine and freshwater brackish sediment samples (B1–2 g), collected along habitats and thereby improve the internal phylogeny the River Colne estuary in southeast England of the group, and (3) to screen existing amplicon and (Hawkins and Purdy, 2007; Dong et al., 2009) and metagenomic data sets for haplosporidians in order (5) Panama (PAN): 12 marine sediment samples to determine whether such databases provide (B1–2 g) from sandy beach sites on the Pacific and reliable representation of haplosporidians. Our Caribbean coasts around Panama City and Portobelo results revealed a previously unappreciated and (Colo´n).
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