INTRODUCTION
Myxozoa are microscopic cnidarians that have undergone extensive morphological simplification and miniaturization as adaptations to parasitism (Okamura et al. Reference Okamura, Gruhl, Bartholomew, Okamura, Gruhl and Bartholomew2015). They have complex life cycles involving primarily aquatic vertebrate and invertebrate hosts and are comprised two classes: the Malacosporea Canning, Curry, Feist, Longshaw & Okamura, 2000 and the Myxosporea Bütschli, 1881. There are some 2400 described species distributed in 64 genera. The great majority are myxosporeans (Fiala et al. Reference Fiala, Bartošová-Sojková, Whipps, Okamura, Gruhl and Bartholomew2015a ).
Malacosporeans have retained certain primitive features, including muscles in active myxoworm stages produced in some species (e.g. Buddenbrockia plumatellae), and epithelia in both myxoworms and the sac-like stages produced in non-motile species. Myxosporeans have lost such tissues and are highly derived. Their trophic stages generally consist of multinucleate plasmodia with many spores or uninucleate pseudoplasmodia that produce one or two spores (Canning and Okamura, Reference Canning and Okamura2004).
Motility in myxozoans has been observed in different stages in both malacosporeans and myxosporeans. Some malacosporeans produce myxoworms whose movement is supported by four sets of muscles whose cells are orientated at 12° with respect to the longitudinal axis of the worm (Gruhl and Okamura, Reference Gruhl and Okamura2012). Muscle contraction results in helical swimming. Motility in myxosporeans is achieved at the cellular level via amoeboid movement and ‘dancing’ (also referred to as twitching) (see Feist et al. Reference Feist, Morris, Alama-Bermejo, Holzer, Okamura, Gruhl and Bartholomew2015 for review). The former involves extensions of the cell membrane, often as pseudopodia or filipodia, and there is direct evidence for the involvement of actin (Alama-Bermejo et al. Reference Alama-Bermejo, Bron, Raga and Holzer2012). Dancing is observed in blood stages of sphaerosporids and is proposed to be achieved by a mobile fold of the plasmalemma that acts like an undulating membrane (Lom et al. Reference Lom, Dyková and Pavlásková1983).
During a survey of fish parasites in rivers of the Amazon basin, Brazil, we observed a myxosporean with unusually shaped, worm-like plasmodia in the gallbladder of Colossoma macropomum (Cuvier, 1816), a serrassalmid fish of great importance to both the local fish market and Brazilian aquaculture (Goudinho and Carvalho, Reference Goudinho and Carvalho1982; MPA, 2012). Here we describe the morphology and motility of this remarkable myxosporean using light, confocal, transmission and scanning electron microscopy and movements captured by video. We also undertake molecular phylogenetic analysis to determine the relationships of this bizarre species to other myxosporeans. Fine details of morphology along with observed movements enable insights on how convergence to an active worm has been achieved at the cellular level. Morphological and molecular data are also used to describe this new vermiform-like myxosporean species.
MATERIAL AND METHODS
Collection of material and morphological analysis
Fifty-three wild C. macropomum specimens were collected from the Tapajós, Amazon and Solimões Rivers in Brazil (Table 1). The catches were authorized by the Brazilian Ministry of the Environment (SISBIO no 44268-4) and fish were transported live to a make-shift field laboratory on the shores of the river, where they were euthanized. The methodology of the present study was approved by the ethics research committee of Federal University of São Paulo (CEUA N 92090802140) in accordance with Brazilian law (Federal Law No. 11794, dated 8 October 2008). All organs and body fluids were examined for myxosporeans and representative material was then collected for morphological and molecular studies (see below). In addition, smears containing free myxospores were air-dried, stained with Giemsa solution and placed in mounting medium on permanent slides. Type specimens were deposited in the collections of the Museum of Zoology ‘Adão José Cardoso’, of State University of Campinas (UNICAMP), Brazil. Morphological and morphometric analyses of myxospores based on Lom and Arthur (Reference Lom and Arthur1989) and following Gunter et al. (Reference Gunter, Whipps and Adlard2009) (with some modification; see Supplementary File Fig. S1) were performed at the Federal University of São Paulo using a computer equipped with AxioVision 4·1 image capture software coupled to an Axioplan 2 Zeiss microscope.
Table 1. Sites and periods of collection of Colossoma macropomum in the Amazon Basin, the number of fish examined, their sizes (total length), and the number parasited by Ceratomyxa vermiformis sp. n. Most fish were immature (⩽55 cm; Costa et al. Reference Costa, Barthem and Bittencourt2001). Size ranges are provided when relevant for distinguishing immature and mature fish
DNA isolation, sequencing and phylogenetic analysis
Bile from gallbladders infected by worm-like plasmodia was preserved in absolute ethanol for molecular analysis. DNA was extracted using a DNeasy® Blood & Tissue Kit (Qiagen, USA), in accordance with the manufacturer's instructions. The concentration of the DNA was measured using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, USA). Polymerase chain reactions (PCRs) were carried out in 25 µL reaction volumes using 100 ng of extracted DNA, 5 × Go Taq Flexi Buffer (Promega Madison, WI, USA), 10 mm dNTP mix, 25 mm MgCl2, 5 mm for each primer ERIB1-ERIB10 (Barta et al. Reference Barta, Martin, Liberator, Dashkevicz, Anderson, Feighner, Elbrecht, Perkins-Barrow, Jenkins, Danforth, Ruff and Profous-Juchelka1997), and 1× Go Taq G2 Flexi DNA polymerase (Promega Madison, WI, USA). The amplification was performed in an Eppendorf AG 22331 Hamburg Thermocycler (Eppendorf, Hamburg, Germany) using a touchdown PCR method (Korbie and Mattick, Reference Korbie and Mattick2008), with initial denaturation at 94 °C for 30 s, followed by nine cycles at 94 °C for 30 s, 75 °C (−1 °C/cycle) for 90 s, 72 °C for 45 s, 36 cycles at 94 °C for 30 s, 65 °C for 40 s, 72 °C for 45 s, and then final elongation at 72 °C for 5 min. PCR products were subjected to electrophoresis in 1·0% agarose gel (BioAmerica, Miami, FL, USA) in TBE buffer (0·045 m Tris-borate, 0·001 m EDTA, pH 8·0), stained with ethidium bromide, and then analysed with a FLA-3000 scanner (Fuji Photo Film, Tokyo, Japan). SSU rDNA was amplified using the primers ERIB1 and ERIB10 (Barta et al. Reference Barta, Martin, Liberator, Dashkevicz, Anderson, Feighner, Elbrecht, Perkins-Barrow, Jenkins, Danforth, Ruff and Profous-Juchelka1997), MYXGEN4f (Diamant et al. Reference Diamant, Whipps and Kent2004) and a specifically designed primer CERATBr (5′-AGAATTTCACCTCTCGCCATC-3′). The sequencing was performed using the BigDye® Terminator v3·1 cycle sequencing kit (Life Technologies Carlsbad, CA, USA) according to the manufacturer's protocol, adapting the reaction end volume to 5 µl in an ABI 3500 DNA sequencing analyser (Applied Biosystems, Foster City, CA, USA) and using the polymer POP-7 (Life Technologies Carlsbad, CA, USA).
A standard nucleotide–nucleotide Basic Local Alignment Search Tool (BLAST) (blastn) search was conducted (Altschul et al. Reference Altschul, Madden, Schaffer, Zhang, Zhang, Miller and Lipman1997). The sequences of all Ceratomyxa species available in GenBank plus Myxodavisia bulani KM273030 and Palliatus indecorus DQ377712 were aligned by ClustalW (Thompson et al. Reference Thompson, Gibson, Plewniak, Jeanmoungin and Higgins1997) using the BioEdit program (Hall, Reference Hall1999). Phylogenetic analysis was conducted using maximum likelihood (ML) in PhyML software (Guindon et al. Reference Guindon, Dufayard, Lefort, Anisimova, Hordijk and Gascuel2010), with NNI search, automatic model selection by SMS (Smart Model Selection), under a GTR + G + I substitution model (with six categories), equilibrium frequencies optimized, transition/transversion ratio estimated, proportion of invariable sites fixed (0·097) and Gamma shape parameter fixed (0·398). To avoid the long branch attraction (LBA) effect (Anderson and Swofford, Reference Anderson and Swofford2004), Maximum Parsimony (MP) analysis (with complete deletion) was conducted using MEGA7 software (Kumar et al. Reference Kumar, Stecher and Tamura2016) on another alignment excluding six long-branching Ceratomyxa species. For comparative purposes, ML analysis was also performed using this same dataset, under a GTR + G + I substitution model (with six categories), equilibrium frequencies optimized, proportion of invariable sites estimated (0·186) and Gamma shape parameter estimated (0·449). Bootstrap analyses (1000 replicates) were employed to assess the relative robustness of internal branches. The malacosporeans Tetracapsuloides bryosalmonae and B. plumatellae were used as an outgroup in both phylogenetic analyses.
Electron and confocal microscopy
For transmission electron microscopy, plasmodia were fixed for at least 12 h in 2·5% glutaraldehyde with 0·1 m cacodylate buffer (pH 7·4), washed in the same buffer and post-fixed with osmium tetroxide (OsO4), all procedures being performed at 4 °C. After dehydration in an ascending ethanol series, the samples were embedded in EMbed 812 resin (Electron Microscopy Sciences, Hatfield, PA, USA). Ultrathin sections, double stained with uranyl acetate and lead citrate, were examined using a LEO 906 transmission electron microscope operating at 60 kV. For scanning electron microscopy, infected bile fixed at 10% formalin in 0·1 m phosphate-buffered saline (PBS) was left for 1 h on a polysine pre-treated round coverslip. Coverslips were then washed in the same buffer, dehydrated in ethanol, critical-point-dried, mounted on stubs, covered with metallic gold, and examined in a Zeiss Ultra Plus scanning electron microscope at 5 kV. For confocal analyses, infected bile fixed at 10% formalin in 0·1 m PBS was left for 30 min. on polysine pre-treated slides. The samples were rinsed three times in PBS and then permeabilized with PBS containing 0·1% Triton X-100 for 1 h. Specimens were then stained with Alexa Fluor® 488 Phalloidin (Invitrogen Eugene, OR, USA) at 0·001 mg mL−1 for 4 h and with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI, Sigma-Aldrich Saint Louis, MO, USA) at 0·004 mg mL−1 for 10 min. The samples were rinsed in PBS and mounted in 90% glycerol, 10% PBS, 0·5% 1,4-diazabicyclo[2·2·2]octane mounting medium. They were examined using a Nikon A1 Confocal Microscope.
RESULTS
Infections in fish
Motile plasmodia of a new myxosporean species were observed in the gallbladders of six of the 53 C. macropomun specimens examined. Five of 36 fish examined from the Tapajós River were infected. The single fish examined from the Amazon River was also infected (Table 1). Prevalences of infection were variable and their estimation compromised by low sample sizes; however, data from the Tapajós River suggest that infection prevalences may vary over time (Table 1).
Taxonomic summary and description
Phylum: Cnidaria Verrill, 1865
Class: Myxosporea Bütschli, 1881
Order: Bivalvulida Shulman, 1959
Family: Ceratomyxidae Doflein, 1899
Genus: Ceratomyxa Thélohan, 1892
Species: Ceratomyxa vermiformis sp. n.
Type host: The fish C. macropomum Cuvier, 1818 (Teleostei: Serrasalmidae).
Location in host: Gallbladder (plasmodia with or without mature spores swimming actively in bile).
Type locality: Tapajós River (Municipality of Santarem, PA), Amazon Basin, Brazil.
Type material: Syntypes–air-dried, stained with Giemsa solution and mounted in mounting medium on permanent slides (accession numbers Zuec Myx 54 and 55).
Etymology: The specific name is based on the form and associated movements of the plasmodia, unprecedented observations for myxosporeans.
Movement and morphology
Plasmodia have an elongate form and showed coordinated, worm-like undulations reminiscent of nematode sinusoidal locomotion (Fig. 1 and Videos S1 and S2). The alternating bending movements result in translocation through the bile as can be seen in the Supplementary Material (Video S1).
Fig. 1. Images taken from a sequence of video frames showing alternating bending associated with translocation of the worm-like plasmodium of Ceratomyxa vermiformis sp. n. Images were obtained from video S1. Due to the poor quality of the video (taken during observation in a makeshift field laboratory and using a manually held camera) the outline of the plasmodium has been manually enhanced and we have imposed a uniform background to clarify and distinguish the plasmodium in each frame. The arrow indicates the direction of the movement. Scale bar = 50 µm.
The plasmodia are characterized by a highly developed cytoplasm that is clearly segregated into an external layer and an internal region. The external layer ranges from about 200–600 nm in thickness (Fig. 2A), has an actin-rich cytoskeleton (Fig. 3) and is bounded by an external membrane that is covered by a secreted glycocalyx-like layer (Fig. 2B). Tubular mitochondria, microtubules, rough endoplasmic reticulum and granular material that may represent ribosomes and/or glycogen are abundant in the external layer (Figs 2 and 4). The elongate mitochondria demonstrate an unusually regular distribution and orientation. Cross-sectional views reveal that the mitochondria are spaced at regular intervals around the periphery of the plasmodia (Fig. 2A), while longitudinal sections demonstrate that their long axis is orientated in parallel with the long axis of the plasmodia (Fig. 4).
Fig. 2. Electron micrograph of plasmodia of Ceratomyxa vermiformis sp. n. from the gallbladder of Colossoma macropomum. (A) Transverse section showing the external layer (el) with regularly situated mitochondria (black arrows) and early sporogonic stages (ss) developing from the external layer towards the internal region (ir), which is occupied by granular/fibrilar material. Note a vegetative nucleus (vn). Scale bar = 2 µm. (B): amplified region of the external layer showing granular material (gr) and the plasmodial membrane (black arrow) covered by a secreted glycocalyx-like layer (white arrow). Scale bar = 0·25 µm. (C) Detail of the external layer (dashed line) showing the external membrane (black arrow), a mitochondrion (m) extending from near the cell membrane and extending across the external layer and granular/fibrilar material (gf) occupying the internal region (ir). Scale bar = 0·5 µm.
Fig. 3. Confocal laser microscopy photomicrographs showing details of the cytoskeleton and development of plasmodia of Ceratomyxa vermiformis sp. n. from the gallbladder of Colossoma macropomum. Stains: blue – DAPI; green – Alex Fluor 488–Phalloidin. (A) Young plasmodium with nuclei (blue) in the growth centre in the anterior pole (ap). Actin is distributed throughout the cytoskeleton (green). Scale bar = 10 µm. (B) Plasmodium showing numerous nuclei (blue) in the growth centre (thin arrow), an actin-rich network throughout the cytoskeleton (green) and developing spores in the interior region of the plasmodium (large arrows). Scale bar = 25 µm. (C) Plasmodia in different developmental stages including an early developmental stage (es), a later but still pre-sporogonic stage (ps) and a more mature plasmodium (p) with internal stages at early stages of spore development (ed) and immature spores (is). Scale bar = 25 µm. (D) Details of the anterior pole of a plasmodium showing numerous nuclei (n) in the growth centre (gc) and nuclei of early sporogonic stages (thin arrows) below. Scale bar = 10 µm. Pp, Posterior pole.
Fig. 4. Electron micrographs of plasmodia of Ceratomyxa vermiformis sp. n. from gallbladder of Colossoma macropomum in longitudinal section. Panel (A) showing corrugated surface (arrows), long tubular mitochondria and part of a vegetative nucleus (vn) and of a young sporogonic stage Scale bar = 1 µm. (B) Amplified micrograph of the area of the upper right corner of panel(A) showing longitudinal sections of microtubules (arrows) and a long tubular mitochondrion (M). Scale bar = 1 µm. (C) Oblique view showing the regular organization of the mitochondria (m) in the external layer (el). Scale bar = 0·5 µm. (D) Detail of marked area of panel (C) showing fragment of a mitochondrion (m) and rough endoplasmic reticulum (rer). Scale bar = 0·25 µm.
The external surface of the plasmodium appears to generally display a series of bulges or ridges that occur as regular transverse bands (Figs 3–6). In certain longitudinal/oblique sections these appear to become highly exaggerated to form a corrugated or peaked surface (Figs 4 and 5) that extends only partially around the circumference of the plasmodia (Fig. 6). sem suggests this corrugated surface may extend along much of the length of the plasmodia (Fig. 6).
Fig. 5. Electron micrograph of plasmodia of Ceratomyxa vermiformis sp. n. from gallbladder of Colossoma macropomum. (A) Longitudinal section showing the early developmental sporogonic forms (ed) associated with the external layer (thin black arrows). Note the regions of the plasmodial surface with (large black arrows) and without (large white arrows) corrugations. Scale bar = 2 µm. (B) Amplified region of panel (A) showing early developmental spore (ed) associated with the external layer (el) (thin black arrows); m: mitochondria; n: nucleus. (C) Transverse section showing a sporogonic cell (sc) developing in proximity to the external layer (el) (thin arrow); n: nucleus, nc: nucleolus. Scale bar: B and C = 0·5 µm.
Fig. 6. Scanning electron microscopy of anterior region of plasmodia of Ceratomyxa vermiformis sp. n. from the gallbladder of Colossoma macropomum. Panel (A) showing the presence of corrugations (arrows) extending a considerable distance posterior from the anterior pole (ap). Despite damage to the specimen the corrugations can be seen to become less regular in posterior direction and eventually disappear (not shown). Scale bar = 20 µm. (B) More detailed view demonstrating the presence (thin arrows) and absence of corrugations on different facets of the plasmodium (large arrow). Scale bar = 2 µm.
The internal region of the plasmodia contains homogeneously distributed granular/fibrillar material. Compared with the external layer, it is less electron-dense and lacks organelles (Figs 2 and 5). Developing spores are present, but are associated with the external layer at all stages of development (Figs 2 and 5). Sporogony is asynchronous and plasmodia contain early sporogonic stages and immature and mature myxospores (Figs 2, 3, 5, 7 and 8). Mature plasmodia containing myxospores had a mean length of 442 µm (s.d. = 44·9, range = 379–520 µm, n = 19) and a mean width of 22·1 µm (s.d. = 2·6, range = 18–26 µm, n = 19) (Fig. 8). One end of the plasmodium is blunt, while the opposite end is very thin at its extremity (Figs 3 and 8). Early sporogonic stages are concentrated in the blunt end, specified here as the anterior pole. TEM and confocal microscopy revealed that numerous cells are present in this anterior end (Figs 2, 3 and 5) and that there is a gradient in maturation of spore developmental stages, with progressively older stages appearing towards the posterior, thin end (the posterior pole) of the plasmodium (Figs 3, 7 and 8). These observations suggest that the cells in the anterior end represent a growth centre.
Fig. 7. Differential interference contrast (DIC) photomicrograph of plasmodia of Ceratomyxa vermiformis sp. n. from the gallbladder of Colossoma macropomum. (A, B) vermiform-shaped plasmodia showing developmental stages (ds) in the anterior pole (ap) and mature myxospores (ms) in the middle and posterior pole (pp). In (B), note the mature spores (ms) and the sporogonic developmental stages (ds) are closely associated with the external layer (black arrows). Polar capsules (white arrows). Scale bars = 40 µm.
Fig. 8. Differential interference contrast (DIC) photomicrograph showing details of plasmodia and spores Ceratomyxa vermiformis sp. n. from the gallbladder of Colossoma macropomum. (A) Anterior pole (ap) of a plasmodium (P) showing the growth centre (gc), wall (black thick arrow) and immature spores (is). Note transverse corrugations (black thin arrows) and the polar capsules (white thin arrow). Scale bar = 20 µm. (B) Early developmental spore stage near the external layer (large arrow) of the plasmodium (P). Scale bar = 10 µm. (C) Mature spores (ms) inside of the plasmodium (P) with thick arrow showing wall and thin arrows showing polar capsules. Scale bar = 20 µm. (D) Mature spore. Scale bar = 10 µm.
The spores are strongly arcuate (Fig. 7 and Fig. S1) with a mean length of 4·5 µm (s.d. = 0·2, range = 4·2–4·8 µm, n = 28), a mean thickness of 8·4 µm (s.d. = 0·4, range = 7·9–9·3 µm, n = 28) and a posterior angle of 30·2° (s.d. = 6·6, range = 22–43°, n = 18). The two elongated valves resemble appendages that are of unequal size and become tapered approximately halfway along their lengths. The mean length of the larger valve = 23·7 µm (s.d. = 0·7, range = 22·1–24·3 µm, n = 28) and that of the smaller valve = 21·9 µm (s.d. = 0·8, range = 20·6–23 µm, n = 23). The two polar capsules are spherical and of equal size with a mean diameter of 2·7 µm (s.d. = 0·1 µm, range = 2·5–2·9 µm, n = 28). The polar filament undergoes three to four turns oblique to the longitudinal axis of the polar capsule and the binucleated sporoplasm occupies the wider region of the spore (Fig. 7 and Fig. S1).
Phylogenetic analysis
A total of 1·601 bases of SSU rDNA was generated from sequencing of this worm-like myxosporean (GenBank Accession No. KX278420) and molecular phylogenetic analysis performed with ML reveal that it is sister to Ceratomyxa amazonensis Mathews et al. Reference Mathews, Naldoni, Maia and Adriano2016 (Fig. 9). Further molecular phylogenetic analyses performed on a dataset excluding the long-branching Ceratomyxa species and using both MP and ML approaches were consistent with this result (Fig. S2). These two species in turn group with Ceratomyxa leatherjacketi Fiala et al. Reference Fiala, Hlavničková, Kodádková, Freeman, Bartošová-Sojková and Atkinson2015b and Ceratomyxa tunisiensis Thabet et al. 2016, forming a lineage with the early diverging M. bulani Fiala et al. Reference Fiala, Hlavničková, Kodádková, Freeman, Bartošová-Sojková and Atkinson2015b . This Myxodavisia/Ceratomyxa clade is sister to the remaining Ceratomyxa clade (+P. indecorus). The two species of the genus Ceratonova Atkinson et al. Reference Atkinson, Foott and Bartholomew2014 cluster together in a separate clade to this large Ceratomyxa lineage (Fig. 9 and Fig. S2).
Fig. 9. Molecular phylogenetic tree based on ML analysis of SSU rDNA showing the position of Ceratomyxa vermiformis sp. n. parasite of gallbladder of Colossoma macropomum. Bootstrap values above 70 are indicated at the nodes. GenBank accession numbers after the species name.
Remarks
The highly arcuate spores of C. vermiformis sp. n. with its long and thin valves resembling appendages are similar to those of Meglitschia mylei Azevedo et al. Reference Azevedo, Ribeiro, Clemente, Casal, Lopes, Matos, Al-Quraishy and Matos2011, a parasite reported from the serrasalmid fish Myleus rubripinnis of the Amazon basin. However, M. mylei exhibits a larger number of polar filament turns and smaller sizes of polar capsules and spores than those exhibited by C. vermiformis sp. n. In addition, the valves of C. vermiformis sp. n. are of unequal sizes whereas they are of a similar size in M. mylei (Azevedo et al. Reference Azevedo, Ribeiro, Clemente, Casal, Lopes, Matos, Al-Quraishy and Matos2011) (for detailed comparison see Table S1). Based in these morphological differences we propose the erection of a new species and assign it to the genus Ceratomyxa, a decision based on molecular phylogenetic data and an earlier suggestion that the basic spore architecture of Meglitschia insolita (Meglitsch, Reference Meglitsch1960) (first described as Ceratomyxa insolita) supports assignment to Ceratomyxa (Meglitsch, Reference Meglitsch1960) as detailed in the discussion section.
DISCUSSION
Fine structure and development in relation to movement
The extraordinary movement displayed by plasmodia of C. vermiformis sp. n., as demonstrated in our videos, is associated with particular features that may support or result from motility and movement. These features variously include: (a) an actin-rich network distributed throughout the cytoskeleton of the plasmodia; (b) the regularly arranged, extremely elongate mitochondria orientated along the longitudinal axis of the worm-like plasmodia, in a peripheral position near the plasmodial membrane; (c) a glycocalyx-like layer secreted externally; (d) regions of the plasmodial surface that demonstrate regular corrugations; (e) microtubules that may function in positioning and contribute to the cytoskeleton (Cooper, Reference Cooper2003; Feist et al. Reference Feist, Morris, Alama-Bermejo, Holzer, Okamura, Gruhl and Bartholomew2015); and (f) segregation to form an electron dense, organelle-rich external layer and an internal region with sporogonic stages but depauperate in organelles. As outlined below, these features provide initial insights on how C. vermiformis sp. n. has achieved convergence to a worm-like form at the cellular level.
The cuticle-like extracellular secretion and components of the external layer may contribute to hardening or strengthening of the plasmodial wall as is suggested for the external and internal secretions of valve cells in spores (see Gruhl and Okamura, Reference Gruhl, Okamura, Okamura, Gruhl and Bartholomew2015 for review). Our combined morphological investigations provide evidence that regions of the wall are highly corrugated (e.g. Figs 5 and 7). It is possible that the corrugations may result from squeezing and shortening during bending that accentuates the ridged surface of the wall – in which case these would be transient developments. An alternate scenario is that the markedly corrugated regions are permanent features and perhaps serve to increase surface area (e.g. for absorption or to facilitate movement). Further study is required to resolve this issue. The peripheral deployment of microtubules, actin and mitochondria results in a highly consolidated cytoskeleton in the external region. The positioning of the elongate mitochondria around the circumference of the plasmodia may be linked with the distribution of actin, which could influence mitochondrial function (Anesti and Scorrano, Reference Anesti and Scorrano2006), for example by shaping, tethering or moving the elongate mitochondria.
An additional and notable feature is axial polarity of the plasmodia. The anterior pole is distinguished by a growth centre from which early sporogonic stages show a clear gradient of development to more mature stages distal to this region (see Figs 3, 7 and 8). Gradients in development have also been observed in large histozoic plasmodia where mature spores are located in the periphery (Naldoni et al. Reference Naldoni, Arana, Maia, Ceccarelli, Tavares, Borges, Pozo and Adriano2009; Azevedo et al. Reference Azevedo, Clemente, Casal, Matos, Oliveira, Al-Quraishy and Matos2013), but axial polarity in development is absent in amorphous plasmodia. At present it is unknown whether only one end of the plasmodia of C. vermiformes sp. n. consistently leads in the direction of movement. The presence of a polarized primary body axis in a motile myxozoan with a tissue-level of organization has been reported for the malacosporean, B. plumatellae (Gruhl and Okamura, Reference Gruhl and Okamura2012).
Amoeboid motility resulting from filipodia has been noted in some Ceratomyxa species infecting the gallbladder (Cho et al. Reference Cho, Kwon, Kim, Nam and Kim2004; Alama-Bermejo et al. Reference Alama-Bermejo, Bron, Raga and Holzer2012), and it is proposed that this motility provides a means of avoiding premature release of immature forms with the bile (Alama-Bermejo et al. Reference Alama-Bermejo, Bron, Raga and Holzer2012). A similar function may be attributed to the motility of C. vermiformis n. sp. Alternatively, swimming may enable the plasmodia to pass through the bile duct into the intestinal tract. In either case, it is striking that very different modes of motility have evolved convergently in cnidarians at the cellular level.
It is notable that certain features associated with bending and contractile movements in protists are also displayed by C. vermiformis sp. n., suggesting convergence of form and function at the cellular level. Thus, regularly situated mitochondria are observed to line up in the cortex of ciliates – in this case below a filamentous sheet that is believed to achieve localized bending (Hausmann et al. Reference Hausmann, Hülsmann and Radek2003). In peritrich ciliates, stalk contraction achieved by the spasmoneme (myoneme) may be antagonized by the extracellular material of the stalk which is proposed to be very elastic (Hausmann et al. Reference Hausmann, Hülsmann and Radek2003). A similar antagonistic function may be achieved by the extracellular glycocalyx secretion of C. vermiformis sp. n. Finally, microtubules in the external layer of C. vermiformis sp. n. are likely to serve a cytoskeletal function and might thus function rather like the stiffening rods, which are proposed to facilitate coiling in peritrichs (Hausmann et al. Reference Hausmann, Hülsmann and Radek2003). An alternative or additional explanation for the function of the glycocalyx is protection from the host's digestive enzymes.
A freshwater ancestral environment of ceratomyxids?
Recently Fiala et al. (Reference Fiala, Hlavničková, Kodádková, Freeman, Bartošová-Sojková and Atkinson2015b ) identified the Ceratomyxa clade as basal to all other marine myxosporean lineages based on molecular phylogenetic analyses using three genes. Ceratomyxa leatherjacketi and M. bulani, in turn, were revealed to form a basal subclade within Ceratomyxa. This basal subclade also now incorporates the newly described C. tunisiensis and C. amazonensis (Mathews et al. Reference Mathews, Naldoni, Maia and Adriano2016) and here we show that C. vermiformis sp. n. groups as sister to C. amazonensis. It is notable that fish parasitized by members of the early diverging Ceratomyxa/Myxodavisia subclade are associated with freshwater environments. Within this subclade, the early diverging M. bulani is a parasite of the amphidromous fish Megalps cyprinoides and C. tunisiensis has been reported infecting Caranx rhonchus, which inhabits brackish-water lagoons and estuaries. C. amazonensis and C. vermiformis sp. n. parasitize, respectively, S. discus and C. macropomum, which live exclusively in freshwater environments (Froese and Pauly, Reference Froese and Pauly2009). Another parasite of the gallbladder of a serrasalmid fish from the Amazon River (M. mylei; Azevedo et al. Reference Azevedo, Ribeiro, Clemente, Casal, Lopes, Matos, Al-Quraishy and Matos2011) is also likely to be a member of this clade (see below discussion on Meglitschia). Although the bootstrap support in our MP analysis is low, the strong support observed in both ML analyses suggests that infection of hosts associated with freshwater environments may have been primitive for ceratomyxids. This would imply a subsequent extensive radiation of ceratomyxids in hosts inhabiting fully marine environments.
The validity of Genus Meglitschia
Zhao et al. (Reference Zhao, Zhou, Kent and Whipps2008) alluded to the general resemblance of Myxodavisia and Ceratomyxa myxospores and we further point out the similarity of myxospores of C. vermiformis sp. n. to those described for the genus Meglitschia Kovaleva (Reference Kovaleva1988). Kovaleva erected this genus to harbor a species originally described as C. insolita (Meglitsch, Reference Meglitsch1960; Kovaleva, Reference Kovaleva1988). In the original description, Meglitsch (Reference Meglitsch1960) argued that although the arcuate spores and large, elongated polar capsules differentiated C. insolita (which forms amorphous plasmodia) from other Ceratomyxa species described at the time, the basic spore architecture supported assignment to Ceratomyxa. Our combined molecular and morphological analyses support Meglitsch's original premise that some Ceratomyxa species produce highly arcuate myxospores with elongated and tapered valves. Thus, the minor morphological differences used to create Meglitschia appear to be insignificant, suggesting that the genus is not supported. Unfortunately, there are no molecular data available for Meglitschia species to help to resolve this issue.
Concluding remarks
The Myxozoa demonstrate how metazoans have evolved to become endoparasites by miniaturization and morphological simplification as descendants of free-living cnidarian ancestors. The highly derived Myxosporea have taken this to the extreme, having effectively converged with parasitic protists to exploit hosts at the cellular level. Here we show that such miniaturization can nevertheless be accompanied by innovations that may promote coordinated movements as plasmodial worms. However, the basis for such movement at the cellular level in C. vermiformis n. sp. remains to be revealed. Whether the remarkable swimming demonstrated by C. vermiformis sp. n. is unique, remains unknown as myxozoan diversity is poorly sampled. Further research is likely to reveal new insights on how myxozoans have evolved abilities to move through and to maintain their positions within their host environments thus illustrating the extraordinary plasticity in lifestyles that can be supported by the cnidarian bauplan.
SUPPLEMENTARY MATERIAL
The supplementary material for this article can be found at https://doi.org/10.1017/S0031182016001852.
ACKNOWLEDGEMENTS
We thank Dr Lincoln Lima Corrêa, Dr Marcos Tavares-Dias, Marcos Oliveira, Luiz Alfredo da Mata, Eduardo Ferraz de Oliveira, and Raimundo Chicó for help with fieldwork and the fishermen of the communities of Jari do Soccorro, Santarém, Pará, Jarilândia, Macapá and Manacapuru, Amazonas for their local knowledge of fish availability and provision of material for study. We also thank Suellen Zatti for initial sequencing attempts that directed subsequent successful sequencing strategies. We are grateful to Alex Gruhl for advice in the interpretation of fine structure and for comments on our draft manuscript from two referees whose reviews have helped us to improve our manuscript.
FINANCIAL SUPPORT
This study was supported by the São Paulo Research Foundation–FAPESP (Procs. No. 2013/21374–6 to E. A. A). E. A. A. was supported by a Post-doctoral scholarship from CNPq (Proc. No. 200514/2015-6). B. O. was supported by the Visiting Researcher Programme–FAPESP (Proc. No. 2015/19463-6).
