Nephromyces, a beneficial apicomplexan symbiont in marine animals

Abstract

With malaria parasites (Plasmodium spp.), Toxoplasma, and many other species of medical and veterinary importance its iconic representatives, the protistan phylum Apicomplexa has long been defined as a group composed entirely of parasites and pathogens. We present here a report of a beneficial apicomplexan: the mutualistic marine endosymbiont Nephromyces. For more than a century, the peculiar structural and developmental features of Nephromyces, and its unusual habitat, have thwarted characterization of the phylogenetic affinities of this eukaryotic microbe. Using short-subunit ribosomal DNA (SSU rDNA) sequences as key evidence, with sequence identity confirmed by fluorescence in situ hybridization (FISH), we show that Nephromyces, originally classified as a chytrid fungus, is actually an apicomplexan. Inferences from rDNA data are further supported by the several apicomplexan-like structural features in Nephromyces, including especially the strong resemblance of Nephromyces infective stages to apicomplexan sporozoites. The striking emergence of the mutualistic Nephromyces from a quintessentially parasitic clade accentuates the promise of this organism, and the three-partner symbiosis of which it is a part, as a model for probing the factors underlying the evolution of mutualism, pathogenicity, and infectious disease.

Fig. 1.

Nephromyces cells from renal sac fluid in M. retortiformis. Arrows indicate spores and flagellated cells. Larger filamentous cells are trophic stages. (Scale bar: 20 μm.)

Fig. 2.

Phylogenetic tree of Apicomplexa, based on 18S ribosomal RNA genes, with representatives of known apicomplexans, and Nephromyces sequences from four Molgula species, including seven cloned sequences of Nephromyces isolated from M. occidentalis (sequences from 18 additional M. occidentalis-Nephromyces clones, not included in the analysis, were identical or nearly identical to clones 4F or 6C). Colpodellids, dinoflagellates, and ciliates were used as outgroups. The topology was constructed using maximum likelihood (PhyML); support values are expressed as percent bootstrap values (greater than 50%) and Bayesian posterior probabilities (>0.5). (Scale bar: 0.1 substitutions per site.) See Table S1 for accession numbers of taxa used in analysis.

Fig. 3.

FISH of Nephromyces and Toxoplasma using apicomplexan-specific (Api-L) and Nephromyces-specific (Neph-1) SSU rRNA probes. Both probes were bound to fluor Cy3 (red). (Scale bars: A, B, D, 20 μm; C, 10 μm; E, 3 μm.) (A) Toxoplasma, Api-L; (B) Nephromyces from M. occidentalis, Api-L. Arrow indicates spores or flagellated cells. All other cells are uncleaved sporangia or trophic stages. (C) Nephromyces from M. manhattensis, Neph-1 (with eubacterial probe, EUB 338, linked to the fluor BODIPY FL (green). Toxoplasma did not bind to the Neph-1 probe. Arrow indicates spores or flagellated cells. All other cells are trophic stages. (D) Nephromyces from M. manhattensis, Api-L [merged with general eukaryote probe, EUK-516, linked to green (Fluos) fluor. Yellow represents binding to both Api-L and EUK-516]. Arrow indicates spores or flagellated cells. All other cells are uncleaved sporangia or trophic stages. (E) Nephromyces spores, Api-L, with EUB 338-BODIPY FL (green), and DAPI (blue).

Fig. 4.

Apicomplexan-like cells in Nephromyces. (A) Infective stage of Nephromyces, in heart of laboratory-raised M. occidentalis, shortly after inoculation with Nephromyces spores. Photo clip from video (Zeiss Axiocam HS camera). (Scale bar: 5 μm.) (B) Sporozoite-like cells in the renal sac of M. retortiformis. (Scale bar: 20 μm.) (C and D) Transmission electron micrographs of two Nephromyces spores from M. occidentalis, with arrows showing rhoptry-like inclusions. (Scale bar: 0.5 μm.)