Conchophthirus curtus is a scuticociliate found in the mantle cavity of unionid bivalve mussels; it is considered by most to be an endocommensal. It was previously redescribed and its morphogenesis has been carefully defined and evaluated. Conchophthirus stomatogenesis along with its enigmatic deep kinetosomal unit as compared with other scuticociliates, peniculines, and peritrichs suggested possible homologies and affinities. Thus, a comparison of Conchophthirus with common molecular markers to all other ciliates was a goal of this study. We collected Conchophthirus spp. in California from two unionid bivalve hosts: Anodonta californiensis in the Pit River and Lake Merced, and Margaritifera falcata from the Trinity River. The ciliates analyzed were predominantly C. curtus although other conchophthirids were present. The small subunit rRNA and the cytochrome c oxidase subunit 1 genes were sequenced. Phylogenetic analyses of these data were analyzed and maximum likelihood for the small submit rRNA dataset and neighbor-joining for the cytochrome c oxidase subunit 1 dataset. The cytochrome c oxidase subunit 1 sequences obtained from M. falcata populations were virtually identical. The analysis placed these sequences with 96% bootstrap support as sister to Dexiotricha sp. The small submit rRNA sequences obtained from populations from both hosts were almost identical, and they showed them to be sister, with 99% support to two unpublished sequences from Chinese populations of Conchophthirus cucumis and C. lamellidens, and sister to a Dexiotricha sp. These data and their analyses confirm Conchophthirus to be a scuticociliate, but not closely related to either philasterine or pleuronematine scuticociliates nor the peniculines or peritrichs. Further analysis awaits additional data. The North American distribution of Conchophthirus, niche analyses, and potential homologous structures are discussed as well as the use of these endocommensals as indicators of water quality and pollution.
The well-known environmental sensitivity of freshwater mussels (Wilbur, 1969) and their symbionts to monitor and assess water quality and pollution (Antipa, 1977) has added to efforts of recent monitoring of lotic and lentic locations for the endemic protistan fauna (Jeelani et al., 2018). Conchophthirus, a single celled organism, generally considered an endocommensal microorganism inhabiting the mantle cavity of fresh water clams or mussels, has been used in this work. Antipa’s work with Conchophthirus has been reviewed (Antipa, 2014) in the selected recent review of ciliates and flagellates. Since Denis Lynn’s monograph on the ciliated protozoa in 2008 (Lynn, 2008), significant progress has been made with use of molecular data to extend the relationships among the protista and especially the ciliated protozoa. In this report it was our objective to provide gene sequences of Conchophthirus curtus, which seems to be a cosmopolitan ciliate of freshwater mussels, and to assess the genetic support for the distinctness of the Conchophthiridae. In this way, should the role of these endocommensals be confirmed for evaluation of environmental degradation, their specific identity can be established. In addition, we use these data to evaluate an evolutionary hypothesis and the consideration of homologies that could ally the scutiociliate conchophthirids with other scuticociliates as well as the peritrichs and/or peniculines.
In our effort to collect Conchophthirus we have extended the range of hosts, and species of Conchophthirus in North America. We have considered the niche for these endosymbionts as have others (Raabe, 1933; Kidder, 1934; Fenchel, 1965; Raabe, 1971). We had hoped to extend our study on the variety and groups of conchophthirids we discovered, but, regretfully, the untimely accident that befell our colleague, (Denis H. Lynn-DHL) prevented us from completing this task in the allotted time. Please consider this to be an initial publication on the way toward our goals of a better understanding of niche specificity, range, and relationships of these endosymbionts to their environment and water quality as well as a furthering of our understanding of ciliate taxonomy and the phylogeny of the conchophthirids.
Sample collection, isolation, and identification
In a search for conchophthirids, the freshwater mussel or unionid Anodonta californiensis, Lea 1852 was collected off the north boating dock of Lake Merced, San Francisco, California with a dredge (37°43’33.1”N 122°29’53.0”W, July 25, 1980). More recently, several specimens of these bivalves were hand collected from each of two other California localities: the Pit River near Burney, CA (40°59’26.3”N 121°30’29.4”W, September 7, 2017) two A. californiensis specimens and the Trinity River near Weaverville, CA (40°44’17.3292”N 123°12’25.1568”W, September 9, 2017), four individuals of Margaritifera falcata Gould 1850. The isolated ciliates were predominantly C. curtus, but there were also specimens of C. anodontae and C. discophorus present in each of the six hosts.
To collect Conchophthirus, the mussels were pried open and filtered environmental water was squirted through the mantle cavity with a Pasteur pipette. The rinse fluid displaced symbionts contained within the mantle cavity including conchophthirids which were collected in a Syracuse dish. Individual cells were removed with a fine bore glass micropipette for observation and/or further preparation. Conchophthirus spp. (Fig. 1) were identified following Protargol protein staining prepared by procedures outlined by Antipa and Small (1971a).
All ciliate samples were fixed in 80% ethanol in preparation for gene sequencing. DNA was successfully extracted from 5 cells of C. curtus from A. californiensis (sample CC1), from 13 cells of a mix of Conchophthirus spp. from A. californiensis (sample CSP2), and from 9, 15, 15, and 30 cells of a mix of Conchophthirus spp. from M. falcata (samples CM1-CM4). Dexiotricha sp. was collected and identified by Dr. Paolo Madoni, Italy, and a sample sent to us in 2006 for a barcoding project. The SSUrRNA gene was amplified using the forward primer 82F (Jerome et al., 1996) and the reverse universal eukaryotic primer B (Medlin et al., 1988). The partial sequence of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene was amplified using the primers F298dT and R1184dT (Strüder-Kypke and Lynn, 2010). The PCR products were purified with a QiaQuick purification kit (Qiagen, Valencia, CA, USA) and sequenced in both directions with a 3730 DNA Analyzer (Applied Biosystems, Burlington, ON, CA), using ABI Prism BigDye Terminator (v. 3.1) and a Cycle Sequencing Ready Reaction kit. Sequencing was done by the NAPS Centre, University of British Columbia.
Gene sequence alignment and phylogenetic analyses
The SSUrRNA gene sequences derived from Conchophthirus found in two host species, as well as the Dexiotricha sequence were assembled into contigs with DNA Dragon v. 1.6.0 (Sequentix; www.DNA-dragon.com), trimmed at the ends, and checked for sequencing errors. Sequences of other scuticociliate and oligohymenophorean genera were downloaded from GenBank and alignment was performed on the MAFFT (v. 7) server (https://mafft.cbrc.jp/alignment/server/; Katoh et al., 2017). The resulting SSUrRNA gene sequence alignment was sent to the Guidance2 server (http://guidance.tau.ac.il/ver2/; Penn et al., 2010; Sela et al., 2015) to calculate alignment confidence scores. Ambiguously aligned, hypervariable regions below a confidence score of 0.95 were removed. Distance data were inferred from complete sequence alignments with only the ends trimmed (130 taxa and 1,887 positions). Pairwise distances were calculated with Mega7 (Kumar et al., 2016), based on the Kimura-2-Parameter model (Kimura, 1980).
The final alignment for phylogenetic analyses comprised 130 taxa and 1,583 nucleotides (83.9% of original alignment). For the SSUrRNA sequences, three different phylogenetic analyses were performed: Maximum Likelihood (ML); Bayesian Inference (BI); Neighbor Joining (NJ). The General Time Reversible Model (GTR) for nucleotide substitution, with gamma distributed substitution rates (Γ) and invariable sites (I), was identified as the best model using jModelTest ver. 2.1.10 (Darriba et al., 2012), AIC Criterion. These parameters were used in ML and BI analyses. jModeltest, RAxML, and MrBayes were performed on the CIPRES Science Gateway (Miller et al., 2010). The ML analysis was run with RAxML-HPC2 on XSEDE (Stamatakis et al., 2008), with 100 rapid bootstrap replicates and a subsequent thorough ML search, using the GTR + I+Γ model. Bayesian Inference was computed with MrBayes ver. 3.2.6. on XSEDE (Ronquist et al., 2012), also using the GTR + I+Γ model. Two parallel runs were performed. The maximum posterior probability of a phylogeny out of 3,000,000 generations, respectively, approximating it with the Markov chain Monte Carlo (MCMC) and sampling every 500th generation was calculated, discarding the first 25% of trees as burn-in. Average standard deviation of split frequencies (<0.01) was used to assess convergence of the two runs. PHYLIP ver. 3.695 (Felsenstein, 2009) was employed to calculate genetic distances with the Kimura-2-Parameter model (Kimura, 1980), using DNADIST. The distance trees were constructed with NEIGHBOR, using the NJ algorithm (Saitou and Nei, 1987). The data were bootstrap re-sampled 1,000 times.
Forward and reverse cox1 gene sequences were obtained for four samples taken from one host species (M. falcata). The sequences were aligned in Mega7 with other scuticociliate and selected hymenostome sequences, based on the translated amino acid sequences. For the cox1 gene, a Neighbor Joining analysis was performed on the complete alignment with only the ends of the sequences trimmed to equal length and using the same parameters as listed above. This alignment consisted of 55 taxa and 702 positions. The data were bootstrap resampled 1,000 times.
Sample collection and identification
At all three sampling sites and within the mantle cavities of all hosts, abundant numbers and multiple species of Conchophthirus were discovered. Like many locations elsewhere, the unionids harbored several species within each mussel (this will be reviewed in the Discussion). Unlike the midwestern USA states, where C. curtus seemed to be the resident endocommensal of unionids (Kelly, 1899; Penn, 1958; Antipa and Small, 1971b), C. curtus, C. anodontae, C. discophorous, and possibly other species commingled within each mussel we examined in California. We followed the detailed descriptions of Antipa and Small (1971a) and Raabe (1971) and others to identify the conchophthirids. The presence of C. discophorus, customarily a commensal of sphaeriid mussels, was found in all unionids we examined and at all three localities.
Genetic data and tree topologies
The partial SSUrRNA gene sequences of the Conchophthirus isolates are 1,661 (CC1), 1,664 (CM3), 1,667 (CSP2), 1,681 (CM2), and 1,691 (CM1) nucleotides in length, respectively. The GC content varies between 42.89-43.01%. The SSU rRNA gene sequences are virtually identical – with 0.18-0.3% divergence to the other available Conchophthirus sequences (C. cucumis and C. lamellidens). This points to conspecifity of all our isolates. All sequences have been submitted to GenBank under the accession numbers MN704274 to MN704278. The SSU rRNA gene sequence of Dexiotricha sp. was amplified during a previous study (Strüder-Kypke and Lynn, 2010) but is yet unpublished. It is 1,654 nucleotides in length with a GC content of 43.29% and has been submitted to GenBank under the accession number MN704273.
The partial mitochondrial cox1 genes of Conchophthirus are 769 (CM1), 835 (CM2), 800 (CM3), and 783 (CM4) nucleotides in length and include an insert that is 324 nucleotides long. This insert is typical for ciliates, and its length fits well into the range reported for oligohymenophorean ciliates (Strüder-Kypke and Lynn, 2010). As in all ciliates, the GC content is low, at 30.63% (CM3) to 31.04% (CM4). The sequences of the four isolates of Conchophthirus were identical. The sequences have been submitted to GenBank under the accession numbers MN702822 to MN702825.
The topologies of the phylogenies derived from the SSUrRNA gene analyses were all similar and grouped Conchophthirus together with Dexiotricha spp. and the astome ciliate Haptophrya planariarum, not within the order Pleuronematida, but at the base of pleurostomatid and philasterine scuticociliates, apostomes, and astomes, and nested within clusters of taxa of the order Loxocephalida. However, support values for all basal branches were very low. The inferred phylogenetic tree (Fig. 2) shows the ML topology with support values for ML/BI/NJ listed at the branches.
In the genetic distance tree inferred from the cox1 gene sequences (Fig. 3), Conchophthirus groups with Dexiotricha, however, this branch clusters with the order Philasterida albeit with low support (68%).
Historical record and geographical locations
Since 1971 there has been no discussion of/or addition to the range of species of Conchophthirus. Ironically, in 1971 both Antipa and Raabe independently and simultaneously published a list of known and accepted species (Antipa and Small, 1971a; Raabe, 1971). They were in agreement in evaluating the ten species of conchophthirids accepted. Conchophthirus probably ranges worldwide within freshwater bivalve habitats supporting unionids and sphaeriids, and most studies seem to show a stable relationship between conchophthirids and their hosts. Both in Europe and North America some species seem devoted to particular unionid host species (Raabe, 1933, 1971; Kidder, 1934) while others are far less localized. In Poland and Eastern and Western North America we commonly found commingling of species (Raabe, 1933; Kidder, 1934; this report) at the same time, in the Mississippi Valley Drainage in Central North America (perhaps housing the largest variety of unionid species in the world) the conchophthirid to infect most if not all species of unionid was C. curtus (Kelly, 1899; Penn, 1958; and Antipa and Small, 1971b). While C. curtus seems to be nearly ubiquitous in the Mississippi Valley Drainage, no commingling or reports of other species of Conchophthirus have yet to be found in Central North America.
Although we did not analyze the frequency of infections as did Fenchel (1965) for single ciliate species Peniculistoma mytili and Ancistrum spp. in Mytilus edulis or as did Antipa et al. (2016) with Mytilophilus pacificae and Ancistrum spp. in Mytilus californianus, we did observe that the frequency of co-habitation does not seem to differ significantly from a prediction based on the occurrence of each species alone. Thus findings support the conclusions of Fenchel (1965) that Ancistrum and P. mytili do not interact and thus Antipa et al. (2016) extend the pattern to Ancistrum and M. pacificae within M. californianus. Kidder (1934) observed that C. anodontae and C. curtus occurred together in multiple unionids as Penculistoma and Ancistrum could coexist within M. edulis. C. magna only seems to be found within Elliptio complanatus, although Kidder (1934) often found it along with C. anodontae. Fenchel (1965, 1966) found a multiplicity of endosymbiotic ciliates in many freshwater and marine Scandinavian molluscs. So, the commingling factor needs to be explored further, and it would seem that further investigations in California could expand this interesting question since all sites we explored discovered three or more conchophthirids symbiotic in all unionids investigated to date.
Our current assumption follows the lead of Fenchel, Kidder, and Raabe who each found abundance in different mantle locations. Kidder (1934) and Raabe (1933) observed that they seemed to find C. anodontae principally near the labial palps while C. curtus were localized adjacent to the lamellar gills. As you can clearly see from Fig. 1 (as well as the thru-focal series in the supplement) the size and shape of each of the conchophthirids we observed is quite different. C. anodontae is nearly tubular in shape while C. curtus and C. discophorous are more leaf-like in overall form yet vastly different in overall size, shape, and volume. Fenchel (1965) discussed the water flow within the mantle cavity, and it seems likely the differences in the size and shape of each of these ciliates would have a preferential location and perhaps food source. This lends itself to an investigation beyond the scope of our current study, one in pursuit of the food source of each species and/or observation with an endoscope-type instrument to make live observations to more fully observe just what is happening with these endosymbionts within the unionid mantle cavity.
Stomatogenesis and inferred homologies
Antipa and Hatzidimitriou (1981) pondered the stomatogenesis of C. curtus as did Foissner (1996) and Lynn (2008). Following the lead of Small (1967) and Corliss (1968), they considered Conchophthirus to be a scuticociliate but could not use their data to firm up further phylogenetic relationships. It was also tempting to consider the DKU to be a homolog of the germinal kinety of peritrichs (Lom, 1964) or the endoral kinety of peniculines (Jones, 1976), and the thigmotactic field to be a homolog to the clavate, or scopular disc of peritrichs (Brown, 1987). Brown’s observations of swimming Vorticella striata telotrochs showed that the scopular end of the telotroch was the effective anterior end and therefore positionally the same as the thigmotactic field of conchophthirids and other “thigmotrichs.” The presence of scopular pegs or clavate cilia (stumpy basal bodies) are used as holdfasts in attachment of the telotroch to be followed by generation of the Vorticella stalk. However, in this study, neither of the molecular markers we used supported this speculation. More and further varying data needs to be collected to extend and/or validate this speculation. The conchophthirids continue to be considered scuticociliates; our data supports this, but at this point does not extend beyond the initial speculation of Small (1967) based on observations of stomatogenesis.
Phylogenetic analyses and taxonomic status of the Conchophthiridae
Based on the result that the SSUrRNA gene sequences of our isolates are identical, and given that isolate CC1 was identified as C. curtus, we conclude that all our isolates belong to the species C. curtus. The cox1 sequences of all isolates from M. falcata are identical and hence, we can exclude the existence of cryptic species at least for our sampling data. However, it is puzzling that, despite the obvious occurence of several morphotypes (see Fig. 1) in each host, we were only able to amplify the DNA of one of them. Either C. curtus dominated the population of conchophtirids in this host, or the DNA of the other species did not amplify with the primers used. We do favour the former explanation, as the cox1 primers are degenerate and the PCR conditions are fairly unspecific during the intial cycles (Strüder-Kypke and Lynn, 2010). Furthermore, the poor reads at the beginning and end of some SSUrRNA sequence data might point to the presence of more than one species within this host.
The phylogenetic trees cluster all Conchophthirus sequences together with Dexiotricha sp. as sister taxon. Unfortunately, no morphological data are available to confirm the assignment of Dexiotricha sp. – however, other published Dexiotricha sequences also cluster basal within this group, although with larger genetic divergences (4.5-4.9% and 9.5-10.9%, respectively). On the other hand, genetic divergences to the species of the Order Pleuronematida – the taxon Conchophthirus was assigned to based on morphological features (Small, 1967; Corliss, 1979) - are at 16-18%, much larger. Interestingly, Antipa (1971, 2014) in his ultrastructural study of C. curtus noted that the longitudinal cortical ridge microtubule of C. curtus appears in the identical location as the microtubule in Dexiotricha colpidiopsis (Peck, 1977). The general topology of the phylogenetic tree from SSUrRNA gene sequence data is congruent with previously published phylogenies (Gao et al., 2013; Antipa et al., 2016; Rataj and Vd’ačný, 2018; Zhang et al., 2019). The basal placement of Conchophthirus with a genus assigned to the order Loxocephalida and the simultaneous paraphyly and basal branching of other members of this order - e.g. Cinetochilum basal to the subclass Apostomatia, Platynematum basal to the subclass Astomatia, Cardiostomatella, Dexiotrichides, and Paratetrahymena basal to all scuticociliates – suggests that this group of ciliates is defined by plesiomorphic characters. This would also explain the morphological and stomatogenetic similarities of Conchophthirus to peniculine and peritrich taxa (Antipa and Hatzidimitriou, 1981). These interpretations, however, need to be considered with caution, since there is no support for the basal branching pattern within the scuticociliate clade.
Additional species of conchophthirids
What does the additional species of conchopthirids mean? It may be time to repeat the Antipa (1977) examination of the sensitivity of endocommensals to organic pollution. In that study there was only C. curtus to challenge against levels of organic pollutants. Now that we seem to have found in California unionids with three or more species of conchophthirids at distances of over 300km, it may be an opportunity to challenge them with organic and/or chemical pollutants to see if there is a variability of the sensitive of each of these individual species, but probably it might be a better idea to first find out about their niche, foodstuffs, and ecological role. Are they really endocommensals or possibly mutualistic symbionts? While there may be a correlation between their presence and the quality of the water and/or pollution present within the waterway that may be detected by these additional endocommensal monitors (Antipa, 1977). Only further study will advance the use of these endocommensals for the assessment of potential environmental impact.
Conclusions and future intentions
The conchophtheriids C. curtus, C. anodontae, and C. discophorous were present and endemic to two different: unionid species at three different localities examined in California.
The use of SSUrRNA gene sequences groups all our samples together as well as close clustering with Dexiotricha sp. as a sister taxon.
Our molecular data confirm and strengthen the scuticociliate lineage of the conchopheriids but does not place them close to the pleuronematids or the philasterines. At this time the molecular data are not complete enough to allow us to elaborate on the possible peritrich/peniculine relationship discussed above.
We discuss and question the symbiotic role and ecological niche of each of the three species we found to be endemic within the mantle cavity of the unionids we examined in California. Only further study, beyond the scope of this study, will establish this.
Following re-establishing our group in the absence of DHL, we intend to collect additional materials as well as new collections from Illinois to determine if the widely spread C. curtus is molecularly identical with its morphotype in California.
Environmental issues and the continuing role of Denis H. Lynn’s legacy
Now that we are aware of the addition of new species of conchophthirids to the flora and fauna of the unionids of California, a more extensive collection of both the conchophthirids and their unionid hosts in the Western parts of North America should provide a better understanding of the frailty of the wetlands. In addition, the opportunity provides for an extension of this study to add to Denis H. Lynn’s contribution to biology, taxonomy, and the betterment of the environment.
We thank Drs. Frank Cipriano, John Dolan, and Mr. Nicholas Irwin for their suggestions based on an early draft of this report, and to noted citizen scientist Mr. Paul Tenczar for support and assistance in collecting mussels. John Dolan prepared the Protargol stained conchophthirids for Fig. 1 and the supplemental files (available online at www.taylorandfrancis.com). The Dexiotricha sp. sample was provided by Dr. Paolo Madoni, Italy. We thank the three reviewers for their consideration and suggestions for modification and improvement of the manuscript.
This investigation was supported, in part, by NSF Grant DEB 78-03550 awarded to GAA and a Natural Science and Engineering Research Council of Canada Discover Grant to DHL.
Supplemental data for this article can be accessed on the publisher’s website.