Support for the coevolution of Neoparamoeba and their endosymbionts, Perkinsela amoebae-like organisms

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Abstract

Some of the species from the genus Neoparamoeba, for example N. perurans have been shown to be pathogenic to aquatic animals and thus have economic significance. They all contain endosymbiont, Perkinsela amoebae like organisms (PLOs). In this study we investigated phylogenetic ambiguities within the Neoparamoeba taxonomy and phylogenetic congruence between PLOs and their host Neoparamoeba to confirm the existence of a single ancient infection/colonisation that led to cospeciation between all PLOs and their host Neoparamoeba. DNA was extracted and rRNA genes from host amoeba and endosymbiont were amplified using PCR. Uncertainties in the Neoparamoeba phylogeny were initially resolved by a secondary phylogenetic marker, the internal transcribed spacer 2 (ITS2). The secondary structure of ITS2 was reconstructed for Neoparamoeba. The ITS2 was phylogenetically informative, separating N. pemaquidensis and N. aestuarina into distinct monophyletic clades and designating N. perurans as the most phylogenetically divergent Neoparamoeba species. The new phylogenetic data were used to verify the tree topologies used in cophylogenetic analyses that revealed strict phylogenetic congruence between endosymbiotic PLOs with their host Neoparamoeba. Strict congruence in the phylogeny of all PLOs and their host Neoparamoeba was demonstrated implying that PLOs are transmitted vertically from parent to daughter cell.

Introduction

Amoebae of the genus Neoparamoeba (Amoebozoa; Flabellinea; Dactylopodida; Vexilliferidae) are ubiquitous in marine environments (Page, 1983, Page, 1987). These small, lobose amoebae form dactylopodiate pseudopodia in their locomotive form and lack the cell-surface structures characteristic of other members of the Dactylopodida (Dyková et al., 2000, Dyková et al., 2005b, Page, 1983). The most discernable morphological feature of Neoparamoeba, shared only with Paramoeba and Janickina amoebae, is the presence of one or more intracellular endosymbionts (Dyková et al., 2000, Dyková et al., 2003). These endosymbionts were initially termed “parasomes” or “Nebenkörper” (Schaudinn 1896) and are kinetoplastid-like, aflagellate, usually binucleated eukaryotic cells with a single, giant kinetoplastid-mitochondrion (Dyková et al. 2003). Morphological similarities between Neoparamoeba “parasomes” and Perkinsiella amoebae, an endosymbiont of the Janickina amoebae (Hollande 1980) led to Neoparamoeba “parasomes” being named Perkinsiella amoebae-like organisms (PLOs) (Dyková et al., 2003, Moreira et al., 2004). Unfortunately the name Perkinsiella was already in use within another taxonomic group, therefore Dyková et al. (2008) renamed the generic name to Perkinsela which resolved the homonymy of its previous name whilst retaining the PLO acronym.

In most cases, when a host harbours an intracellular endosymbiont it profits from its biosynthetic capabilities (Moran et al. 2008). The advantages that Neoparamoeba and/or PLOs receive from their association are not known. However, as Neoparamoeba trophozoites always retain at least one PLO in intimate association with their nucleus, their symbiotic relationship is assumed to be obligatory and considered mutualistic (Dyková et al., 2000, Dyková et al., 2003). Furthermore, the available phylogenetic data suggest that, a strong coevolutionary relationship exists between endosymbiotic PLOs and Neoparamoeba pemaquidensis, Neoparamoeba branchiphila and Neoparamoeba aestuarina (Caraguel et al., 2007, Dyková et al., 2003, Dyková et al., 2008) suggesting that their symbiotic relationship is hereditary. The relationship between Neoparamoeba perurans and their PLOs has yet to be assessed. N. perurans are distinct from other Neoparamoeba as they are the only pathogenic species and confirmed aetiological agents of amoebic gill disease (AGD) (Crosbie et al., 2012, Young et al., 2007, Young et al., 2008). It is unclear why only N. perurans elicits AGD even though other Neoparamoeba species have been shown to cohabit the gills of AGD-affected fish (Dyková et al., 2005b, Dyková et al., 2007Fiala and Dyková, 2003, Kent et al., 1988).

Definitive taxonomic classification of Neoparamoeba and their endosymbiotic PLOs is currently based on 18S ribosomal RNA gene phylogenetic inference (Dyková et al., 2003, Dyková et al., 2005b, Dyková et al., 2007, Dyková et al., 2008Fiala and Dyková, 2003, Steinum et al., 2008, Young et al., 2007). With the addition of new Neoparamoeba species and strains the relationships among taxa are uncertain. It is for example unclear if N. perurans or N. branchiphila represent a basal lineage (Young et al. 2007) and what are the relationships between the two morphologically distinct species, Neoparamoeba pemaquidensis and Neoparamoeba aestuarina and other taxa of the group (Dyková et al., 2008, Steinum et al., 2008, Young et al., 2007). In this study, we expanded the 18S rRNA gene phylogeny of genus Neoparamoeba, by including two new Neoparamoeba isolates. Furthermore, we examined the usefulness of the rRNA internal transcribed spacer region (ITS) as an additional phylogenetic marker providing higher phylogenetic resolution. The new phylogenetic data were used in cophylogenetic analyses to assess the degree of phylogenetic congruence between endosymbiotic PLOs and their host Neoparamoeba.

Section snippets

Acquisition of clonal cultured and non-cultured gill-derived (NCGD) amoebae

Two new strains of Neoparamoeba were collected from Tasmania (Atlantic salmon Salmo salar) and Norway (red alga Palmaria palmata) for morphological and molecular characterisation (Table 1). Clonal, cultured amoebae in hanging drop preparations were examined using Nomarski differential interference contrast (DIC) light microscopy. Previous identification of strains was based on phylogenetic analyses and 18S rRNA gene-specific PCR (Dyková et al., 2003, Dyková et al., 2005c, Dyková et al., 2007,

Trophozoite morphology and 18S rRNA phylogeny of two new amoeba strains

The trophozoites of the two new strains were indistinguishable by light microscopy. Trophozoites contained one or more PLOs (Fig. 1, white arrow). To discriminate between the morphologically similar Neoparamoeba species the taxonomic positions of PAL2 and ASL1 were inferred from their 18S rRNA gene phylogeny. The Neoparamoeba 18S rRNA gene alignment consisted of 2205 nucleotide sites, and of these, 1871 (85%) were retained and 713 were parsimony-informative. The ML and BI analyses yielded three

Phylogeny of Neoparamoeba

As 18S SSU phylogeny did not resolve the relationship between N. pemaquidensis and N. aestuarina, the ITS was assessed as an additional phylogenetic marker to assist in the taxonomic classification of Neoparamoeba. ITS1 and ITS2 nucleotide sequences are removed via splicing during the process of transcription and therefore thought to be subject to mild functional constraints leading to nucleotide and length variation (Álvarez and Wendel 2003). Whilst ITS1 and ITS2 display a high degree of

Acknowledgements

This work formed part of a project of Aquafin CRC and received funds from the Australian Government CRCs Programme, the Fisheries R & D Corporation, other CRC participants, ARC/NHMRC Research Network for Parasitology and the Czech Science Foundation (206/05/2384).

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