Integrative and Comparative Biology Advance Access originally published online on June 27, 2007
Integrative and Comparative Biology 2007 47(5):752-758; doi:10.1093/icb/icm026
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Origin and evolution of a myxozoan worm
*Department of Zoology, University of Oxford, South Parks Road, Oxford OX1 3PS, UK; School of Biological Sciences, University of Reading, Whiteknights, Reading RG6 6BX, UK
Correspondence: 1E-mail: peter.holland{at}zoo.ox.ac.uk
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Buddenbrockia plumatellae is an active, muscular, worm-shaped parasite of freshwater bryozoans. This rare and enigmatic animal has been assigned to the Myxozoa on the basis of 18S ribosomal DNA sequences and the presence of malacosporean spores. Here we report cloning of four homologous protein-coding genes from Buddenbrockia worms, the putatively conspecific sac-shaped parasite originally described as Tetracapsula bryozoides and the related sac-shaped parasite Tetracapsuloides bryosalmonae, the causative agent of proliferative kidney disease in salmonid fish. Analyses are consistent with the hypothesis that Buddenbrockia is indeed a malacosporean myxozoan, but do not provide support for conspecificity with either T. bryozoides or T. bryosalmonae. Implications for the evolution of worm-like body plans in the Myxozoa are discussed.
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Introduction |
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SynopsisIntroductionMethodsResults and discussionAre worms and sacs...The evolution of worms:...AcknowledgmentsReferences |
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It is now over 150 years since the first report of parasitic worms within the body cavity of freshwater bryozoans. Dumortier and van Beneden (1850
) described motile worms with rounded bodies inside colonies of Alcyonella (now Plumatella) fungosa from Belgium, and observed the worms undergoing extensive movement within the bryozoan body cavity. Although van Beneden was a zoologist with a wide knowledge of animal diversity, and also credited with the first serious study of mesozoans, he did not speculate on the systematic position of the worms, nor did he assign a taxonomic name. Schröder (1910) later discovered worm-shaped parasites, ranging from 0.05 to 3 mm in length, from the body cavity of colonies of Plumatella repens from Germany and named them Buddenbrockia plumatellae. Later histological examinations by Schröder revealed four sets of longitudinal muscles present in the larger specimens, these muscle blocks developing between inner and outer cell layers (Schröder 1912). There was no evidence for a gut, or for a central nervous system. In his original paper, Schröder (1910) suggested that Buddenbrockia was a mesozoan, but after describing the longitudinal muscle blocks he changed his opinion and proposed a relationship to nematodes (Schröder 1912). Braem (1911) confirmed Schröder's morphological description but suggested that the worms were the larvae of parasitic platyhelminths.
Throughout the rest of the 20th century, there were only sporadic reports of Buddenbrockia worms and no further morphological or developmental studies to build on the original descriptions of Schröder. Although the parasitic worms were rarely observed, despite many extensive studies of freshwater bryozoans, the scattered reports that were published suggested a very widespread geographic distribution. Buddenbrockia was reported to parasitize a range of bryozoans, including Plumatella, Stolella, Hyalinella and Lophopodella, from Brazil, Bulgaria, Japan and Austria (Marcus 1941; Grancarova 1968; Oda 1972, 1978; Wöss 2000). More recent papers have reported Buddenbrockia worms from the UK, France, Italy and the US (Canning et al. 2002; Okamura et al. 2002; Monteiro et al. 2002; Morris et al. 2002; Tops et al. 2005; Fig. 1). Buddenbrockia is clearly a widespread animal, but one that is relatively rare or difficult to find.
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In 2002, almost a century after Schröder's original description, our understanding of the anatomy and evolution of Buddenbrockia was extensively revised. Ultrastructural studies using transmission electron microscopy confirmed several of the original observations, notably the presence of four longitudinal muscle blocks and the apparent absence of a gut and a centralized nervous stem. However, these studies also forced a significant correction to the original description. Schröder (1910
, 1912) had described what he believed were multicellular embryos developing inside the elongated worms, which he suggested were then released into the bryozoan by degeneration of the outer cell layer of Buddenbrockia. The recent studies have demonstrated that these structures are not embryos, but are multicellular spores bearing polar capsules (intracellular organelles capable of firing an attachment thread). Indeed, the structure of the spores and the polar capsules were shown to be remarkably similar to those already described for another group of parasitic animals: the malacosporean myxozoans (Morris et al. 2002; Okamura et al. 2002). Further evidence that Buddenbrockia is indeed a malacosporean myxozoan came from sequencing of the 18S ribosomal genes, which revealed almost identical DNA sequences in Buddenbrockia worms and in one of the described species of malacosporean myxozoans, Tetracapsula bryozoides (Monteiro et al. 2002).
The analyses of spore structure and ribosomal DNA sequence seemed to solve the phylogenetic enigma that is Buddenbrockia. This rare, motile, parasitic worm is a malacosporean myxozoan. Yet, this conclusion raises several important questions. First, should the evidence really be taken as incontrovertible? After all, no other myxozoan has morphology remotely like the active, vermiform B. plumatellae. All other described species of myxozoans (more than a thousand species) exist as single cells, small cellular sacs or plasmodia. Second, is the motile worm a life-cycle stage, or a facultatively produced form, of a sac-shaped malacosporean? This has been suggested based on the remarkably high level of similarity of 18S rDNA sequences (Monteiro et al. 2002), and indeed a subsequent paper formally synonymized the species names B. plumatellae and T. bryozoides (Canning et al. 2002). Third, if Buddenbrockia is a myxozoan, what can we deduce about the evolution of worm-like forms in the animal kingdom?
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Methods |
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We collected colonies of Cristatella mucedo infected with sacs currently attributed to B. plumatellae (Canning et al. 2002
) from Burghfield Lake, Berkshire, UK. For the purposes of clarity we will refer to these as "Tetracapsula bryozoides" sacs, thus distinguishing them from B. plumatellae worms. The sacs were collected by depressing the body wall of C. mucedo in order to increase internal pressure in the body cavity and the release of sacs. DNA was extracted from specimens preserved in RNAlater (Ambion) or ethanol using TRI Reagent (Sigma) or Chelex100 resin (Bio-Rad). Buddenbrockia plumatellae (worms) were collected from infected Hyalinella punctata colonies in Cowan Lake, OH, USA; worms were collected individually by pipette after dissecting infected colonies. We never observed "sacs" in material from Cowan Lake, nor "worms" in material from Burghfield Lake. RNA was extracted using TRI Reagent (Sigma), a SMART cDNA library constructed and cDNA clones sequenced as described elsewhere (Jiménez-Guri, Philippe, Okamura and Holland, manuscript submitted). DNA from the same collection of worms was confirmed to be free from host contamination by PCR using universal 18S ribosomal DNA primers followed by cloning and sequencing (50/50 clones were myxozoan). Tetracapsuloides bryosalmonae (PKD) sacs were collected by shredding infected colonies of Fredericella sultana from the River Cerne, Dorset, UK Cloning of homologous genes from the three samples was based on PCR, with primers designed to cDNA sequences obtained from B. plumatellae. Primer sequences used successfully were (5' to 3'): A1P6AA (ATHAAYTGYGCNGAYAAYAC and GCRCAYTCYTTNGCNAC), P2D5 (TATCATTCTTGCTGGTCG and ACGTGCATTAGATACAGC), H7P1 (CAYCCNGAYAARATHTGYGA and RTCNGTNGCRTANCCRAACAT), B3P3 (ATHGARACNGGNWSNATHACNGA and SWRTCRTADATYTTRCADAT). Phylogenetic analysis was performed using PhyML. Different substitution models for each gene were used, as appropriate to the data. Unrooted trees were displayed using the TreeExplorer program. |
Results and discussion |
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Buddenbrockia is a malacosporean myxozoan
The Phylum Myxozoa is divided into two classes: Myxosporea and Malacosporea. There are over 2000 species in the Myxosporea, with most described species being parasites of teleost fish (Lom and Dyková 2006
). Approximately 30 species are known to cycle between fish and annelid worm hosts alternating between actinosporean and myxosporean stages (Lom and Dyková 2006). The class Malacosporea, in contrast, has very few described species, all of which infect freshwater bryozoans and whose life cycles are incompletely understood (Canning and Okamura 2004). A commercially important disease of salmon and trout, proliferative kidney disease (PKD), is caused by a member of this class, originally known simply as the PKX organism and later described as T. bryosalmonae (renamed Tetracapsuloides bryosalmonae by Canning et al. 2002). The originally described malacosporean species, T. bryozoides (Canning et al. 1996; currently synonymized with B. plumatellae; Canning et al. 2002), has a very similar sac-like form and is also found in freshwater bryozoans. It has not been detected in fish. Analysis of 18S rDNA sequences suggest there are also two additional species of malacosporean myxozoans (Tops et al. 2005). One occurs as sacs inside the bryozoan Lophopus crystallinus (Tops et al. 2005) and has recently been described and named (Canning et al. 2007). The other is found as worms in the bryozoan Fredericella sultana (Tops et al. 2005). Two lines of evidence already support the view that the vermiform parasite B. plumatellae is a member of the Class Malacosporea in the Phylum Myxozoa: the presence of typical malacosporean spores developing within the worm, and analysis of 18S rDNA sequences. We elected to examine protein-coding gene sequences to further test this contention. Starting with a set of cDNA sequences cloned from vermiform B. plumatellae (Jiménez-Guri, Philippe, Okamura and Holland, manuscript submitted), we selected 30 genes for targeted cloning in the sac-shaped "T. bryozoides" and in the sac-shaped PKD agent T. bryosalmonae. Degenerate primers were designed to the deduced protein coding regions of these genes, and PCR amplification reactions used with genomic DNA extracted from the two target species. All major amplified bands were cloned into plasmid vectors and multiple recombinant clones sequenced for each band from these reactions. We successfully isolated six of these protein-coding genes from one or both of the target species. These genes were rad51, rpl23a, rpl27e, AdoMet synthetase, actin and hsp70. The actin and hsp70 genes are part of multigene families, and preliminary phylogenetic analyses suggested problems in distinguishing paralogues and orthologues; these two genes were therefore not analysed further.
The rad51 gene has been cloned from a diverse range of eukaryotes and encodes a well conserved protein involved in homologous recombination and repair of double-stranded DNA breaks. We included the rad51-deduced protein sequence from vermiform Buddenbrockia and from the PKD organism in a molecular phylogenetic analysis, including sequences from three chordates, one nematode, four arthopods, one echinoderm, three lophotrochozoans and one cnidarian. The resulting tree has little deep-level resolution, not surprisingly considering the level of protein conservation (Fig. 2). There is a well-supported sister-group relationship between Buddenbrockia and T. bryosalmonae.
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The AdoMet synthetase gene codes for the enzyme S-adenosylmethionine synthetase which catalyzes the formation of adenosylmethionine from methionine and ATP. We included the S-adenosylmethionine-synthetase-deduced protein sequence from vermiform Buddenbrockia and from the PKD organism in a molecular phylogenetic analysis, including sequences from three chordates, five nematodes, four arthropods, one lophotrochozoan and three non-animal eukaryotes. The resulting tree has limited resolution, but there is a well-supported sister-group relationship between the vermiform Buddenbrockia and T. bryosalmonae (Fig. 2).
The rpl27e gene encodes a protein from the large subunit of eukaryotic ribosomes. We included the rpl27e-deduced protein sequence from vermiform Buddenbrockia and from the sac-shaped "T. bryozoides" in a molecular phylogenetic analysis with a diversity of other animal taxa. The resulting tree has limited resolution, but there is a well-supported and distinct sister-group relationship between the vermiform Buddenbrockia and the sac-producing "T. bryozoides" (Fig. 2). The other well-supported clades are all sensible phylogenetic groupings (an arthropod clade, a hydrozoan clade and a chordate clade).
The rpl23a gene encodes another protein found in the large subunit of eukaryotic ribosomes. We cloned this protein-coding gene from all three target species. Phylogenetic analysis recovers a clade containing Buddenbrockia, T. bryosalmonae and "T. bryozoides" sacs (Fig. 2). The other well-supported clades in these trees are phylogenetically sensible.
Although too much emphasis should not be placed on phylogenetic analyses from single genes, taken collectively these results suggest that the vermiform B. plumatellae is more closely related to Myxozoa than to other animal taxa. This is consistent with the previous proposal that Buddenbrockia is a malacosporean myxozoan.
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Are worms and sacs conspecific? |
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On the basis of extremely similar 18S rDNA sequences and spore morphologies, Monteiro and others (2002
) stated that B. plumatellae and T. bryozoides are congeneric, and possibly even conspecific. Specifically, Monteiro et al. suggested the worm-shaped Buddenbrockia could be an alternative form of the sac-shaped T. bryozoides, perhaps produced facultatively under certain (rare) environmental conditions. Indeed, Canning et al. (2002) proposed a taxonomic revision involving T. bryozoides being synonymized with B. plumatellae (the latter name being used due to taxonomic priority), and a new genus Tetracapsuloides being erected for the PKD organism T. bryosalmonae.
More recently, however, Tops et al. (2005) published data that cast a degree of doubt upon the conspecificity, and hence upon the taxonomic revision. In an extensive survey of 18S rDNA sequences from worms and sacs, Tops et al. (2005) found a very slight, but consistent, sequence difference between worms and sacs. The level of sequence similarity was still extremely high but phylogenetic analysis produced a weakly supported separation of worms and sacs. Although Tops et al. (2005) did not propose that these are separate species, it is notable that vermiform samples from Berkshire (UK), Cowan Lake (USA), and Brittany (France) clustered together, separate from sac-like forms collected from Berkshire (UK), Maine et Loire (France) and Bavaria (Germany).
To investigate this issue further, we examined the protein-coding genes described earlier. One of the two ribosomal protein genes, rpl23a, gave the clearest information. In the phylogenetic analysis of rpl23a, we recovered a trichotomy within the clade containing vermiform Buddenbrockia, sac-shaped "T. bryozoides" and sac-shaped T. bryosalmonae. This trichotomy is supported by a bootstrap value of 89%. Within the clade, there is tentative support (57%) for a sister group containing vermiform Buddenbrockia and sac-shaped "T. bryozoides", but this is not significantly stronger support than seen between any two of the three samples. There is no indication that Buddenbrockia worms and "T. bryozoides" sacs are conspecific. Examination of the protein sequence alignments reveals clear divergences of protein sequences among the three species. The vermiform Buddenbrockia and the sac-shaped "T. bryozoides" share just 74 identical amino acids over 94 sites of the rpl23a alignment (78.7% identity), while vermiform Buddenbrockia and sac-shaped T. bryosalmonae share 74 of a 95 site alignment.
These data, therefore, provide no support for the hypothesis that vermiform B. plumatellae from Ohio, United States, and sac-shaped "T. bryozoides" from Berkshire, UK, are the same species. It is possible that B. plumatellae/"T. bryozoides" comprises several species (or geographically isolated populations) each with worm and sac forms, and we have sampled from a worm of one species and a sac of another. Alternatively, B. plumatellae consists of one (or more) worm species, distinct from one (or more) sac-like species. Our data do not distinguish between these alternatives, although taken in concert with the ribosomal DNA data of Tops et al. (2005), the latter seems more likely. Additional sampling of protein-coding gene sequences from worms and sacs collected from a range of additional sites would resolve between these alternatives.
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The evolution of worms: convergent evolution, atavism or plesiomorphy? |
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The data from protein-coding genes provide no evidence to support the view that vermiform B. plumatellae and sac-like "T. bryozoides" are the same species. Instead, we suggest that the malacosporean clade of myxozoans contains at least three species of sac-shaped parasites, T. bryosalmonae, "T. bryozoides" and the sac-like form found in Lophopus (Tops et al. 2005
; Canning et al. 2007), plus at least two species of motile muscular worm, B. plumatellae, and the worm form found in Fredericella (Tops et al. 2005). How could forms as fundamentally distinct as small hollow sacs and muscular worms be present in the same class of animals? We do not have an answer to this question, but we can at least formulate the necessary lines of enquiry.
First, it would be helpful to elucidate where in the animal kingdom the myxozoans should be placed. This has been a topic of intense analysis and discussion in the literature (Smothers et al. 1994; Siddall et al. 1995; Schlegel et al. 1996; Zrzav
and Hypsa 2003; Canning and Okamura 2004), and will not be revisited here. It suffices to say that the two most commonly proposed phylogenetic placements are related to the Cnidaria or amongst the bilaterian animals. Resolving this controversy would allow one to deduce what developmental and cellular characters the ancestors of myxozoans possessed (including which regulatory genes), and therefore speculate on how a worm form could be generated in the development of these animals. For example, is the vermiform Buddenbrockia an example of convergent evolution with nematodes, or retention of an ancestral (plesiomorphic) condition?
Second, it is necessary to ascertain the extent of geographical and intrapopulation variation in the sequence of these protein-coding genes to assess how far the conclusions drawn here may be extrapolated.
Third, such analyses should also be conducted for the new species of malacosporeans noted by Tops et al. (2005). Considering the scarcity of these animals, and the ever-present contamination problems that plague molecular analysis of parasites, this will not be a simple task. If it is found that all the vermiform species form a clade within malacosporeans, then one could postulate that the worm form is secondarily derived from a sac-like ancestor. Alternatively, if the worms do not form a single clade within the diversity of malacosporeans, then it would be reasonable to deduce that the worm form may be ancestral within the malacosporeans. In either example, it is possible that the myxozoan ancestor was not in fact a true worm, but possessed characters (such as muscle blocks) displayed by living vermiform myxozoans, and that these were highly modified during the evolution of malacosporeans, or possibly even lost entirely and regained atavistically during evolution.
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Acknowledgments |
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SynopsisIntroductionMethodsResults and discussionAre worms and sacs...The evolution of worms:...AcknowledgmentsReferences |
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The authors would like to thank Sylvie Tops and Herve Philippe for useful discussions, Viv Rimmer, and Louise Evans for assistance, Sylvie Tops for provision of material, Tim Wood, Wright State University, for help in fieldwork and provision of laboratory space in Ohio, and two referees for constructive suggestions. This research was funded by the BBSRC and NERC (grant NER/A/S/1999/00075).
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Footnotes |
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From the symposium "Key Transitions in Animal Evolution" presented at the annual meeting of the Society for Integrative and Comparative Biology, January 3–7, 2007, at Phoenix, Arizona.
2Present address: Department of Zoology, National History Museum, Cromwell Road, London SW7 5BD, UK 
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References |
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