Histology, fatty acid composition, antioxidant and glycolipid metabolism, and transcriptome analyses of the acute cold stress response in Phoxinus lagowskii.

Water temperature is a crucial environmental factor that significantly affects the physiological and biochemical processes of fish. Due to the occurrence of cold events in aquaculture, it is imperative to investigate how fish respond to cold stress. This study aims to uncover the mechanisms responds to acute cold stress by conducting a comprehensive analysis of the histomorphology, glycolipid metabolic and antioxidant enzymes, fatty acid composition and transcriptome at three temperatures (16 °C, 10 °C and 4 °C) in Phoxinus lagowskii. Our results showed that cold stress not damaged muscle microstructure but caused autophagy (at 10 °C). In addition, serum glucose (Glu) and triglycerides (TG) increased during cold stress. The activities of reactive oxygen species (ROS), superoxide dismutase (SOD), catalase (CAT), fructose phosphokinase (PFK), hexokinase (HK), pyruvate kinase (PK), and malondialdehyde (MDA) content in muscle were measured and analyzed. During cold stress, superoxide dismutase and catalase activities increased, reactive oxygen species content decreased. No significant difference in Glutathione peroxidase (GPx) activity, malondialdehyde and total cholesterol (T-CHO) contents among groups. Phosphokinase and pyruvate kinase activities decreased, and HK activity increased during cold stress. Our study resulted in the identification of a total of 25,400 genes, with 2524 genes showing differential expression across different temperature treatments. Furthermore, KEGG pathway indicated that some pathways upregulated during light cold stress (at 10 °C, including autophagy, and AMP-activated protein kinase (AMPK) signaling pathway. Additionally, circadian rhythm is among the most enriched pathways in genes up-regulated during severe cold stress (at 4 °C). Our findings offer valuable insights into how cold-water fish respond to cold stress.

Transcriptome, histological, and physiological responses of Amur sleeper (Perccottus glenii) during cold stress, freezing, and recovery.

Freeze tolerance is a survival strategy employed by some ectotherms living in extremely cold environments. Some fish in extremely cold areas can recover from their frozen state, but they also have to endure cold stress. Amur sleeper (Perccottus glenii) can recover from a completely frozen state. To explore the response of freeze-resistant fish to low temperatures, we analyzed histological alterations, and antioxidant and carbohydrate-lipid metabolizing enzymes of P. glenii under low temperatures. So far, sensory genes regulating P. glenii during cold stress, freezing, and recovery have not been identified. Ultrastructure results indicated that glycogen content and mitochondrial ridge decreased during cold stress and freezing, whereas the number of endoplasmic reticulum increased during recovery. Plasma glucose and glycerol levels of the three treatment groups significantly increased. Lactate dehydrogenase and pyruvate kinase levels significantly increased during cold stress and freezing, and hexokinase levels significantly increased during cold stress. In total, 30,560 unigenes were found (average length 1724 bp, N50 2843 bp). In addition, 7370 differentially expressed genes (DEGs; including 2938 upregulated genes and 4432 downregulated genes) were identified. KEGG analysis revealed that the DEGs were enriched in carbohydrate and lipid metabolism, lipid synthesis, immune system, and anti-apoptosis. Genes involved in glycolysis and phospholipid metabolism were significantly upregulated during cold stress; genes related to circadian rhythm, oxidative phosphorylation, and lipid synthesis were significantly upregulated during freezing; and genes involved in the immune system and anti-apoptosis were significantly upregulated during recovery. Our results attempt to offer new insights into the physiological mechanisms of complex adaptation in P. glenii and provide useful information for future studies on the mechanism underlying freezing/recovery in animals.

Cloning, tissue distribution, and effects of different circadian rhythms on the mRNA expression levels of circadian clock genes Per1a and Per1b in Phoxinus lagowskii.

This study describes the cloning and characterization of Period 1a and Period 1b genes and the analysis of their mRNA and protein expression in Amur minnow (Phoxinus lagowskii) after exposure to different light cycles. The full-length P. lagowskii Per1a and Per1b genes encode proteins consisting of 1393 and 1409 amino acids, and share high homology with the per1 genes of other freshwater fish species. The Per1a and Per1b genes were widely expressed within the brain, eye, and peripheral tissues. The acrophase of the Per1a gene in the pituitary gland occurred during the dark phase at ZT15 (zeitgeber time 15, 12 L: 12 D) and ZT18 (8 L, 16 D), whereas the acrophase of the Per1b gene in the pituitary gland was observed during the light phase. Our study suggests that the expression of Per1a and Per1b in P. lagowskii varied depending on differences in circadian rhythm patterns. The results of our dual-luciferase reporter assays demonstrated that the P. lagowskii Per1b gene enhances the activation of NF-κB. This study is the first to examine the circadian clock gene Per1a and Per1b in the high-latitude fish P. lagowskii, offering valuable insights into the effects of different light periods on this fish species.

Small-molecular cyclic peptide exerts viability suppression effects on HepG2 cells via triggering p53 apoptotic pathways.

Cyclic peptides have become an attractive modality for drug development due to their high specificity, metabolic stability and higher cell permeability. In an effort to explore novel antitumor compounds based on natural cyclopeptide from the phakellistatin family, we found an isoindolinone-containing analog (S-PK6) of phakellistatin 6 capable of suppressing the viability and proliferation of HepG2 cells. The aim of the present study is to shed light on the mechanism of action of this novel compound. We have detected differences in gene expression before and after treatment with S-PK6 in human hepatocellular carcinoma HepG2 cell line by transcriptome sequencing. To further investigate biological effects, we have also extensively investigated the tumor cell cycle, mitochondrial membrane potential, and intracellular Ca2+ concentration after S-PK6 treatment. Based on the finding that the apoptosis was associated with the p53 signaling pathway and MAPK signaling pathway, western blotting tests were used to assess the expression level of p53 protein and its degenerative regulator MDM2 protein, which showed that S-PK6 could increase p53 levels efficiently. In summary, our results demonstrate the mechanism of action of a small-molecule cyclopeptide, which could be very useful for examining of the possible mechanisms of natural cyclopeptides.

The effects of sustained and diel-cycling hypoxia on high-latitude fish Phoxinus lagowskii.

High-latitude fish are subjected to sustained and diel-cycling hypoxia. Oxygen deficiency could pose a serious threat to fish, but little information is available regarding the response mechanisms employed by high-latitude fish to sustained and diel-cycling hypoxia. In this study, a combination of transcriptomics and metabolomics were used to examine the molecular response mechanisms actioned by sustained and diel-cycling hypoxia in the high-latitude fish, Phoxinus lagowskii. P. lagowskii was divided into normoxic control (6.0-7.0 mg/L dissolved oxygen), sustained (1.5 mg/L dissolved oxygen), and diel-cycling hypoxic treatment (6.0-7.0 mg/L between 07:00-21:00, and 3.0-4.0 mg/L between 21:00-07:00) tanks for 28 days. Differentially expressed genes (DEGs) and significantly different metabolites (DMs) related to digestive proteases, lipid metabolism, estrogen signaling pathway, steroid hormone biosynthesis, glutathione metabolism, and tryptophan metabolism were identified from comparative metabolomic and transcriptomic data expression profiles within the liver. The current study found that P. lagowskii had significantly different responses between sustained and diel-cycling hypoxia. P. lagowskii faced with sustained hypoxia may enhance their tolerance capacity through phospholipid and glutathione metabolism. Our data provide new insights into the high latitude fish coping with changes in hypoxia and warrants further investigation into these potentially important genes and metabolites.

Molecular cloning, characterization and expression analysis of P53 from high latitude fish Phoxinus lagowskii and its response to hypoxia.

As an intermediate link between multiple cellular stresses and cellular responses, p53, together with its upstream and downstream regulators and related genes, constitutes a complex network that regulates cellular stresses and cellular responses. However, no studies have investigated p53 in Phoxinus lagowskii, particularly the expression of p53 under different hypoxic conditions. In the present study, the cDNA of p53 from the Phoxinus lagowskii was cloned by the combination of homology cloning and rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of Pl-p53 was 1878 bp, including an open reading frame (ORF) of 1116 bp encoding a polypeptide of 371 amino acids with a predicted molecular weight of 41.22 kDa and a theoretical isoelectric point of 7.38. Quantitative real-time (qRT) PCR assays revealed that Pl-p53 was commonly expressed in all tissues examined, with highest expression in the heart. In addition, we investigated the expression of Pl-p53 in different tissues under different hypoxic conditions. In the short-term hypoxia group, Pl-p53 expression was down-regulated in both the brain and heart. The Pl-p53 expression was significantly elevated at 6 h in the muscle and liver, and was significantly up-regulated at 24 h in spleen. These results suggest that Pl-p53 plays different regulatory roles and provide a theoretical basis for the changes of p53 in fish facing hypoxic environments.

A Multifunctional Nanoplatform Based on Fenton-like and Russell Reactions of Cu, Mn Bimetallic Ions Synergistically Enhanced ROS Stress for Improved Chemodynamic Therapy.

Chemodynamic therapy (CDT) based intracellular chemical reactions to produce highly cytotoxic reactive oxygen species has received wide attention. However, low efficiency of single CDT in weakly acidic pH and glutathione (GSH) overexpressed tumor cells has limited its clinical application. For this study were prepared two-dimensional metal-organic framework (MOF) to improve CDT efficiency based on the combined action of bimetallic CDT, consumption of overexpressed glutathione (GSH) in cells, folic acid (FA) induced tumor targeting and triphenylphosphine (TPP) induced mitochondrial targeting. With the use of Cu(II) as the central ion and tetrakis(4-carboxyphenyl)porphyrin (TCPP) as the ligand, two-dimensional Cu-MOF nanosheets were prepared, which were surface modified by manganese dioxide based on the in situ redox reaction between poly(allylamine hydrochloride) (PAH) and KMnO4 to obtain Cu-MOF@MnO2. Then FA and TPP were coupled with the nanosheets to form the title nanoplatform. Comprehensive physiochemical research has suggested that Cu(II) and MnO2 constituents in the nanoplatform could consume intracellular GSH and hydrogen peroxide to generate hydroxyl radicals through a Fenton-like reaction; meanwhile Cu(II) could undergo a Russell reaction to produce cytotoxic singlet oxygen. Detailed in vitro and in vivo biological experiments have revealed a good biosafety profile and a high tumor suppression effect. Therefore, the present research has realized multiple and efficient CDT effects with the aid of the sequential targeting of FA/TPP, also providing a strategy for the development of CDT drugs based on polymetallic organic frameworks.

Histology, physiology, and glucose and lipid metabolism of Lateolabrax maculatus under low temperature stress.

Spotted sea bass (Lateolabrax maculatus) is a popular and important commercial fish throughout the world, but it is unknown whether introducing domesticated fish to locations that experience cold weather might alter physiological performance. In this study, we evaluated the behavior, fatty acid content, histological analysis of liver and gills, liver enzymatic activity in carbohydrate and lipid metabolism, and gene expression in liver related to carbohydrate and lipid metabolism of spotted sea bass acclimatized at 22 °C (control), 16 °C, 10 °C, 8 °C, and 4 °C for 24 h, and 8 °C for 4 days. When L. maculatus was exposed to acute cold stress for 24 h, the gill showed curling, lamellar disorganization, lamellar epithelium hyperplasia, and formed aneurysms inside of the secondary lamellae. Long term stress over four days resulted in severe lamellar epithelium hyperplasia and curling. Continued extreme cold exposure (4 °C) in L. maculatus caused liver HK, PK levels and LDH activities to achieve a peak value at 0 h, and decreased over time. These indicated that glucose metabolism might play critical roles in the initial time of stress. Results of carbohydrate and lipid metabolism showed that lipids appear to play roles in prolonged cold stress. The constitutive transcriptional levels of six genes related to glucose (G6Pase) and lipid metabolism (PPAR-α, PPAR-γ) and mTOR signal pathway (eif4ebp1, eif4ebp2, mlst8) genes increased significantly in most groups during cold stress.

Comparison of effects in sustained and diel-cycling hypoxia on hypoxia tolerance, histology, physiology and expression of clock genes in high latitude fish Phoxinus lagowskii.

Phoxinus lagowskii is a popular fish in Chinese cuisine. Though it is found mainly in China's high-latitude regions, where diel-cycling hypoxia (DCH) is known to have unique impacts on aquatic organisms, there is little known about its response to hypoxia. Currently, nothing is known about the changes in blood parameters, gill and liver morphology, glucose and lipid metabolism, or expression of genes involved in clock and glucose metabolism in response to sustained hypoxia (SH) and diel-cycling hypoxia (DCH). To elucidate the influence of sustained and diel-cycling hypoxia on fish hypoxia tolerance, resting oxygen consumption (MO2) analysis was performed after ten days of hypoxia. This analysis revealed that hypoxia tolerance profoundly improved after ten days of either sustained or diel-cycling hypoxia acclimation, with DCH groups showing greater improvements than SH groups. Additionally, an increase in RBCs was found in P. lagowskii, suggesting an increase in the O2-carrying capacity of the blood to tolerate hypoxia. Hemoglobin (Hb) concentrations in P. lagowskii were increased at four days of diel-cycling hypoxia, confirming that physiological and metabolic adaptation to hypoxia is based on the duration of O2 exposure. Increased Hb and hematocrit (Hct) were found in DCH-exposed fish, both of which have been directly linked to high-latitude hypoxia tolerance. In the gills, lamella surface area increased in SH-exposed fish more than DCH-exposed fish, and these increases were accompanied by a decrease in the volume of interlamellar cell mass (ILCM). Histology changes in the liver showed a higher frequency of cytoplasmic vacuolization in DCH-exposed fish. PK increases in SH-exposed fish suggest that fish can use more energy sources in persistent hypoxia. Meanwhile, DCH-exposed fish use TG as an energy source. In SH-exposed fish, self-regulation of Cry1a was observed, whereas Cry1b gene was up-regulated significantly. In DCH-exposed fish, three of eight clock genes studied had increased expression, including Per1a, Clocka, and Cry1b, suggesting that SH and DCH result in different hypoxic responses. This study presents a novel approach to the study of fish responses to hypoxia in high latitude and shows that sustained hypoxia and diel-cycling hypoxia induce large differences in fish physiology.

Histopathological, hematological, and biochemical changes in high-latitude fish Phoxinus lagowskii exposed to hypoxia.

Hypoxia is one of the most significant threats to biodiversity in aquatic systems. The ability of high-latitude fish to tolerate hypoxia with histological and physiological responses is mostly unknown. We address this knowledge gap by investigating the effects of exposures to different oxygen levels using Phoxinus lagowskii (a high-latitude, cold-water fish) as a model. Fish were exposed to different oxygen levels (0.5 mg/L and 3 mg/L) for 24 h. The loss of equilibrium (LOE), an indicator of acute hypoxia tolerance, was 0.21 ± 0.01 mg/L, revealing the ability of fish to tolerate low-oxygen conditions. We sought to determine if, in P. lagowskii, the histology of gills and liver, blood indicators, enzyme activities of carbohydrate and lipid metabolism, and antioxidants changed to relieve stress in response to acute hypoxia. Notably, changes in vigorous jumping behavior under low oxygen revealed the exceptional hypoxia acclimation response compared with other low-latitude fish. A decrease in blood parameters, including RBC, WBC, and Hb, as well as an increase in MCV was observed compared to the controls. The increased total area in lamella and decreased ILCM volume in P. lagowskii gills were detected in the present study. Our results also showed the size of vacuoles in the livers of the hypoxic fish shrunk. Interestingly, an increase in the enzyme activity of lipid metabolism but not glucose metabolism was observed in the groups exposed to hypoxia at 6 h and 24 h. After combining histology and physiology results, our findings provide evidence that lipid metabolism plays a crucial role in enhancing hypoxia acclimation in P. lagowskii. Additionally, SOD activity significantly increased during hypoxia, suggesting the presence of an antioxidant response of P. lagowskii during hypoxia. High expression levels of lipogenesis and lipolysis-related genes were detected in the 6 h 3 mg/L and 24 h 3 mg/L hypoxia group. Enhanced expression of lipid-metabolism genes (ALS4, PGC-1, and FASN) was detected during hypoxia exposure. Together, these data suggest that P. lagowskii's ability to tolerate hypoxic events is likely mediated by a comprehensive strategy.

Redescription of a Hymenostome Ciliate, Tetrahymena setosa (Protozoa, Ciliophora) Notes on its Molecular Phylogeny.

In recent years, Tetrahymena species have been used as model organisms for research in a wide range of fields, highlighting the need for a fuller understanding of the taxonomy of this group. It is in this context that this paper uses living observation and silver staining methods to investigate the morphology and infraciliature of one Tetrahymena species, T. setosa (Schewiakoff 1892 Verh. Naturh. Med. Ver. Heidelb., 4:544) McCoy (1975) Acta Protozool., 14:253; the senior subjective synonym of T. setifera Holz and Corliss (1956) J. Protozool., 3:112; isolated from a freshwater pond in Harbin, north-eastern China. This organism can be distinguished from other described Tetrahymena species mainly by its single caudal cilium, which is about twice the length of the somatic ciliature. While the Harbin isolate appears similar to the population described by Holz and Corliss (1956) J. Protozool., 3:112, an improved diagnosis for T. setosa is given based on the previous descriptions and the Harbin population. In summary, this species can be recognized mainly by the combination of the following characters: body in vivo approximately 40 µm × 25 µm, 21-26 somatic kineties, one to four contractile vacuole pores associated with meridians 6-11 and a single caudal cilium. The small subunit ribosomal (SSU) rRNA gene and the cox1 gene sequences of Harbin population are also characterized in order to corroborate that the isolated species branches in phylogenetic trees as a T. setosa species. The phylogenetic analysis also indicated that sequences of populations of Tetrahymena species should be published with detailed morphological identifications.

Transcriptome analysis on chlorpyrifos detoxification in Uronema marinum (Ciliophora, Oligohymenophorea).

Chlorpyrifos (CPF) pollution has drawn widespread concerns in aquatic environments due to its risks to ecologic system, however, the response mechanisms of ciliates to CPF pollution were poorly studied. In our current work, the degradation of CPF by ciliates and the morphological changes of ciliates after CPF exposure were investigated. In addition, the transcriptomic profiles of the ciliate Uronema marinum, with and without exposure with CPF, were detected using digital gene expression technologies. De novo transcriptome assembly 166,829,634 reads produced from three groups (untreated, CPF treatment at 12 h and 24 h) by whole transcriptome sequencing (RNA-Seq). Gene ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathways were analyzed in all unigenes and different expression genes to identify their biological functions and processes. Furthermore, the results indicated that genes related to the stress response, cytoskeleton and cell structure proteins, and antioxidant systems might play an important role in the resistance mechanism of ciliates. The enzyme activities of SOD and GST after CPF stress were also analyzed, and the result showed the good antioxidant capacity of SOD and GST in ciliates inferred from the increase of the activities of the two enzymes. The ciliate Uronema marinum showed a resistance response to chlorpyrifos stress at the transcriptomic level in the present work, which indicates that ciliates can be considered as a potential bioremediation agent.

Morphology and systematics of two freshwater Frontonia species (Ciliophora, Peniculida) from northeastern China, with comparisons among the freshwater Frontonia spp.

The morphology and infraciliature of two Frontonia species, F. shii spec. nov. and F. paramagna Chen et al., 2014, isolated from a freshwater pond in northeastern China, were investigated using living observation and silver staining methods Frontonia shii spec. nov. is recognized by the combination of the following characters: freshwater Frontonia, size in vivo about 220-350 × 130-250 µm, elliptical in outline; 128-142 somatic kineties; three or four vestibular kineties, six or seven postoral kineties; peniculi 1-3 each with four kineties; single contractile vacuole with about 10 collecting canals. The improved diagnosis for F. paramagna is based on the current and previous reports. Comparisons among freshwater Frontonia are also provided. The small subunit ribosomal rRNA gene (SSU rDNA) sequences of the two species are characterized and phylogenetic analyses based on these sequences show that both species fall into the core clade of the genus Frontonia, and this genus is not monophyletic.

Toxicological effects of cadmium and lead on two freshwater diatoms.

In recent years, there have been significant advances in the knowledge and understanding of the pollution attributed to effects of aquatic toxic metals on fresh water benthic diatoms. In this study, the cell growth, chlorophyll a content and superoxide dismutase activity in Halamphora veneta (Kützing) Levkov and Surirella crumena Brébisson exposed to cadmium and lead, were investigated. Furthermore, in order to explore the potential function of H. veneta on environmental monitoring and environmental remediation, expression of two genes (psbA, psaB) and morphological analysis of H. veneta were carried out. The cells growth of H. veneta and S. crumena were generally inhibited with cadmium and lead exposure during 96 h, while cells density of H. veneta was significantly increased under the low concentration at 24 h cadmium exposure. Our results indicated that H. veneta had a certain tolerance to toxic metals at initial treated time. In addition, the significantly changes of chlorophyll a content and SOD activities in H. veneta and S. crumena indicated that both photosynthetic system and the antioxidant system in benthic diatom might play important roles on the toxic metals tolerant mechanism. Meanwhile, it can be confirmed that the diatom photosynthetic systems play roles on toxic metals resistance inferred from the gene expression of psbA and psaB in H. veneta. Finally, the results of scanning electron microscopy showed that there was a slightly deformation on cells following the cadmium exposure in H. veneta, while obvious deformation with cell greatly widened after lead exposure. The present work will be helpful to understand the effect mechanisms of toxic metal by comparing two kinds of diatom on cell inhibition, biological response and morphological changes, which will provide more information for possible use of benthic diatoms in bioremediation.

The oxidative stress response of oxytetracycline in the ciliate Pseudocohnilembus persalinus.

Oxytetracycline (OTC) is commonly employed in fish farms to prevent bacterial infections in China, and because of their widely and intensive use, the potential harmful effects on organisms in aquatic environment are of great concern. Ciliates play an important role in aquatic food webs as secondary producers, and Pseudocohnilembus persalinus, is one kind of them which are easily found in fish farms, surviving in polluted water. Therefore, using P. persalinus as experimental models, this study investigated the effects of oxytetracycline (OTC) on the growth, antioxidant system and morphological damage in pollution-resistant ciliates species. Our results showed that the 96-h EC50 values for OTC of P. persalinus was 21.38mgL-1. The increased level of SOD and GSH during 96h OTC stress was related to an adaptive response under oxidative stress induced in ciliates. Additionally, sod1, sod2 and sod3 exhibited a significant increased expression level compared to control group at 24h treatment, indicating a promoting of dense system in ciliates at this exposure time. However, only sod1 and sod2 showed raised expression level at 48h stress, showing the different sensitive of gene isoforms to some extent. With OTC treatment, damage of regular wrinkles, shrunk, twisted on the cell surface, even forming cyst of scuticociliatid ciliate cells were firstly observed by SEM (scanning electron microscope) in this study. Overall, physiological, molecular and morphological information on the toxicological studies of ciliates and more information on possibility of ciliates as indicators of contamination were provided in this study.

Response of the freshwater diatom Halamphora veneta (Kützing) Levkov to copper and mercury and its potential for bioassessment of heavy metal toxicity in aquatic habitats.

This study investigates the effects of copper and mercury on growth rate, chlorophyll a content, superoxide dismutase (SOD) activity, SOD mRNA gene expression, and frustule morphology of the benthic freshwater diatom Halamphora veneta (Kützing) Levkov and the potential utility of each for toxicity assessment in aquatic habitats. Results showed the following: (1) Compared to mercury, exposure to copper resulted in greater growth inhibition of H. veneta even at low concentrations and after short durations of exposure; (2) high accumulation of chlorophyll a in H. veneta is a stress response to the presence of heavy metals; (3) SOD activity and SOD gene expression varied in H. veneta according to the concentration, exposure time, and type of heavy metal; and (4) exposure to mercury resulted in deformity in the shape and an increase in size of the frustule of H. veneta. Growth rate, chlorophyll a content, SOD activity and gene expression, and frustule morphology of H. veneta are all potential candidates for the toxicological assessment of copper and mercury in aquatic habitats.

One freshwater species of the genus Cyclidium, Cyclidiumsinicum spec. nov. (Protozoa; Ciliophora), with an improved diagnosis of the genus Cyclidium.

The morphology and infraciliature of one freshwater ciliate, Cyclidium sinicum spec. nov., isolated from a farmland pond in Harbin, northeastern China, was investigated using living observation and silver staining methods. Cyclidium sinicum spec. nov. could be distinguished by the following features: body approximately 20-25×10-15 µm in vivo; buccal field about 45-50 % of body length; 11 somatic kineties; somatic kinety n terminating sub-caudally; two macronuclei and one micronucleus; M1 almost as long as M2; M2 triangle-shaped. The genus Cyclidium is re-defined as follows: body outline usually oval or elliptical, ventral side concave, dorsal side convex; single caudal cilium; contractile vacuole posterior terminal; adoral membranelles usually not separated; paroral membrane 'L'-shaped, with anterior end terminating at the level of anterior end of M1; somatic kineties longitudinally arranged and continuous. Phylogenetic trees based on the SSU rDNA sequences showed that C. sinicum spec. nov. clusters with the type species, Cyclidiumglaucoma, with full support. Cyclidium is not monophyletic with members of the clade of Cyclidium+Protocyclidium+Ancistrum+Boveria.

Epidermal growth factor-induced stimulation of proliferation and gene expression changes in the hypotrichous ciliate, Stylonychia lemnae.

Epidermal growth factor (EGF) induces proliferation of epidermal and epithelial tissues in mammals. However, the effect of EGF on the single-celled eukaryotes is not well characterized, especially in the protists. Ciliates, an important group of protists, are well characterized as both pollution indicators and model organisms for research. Stylonychia lemnae, is one of the most common free-living ciliates, widely distributed in ponds, rivers and marshes. Here, we report the role of EGF on cell proliferation stimulation in S. lemnae. The growth curve of S. lemnae was established, and the stimulation effect of EGF on the proliferation of S. lemnae was investigated. Based on the results, potential EGF receptors were identified in S. lemnae according to the conserved domains and gene expression. Differential gene expression revealed that EGF-induced genes in other organisms (e.g. antioxidant) also up-regulated in S. lemnae cells at propagation stages. In addition, our results showed that EGF could up-regulate the signal transduction-related processes in the decline stage of S. lemnae cells, indicating its potential function in apoptosis inhibition. In summary, this study reports findings of the first investigation of EGF effects in hypotrich ciliates, and establishes an additional system for the study of the molecular mechanisms of EGF actions in eukaryotic cell division and proliferation.

HIFs genes expression and hematology indices responses to different oxygen treatments in an ovoviviparous teleost species Sebastes schlegelii.

Hypoxia-inducible factors (HIFs) are transcription factors considered as a respond factor to oxygen tension. By using quantitative real-time PCR, expression files of HIF-1α and HIF-2α mRNA were detected in the Korean rockfish ovary, liver, gill and spleen after 30 min and 60 min acute hypoxia exposure. Meanwhile, the cortisol levels, white blood cells and several serum biochemical values of Korean rockfish under different oxygen concentration treatments were also detected. All the results might be helpful for further understanding of the potential effect of hypoxia in ovoviviparous fish.

Identification, phylogeny and expression analysis of suppressors of cytokine signaling in channel catfish.

The suppressors of cytokine signaling (SOCS) family genes play important roles in regulating a variety of signal transduction pathways that are involved in immunity, growth and development. Because of their importance, they have been extensively studied in mammalian species, but they have not been systematically studied among teleost fish species. In this study, a total of 12 SOCS genes were characterized to understand the molecular mechanisms of SOCS function in channel catfish. Phylogenetic analyses suggested that all SOCS were clustered into two main clusters. Further syntenic analysis confirmed the phylogenetic analyses and allowed the annotation of SOCS genes in channel catfish. This work, for the first time, determined the expression profiles of the 12 SOCS genes after bacterial infections with Flavobacterium columnare and Edwardsiella ictaluri in channel catfish. The SOCS1a and SOCS3a were significantly up-regulated at 4h after F. columnare challenge in the gill, but were down-regulated at later stages of pathogenesis. Similarly, SOCS1a and CISH were significantly up-regulated at 3h in intestine under E. ictaluri infection, but were down-regulated at later stages of pathogenesis at 24h and 3 days after infection. These expression patterns may indicate that SOCS genes could be induced in acute immune responses after bacterial infections, but the massive cytokine expression, especially chemokine expression after the first day of infection may have had negative feedback leading to the overall down-regulation of the expression of SOCS genes. Moreover, the differential expression patterns of SOCS genes in the catfish gill and intestine after F. columnare and E. ictaluri infection demonstrated that the regulation of SOCS gene expression was both tissue-specific and time-dependent. Taken together, these results suggested that SOCS genes were involved in immune responses to bacterial invasions, and these results set the foundation for future studies of SOCS gene functions.