Morphological and Molecular Characterization of Two New Species of Ceratomyxa Thélohan, 1892 (Cnidaria: Myxosporea) from the Marine Ornamental Fish Zanclus cornutus (Linnaeus, 1758) off Lakshadweep Islands, Arabian Sea.

PURPOSE: The present study provides the complete morphological and molecular description of two new species of myxosporeans, Ceratomyxa zancli n. sp. and Ceratomyxa cornuti n. sp. infecting the gallbladder of Zanclus cornutus from the Lakshadweep Islands, Arabian Sea. METHODS: Zanclus cornutus were screened for the presence of myxosporeans, and the recovered myxospores were morphologically characterized using Nomarski Differential Interference Contrast (DIC) optics. The sequences of SSU rDNA were employed for molecular and phylogenetic studies. RESULTS: Both the parasites exhibited a prevalence of 21% each. C. zancli n. sp. is characterized by broadly cresentic myxospores with convex anterior and slightly concave to straight posterior margins and rounded ends. Spore valves two, unequal, measured 9.6 ± 0.7 µm × 25.2 ± 1.3 µm. Polar capsules two, unequal, spherical, measured 4 ± 0.6 µm × 3.5 ± 0.6 µm. Polar filament exceptionally long and arranged irregularly. Myxospores of C. cornuti n. sp. are elongated with convex anterior and slightly concave to straight posterior margins. Spore valves two, unequal, measured 7.00 ± 0.4 µm × 26.56 ± 1.8 µm. Polar capsules spherical, unequal, measured 3.52 ± 0.2 × 3.36 ± 0.35. Molecular analysis of C. zancli n. sp. (ON818297) and C. cornuti n. sp. (ON818298) resulted in 1469 and 1491 bp long SSU rDNA sequences, respectively. Molecularly C. zancli n. sp. is close to C. diplodae and C. barnesi with 91.39% similarity, while C. cornuti n. sp. appears closer to C. robertsthomsoni with 97.46% similarity. In phylogenetic analyses, C. zancli n. sp. branched separately within the Ceratomyxa clade while C. cornuti n. sp. clustered with C. robertsthomsoni and C. thalassomae. CONCLUSION: Based on the differences in morphological, morphometric, molecular, and phylogenetic characteristics, as well as differences in the host and geographic location, the above two species of myxosporeans are considered novel. The study forms the first report of a species of Ceratomyxa from Z. cornutus.

Hepatobiliary Ascariasis in a Piglet.

PURPOSE: Ascariasis caused by the helminth Ascaris suum is the most common parasitosis of swine worldwide and it may involve all age categories of pigs. The present study reports an unusual localization of A. suum worms in the biliary system of a piglet slaughtered for human consumption. METHODS: The liver was subjected to ultrasound scan and pathological examination. The isolated worms were morphologically examined and the DNA was extracted for the molecular identification of the species involved. RESULTS: A total of 43 preadult nematodes were found within the gallbladder and the bile ducts. Parasites were morphologically identified as belonging to the genus Ascaris and molecularly as A. suum. At gross examination, the liver was moderately enlarged, with the bile ducts severely dilated. A chronic inflammatory infiltrate was noted, often centered around ectatic bile ducts (up to 5 mm in diameter), lined by hyperplastic epithelium and filled with sections of nematodes. The worm sections showed smooth cuticle, coelomyarian musculature, and an intestinal tract lined by columnar, uninucleated cells within a pseudocoelom. The ex vivo ultrasonographic examination of the liver allowed the visualization of several nematodes in the bile duct lumen and could be suggested for in vivo diagnosis. Unfortunately, the absence of the intestine did not allow to define the pathogenesis of the infection. CONCLUSION: Although, given the unusual nature of this finding, it is difficult to identify predisposing factors for this A. suum localization, it suggests that ascariasis should be considered in the differential diagnosis of pigs with hepatobiliary disease.

Occurrence of two myxosporean parasites in the gall bladder of white seabream Diplodus sargus (L.) (Teleostei, Sparidae), with the morphological and molecular description of Ceratomyxa sargus n. sp.

Myxosporeans are widespread cnidarian parasites that usually parasitize fish as part of their complex life cycle, thus constituting a potential threat for the aquaculture industry. White seabream Diplodus sargus (L.) is a commercially valuable sparid fish reared in Southern European aquacultures. Nonetheless, knowledge on myxosporean infections potentially harming the sustainable production of this fish is extremely limited. In this study, a myxosporean survey was conducted on D. sargus specimens reared in two Southern Portuguese fish farms. Two coelozoic myxosporeans were detected infecting the gall bladder, and are herein reported based on microscopic and molecular procedures: Ceratomyxa sargus n. sp. and Zschokkella auratis Rocha et al., 2013, previously described from reared stocks of gilthead seabream Sparus aurata in the same geographic locality. Ceratomyxa sargus n. sp. is the 12th species of the genus to be reported from Southern European sparids, reinforcing a substantial radiation of Ceratomyxa within this fish family and geographic region. SSU rRNA-based Bayesian inference and maximum likelihood analyses revealed C. sargus n. sp. positioned separately from other sparid-infecting Ceratomyxa spp. reported from Southern European countries, demonstrating that this species does not share a more immediate common ancestor with its closest relatives based on host affinity and geography. The recognition of a novel sparid-infecting lineage within the Ceratomyxa clade strengthens the contention that this genus entered sparid fish multiple times, namely in the Southern European region. The identification of Zschokkella auratis infections in D. sargus demonstrates that host shift has occurred among sparids reared in the Southern Portuguese coast. This agrees with the broad host specificity that is usually attributed to this genus, and that may be suggested to be the outcome of the capacity of the Zschokkella morphotype to undergo host shift/switch based on our findings and the limited molecular data available for this genus. Thus, a better understanding of Zschokkella host-associated diversification and dispersal mechanisms requires the increasing availability of molecular data from infections of the same species occurring in multiple hosts and geographical locations.

Myxidium tunisiensis n. sp. (Myxosporea: Myxidiidae) infecting the rough skate Raja radula Delaroche, 1908 (Rajiformes: Rajidae) from North East Tunisia.

A new marine myxosporean, Myxidium tunisiensis n. sp. (Myxosporea: Myxidiidae), is described infecting the gallbladder of the rough skate Raja radula Delaroche, 1908 (Chondrichthyes: Rajidae) harvested from the Bay of Bizerte, Tunisia. Observed stages of the parasite were floating free in the bile. Plasmodia, disporic or polysporic, contain disporic pansporoblasts measuring 23.0-27.2 µm in maximum diameter and 21.0-25.5 µm in minimum diameter. Mature myxospores, which are elongated, typically fusiform measured 18.6 ± 1.7 (17.2-22.0) µm in length, 8.6 ± 1.2 (7.2-11.2) µm in width and 8.2 ± 0.9 (6.4-10.1) in thickness. Myxospore valves are symmetrical with 9-10 longitudinal striations. Two polar capsules, pyriform, were equal in size, measuring 5.8 ± 0.4 (5.2-6.7) µm in length and 3.1 ± 0.2 (3.0-3.2) µm in width, with 5-6 polar filament turns. The infection was prevalent throughout the year with the highest prevalence in autumn (81.8%) and the lowest in winter (32.3%). The seasonal prevalence of M. tunisiensis n. sp. in the Bay of Bizerte appeared to fluctuate according to seawater temperature. The morphological and morphometrical differences with all Myxidium spp. described so far, complemented by molecular data, prove clearly that the present species is a new member of the genus Myxidium. The SSU rDNA phylogenetic analyses revealed that Myxidium tunisiensis n. sp. clustered within the oligochaete-gallbladder clade in a basal position to the marine subclade of Myxidium baueri and Myxidium coryphaenoideum. While the new species branching pattern is stable in the ML and the BI analyses, it changed in the MP analysis. This is the first molecular study of a Myxidium species infecting an elasmobranch host inhabiting the Mediterranean Sea.

Occurrence of the host-parasite system Rhaphiodon vulpinus and Ceratomyxa barbata n. sp. in the two largest watersheds in South America.

While around world, species of the genus Ceratomyxa parasite majority marine hosts, growing diversity has been reported in South American freshwater fish. The present study reports Ceratomyxa barbata n. sp. parasitizing the gallbladder of the Rhaphiodon vulpinus fish from the Amazon and La Plata basins. Morphological (light and transmission electron microscopy), molecular (sequencing of small subunit ribosomal DNA - SSU rDNA), and phylogenetic analyses were used to characterize the new species. Worm-like plasmodia endowed with motility were found swimming freely in the bile. The myxospores were elongated, lightly arcuate, with rounded ends and had polar tubules with 3 coils in the polar capsules. Ultrastructural analysis revealed plasmodia composed of an outer cytoplasmic region, where elongated tubular mitochondria, a rough endoplasmic reticulum, sporogonic stages, and a large vacuole occupying the internal area were observed. Phylogenetic analysis, based on SSU rDNA, found that among all South America freshwater Ceratomyxa species, C. barbata n. sp. arises as an earlier divergent species. The present study reveals the occurrence of this host-parasite system (R. vulpinus/C. barbata n. sp.) in the two largest watersheds on the continent.

A novel Ceratomyxa species (Myxozoa: Cnidaria) infecting an Amazonian catfish.

Parasites are important organisms for the health of ecosystems. While the Amazon Basin is home to a great diversity of ichthyofauna, our knowledge of myxozoan diversity in the biome remains relatively limited. The present study describes a new myxozoan species, Ceratomyxa mandii n. sp., parasitizing the gallbladder of the Amazonian catfish Pimelodina flavipinnis (Pimelodidae) from the Solimões River, in the region of Manaus, Brazil. Light and electron microscopy,  small subunit ribosomal DNA (SSU rDNA) sequencing and phylogenetic analysis were performed. The new species exhibited worm-like plasmodia with undulatory motility. The SSU rDNA based phylogenetic analysis revealed it to be a sister taxon of C. gracillima, which also parasitizes an Amazonian pimelodid fish, possibly reflecting a host-parasite co-speciation process. This study contributes to our understanding of this little sampled group of organisms.

Sphaeromyxa azevedoi n. sp. (Myxozoa: Sphaeromyxidae) infecting the gall bladder of Gobioides grahamae (Perciformes: Gobiidae) in the Amazon region.

Sphaeromyxa azevedoi n. sp. is described from the gall bladder of the goby Gobioides grahamae (Gobiidae) captured on the Paracauari River in Salvaterra, on Marajó Island, northern Brazil. A total of 50 G. grahamae specimens were analysed, and 15 (30%) were parasitised by the plasmodia and myxospore of Sphaeromyxa azevedoi n. sp. Large plasmodia were observed floating in the bile. These plasmodia were flat, rounded, oval or elongated, and of varying sizes. The mature myxospores, found singly or in pairs, were 27.1 ± 2.7 (20.5-30.1) µm Length and 3.8 ± 0.2 (3.5-4.4) µm Width in the valvular view. The myxospore has two polar capsules of equal size, 8.1 ± 0.6 (7.4-9.4) µm in length and 2.9 ± 0.2 (2.3-3.3) µm in width. A polar tubule was observed in each capsule, arranged perpendicularly to the principal axis, with three or four coils. The histological analysis showed that the plasmodia and myxospore are located in the lumen of the gall bladder, arranged in pairs, and the epithelium of the gall bladder presented multifocal necrosis. The SSU rDNA of Sphaeromyxa azevedoi n. sp. clusters in the 'balbianii' group of the Sphaeromyxa clade. The morphological characteristics and molecular phylogeny of Sphaeromyxa azevedoi n. sp. support its classification as a new species of the genus Sphaeromyxa, which represents an important advancement in the understanding of the diversity of the myxozoan parasite fauna of Brazilian fishes, especially considering that the new species may be detrimental to the host, a commercially important Brazilian fish species.

Morphological Redescription and Phylogenetical Position of Ceratomyxa truncata Thelohan (1895) and Coccomyxa morovi Léger and Hesse, 1907 (Myxozoa: Myxosporea) Infecting the Gall Bladder of Sardina pilchardus (Walbaum) from Tunisian Coast.

PURPOSE: Two myxosporean species have been, so far, independently reported from the gallbladder of the European pilchard, Sardina pilchardus (Walbaum) (synonym Clupea pilchardus) in the Northern shore of the Mediterranean Sea; Ceratomyxa truncata Thélohan, and Coccomyxa morovi Léger and Hesse, 1907. The two species were described with incomplete morphological data and based only on line drawings of their mature myxospores. METHODS: During a parasitological survey in the Southern shores of the Mediterranean coast in the gulf of Gabès off Tunisia, two coelozoic myxosporean species were found in the European pilchard and described using morphological and molecular phylogenetic tools. Morphological characterization was based on the mature myxospore study and some vegetative stages. The SSU rDNA sequences were performed for molecular and phylogenetic study. RESULTS: The most frequently encountered species belongs to the genus Ceratomyxa Thelohan, 1892. The second species belongs to the genus Coccomyxa. Morphological examinations, allowed us to match these two recorded species with Ceratomyxa truncata and Coccomyxa morovi, respectively, as previously described in the same host species referring to the original manuscripts instead of some morphological differences. Molecular analyses based on the partial SSU rDNA sequences did not much with any of the previously reported myxozoan sequences. Phylogenetic analysis positioned C. truncate in a well-supported clade including Ceratomyxa ssp. from Mediterranean Sea, while C. morovi was positioned on the basis of the subclade grouping all Coccomyxidae species. CONCLUSION: We provided herein a first morphological redescription of Ceratomyxa truncata and Coccomyxa morovi parasite of Sardina pilchardus from the Southern shores of the Mediterranean Sea and we successfully obtained the SSU rDNA sequences of these two species and positioned them in the phylogenetic tree.

GENETIC DIVERSITY OF CYSTODISCUS SPECIES IN AMPHIBIANS IN THE SOUTHERN UNITED STATES.

Myxosporean species in the genus Cystodiscus are parasites of amphibians and have been reported from several continents. Typically used for the identification of myxozoans, the spores produced by these species are similar to one another, possessing 2 polar capsules and being ovoid. The number of transverse depressions on the spore can be useful for delineating species, but these can sometimes be difficult to distinguish. In North America, Cystodiscus serotinus and Cystodiscus melleni have been described, and for C. serotinus in particular, numerous reports and a wide range of hosts have been associated with this species. Given the challenges of identifying some of these species, we questioned whether all encounters of Cystodiscus species can be attributed to these 2 described species, or if there may be additional undescribed species or cryptic species. Over 7 yr, 383 amphibians representing 13 species of toads, frogs, and salamanders were collected from sites in Oklahoma and Arkansas. Cystodiscus infections were found in 56 individuals (14.6%). Tissues from these infected individuals were preserved in alcohol for genetic analysis. The small subunit (SSU) and large subunit (LSU) ribosomal RNA genes were partially sequenced and analyzed phylogenetically. Nine distinct SSU sequence types and 7 distinct LSU sequence types were identified. Phylogenetically, sequence types were attributable to C. serotinus, C. melleni, Cystodiscus axonis, and an undescribed species. For the previously described species, there were multiple SSU sequence types: 4 for C. serotinus and 2 for both C. melleni and C. axonis. Phylogenetic patterns were similar for the LSU sequence analysis using a shorter sequence than the SSU, and we propose that the LSU is useful for initial barcoding of Cystodiscus species in any future surveys. In our qualitative assessment of sequence types compared to geography and host species, SSU types C1 and C2 (C. axonis) were only found in Union County, Arkansas, and McCurtain County, Oklahoma, respectively. Also, salamanders were only infected with SSU types B or D (C. melleni), and type B was only found in salamanders. Our finding of C. axonis in North America is notable because this species was described in Australia and is associated with host pathology. Our work reveals that there are cryptic species of Cystodiscus in the United States, one of which may be a pathogen, highlighting the importance of genetic analysis for future surveys of these species.

First description of Chloromyxum squali Gleeson and Adlard, 2012 (Myxozoa) in the Mediterranean Sea in a new host Squalus blainville (Chondrichthyes: Squalidae): morphological, ultrastructural and phylogenetic data.

Chloromyxum squali Gleeson and Adlard, 2012 originally described from Squalus acanthias Linnaeus, 1758 is reported for the first time from the gallbladder of Squalus blainville (Risso, 1827) caught from the eastern coast of Tunisia. In the present study, this chloromyxid was described based on morphological and ultrastructural features combined with molecular analysis of 18S rDNA. Young plasmodia were found attached to the gallbladder, while mature plasmodia and myxospores were observed floating free in the bile. Mature plasmodia were polysporic, subspherical in shape, measured 97.8 ± 0.5 µm long and 63.4 ± 0.4 µm wide. Mature myxospores were ovoid with a pointed anterior end, measuring 10.2 ± 0.5 µm long and 8.3 ± 0.5 µm wide. Two asymmetrical shell valves adhered together along an S-shaped suture line. Each valve has 5-7 elevated surface ridges parallel to suture line. A bundle of long caudal filaments extended from the basal end of shell valves. Four pyriform polar capsules equal in size, measuring 3.1 ± 0.4 µm long and 2.5 ± 0.3 µm wide, were situated at the same level in the anterior pole of the myxospore, each with a polar filament coiled in 7-8 turns. Pairwise comparisons among the SSU rDNA sequences revealed significant similarity between Chloromyxum squali infecting S. acanthias with the sequence obtained in this study. Phylogenetic analysis revealed that C. squali clustered in the clade of Chloromyxum species infecting the gallbladder of marine Chondrichthyes. Chloromyxum squali showed a seasonal variation of prevalence with significantly higher prevalence noted in summer and in autumn and absence of infection in winter.

Morphological and molecular characterization of Ceratomyxa xanthopteri n. sp. (Myxosporea: Ceratomyxidae) from the marine ornamental fish Acanthurus xanthopterus Valenciennes 1835 (Acanthuridae) off Vizhinjam coast, Kerala.

A new species of Ceratomyxa infecting the gallbladder of the marine ornamental fish Acanthurus xanthopterus collected from the Vizhinjam coast of Kerala is described. The parasite exhibited a prevalence of 100%. Mature spores recovered from the gallbladder were slightly crescentic with rounded lateral extremities and possessed convex anterior and slightly concave to straight posterior margins. Spore valves two, equal, joined by a straight and prominent suture. Myxospores measured 5.5 ± 0.6 µm in length and 15.9 ± 2.3 µm in thickness. Polar capsules two, equal, spherical, positioned anteriorly on either sides of the suture, 2.3 ± 0.2 µm long and 2.2 ± 0.2 µm wide. Polar filament with four to five coils, 21.2 ± 0.6 µm when extruded. Posterior angle 173.6 ± 5.2°. Early sporogonic stages and monosporic, disporic, and multisporic plasmodial stages were spherical to irregular in shape, with or without filopodia. Histopathologic analysis revealed that spores and developing stages were attached to the gallbladder wall as well as found free in the lumen. Morphologic and morphometric comparison of the present parasite with known species of Ceratomyxa indicated significant differences. In molecular and phylogenetic analyses, the present myxosporean revealed high divergence with related forms and occupied an independent position within the Ceratomyxa clade with high nodal support. Considering the morphological, morphometric, molecular, and phylogenetic dissimilarities with the previously described species of Ceratomyxa and the differences in host and geographic locations, the present species of myxosporean is treated as new and is named Ceratomyxa xanthopteri n. sp.

A novel myxozoan parasite, Ellipsomyxa boleophthalmi sp. nov. (Myxozoa: Ceratomyxidae) in the brackishwater fish, Boleophthalmus dussumieri Valenciennes, 1837 (Perciformes: Gobiidae) from India.

A novel myxozoan parasite is identified and described from mudskipper, Boleophthalmus dussumieri, collected from a brackishwater ecosystem in Maharashtra, India. Ellipsomyxa boleophthalmi sp. nov. was found in the gallbladder of 58 of 60 fish examined (96.7%). The parasite formed disporous plasmodia that varied in size and shape, and the thin-walled, ellipsoidal and elongated myxospores measured 9.0-10.7 × 6.0-7.8 µm. The two, spherical polar capsules measured 2.7 µm in diameter and enclosed 3-4 coils of polar tubules. Histological observations of infected gallbladder revealed the attachment of disporous plasmodial stages of the parasite to the gallbladder wall with fine pseudopodia. Under the scanning electron microscope (SEM), the myxospores showed a distinct central sutural line and two distinct depressions on the opposite sides at the openings of polar capsules. SEM also revealed the engulfment of microvilli of gallbladder wall by pseudopodia of the plasmodial stages. Analysis of the partial fragment of the SSU rDNA region (1386 bp) showed less than 98% sequence similarity with the other reported Ellipsomyxa spp. In the phylogenetic tree, the present species formed as a distinct subclade within the major clade of Ellipsomyxa spp. The unique morphological and morphometric features of the myxospore, together with the molecular analysis, allowed us to conclude that the present myxozoan is a new species and is named Ellipsomyxa boleophthalmi sp. nov., after the generic name of the host. This is the first report on the occurrence of the genus Ellipsomyxa in B. dussumieri.

Zschokkella epinepheli n. sp. (Myxosporea: Myxidiidae) infecting the gallbladder of the white grouper Epinephelus aeneus (Serranidae) from Tunisian waters.

A new coelozoic myxosporean species, Zschokkella epinepheli n. sp., collected from the gallbladder of the white grouper Epinephelus aeneus (Perciformes: Serranidae) from the bay of Bizerte, Tunisia, is described based on morphological and molecular characteristics. Myxospores and plasmodia were observed floating free in the bile. Mature plasmodia were polysporic and subspherical in shape, measuring 85.0-94.0 µm long and 70.0-82.0 µm wide. Mature myxospores were ovoid in valvular view, measuring 10.0 ± 1.7 (8.0-11.0) µm in length and 7.0 ± 0.3 (6.6-7.5) µm in width. Polar capsules were pyriform and equal in size, measuring 3.0 ± 0.2 (2.8-3.6) µm in length and 2.3 ± 0.3 (1.8-2.7) µm in width. Myxospore valves had 12-14 longitudinal striations. Based on the small subunit rDNA, the new species Z. epinepheli n. sp. differs from all other Zschokkella species for which there is a DNA sequence deposited in GenBank. Phylogenetic analysis revealed that Z. epinepheli n. sp. clustered in the marine subclade of Zschokkella species within the biliary tract IV clade. This is the first report of a Zschokkella species from the gallbladder of an epinephelin fishes.

Pitfalls in Morphologic Diagnosis of Pathogens: Lessons Learned From the Pseudo-Cystoisospora Epidemic.

Multiple groups have recently reported involvement of the gallbladder mucosa of immunocompetent patients by cystoisospora organisms. However, this has recently been disproved with the support of molecular and ultrastructural studies. Here we present a summary of these events, recounting how this pseudo-Cystoisospora epidemic began and ended. This review also highlights the important role played by ancillary techniques in supplementing the morphologic diagnosis of pathogens.

The first report of two Sphaeromyxa species (Myxozoa: Bivalvulida) from the South China Sea: Sphaeromyxa scorpaena n. sp. from long-fingered scorpionfish (Scorpaenodes albaiensis) and Sphaeromyxa theraponi from tiger perch (Terapon jarbua).

Two myxosporean species of the genus Sphaeromyxa were isolated from the gallbladders of marine fish in the South China Sea. Sphaeromyxa scorpaena n. sp. was collected from Scorpaenodes albaiensis Evermann and Seale, 1907. The mature myxospores were arcuate-shaped with tapered to pointed ends, and a length of 14.1 ± 0.7 (13.8-15.1) µm and a width of 5.2 ± 0.3 (4.9-5.8) µm. The polar capsules (PCs) were pyriform with a length of 3.2 ± 0.2 (3.1-3.5) µm and a width of 1.6 ± 0.1 (1.4-1.8) µm, and containing ribbon-like polar filaments irregularly folded 1.5-2.5 turns. Molecular characteristics and phylogenetic analysis based on 18S rDNA as well as morphological comparison confirmed that S. scorpaena n. sp. was a previously undescribed species. Sphaeromyxa theraponi, isolated from Terapon jarbua Forsskål, 1775, was reported for the first time from the South China Sea. The mature myxospores were slightly arched, tapering to bluntly rounded ends, with a length of 17.3 ± 0.9 (15.5-19.4) µm and a width of 4.8 ± 0.3 (4.1-5.3) µm. A sporoplasm was situated in the space between PCs in the myxospore. The PCs were pyriform, which contained ribbon-like polar filaments irregularly folded by 2-3 turns, with a length of 7.0 ± 0.5 (5.8-8.1) µm and a width of 2.6 ± 0.2 (2.2-3.0) µm. Our morphological and phylogenetic analyses suggest that the pointed ends of S. scorpaena n. sp. might be a secondarily acquired characteristic rather than an ancestral trait.

Biliary Microbiota and Bile Acid Composition in Cholelithiasis.

BACKGROUND: A functional interplay between BAs and microbial composition in gut is a well-documented phenomenon. In bile, this phenomenon is far less studied, and with this report, we describe the interactions between the BAs and microbiota in this complex biological matrix. Methodology. Thirty-seven gallstone disease patients of which twenty-one with Opisthorchis felineus infection were enrolled in the study. The bile samples were obtained during laparoscopic cholecystectomy for gallstone disease operative treatment. Common bile acid composition was measured by LC-MS/MS. Gallbladder microbiota were previously analyzed with 16S rRNA gene sequencing on Illumina MiSeq platform. The associations between bile acid composition and microbiota were analyzed. RESULTS: Bile acid signature and Opisthorchis felineus infection status exert influence on beta-diversity of bile microbial community. Direct correlations were found between taurocholic acid, taurochenodeoxycholic acid concentrations, and alpha-diversity of bile microbiota. Taurocholic acid and taurochenodeoxycholic acid both show positive associations with the presence of Chitinophagaceae family, Microbacterium and Lutibacterium genera, and Prevotella intermedia. Also, direct associations were identified for taurocholic acid concentration and the presence of Actinomycetales and Bacteroidales orders, Lautropia genus, Jeotgalicoccus psychrophilus, and Haemophilus parainfluenzae as well as for taurochenodeoxycholic acid and Acetobacteraceae family and Sphingomonas genus. There were no differences in bile acid concentrations between O. felineus-infected and noninfected patients. Conclusions/Significance. Associations between diversity, taxonomic profile of bile microbiota, and bile acid levels were evidenced in patients with cholelithiasis. Increase of taurochenodeoxycholic acid and taurocholic acid concentration correlates with bile microbiota alpha-diversity and appearance of opportunistic pathogens.

Growing diversity supports radiation of an Ellipsomyxa lineage into the Amazon freshwater: Description of two novel species parasitizing fish from Tapajós and Amazon rivers.

Species of the genus Ellipsomyxa Køie, 2003, parasitize mostly marine and brackish fish around the world. In the present study, we describe two novel species of Ellipsomyxa: Ellipsomyxa plagioscioni n. sp. parasitizing the gall bladder of Plagioscion squamosissimus (Sciaenidae), a freshwater fish but commonly found in brackish water in the Amazonian estuarine environment; and Ellipsomyxa paraensis n. sp. infecting Cichla monoculus (Cichlidae), a strictly freshwater fish. The host specimens were caught from the Amazon and Tapajós rivers, in the municipal region of Santarém, in the State of Pará, Brazil. The study was performed using a combination of morphological, biological, and SSU rDNA-based phylogeny, which suggested that marine transgressions of the Miocene epoch, in the central region of South America, were a pathway for the adaptation and radiation of these cnidarian parasites in the freshwater environment. Both disporic plasmodia and mature myxospores were found floating freely in the bile. Mature myxospores from both species were ellipsoidal in the valvular and sutural views, with thin smooth valves elongated in the direction perpendicular to the plane of the transverse sutural line. Ellipsomyxa plagioscioni n. sp. myxospores measured 11.1 (10.2-12.8) µm in length and 6.6 (5.6-7.6) µm in width. Two pyriform polar capsules discharging on opposite sides, some distance from both the sutural line and the spore ends, measured 3.8 (3.2-4.4) µm in length and 2.8 (2.3-3.3) µm in width, with 5-6 coil polar tubules. Ellipsomyxa paraensis n. sp. myxospores measured 11.5 (10.5-12.4) µm in length and 7.5 (6.6-8.6) µm in width. Two pyriform polar capsules which discharged on opposite sides some distance from both the sutural line and spore ends, measured 3.2 (2.1-3.9) µm in length and 2.6 (2.0-3.3) µm in width, with 2-3 coil polar tubules. Valvular protrusions were observed, associated with the tips of the polar capsules. Molecular analysis based on the SSU rDNA sequences indicated that the two novel Ellipsomyxa species were distinct from all other sequences deposited in the GenBank database. The phylogenetic trees clustered E. plagioscioni n. sp. as a basal species of a lineage of the marine/estuarine Ellipsomyxa, while E. paraensis n. sp. clustered together with other Amazonian species.

Morphological and molecular descriptions of Sphaeromyxa sevastopoli (Cnidaria) from host fishes from Sinop on the Black Sea coast.

Members of the genus Sphaeromyxa Thélohan, 1892 have been reported from a wide variety of fish species worldwide. In the present study, specimens of rusty blenny, Parablennius sanguinolentus, collected from Sinop on the Turkish Black Sea coast were investigated for myxosporean parasites by using both conventional and molecular methods. Sphaeromyxa sevastopoli Naidenova 1970 was the only myxosporean parasite found in the gall bladder of host fishes. The morphology peculiarities of obtained S. sevastopoli spores are in good agreement with those of original description and the morphometric data overlapped in spore length and width but differed in polar capsule length and width; however, they were within the ranges previously reported from 18 host fish species. Moreover, in the present study, molecular analysis of the 18S rDNA gene of S. sevastopoli isolate in our P. sanguinolentus as well as isolates from shore rockling Gaidropsarus mediterraneus and knout goby Mesogobius batrachocephalus which were previously morphologically identified and reported by Okkay and Özer (Acta Zool Bulg 72(1):123-130, 2020) was done for the first time and our three S. sevastopoli genotypes were allocated to the "balbianii" group which is characterized by straight or slightly curved and fusiform or ovoid spores with ovoid polar capsules.

Morphology, seasonality and molecular characterization of Ceratomyxa draconis n. sp. parasite of Trachinus draco (L.) from the Bay of Bizerte, Tunisia.

The specimens of Trachinus draco collected from the Bay of Bizerte were found to be infected with a new Ceratomyxa species described as Ceratomyxa draconis n. sp. The sequence of small subunit ribosomal RNA gene obtained in this study differs from other Ceratomyxa sequences available in GenBank. Mature spores of this species were elongated and crescent-shaped in sutural view, measuring 7.4 ± 0.77 (6.4-8.0) µm in thickness and 30.8 ± 1.65 (28.8-32.8) µm in width. The polar capsules were spherical, equal in size, and measuring 3.3 ± 0.2 (3.6-4.0) µm in diameter. The Ceratomyxa draconis n. sp. showed a clearly seasonal variation of prevalence with highest prevalence noted during summer months.

Prosthenhystera gattii n. sp. (Digenea: Callodistomidae), a gallbladder parasite of Bryconamericus ikaa from the lower Iguazú River, described based on combined molecular and morphological evidence.

Adult forms of members of the Callodistomidae always parasitize the gallbladder of freshwater fishes and occur in Africa and America. This study provides a description of a new South American species belonging in Prosthenhystera from the gallbladder of a characid fish (Bryconamericus ikaa), and ribosomal gene sequences (28S rDNA and ITS1-5.8S-ITS2) are used to demonstrate molecular differences between the new species and congeners as well as explore interrelationships among congeners. Additionally, the first cytological analysis is conducted for a member of the family to determine chromosome number and arrangement. Prosthenhystera gattii n. sp. most closely resembles Prosthenhystera caballeroi in morphology, but the vitellarium is more extensive reaching anterior to the caecal bifurcation in the new species and the uterus is confined to the hindbody in P. gattii n. sp., whereas it extends to the level of the pharynx in P. caballeroi. Also, the testes, cirrus sac, seminal receptacle and the ratio of body length to width are larger in P. gattii n. sp. Independent Bayesian inference analyses of 28S rDNA and ITS1-5.8S-ITS2 sequence fragments produced phylograms that showed P. gattii n. sp. is more similar to Prosthenhystera obesa + Prosthenhystera oonastica than P. caballeroi + two unidentified species of Prosthenhystera, but with poor posterior probability support for the node in the ITS1-5.8S-ITS2-based phylogram. Further, the genetic distance between P. oonastica and P. gattii n. sp. are the largest among Prosthenhystera spp. Cytological analysis revealed ten metacentric chromosomes, which is fewer than the 12-18 chromosomes present in species from the closely related Gorgoderidae.