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.

Comparison of droplet digital PCR and real-time quantitative PCR for quantitative detection of the parasitic ciliate Ichthyophthirius multifiliis in the water environment.

Ichthyophthiriasis, caused by the parasitic ciliate Ichthyophthirius multifiliis (Ich), is considered one of the most harmful diseases affecting freshwater fish globally. It can cause mass mortalities of fish in intensive farming systems. In such systems, it is thus necessary to detect and quantify the number of Ich in the water so that control measures can be implemented before Ichthyophthiriasis breaks out. In recent years, molecular diagnostic methods have become increasingly important in aquaculture. Real-time quantitative polymerase chain reaction (qPCR) and droplet digital polymerase chain reaction (ddPCR) have become robust assays for detecting pathogens. In this study, a set of specific primers and a TaqMan-minor groove binder probe targeting the small-subunit rDNA (SSU rDNA) of Ich were developed. They were used in qPCR and ddPCR assays to compare the performance of these two different methods in quantitatively detecting Ich. After optimizing the reaction conditions, both qPCR and ddPCR assays were found to have high linearity and quantitative correlations for standard plasmid DNA. When used for the detection of Ich eDNA in water samples, the qPCR assay had a wider detection range, making it a suitable method to screen for the prevalence of Ichthyophthiriasis. However, the ddPCR approach had higher sensitivity, which would help provide advance notice of the disease in complex water environmental samples.

Population growth of Gyrodactylus kobayashii in goldfish (Carassius auratus) associated with host density.

Host density is a key regulatory factor in parasite transmission. The goldfish (Carassius auratus)-Gyrodactylus kobayashii model was used to investigate effects of host density on population growth of gyrodactylids. A donor fish infected by five gravid gyrodactylids was mixed with 11 parasite-free goldfish at five host densities. There was a significant positive correlation between host density and mean abundance of G. kobayashii throughout the 58-day experiment. During early infection (days 15­24), mean abundance in medium high (0.5 fish L−1) and high host density groups (1 and 2 fish L−1) was significantly higher than that in the low host density groups (0.125 and 0.25 fish L−1). At high host density, prevalence increased more rapidly, and the peak prevalence was higher. Fitting of an exponential growth model showed that the population growth rate of the parasite increased with host density. A hypothesis was proposed that higher host density contributed to increased reinfection of detached gyrodactylids. A reinfection experiment was designed to test this hypothesis. Both mean abundance and prevalence at a host density of 1 fish L−1 were significantly higher than those at 0.25 fish L−1 on days 1 and 3, which suggested that more reinfections of G. kobyashii occurred at the higher host density. Density-dependent transmission during the early infection was an important contributor of population growth of G. kobayashii, as well as density-dependent reinfection of the detached gyrodactylids.

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.

Bacillus licheniformis FA6 Affects Zebrafish Lipid Metabolism through Promoting Acetyl-CoA Synthesis and Inhibiting ß-Oxidation.

The intestinal microbiota contributes to energy metabolism, but the molecular mechanisms involved remain less clear. Bacteria of the genus Bacillus regulate lipid metabolism in the host and are thus commonly used as beneficial probiotic supplements. In the present study, Bacillus licheniformis FA6 was selected to assess its role in modulating lipid metabolism of zebrafish (Danio rerio). Combining 16S rRNA high-throughput sequencing, micro-CT scan, metabolic parameters measurement, and gene expression analysis, we demonstrated that B. licheniformis FA6 changed the gut microbiota composition of zebrafish and increased both the Firmicutes/Bacteroidetes ratio and lipid accumulation. In terms of metabolites, B. licheniformis FA6 appeared to promote acetate production, which increased acetyl-CoA levels and promoted lipid synthesis in the liver. In contrast, addition of B. licheniformis lowered carnitine levels, which in turn reduced fatty acid oxidation in the liver. At a molecular level, B. licheniformis FA6 upregulated key genes regulating de novo fatty acid synthesis and downregulated genes encoding key rate-limiting enzymes of fatty acid ß-oxidation, thereby promoting lipid synthesis and reducing fatty acid oxidation. Generally, our results reveal that B. licheniformis FA6 promotes lipid accumulation in zebrafish through improving lipid synthesis and reducing ß-oxidation.

Identification of Intracellular Bacteria in the Ciliate Balantidium ctenopharyngodoni (Ciliophora, Litostomatea).

The ciliate Balantidium ctenopharyngodoni is the most prominent protist in the guts of grass carp, where it mainly inhabits the creamy luminal contents of the hindgut. Ciliates are generally colonized by microorganisms via phagotrophic feeding. In order to study the intracellular bacteria in this ciliate, we have successfully established it in in vitro culture. Herein, we investigated and compared the bacterial community structures of cultured and freshly collected B. ctenopharyngodoni. The results showed that these two groups exhibited different bacterial communities. The most abundant bacterial family in freshly collected samples was Enterobacteriaceae, while in cultured samples it was Fusobacteriaceae. In addition, a key intracellular bacterium, Cetobacterium somerae, was identified in the cytoplasm of cultured ciliates using fluorescence in situ hybridization (FISH). This study shows that ciliates can retain the intracellular bacteria acquired in the natural habitat for quite a long time, but the bacterial community structure of ciliates eventually changes after a long period of cultivation.

Effects of Bacillus licheniformis on the growth, antioxidant capacity, intestinal barrier and disease resistance of grass carp (Ctenopharyngodon idella).

To study the effect of dietary supplementation of Bacillus licheniformis FA6 on the growth, survival and intestinal health of grass carp, we assessed the antioxidant capacity, intestinal barrier, expression levels of immune genes, and the resistance to Aeromonas hydrophila AH-1 infection. Experimental setup comprised three groups (90 specimens each; average initial weight = 16.5 g): the control group was fed the basal diet without B. licheniformis, the low-dose (LD) group was supplemented with B. licheniformis at the concentration of 1 × 105 cfu/g, and the high-dose (HD) group with 1 × 106 cfu/g. After 56 days of growth trial, the challenge test with A. hydrophila AH-1 was conducted for 14 days. The results revealed that the grass carp in LD group and HD group had significantly (p < 0.05) improved percent weight gain (PWG) and specific growth rate (SGR) parameters. Additionally, the antioxidant status was improved, which included increased superoxide dismutase (SOD) activity and decreased malondialdehyde (MDA) levels in the serum, and upregulated mRNA levels of antioxidant enzymes MnSOD and catalase (CAT) in the intestine. Meanwhile, B. licheniformis FA6 supplementation groups exhibited a decreased mRNA expression of proinflammatory cytokines (such as IL-1ß and TNF-α) and increased the expression of anti-inflammatory cytokine IL-10. Histological (villi length was increased) and gene expression (qPCR: upregulated ZO-1, occludin, and claudin-c) analyses suggested improved functioning of the intestinal barrier. Post-challenge mortality rates in LD and HD groups were significantly lower (56.6% and 70% respectively) than in the control group (100%). Overall, these results indicated that dietary supplementation of B. licheniformis FA6 can improve growth, antioxidant capacity, intestinal barrier functions and disease resistance of grass carp.

Morphological Redescription of Opalina undulata Nie 1932 from Fejervarya limnocharis with Molecular Phylogenetic Study of Opalinids (Heterokonta, Opalinea).

The redescription of Opalina undulata Nie 1932, collected from the rectum of the frog Fejervarya limnocharis, is presented in this paper based on detailed morphological information and molecular data. Our results revealed that specimens collected from Diaocha Lake in late August were larger and had more nuclei than those collected from the same site in early May. We sequenced their SSU rDNA-ITS1-5.8S rDNA-ITS2-LSU rDNA (5' end) and found that they were completely identical, which means that the two populations belonged to the same species. These facts gave us a hint that body dimension and number of nuclei are not reliable taxonomic parameters for opalinids during their life cycle. Therefore, we recommended that the specific identification of opalinids based on morphological features should be carried out during seasons except spring. Meanwhile, our molecular phylogenetic analysis confirmed the monophyly of Opalinata. Within Opalinata, Opalinea were monophyletic with all opalinid species grouping together. Karotomorpha and Proteromonas did not group together confirming the paraphyly of Proteromonadea.

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.

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.

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.

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.

Distribution and influencing factors of antibiotic resistance genes of crayfish (Procambarus clarkii) intestine in main crayfish breeding provinces in China.

The propagation of antibiotic resistance genes (ARGs) has become a global public health concern. However, the distribution and influencing factors of ARGs, especially high-risk ARGs, in the gut of aquaculture animals remain unclear. Here, we employed 16S rRNA gene sequencing and high-throughput quantitative PCR techniques to determine crayfish gut microbiota and ARGs collected from 40 culture ponds in major crayfish farming provinces of China. We detected 74 ARGs in crayfish gut. Among them, the beta-lactamase and tetracycline resistance genes were dominant. The total ARG abundance was the highest in Hubei Province. High-risk ARGs were also found in crayfish gut, and ermB had the highest abundance and distributed in Anhui, Hubei, Henan and Jiangxi Province. In addition, opportunistic pathogens (Streptococcus, Aeromonas and Acinetobacter) might be potential hosts for ARGs, including high-risk ARGs. Finally, habitat, environmental factors (NO3-N, pH and temperature), microbial alpha diversity and mobile genetic elements (MGEs) showed significant influence on ARGs profiles. Generally, our results illustrate that ARGs are prevalent in crayfish gut and may pose potential risk to human health, which will help develop targeted strategies for the risk management and assessment of ARGs in the aquaculture.

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.

Fate of high-risk antibiotic resistance genes in large-scale aquaculture sediments: Geographical differentiation and corresponding drivers.

Antibiotic resistance genes (ARGs), emerging environmental contaminants, have become challenges of public health security. However, the distribution and drivers of ARGs, especially high-risk ARGs, in large-scale aquaculture sediments remain unknown. Here, we collected sediment samples from 40 crayfish ponds in seven main crayfish culture provinces in China and then investigated the distribution and risk of ARGs based on high-throughput sequencing and quantitative PCR techniques. Our results suggested that aquaculture sediment was potential reservoir of ARGs and the abundance of aadA-02 was the highest. High-risk ARG (floR) was also prevalent in the sediment and was the most abundant in Jiangsu Province, where opportunistic pathogens were also enriched. The abundance of floR was positively correlated with different environmental factors, such as total phosphorus in water and total carbon in sediment. In addition, Mycobacterium sp., opportunistic pathogenic bacteria, might be potential host for floR. Furthermore, the potential propagation pathway of ARGs was from sediment to crayfish gut, and Bacteroidetes and Proteobacteria might be the main bacterial groups responsible for the proliferation of ARGs. Generally, our results illustrate that pond sediment may be an ARG reservoir of aquatic animals. Meanwhile, our study helps develop valuable strategies for accessing risks and managing ARGs.

Characterization of bacterial community in the stomach of yellow catfish (Pelteobagrus fulvidraco).

In this study, the traditional culture-based technique and the 16S rDNA sequencing method were used to investigate the characterization of bacterial community in the stomach contents and mucus of yellow catfish (Pelteobagrus fulvidraco). The culture-based technique disclosed that the average bacterial numbers in the gastric contents and mucus were 5.79 × 10(7) cfu/g (cfu: colony forming unit) and 1.89 × 10(5) cfu/g, respectively. Several different bacteria were obtained from gastric contents, including species from genera Bradyrhizobium, Phyllobacterium, Plesiomonas, Hafnia, Edwardsiella, Pseudomonas, and Bacillus. However, only two species were isolated from the gastric mucus, including species from genera Plesiomonas and Aeromonas. Forty-five phylotypes were observed from the 65 positive clones from the stomach contents (library SC); nineteen phylotypes were detected from the 45 clones from the stomach mucus (library SM). Further analyses revealed that the fish stomach harbored characteristic microbiota, where Firmicutes was dominant, followed by Proteobacteria and Bacteroidetes and Fusobacteria. This characterization of bacterial community is markedly different from that of the fish intestine, where Proteobacteria is predominant, followed by Fusobacteria and Firmicutes. Chloroflexi (1.5%) was only found in the library SC, while Actinobacteria (4.4%) was only found in the library SM, suggesting that microbiota of GI contents was quite different from that of GI mucus. In addition, several species of bacteria found in the stomach may be potentially opportunistic pathogens, indicating that fish digestive tract is a reservoir for many nosocomial pathogens.

The Role of Intestinal Microbiota in Regulating the Metabolism of Bile Acids Is Conserved Across Vertebrates.

In mammals, bile acid (BA) concentrations are regulated largely by the gut microbiota, and a study has shown that some metabolic responses to the gut microbiota are conserved between zebrafish and mice. However, it remains unknown whether the influence of specific intestinal microbes on BA metabolism is conserved between higher and lower vertebrates (i.e., mammals and fish). In the present study, Citrobacter freundii GC01 isolated from the grass carp (Ctenopharyngodon idella) intestine was supplemented to the fish and mice feed. We found the changes in the bile acid profile, especially significant changes in secondary BAs in both grass carp and mice fed on C. freundii. Also, lipid metabolism was significantly affected by C. freundii. Analysis of liver transcriptome sequencing data and validation by RT-qPCR revealed that the CYP7A1 gene was significantly up-regulated in both grass carp and mice. In addition, the overexpression of HNF4B from grass carp resulted in a significant increase in the expression level of CYP7A1. Generally, our results suggest that the metabolism of BAs by intestinal microbiota is conserved across vertebrates. Furthermore, specific intestinal bacteria may regulate the bile salt synthesis through CYP7A1 and that HNF4B might be an important regulator of BA metabolism in fish.

Description of Gyrodactylus banmae n. sp. (Monogenea, Gyrodactylidae) parasitic on zebrafish, Danio rerio.

A new species of Gyrodactylus was described on the body surface of zebrafish (Danio rerio) in China. Basing on morphological characteristics and ITS sequence, we identified the parasite as a new member of the Gyrodactylus-wageneri group. Morphologically, Gyrodactylus sp. nov. is greatly similar to "G. zebrae", another species parasitic on zebrafish: both have moderately stout hamulus, marginal hook sickle with a prominent heel and toe, as well as a curved blade. However, distinct haptoral shape differences were detected between these two species. The dorsal bar is straight in Gyrodactylus sp. nov. but strongly curved in "G. zebrae", and the sickle shaft in Gyrodactylus sp. nov. is approximately perpendicular to the base, but in "G. zebrae" it is slanted downwards. The ITS1-5.8S-ITS2 sequence also indicate that Gyrodactylus sp. nov. exhibits the highest similarity to "G. zebrae": 95.7% sequence identity suggests interspecific differentiation. Phylogenetic analysis of the ITS1-ITS2 sequence showed that Gyrodactylus sp. nov. formed a sister clade with "G. zebrae", and exhibited a relatively close phylogenetic relationship with G. kobayashii, G. gurleyi, and G. longoacuminatus, all of which parasitise on goldfish, Carassius auratus. To test the susceptibility of zebrafish and goldfish to the Gyrodactylus sp. nov., ten gyrodactylids were inoculated to the caudal fin of zebrafish and goldfish. The gyrodactylids exhibited the ability to attach themselves to the goldfish, and some gyrodactylids reproduced a few days after the inoculation. On day 9, however, the mean abundance sharply decreased to zero on goldfish and increased to more than 30 on zebrafish. The result suggested that golfish is an unsuitable host for Gyrodactylus sp. nov. Therefore, on the basis of morphology, molecular sequence similarity, and host susceptibility, we conclude that the gyrodactylid found on the zebrafish is a new species, which we named Gyrodactylus banmae.

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.

Sindiplozoon coreius n. sp. (Monogenea: Diplozoidae) from the gills of Coreius guichenoti (Cyprinidae) in China.

Sindiplozoon coreius n. sp. is described from the gills of Coreius guichenoti in Sichuan province, China. There is a smooth tegument and a cup-like widened area in the posterior part of the worm body, which are particular features of the genus Sindiplozoon. There are no branched intestinal caeca before the widened area, but some branches reach the fourth clamp in the hind body; there was no cross striation on the anterior arch of the anterior clamp jaw and medial part of the posterior jaw, which are distinguished from the other species in Sindiplozoon. In addition, S. coreius n. sp. shared the highest ITS2 sequence identity (96.0%) with S. ctenopharyngodoni. The established phylogenetic tree showed that the two species of Sindiplozoon formed a sister group. The k2p genetic distance between the new species and other diplozoids was higher than 3.4%, which suggested interspecific differentiation.