Significance Mitochondrial respiration is an ancient characteristic of eukaryotes. However, it was lost independently in multiple eukaryotic lineages as part of adaptations to an anaerobic lifestyle. We show that a similar adaptation occurred in a member of the Myxozoa, a large group of microscopic parasitic animals that are closely related to jellyfish and hydroids. Using deep sequencing approaches supported by microscopic observations, we present evidence that an animal has lost its mitochondrial genome. The myxozoan cells retain structures deemed mitochondrion-related organelles, but have lost genes related to aerobic respiration and mitochondrial genome replication. Our discovery shows that aerobic respiration, one of the most important metabolic pathways, is not ubiquitous among animals.

Abstract Although aerobic respiration is a hallmark of eukaryotes, a few unicellular lineages, growing in hypoxic environments, have secondarily lost this ability. In the absence of oxygen, the mitochondria of these organisms have lost all or parts of their genomes and evolved into mitochondria-related organelles (MROs). There has been debate regarding the presence of MROs in animals. Using deep sequencing approaches, we discovered that a member of the Cnidaria, the myxozoan Henneguya salminicola, has no mitochondrial genome, and thus has lost the ability to perform aerobic cellular respiration. This indicates that these core eukaryotic features are not ubiquitous among animals. Our analyses suggest that H. salminicola lost not only its mitochondrial genome but also nearly all nuclear genes involved in transcription and replication of the mitochondrial genome. In contrast, we identified many genes that encode proteins involved in other mitochondrial pathways and determined that genes involved in aerobic respiration or mitochondrial DNA replication were either absent or present only as pseudogenes. As a control, we used the same sequencing and annotation methods to show that a closely related myxozoan, Myxobolus squamalis, has a mitochondrial genome. The molecular results are supported by fluorescence micrographs, which show the presence of mitochondrial DNA in M. squamalis, but not in H. salminicola. Our discovery confirms that adaptation to an anaerobic environment is not unique to single-celled eukaryotes, but has also evolved in a multicellular, parasitic animal. Hence, H. salminicola provides an opportunity for understanding the evolutionary transition from an aerobic to an exclusive anaerobic metabolism.

The acquisition of the mitochondrion was a fundamental event in the evolution of eukaryotes, and most extant eukaryotes cannot survive without oxygen. Interestingly, the loss of aerobic respiration has occurred independently in several eukaryotic lineages that adapted to low-oxygen environments and replaced the standard mitochondrial (mt) oxidative phosphorylation pathway with novel anaerobic metabolic mechanisms (Fig. 1) (1, 2). Such anaerobic metabolism occurs within mitochondria-related organelles (MROs), which have often lost their cristae, and include hydrogenosomes and mitosomes (1, 2). There is debate regarding the existence of exclusively anaerobic animals and accompanying MROs (3). Although it was reported that some loriciferans found in anoxic conditions possess hydrogenosomes (4, 5), genomic data are not yet available for these organisms, and alternative explanations have been proposed (3). Here, we show that a myxozoan parasite (Cnidaria) has lost both its mt genome and aerobic metabolic pathways, and has a novel type of anaerobic MRO. Myxozoans are a large group of enigmatic, parasitic, cnidarians with complex life cycles that require two hosts, usually a fish and an annelid (6). They have a substantial negative economic impact on fisheries and aquaculture (7). Myxozoan mitochondria have highly divergent genome structures, with large multipartite circular mt chromosomes and unusually high evolutionary rates (8, 9). To gain further insight into the evolution of the myxozoan mt genome, we studied two closely related freshwater species, Henneguya salminicola and Myxobolus squamalis (SI Appendix, Fig. S1), both of which are parasites of salmonid fish (10⇓–12).

Fig. 1. Eukaryote phylogenetic relationships inferred from a supermatrix of 9490 amino acid positions for 78 species. Bayesian majority-rule consensus tree reconstructed using the CAT + Γ model from two independent Markov-chain Monte Carlo chains. Branches with low node support (posterior probabilities PP < 0.7) were collapsed. Most nodes were highly supported (PP > 0.98), and PP are only indicated for nodes with PP < 0.98. The eukaryote classification is based on Adl et al. (47). Species known to have lost their mt genome are indicated in bold with an asterisk. Myxozoan species form a well-supported group (PP = 1.0) and our reconstructions agree with previous studies (14), which show monophyly of the fresh-water/oligochaete host lineage (10).

Discussion Structurally, H. salminicola has lost its mt genome, but has retained an organelle that resembles a mitochondrion. However, as mitochondria are defined based on the use of oxygen as electron acceptor (21), and usually the presence of an mt genome (but see ref. 22), we conclude that H. salminicola possesses MROs rather than true mitochondria. Although MROs have evolved several times independently, some of them present striking similarities (1, 2). Not only have MROs often lost the same mt pathways (e.g., pyruvate dehydrogenase or electron transport chain enzymes) but also, in several cases, homologous enzymes, such as hydrogenases or pyruvate formate lyases, have been acquired independently by horizontal gene transfer. These enzymes allow ATP production by anaerobic pyruvate metabolism and H 2 synthesis. MROs with such abilities are called hydrogen-producing mitochondria or hydrogenosomes, the latter having lost their ability to utilize oxygen (Fig. 3C) (1, 2). As our H. salminicola assemblies did not contain any hydrogenase or other genes of prokaryotic origin (Fig. 3C and Dataset S5), we conclude that the MROs in H. salminicola are not hydrogenosomes. The presence of cristae in H. salminicola’s MRO is surprising since these membrane invaginations are usually absent in anaerobic MROs (1, 16, 17). However, we note that the MROs of H. salminicola share these characteristics with the MRO of the apicomplexan Cryptosporidium muris, which has also lost complexes I, III, and IV, but possesses an alternative oxidase and retains cristae (Fig. 3C) (23). The presence of cristae together with the identification of pseudogenes suggest that the loss of mtDNA and aerobic respiration may be a recent evolutionary event in the Henneguya lineage. Future experiments are needed to better characterize the metabolic energy pathways of H. salminicola. However, such experiments are challenging because it is currently not possible to culture H. salminicola in the laboratory. Similar to most Myxozoa, H. salminicola likely alternates between two hosts (6). In its fish host, it undergoes proliferation and sporogenesis in pseudocysts within the white muscle (11), a tissue known to have anaerobic metabolism (24). While the obligate invertebrate host of H. salminicola is unknown, it is probably an annelid from the family Naididae, based on known life cycles of related myxozoans (25). Members of the Naididae can grow and reproduce in anoxic environments (26). As all protists that have lost their mt genomes live in anaerobic environments, we speculate that the loss of the mt genome in H. salminicola was driven by low-oxygen environments in both of its hosts. Loss of superfluous genes likely conveys an evolutionary advantage, as it has been shown that the bioenergetic cost of a gene is higher in small genomes (27). Myxozoans have smaller genomes [22 to 180 Mb (14, 18)] than free-living Cnidaria [>250 Mb (28, 29)]. Therefore, the loss of the mt genome and associated nuclear genes involved in its replication and electron pathways may be advantageous for a myxozoan living in anaerobic environments. However, the loss of useless genes by random drift cannot be excluded. Interestingly, our results also open the way to new treatment options against this pathogen, since anaerobic protists are known to be sensitive to specific drugs (30). Myxozoans have gone through outstanding morphological and genomic simplifications during their adaptation to parasitism from a free-living cnidarian ancestor (31). It is remarkable that these myxozoan simplifications do not appear to be ancestral, but rather the result of secondary losses (14). Here we show that at least one myxozoan species has lost a core animal feature: the genetic basis for aerobic respiration in its mitochondria. As a highly diverse group with >2,400 species, which inhabit marine, freshwater, and even terrestrial environments (32), evolutionary loss and simplification has clearly been a successful strategy for Myxozoa, which shows that less is more (33).

Acknowledgments We thank Mark Dasenko and the Center for Genome Research and Biocomputing at Oregon State University (OSU), and Teresa Sawyer of the OSU Electron Microscope Facility for their assistance. This work was supported by the Binational Science Foundation (Grant No. 2015010 to D.H. and P.C.).

Footnotes Author contributions: P.C., J.L.B., and D.H. designed research; D.Y., S.D.A., and D.H. performed research; D.Y., J.L.B., and D.H. contributed new reagents/analytic tools; D.Y., M.N., E.S.C., H.P., and D.H. analyzed data; and D.Y., S.D.A., and D.H. wrote the paper.

The authors declare no competing interest.

This article is a PNAS Direct Submission.

Data deposition: Voucher paratype material was deposited at the US National Parasite Collection, Smithsonian Institution, Washington, DC (https://collections.nmnh.si.edu/search/iz/) under the following accession numbers: Henneguya salminicola myxospores: USNM1611578 (from genome sample) and USNM1611579 (from transcriptome sample); Myxobolus squamalis myxospores: USNM1611580 (from genome sample); M. squamalis myxospores and developmental stages: USNM1611581 (from transcriptome sample). All sequence data have been deposited in the National Center for Biotechnology Information (NCBI) database (https://www.ncbi.nlm.nih.gov/). The H. salminicola data are available under the BioProject accession number PRJNA485580. The SSU rDNA sequence was deposited under MK480607. The raw transcriptome and genome reads are available under accession numbers SRR7754566 and SRR7754567, respectively. The transcriptome and genome shotgun assembly projects were deposited at under the accession GHBP00000000 and SGJC00000000, respectively. The M. squamalis data are available under the BioProject accession number PRJNA485581. The SSU rDNA sequence was deposited under MK480606. The raw transcriptome and genome reads are available under the accession numbers SRR7760054 and SRR7760053, respectively. The transcriptome and genome shotgun assembly projects were deposited under accession numbers GHBR00000000 and QWKW00000000, respectively. The mt genome of M. squamalis was deposited under the accession number MK087050. These accession numbers are provided in Dataset S6. The Bayesian trees and all alignments were deposited in the Dryad Digital Repository (https://doi.org/10.5061/dryad.v15dv41sm). Finally, the uncropped images underlying Fig. 2 and SI Appendix, Figs. S7 and S8 were deposited in the Figshare repository (https://doi.org/10.6084/m9.figshare.8300003, https://doi.org/10.6084/m9.figshare.9897284, https://doi.org/10.6084/m9.figshare.9897320).

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