Mitochondrial metabolism of the facultative parasite Chilodonella uncinata (Alveolata, Ciliophora).

BACKGROUND: Chilodonella uncinata is an aerobic ciliate capable of switching between being free-living and parasitic on fish fins and gills, causing tissue damage and host mortality. It is widely used as a model organism for genetic studies, but its mitochondrial metabolism has never been studied. Therefore, we aimed to describe the morphological features and metabolic characteristics of its mitochondria. METHODS: Fluorescence staining and transmission electron microscopy (TEM) were used to observe the morphology of mitochondria. Single-cell transcriptome data of C. uncinata were annotated by the Clusters of Orthologous Genes (COG) database. Meanwhile, the metabolic pathways were constructed based on the transcriptomes. The phylogenetic analysis was also made based on the sequenced cytochrome c oxidase subunit 1 (COX1) gene. RESULTS: Mitochondria were stained red using Mito-tracker Red staining and were stained slightly blue by DAPI dye. The cristae and double membrane structures of the mitochondria were observed by TEM. Besides, many lipid droplets were evenly distributed around the macronucleus. A total of 2594 unigenes were assigned to 23 functional classifications of COG. Mitochondrial metabolic pathways were depicted. The mitochondria contained enzymes for the complete tricarboxylic acid (TCA) cycle, fatty acid metabolism, amino acid metabolism, and cytochrome-based electron transport chain (ETC), but only partial enzymes involved in the iron-sulfur clusters (ISCs). CONCLUSIONS: Our results showed that C. uncinata possess typical mitochondria. Stored lipid droplets inside mitochondria may be the energy storage of C. uncinata that helps its transmission from a free-living to a parasitic lifestyle. These findings also have improved our knowledge of the mitochondrial metabolism of C. uncinata and increased the volume of molecular data for future studies of this facultative parasite.

Morphological and molecular characterization of a new ciliate Nyctotheroides grimi n. sp. (Armophorea, Clevelandellida) from Chinese frogs.

A new species of clevelandellid ciliate, Nyctotheroides grimi n. sp., is described from the frog Fejervarya limnocharis. Light and scanning electron microscopy were used for the morphological studies, and the DNA encoding the SSU rRNA gene (SSU rDNA) and the ITS1-5.8S subunit rRNA-ITS2 region (ITS) were sequenced for genetic comparisons and phylogenetic analysis. The main distinctive morphological feature is a knob-like projection in the left-posterior end; other differential characters are the cell size, the length of the oral groove and the shape of the infundibulum. Nyctotheroides grimi possess an apical suture line in the left and right side of the anterior end and in the left side of the caudal end. In the phylogenetic analyses, the new species engroups with other Nyctotheroides species forming a monophyletic group. The high similarity in the SSU rDNA and ITS sequences between Nyctotheroides species suggests a relative recent divergence. The genetic data and the different host range support the separation of Nyctotheroides and Nyctotherus; however the morphological criterion based on the presence (in Nyctotheroides)/absence (in Nyctothterus) of an apical kinetal suture line should be modified to consider the presence of kinetal suture lines in the apical and/or the caudal left side in Nyctotheroides.

Redescription of Opalina triangulata (Heterokonta, Opalinea) from Fejervarya limnocharis based on morphological and molecular data.

Opalinids are a large group of anaerobic protists, mainly inhabiting the cloacae of amphibians (frogs and toads). The classification of this group has not been fully resolved, because of a lack of molecular information. Here, we give a redescription of Opalina triangulata Metcalf, 1923, collected from the rectum of the frog Fejervarya limnocharis, based on detailed morphological and molecular data. Our phylogenetic analyses confirmed the monophyly of Opalinata. Within it, Opalinea were monophyletic with O. triangulata and O. undulata as well as two Protoopalina species grouping together. Karotomorpha and Proteromonas did not group together confirming the paraphyly of Proteromonadea. Meanwhile, the ITS2 secondary structural similarities as well as G-C content revealed greater similitudes between Opalina species and P. lacertae than with Blastocystis hominis, which is in accordance with their position as sister clades in the SSU rDNA-based phylogenies.

Epidemiology and identification of two species of Chilodonella affecting farmed fishes in China.

The genus Chilodonella includes free-living ciliated protozoa as well as pathogenic species for freshwater fish, with Chilodonella hexasticha and Chilodonella piscicola being the most important ones. These parasites cause outbreaks with high mortalities among farmed freshwater fishes with great economic losses. There are few reports of these species in China, and their identification has been based mostly on their morphological characteristics. In the present work, the parasites causing five outbreaks occurring in China between 2014 and 2017 have been identified by morphological and genetic analysis. We provide the first records of Ctenopharingodon idella and Siniperca chuatsi as hosts of C. hexasticha, and of Procypris rabaudi and Schizothorax wangchiachii as hosts of C. piscicola. There are no differences in the gross pathological findings produced by C. hexasticha and C. piscicola, consisting in desquamation and necrosis of epithelial cells in the skin and gills and in severe fusion of gill lamellae. However, both species differ in their geographic distribution: C. piscicola was found in farms located at altitudes over 1500 m above sea level and with a water temperature ≤18 °C, while C. hexasticha was found in farms located at altitudes under 50 m above sea level and with a water temperature ≥21 °C. Present results confirm that C. hexasticha and C. piscicola are two different species that can be differenced by their morphology; however, their biological variability may lead to erroneous identifications and the diagnosis should be preferably based in genetic analysis including nuclear LSU rDNA and mitochondrial SSU rDNA sequences.

Dietary Bile Salt Types Influence the Composition of Biliary Bile Acids and Gut Microbiota in Grass Carp.

Lipid metabolism can influence host's health. There is increasing evidence for interplay between two key regulating factors in lipid metabolism: bile acids (BAs) and gut microbiota. However, very little is known about how types of different diet-supplemented bile salts (BS) influence this interaction in vivo. We sought to explore these relationships using grass carp (Ctenopharyngodon idellus), which often suffers functional disorder of liver and gallbladder. We studied fluctuations of BAs in the gall and changes of microbial communities in the gut in response to seven different diets: five different BS, chelating BS agent, and control. The BS comprised two primary BS [sodium taurochololate (TCAS) and sodium taurochenodeoxycholate (TCDCAS)], sodium tauroursodeoxycholate (TUDCAS), and two secondary BS [sodium taurodeoxycholate (TDCAS) and sodium taurolithocholate (TLCAS)]. Supplementation of primary BS caused a more significant fluctuation of biliary BAs than secondary BS, and TCAS caused a more prominent increase than TCDCAS and TUDCAS. For the gut microbiota, primary BS tended to increase their diversity and induce community succession, secondary BS resulted in a higher firmicutes/bacteroidetes ratio, while TUDCAS had no significant effects. Changes of the gut microbiota triggered by different types of BS caused alteration in BAs biotransformation. Two-obesity-associated families, Lachnospiraceae and Ruminococcaceae were positively correlated with biliary cholic acid (CA), taurochenodeoxycholic acid (TCDCA), and deoxycholic acid (DCA). As both primary and secondary BS resulted in increased synthesis of toxic secondary Bas by the gut microbiota, future studies should pay closer attention to gut microbiota when considering BA treatment.

Altered gut microbiota associated with intestinal disease in grass carp (Ctenopharyngodon idellus).

Gut microbiota plays a crucial importance in their host. Disturbance of the microbial structure and function is known to be associated with inflammatory intestinal disorders. Enteritis is a significant cause of high mortality in fish species, including grass carp (Ctenopharyngodon idellus). Study regarding the association between microbial alternations and enteritis in grass carp is still absent. In this study, changes in the gut microbiota of grass carp suffering from enteritis were investigated using NGS-based 16S rRNA sequencing. Six healthy and ten abnormal fish (showing reddening anus, red odiferous fluid accumulating in the abdominal capacity, and flatulence and haemorrhage in the intestine) were collected from a fish farm in Huanggang Fisheries Institute (Hubei, China). Our results revealed that the diversity, structure, and function of gut microbiota were significantly different between diseased and healthy fish (P < 0.05). Particularly, members of the genera Dechloromonas, Methylocaldum, Planctomyces, Rhodobacter, Caulobacter, Flavobacterium, and Pseudomonas were significantly increased in diseased fish compared with that in healthy fish (P < 0.05). Predicted function indicated that microbiota significantly changed the specific metabolic pathways (related to amino acid metabolism, xenobiotics biodegradation and metabolism, and carbohydrate metabolism) in diseased fish (P < 0.05). Taken together, our findings point out the association between changes of the gut microbiota and enteritis in grass carp, which provide basic information useful for diagnoses, prevention, and treatment of intestinal diseases occurring in cultured fish.

Succession and Fermentation Products of Grass Carp (Ctenopharyngodon idellus) Hindgut Microbiota in Response to an Extreme Dietary Shift.

Dietary intake affects the structure and function of microbes in host intestine. However, the succession of gut microbiota in response to changes in macronutrient levels during a long period of time remains insufficiently studied. Here, we determined the succession and metabolic products of intestinal microbiota in grass carp (Ctenopharyngodon idellus) undergoing an abrupt and extreme diet change, from fish meal to Sudan grass (Sorghum sudanense). Grass carp hindgut microbiota responded rapidly to the diet shift, reaching a new equilibrium approximately within 11 days. In comparison to animal-diet samples, Bacteroides, Lachnospiraceae and Erysipelotrichaceae increased significantly while Cetobacterium decreased significantly in plant-diet samples. Cetobacterium was negatively correlated with Bacteroides, Lachnospiraceae and Erysipelotrichaceae, while Bacteroides was positively correlated with Lachnospiraceae. Predicted glycoside hydrolase and polysaccharide lyase genes in Bacteroides and Lachnospiraceae from the Carbohydrate-Active enZymes (CAZy) database might be involved in degradation of the plant cell wall polysaccharides. However, none of these enzymes was detected in the grass carp genome searched against dbCAN database. Additionally, a significant decrease of short chain fatty acids levels in plant-based samples was observed. Generally, our results suggest a rapid adaption of grass carp intestinal microbiota to dietary shift, and that microbiota are likely to play an indispensable role in nutrient turnover and fermentation.

Morphology of Nyctotheroides hubeiensis Li et al. 1998 from Frog Hosts with Molecular Phylogenetic Study of Clevelandellid Ciliates (Armophorea, Clevelandellida).

The morphology of Nyctotheroides hubeiensis (Acta Hydrobiol. Sin. 1998, 22(suppl.):187), collected from the rectum of Phelophylax nigromaculatus, is presented in this paper based on detailed morphological information and molecular data. Our phylogenetic analysis showed that N. hubeiensis fell into the Nyctotheroides clade, which was strongly supported as monophyletic and clustered as basal to the genera Nyctotherus and Clevelandella. Also, the monophyly of the Order Clevelandellida and the affinity of parasitic nyctotherids and free-living metopids were indicated in our work. The origin of clevelandellid ciliates as well as their possible evolutionary history was also discussed here; however, the analysis of more species from other vertebrate hosts (fish, reptiles) should be made before a well-supported conclusion can be drawn.

The complete mitochondrial genome of Gyrodactylus kobayashii (Platyhelminthes: Monogenea).

The complete mitochondrial genome of Gyrodactylus kobayashii was 14 786 bp in length, containing 12 protein-coding genes (lacking Atp8), 22 tRNA genes, two rRNA genes and two major non-coding regions (NC1 and NC2). The overall A + T content of mitochondrial genome was 71.6%. A close relationship between G. kobayashii and the three Gyrodactylus species (G. salaris, G. thymalli and G. derjavinoides) was uncovered in the phylogenetic tree based on amino acid sequences.

Intestinal microbiota of gibel carp (Carassius auratus gibelio) and its origin as revealed by 454 pyrosequencing.

The intestinal microbiota has received increasing attention, as it influences growth, feed conversion, epithelial development, immunity as well as the intrusion of pathogenic microorganisms in the intestinal tract. In this study, pyrosequencing was used to explore the bacterial community of the intestine in gibel carp (Carassius auratus gibelio), and the origin of these microorganisms. The results disclosed great bacterial diversities in the carp intestines and cultured environments. The gibel carp harbored characteristic intestinal microbiota, where Proteobacteria were predominant, followed by Firmicutes. The analysis on the 10 most abundant bacterial operational taxonomic units (OTUs) revealed a majority of Firmicutes in the intestinal content (by decreasing order: Veilonella sp., Lachnospiraceae, Lactobacillales, Streptococcus sp., and Lactobacillus sp.). The second most abundant OTU was Rothia sp. (Actinobacteria). The most likely potential probiotics (Lactobacillus sp., and Bacillus sp.) and opportunists (Aeromonas sp., and Acinetobacter sp.) were not much abundant. Bacterial community comparisons showed that the intestinal community was closely related to that of the sediment, indicating the importance of sediment as source of gut bacteria in gibel carp. However, 37.95 % of the OTUs detected in feed were retrieved in the intestine, suggesting that food may influence markedly the microbiota of gibel carp, and therefore may be exploited for oral administration of probiotics.

Supplementary studies on Henneguya doneci Schulman, 1962 (Myxozoa: Myxosporea) infecting the gill filaments of Carassius auratus gibelio (Bloch) in China: histologic, ultrastructural, and molecular data.

Henneguya doneci Schulman, 1962 was collected from the gill filaments of Carassius auratus gibelio (Bloch) in Hubei Province, China. The plasmodia located on the surface of the gill arches deformed the neighboring gill filaments. The size of the plasmodia ranged from 0.6 to 4.5 mm in diameter in different months. The myxospores in the plasmodia measured 10.1 (9.2-11.5) µm long × 8.0 (7.5-8.5) µm wide × 7.5 (7-8) µm thick, with two equal capsules at 4.7 (4-5.5) µm long × 3.3 (2.5-4) µm wide, and two caudal processes 32.7 (24-38.5) µm long, respectively. Polar filaments were coiled 5-6 turns. Ultrastructural observation of the plasmodia showing the capsulogenesis of H. doneci is described briefly. The external tubule initially invaginated into the polar capsule. The rudimentary polar filaments were observed to undergo a series of considerable modification, finally developing into mature polar filaments. Molecular analysis demonstrated that although the myxosporean species were collected from different tissues of hosts in various geographic locations, they clustered with the Cyprinidae-infecting myxosporean species in the phylogenetic tree.

Khawia saurogobii N. Sp. (Cestoda: Caryophyllidea) from freshwater fish Saurogobio Spp. (Cyprinidae) in China.

Khawia saurogobii n. sp. is described from the intestine of the cyprinid fishes Saurogobio dabryi and Saurogobio dumerili in China. The new species belongs to the Lytocestidae and is placed in Khawia because of its afossate scolex, vitelline follicles in the cortical parenchyma, uterus not looping anterior to the cirrus sac, gonopores separate but close together in distinct genital atrium, external seminal vesicle absent, and postovarian follicles present. The new species differs from other congeneric species by the shape of the body markedly tapering posteriorly from the end of the anterior third, the shape of the scolex that is very short and markedly wider than the neck, spatulate without incisions, but shallow superficial grooves, vitelline follicles and testes starting immediately posterior to the scolex, and an ovary with long, posterior arms bent medially, thus giving the shape of an inverted A.

Development of Eustrongylides ignotus (Nematoda: Dioctophmida) in domestic ducks (Anas platyrhynchos domestica (L.)).

Investigating the development of Eustrongylides ignotus in its definitive host would enable us to trace the complete life cycle of this nematode. Fourth-stage larvae isolated from naturally infected swamp eels (Monopterus albus) were used to infect domestic ducks (Anas platyrhynchos domestica [L.]). We observed that male and female worms exhibited different developmental patterns in host ducks. In males, the fourth molt occurred at day 1-2 post-infection (PI), after which they attained maturity on day 4 PI and died between day 7 and 9 PI. However, females underwent the fourth molt at day 2-4 PI, produced eggs from day 9 to 17 PI, and then degenerated and died. When compared to fourth-stage female larvae, adult females demonstrated a considerable increase in total body size with a 151% increase in average body width and a 17% increase in average body length. However, the increase in size of the male larvae was not as significant as that in females. The average body width in adult males exhibited only a 45% increase over that in the larval stage.

Molecular characteristics of Camallanus Spp. (Spirurida: Camallanidae) in fishes from China based on its rDNA sequences.

In the paper, we explored the intra- and interspecific evolutionary variation among species of Camallanus collected from different fish species in various regions of China. We determined the internal transcribed spacers of ribosomal DNA (ITS rDNA) sequences of these nematodes. The divergence (uncorrected p-distance) of ITS1, ITS2, and ITS rDNA data sets confirmed 2 valid species of Camallanus in China, i.e., C. cotti and C. hypophthalmichthys. The 2 species were distinguished not only by their different morphologies and host ranges but also by a tetranucleotide microsatellite (TTGC)n present in the ITS1 region of C. cotti. Phylogenetic analyses of the nematodes disclosed 2 main clades, corresponding to different individuals of C. cotti and C. hypophthalmichthys from different fish species in various geographical locations, although the interior nodes of each clade received poor support.