Genome assembly of a symbiotic balantidia (Balantidium ctenopharyngodoni) in fish hindgut.

Balantidium ctenopharyngodoni is identified as the sole ciliate species that exclusively resides within the hindgut of grass carp with high prevalence and intensity. In this study, the successful cultivation of B. ctenopharyngodoni enabled us to collect enough cells for genome sequencing. Consequently, we acquired a high-quality genome assembly spanning 68.66 Mb, encompassing a total of 22,334 nanochromosomes. Furthermore, we predicted 29,348 protein-coding genes, and 95.5% of them was supported by the RNA-seq data. The trend of GC content in the subtelomeric regions of single-gene chromosomes was similar to other ciliates containing nanochromosomes. A large number of genes encoding carbohydrate-binding modules with affinities for starch and peptidoglycans was identified. The identification of mitochondrion-related organelles (MROs) within genome indicates its well-suited adaptation to the anaerobic conditions in the hindgut environment. In summary, our results will offer resources for understanding the genetic basis and molecular adaptations of balantidia to hindgut of herbivorous fish.

Immune response and apoptosis of gibel carp (Carassius auratus gibelio) gills to Chilodonella hexasticha infection: Insights from histopathological and multi-omics analyses.

Gibel carp (Carassius auratus gibelio) is an important economically farmed fish in China. Chilodonella hexasticha parasitizes on the gills and fins of host fish, causing disruption to their normal respiration and movement, ultimately resulting in death of the fish. In this study, a combination of histopathological, immunohistochemical, transferase dUTP nick end labeling (TUNEL), multi-omics, and molecular approaches were employed to identify the immune reaction and cell apoptosis in gill tissue in response to C. hexasticha infection. Significant lamellae fusion, hyperplasia, hyperemia, necrosis, and desquamation of infected gibel carp gills were observed. In total, the expression of 3619 genes was higher, and 3143 lower, for gills in the infected group compared to the control group. Furthermore, 76 metabolites were significantly increased and 105 were significantly decreased in the infected group compared with the control group. From the qRT-PCR analysis results, immune system-related genes encoding IL-8, CXCL8a, and CXC11 were significantly up-regulated in infected gibel carp, while ZAP70 was significantly down-regulated. Immunohistochemical results also showed the down-regulated ZAP70 in the infected group. Apoptosis-related genes encoding CASP3 and Mcl-1b were up-regulated in response to C. hexasticha infection. These genes indicate the activation of CASP family-related apoptosis and Bim-mediated mitochondrial apoptotic pathways. TUNEL assays also revealed severe apoptosis in the infected group. Based on this study's results, it can be concluded that C. hexasticha infection leads to histopathological changes in the gills of infected fish, and induces both a significant immune response and apoptosis.

Investigation of Ichthyophthirius multifiliis infection in fish from natural water bodies in the Lhasa and Nagqu regions of Tibet.

This study aimed to examine the prevalence of Ichthyophthirius multifiliis in fish inhabiting natural water bodies in the Lhasa and Nagqu regions of Tibet in September 2020 and August 2021. The results showed that Schizopygopsis selincuoensis had the highest prevalence of I. multifiliis at 33.73% (56/166), followed by Triplophysa tibetana at 30.00% (6/20), Triplophysa brevicauda at 27.91% (12/43) and Schizopygopsis thermalis at 23.66% (31/131). No infection with I. multifiliis was observed in exotic fish species. In addition, the prevalence of I. multifiliis in Boqu Zangbo (river), Selincuo Lake and Cuona Lake in the Nagqu region was found to be significantly higher than that in Lalu Wetland and Chabalang Wetland in the Lhasa region (P < 0.05). The study revealed a significantly lower prevalence in Lhasa River than in Cuona Lake (P < 0.05). Notably, our findings revealed instances of I. multifiliis infections even in saline water bodies, thereby emphasizing the potential threat that this parasite poses to the preservation of indigenous fish resources in Tibet. Consequently, immediate and effective countermeasures are imperative. This study represents the first systematic investigation of I. multifiliis infection in natural water bodies in Tibet.

Interactions between Balantidium ctenopharyngodoni and microbiota reveal its low pathogenicity in the hindgut of grass carp.

BACKGROUND: Hosts, parasites, and microbiota interact with each other, forming a complex ecosystem. Alterations to the microbial structure have been observed in various enteric parasitic infections (e.g. parasitic protists and helminths). Interestingly, some parasites are associated with healthy gut microbiota linked to the intestinal eubiosis state. So the changes in bacteria and metabolites induced by parasite infection may offer benefits to the host, including protection from other parasitesand promotion of intestinal health. The only ciliate known to inhabit the hindgut of grass carp, Balantidium ctenopharyngodoni, does not cause obvious damage to the intestinal mucosa. To date, its impact on intestinal microbiota composition remains unknown. In this study, we investigated the microbial composition in the hindgut of grass carp infected with B. ctenopharyngodoni, as well as the changes of metabolites in intestinal contents resulting from infection. RESULTS: Colonization by B. ctenopharyngodoni was associated with an increase in bacterial diversity, a higher relative abundance of Clostridium, and a lower abundance of Enterobacteriaceae. The family Aeromonadaceae and the genus Citrobacter had significantly lower relative abundance in infected fish. Additionally, grass carp infected with B. ctenopharyngodoni exhibited a significant increase in creatine content in the hindgut. This suggested that the presence of B. ctenopharyngodoni may improve intestinal health through changes in microbiota and metabolites. CONCLUSIONS: We found that grass carp infected with B. ctenopharyngodoni exhibit a healthy microbiota with an increased bacterial diversity. The results suggested that B. ctenopharyngodoni reshaped the composition of hindgut microbiota similarly to other protists with low pathogenicity. The shifts in the microbiota and metabolites during the colonization and proliferation of B. ctenopharyngodoni indicated that it may provide positive effects in the hindgut of grass carp.

Alterations of gut microbiota and short-chain fatty acids induced by Balantidium polyvacuolum in the hindgut of Xenocyprinae fishes providing new insights into the relationship among protozoa, gut microbiota and host.

Introduction: Parasitic ciliates are protozoans with a global distribution. Along with the gut microbiota, they have formed a micro-ecosystem that affects the host's nutrition, metabolism, and immunity. The interactions and relationships among the three components of this microecosystem (protozoa, gut microbiota, and host) remain only partially understood. Xenocypris fish and the unique ciliate Balantidium polyvacuolum in its hindgut are good materials to study the interplay. Methods: In this study, 16S rRNA gene amplicon sequencing and short-chain fatty acids (SCFAs) identification were used. Network was also constructed to understand their relationships. Results: We found that the gut microbiota of B. polyvacuolum-infected X. davidi and X. argentea had higher diversity, richness, and evenness than uninfected ones. B. polyvacuolum could lead to an increase of Fusobacterium and Chloroflexi in both X. davidi and X. argentea, while significantly increase the abundance of genera Romboutsia and Clostridium in X. argentea. Besides, B. polyvacuolum could significantly increase the content of total SCFAs and acetic acid in X. davidi and increase the concentrations of propionic, isobutyric and butanoic acids in X. argentea. Furthermore, correlation analyses showed that B. polyvacuolum may alter SCFAs by affecting key SCFAs-producing bacteria such as Clostridium and Cetobacterium. Discussion: This study greatly expands our understanding of relationships among B. polyvacuolum, gut microbiota and host Xenocypris fish, which sheds new insights into the mechanism of interaction among protozoa, gut microbiota and host.

The energy metabolism of Balantidium polyvacuolum inhabiting the hindgut of Xenocypris davidi.

Anaerobic parasitic ciliates are a specialized group of ciliates that are adapted to anoxic and oxygen-depleted habitats. Among them, Balantidium polyvacuolum, which inhabits the hindgut of Xenocyprinae fishes, has received very limited scientific attention, so the molecular mechanism of its adaptation to the digestive tract microenvironment is still unclear. In this study, transmission electron microscopy (TEM) and single-cell transcriptome analysis were used to uncover the metabolism of B. polyvacuolum. Starch granules, endosymbiotic bacteria, and multiple specialized mitochondrion-related organelles (MROs) of various shapes were observed. The MROs may have completely lost the electron transport chain (ETC) complexes I, III, IV, and V and only retained succinate dehydrogenase subunit A (SDHA) of complex II. The tricarboxylic acid (TCA) cycle was also incomplete. It can be inferred that the hypoxic intestinal environment has led to the specialization of the mitochondria in B. polyvacuolum. Moreover, carbohydrate-active enzymes (CAZymes), including carbohydrate esterases, enzymes with a carbohydrate-binding module, glycoside hydrolases, and glycosyltransferases, were identified, which may constitute evidence that B. polyvacuolum is able to digest carbohydrates and starch. These findings can improve our knowledge of the energy metabolism and adaptive mechanisms of B. polyvacuolum.

Comparative Transcriptomics of Chilodonella hexasticha and C. uncinata Provide New Insights into Adaptations to a Parasitic Lifestyle and Mdivi-1 as a Potential Agent for Chilodonellosis Control.

Chilodonella hexasticha is a harmful parasitic ciliate that can cause severe damage to fish and high mortalities worldwide. Its congeneric species, C. uncinata, is a facultative parasite that not only can be free-living but also can parasitize on fish gills and fins. In this study, single-cell transcriptomes of these two species were assembled and characterized. Numerous enzymes related to energy metabolism and parasitic adaption were identified through annotation in the Non-Redundant (NR), Clusters of Orthologous Genes (COG), Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. The expression of isocitrate dehydrogenase (IDH), cytochrome c oxidase subunit 1 (Cox1) and ATP synthase F1, delta subunit (ATP5D) was up-regulated in C. hexasticha compared with C. uncinata. The oxidative phosphorylation process was also enriched in C. hexasticha. The main mitochondrial metabolic pathways in C. hexasticha were depicted and enzymes related to energy metabolism pathways were compared between these two species. More importantly, mitochondrial division inhibitor 1 (mdivi-1) proved to be very effective in killing both C. hexasticha and C. uncinata, which could be a novel drug for Chilodonellosis control. This study can help us better understand the energy metabolisms of C. hexasticha and C. uncinata and provide new insight into novel targets for chilodonellosis control. Meanwhile, the transcriptome data can also facilitate genomic studies of these two species in the future.

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.

The Genome of the Mitochondrion-Related Organelle in Cepedea longa, a Large Endosymbiotic Opalinid Inhabiting the Recta of Frogs.

Mitochondrion-related organelles (MROs) are loosely defined as degenerated mitochondria in anaerobic and microaerophilic lineages. Opalinids are commonly regarded as commensals in the guts of cold-blooded amphibians. It may represent an intermediate adaptation stage between the conventional aerobic mitochondria and derived anaerobic MROs. In the present study, we sequenced and analyzed the MRO genome of Cepedea longa. It has a linear MRO genome with large inverted repeat gene regions at both ends. Compared to Blastocystis and Proteromonas lacertae, the MRO genome of C. longa has a higher G + C content and repeat sequences near the central region. Although three Opalinata species have different morphological characteristics, phylogenetic analyses based on eight concatenated nad genes indicate that they are close relatives. The phylogenetic analysis showed that C. longa clustered with P. lacertae with strong support. The 18S rRNA gene-based phylogeny resolved the Opalinea clade as a sister clade to Karotomorpha, which then further grouped with Proteromonas. The paraphyly of Proteromonadea needs to be verified due to the lack of MRO genomes for key species, such as Karotomorpha, Opalina and Protoopalina. Besides, our dataset and analyses offered slight support for the paraphyly of Bigyra.

Transcriptomic Differences between Free-Living and Parasitic Chilodonella uncinata (Alveolata, Ciliophora).

Chilodonella uncinata is a facultatively parasitic ciliate, which can opportunistically parasitize on fish gills and fins, and sometimes even cause host mortality. Previous molecular studies of C. uncinata mainly focused on genetic diversity and molecular evolution. There are currently no transcriptome reports studying differences between free-living and parasitic C. uncinata. We addressed this by sequencing transcriptomes of these two C. uncinata lifestyle types using Smart-seq2 and Illumina HiSeq technologies. In total, 1040 differentially expressed genes (DEGs) were identified. Compared with the free-living type, 494 genes of the parasitic type were downregulated and 546 genes were upregulated. These DEGs were identified through BLAST with NCBI-nr, Swiss-Port, and Pfam databases and then annotated by GO enrichment and KEGG pathway analysis. The results showed that parasitism-related genes such as heat shock proteins (HSPs), actin I, and leishmanolysin were significantly upregulated in parasitic C. uncinata. The ciliary-related dynein heavy chain also had a higher expression in parasitic C. uncinata. Furthermore, there were significant differences in the amino acid metabolism, fatty acid metabolism, lipid metabolism, and TCA cycle. This study increases the volume of molecular data available for C. uncinata and contributes to our understanding of the mechanisms underlying the transition from a free-living to a parasitic lifestyle.