Zhigang Zhou

Transcriptome Analysis of the Model Protozoan, Tetrahymena thermophila, Using Deep RNA Sequencing

Background The ciliated protozoan Tetrahymena thermophila is a well-studied single-celled eukaryote model organism for cellular and molecular biology. However, the lack of extensive T. thermophila cDNA libraries or a large expressed sequence tag (EST) database limited the quality of the original genome annotation. Methodology/Principal Findings This RNA-seq study describes the first deep sequencing analysis of the T. thermophila transcriptome during the three major stages of the life cycle: growth, starvation and conjugation. Uniquely mapped reads covered more than 96% of the 24,725 predicted gene models in the somatic genome. More than 1,000 new transcribed regions were identified. The great dynamic range of RNA-seq allowed detection of a nearly six order-of-magnitude range of measurable gene expression orchestrated by this cell. RNA-seq also allowed the first prediction of transcript untranslated regions (UTRs) and an updated (larger) size estimate of the T. thermophila transcriptome: 57 Mb, or about 55% of the somatic genome. Our study identified nearly 1,500 alternative splicing (AS) events distributed over 5.2% of T. thermophila genes. This percentage represents a two order-of-magnitude increase over previous EST-based estimates in Tetrahymena. Evidence of stage-specific regulation of alternative splicing was also obtained. Finally, our study allowed us to completely confirm about 26.8% of the genes originally predicted by the gene finder, to correct coding sequence boundaries and intron-exon junctions for about a third, and to reassign microarray probes and correct earlier microarray data. Conclusions/Significance RNA-seq data significantly improve the genome annotation and provide a fully comprehensive view of the global transcriptome of T. thermophila. To our knowledge, 5.2% of T. thermophila genes with AS is the highest percentage of genes showing AS reported in a unicellular eukaryote. Tetrahymena thus becomes an excellent unicellular model eukaryote in which to investigate mechanisms of alternative splicing.

Complete Genome Sequence of Aeromonas veronii Strain B565

Aeromonas veronii strain B565 was isolated from aquaculture pond sediment in China. We present here the complete genome sequence of B565 and compare it with 2 published genome sequences of pathogenic strains in the Aeromonas genus. The result represents an independent stepwise acquisition of virulence factors of pathogenic strains in this genus.

New phylogenomic and comparative analyses provide corroborating evidence that Myxozoa is Cnidaria.

Myxozoa, a diverse group of morphologically simplified endoparasites, are well known fish parasites causing substantial economic losses in aquaculture. Despite active research, the phylogenetic position of Myxozoa remains ambiguous. After obtaining the genome and transcriptome data of the myxozoan Thelohanellus kitauei, we examined the phylogenetic position of Myxozoa from three different perspectives. First, phylogenomic analyses with the newly sequenced genomic data strongly supported the monophyly of Myxozoa and that Myxozoa is sister to Medusozoa within Cnidaria. Second, we detected two homologs to cnidarian-specific minicollagens in the T. kitauei genome with molecular characteristics similar to cnidarian-specific minicollagens, suggesting that the minicollagen homologs in T. kitauei may have functions similar to those in Cnidaria and that Myxozoa is Cnidaria. Additionally, phylogenetic analyses revealed that the minicollagens in myxozoans and medusozoans have a common ancestor. Third, we detected 11 of the 19 proto-mesodermalgenes in the T. kitauei genome, which were also present in the cnidarian Hydra magnipapillata, indicating Myxozoa is within Cnidaria. Thus, our results robustly support Myxozoa as a derived cnidarian taxon with an affinity to Medusozoa, helping to understand the diversity of the morphology, development and life cycle of Cnidaria and its evolution.

Abrupt suspension of probiotics administration may increase host pathogen susceptibility by inducing gut dysbiosis

In this study, we investigated the risk associated with suspension of probiotics administration in tilapia, an animal model that may mimic immune-compromised conditions in humans. Tilapias were fed for 14 days using a probiotics-supplemented diet, followed by a three-day suspension of probiotics treatment and a subsequent challenge by Aeromonas hydrophila. Unexpectedly, the suspension of a probiotic strain Lactobacillus plantarum JCM1149 significantly triggered susceptibility of the host to A. hydrophila. We further observed that suspension of JCM1149 resulted in host gut microbiota dysbiosis and the subsequent disorder in the intestinal metabolites (bile acids, amino acids, and glucose) and damage in the intestinal epithelium, giving rise to a condition similar to antibiotics-induced gut dysbiosis, which collectively impaired tilapia’s gut health and resistance to pathogenic challenges. Additionally, we determined that JCM1149 adhered relatively poorly to tilapia intestinal mucosa and was rapidly released from the gastrointestinal tract (GIT) after suspension, with the rapid loss of probiotic strain probably being the direct cause of gut dysbiosis. Finally, three other probiotic Lactobacillus strains with low intestinal mucosa binding activity showed similar rapid loss phenotype following administration suspension, and induced higher host susceptibility to infection, indicating that the risk is a generic phenomenon in Lactobacillus.

A Comparison of the Beneficial Effects of Live and Heat-Inactivated Baker’s Yeast on Nile Tilapia: Suggestions on the Role and Function of the Secretory Metabolites Released from the Yeast

Yeast is frequently used as a probiotic in aquaculture with the potential to substitute for antibiotics. In this study, the involvement and extent to which the viability of yeast cells and thus the secretory metabolites released from the yeast contribute to effects of baker’s yeast was investigated in Nile tilapia. No yeast, live yeast or heat-inactivated baker’s yeast were added to basal diets high in fishmeal and low in soybean (diet A) or low in fishmeal and high in soybean (diet B), which were fed to fish for 8 weeks. Growth, feed utilization, gut microvilli morphology, and expressions of hsp70 and inflammation-related cytokines in the intestine and head kidney were assessed. Intestinal microbiota was investigated using 16S rRNA gene pyrosequencing. Gut alkaline phosphatase (AKP) activity was measured after challenging the fish with Aeromonas hydrophila. Results showed that live yeast significantly improved FBW and WG (P < 0.05), and tended to improve FCR (P = 0.06) of fish compared to the control (no yeast). No significant differences were observed between inactivated yeast and control. Live yeast improved gut microvilli length (P < 0.001) and density (P < 0.05) while inactivated yeast did not. The hsp70 expression level in both the intestine and head kidney of fish was significantly reduced by live yeast (P < 0.05) but not inactivated yeast. Live yeast but not inactivated yeast reduced intestinal expression of tnfα (P < 0.05), tgfβ (P < 0.05 under diet A) and il1β (P = 0.08). Intestinal Lactococcus spp. numbers were enriched by both live and inactivated yeast. Lastly, both live and inactivated yeast reduced the gut AKP activity compared to the control (P < 0.001), indicating protection of the host against infection by A. hydrophila. In conclusion, secretory metabolites did not play major roles in the growth promotion and disease protection effects of yeast. Nevertheless, secretory metabolites were the major contributing factor towards improved gut microvilli morphology, relieved stress status, and reduced intestinal inflammation of Nile tilapia fed diets supplemented with baker’s yeast.

The Genome of the Myxosporean Thelohanellus kitauei Shows Adaptations to Nutrient Acquisition within Its Fish Host

Members of Myxozoa, a parasitic metazoan taxon, have considerable detrimental effects on fish hosts and also have been associated with human food-borne illness. Little is known about their biology and metabolism. Analysis of the genome of Thelohanellus kitauei and comparative analysis with genomes of its two free-living cnidarian relatives revealed that T. kitauei has adapted to parasitism, as indicated by the streamlined metabolic repertoire and the tendency toward anabolism rather than catabolism. Thelohanellus kitauei mainly secretes proteases and protease inhibitors for nutrient digestion (parasite invasion), and depends on endocytosis (mainly low-density lipoprotein receptors-mediated type) and secondary carriers for nutrient absorption. Absence of both classic and complementary anaerobic pathways and gluconeogenesis, the lack of de novo synthesis and reduced activity in hydrolysis of fatty acids, amino acids, and nucleotides indicated that T. kitauei in this vertebrate host–parasite system has adapted to inhabit a physiological environment extremely rich in both oxygen and nutrients (especially glucose), which is consistent with its preferred parasitic site, that is, the host gut submucosa. Taking advantage of the genomic and transcriptomic information, 23 potential nutrition-related T. kitauei-specific chemotherapeutic targets were identified. This first genome sequence of a myxozoan will facilitate development of potential therapeutics for efficient control of myxozoan parasites and ultimately prevent myxozoan-induced fish-borne illnesses in humans.

pO157_Sal, a Novel Conjugative Plasmid Detected in Outbreak Isolates of Escherichia coli O157:H7

ABSTRACT In addition to the large virulence plasmid pO157, a novel 38-kb conjugative plasmid, pO157_Sal, was identified and sequenced from an Escherichia coli O157:H7 outbreak-associated Chinese isolate that shares high similarity with a plasmid in Salmonella enterica serovar Agona. The plasmid was found in 15 of 326 isolates, 12 of which were of the same pulsed-field gel electrophoresis type.

Thymol and Carvacrol Affect Hybrid Tilapia through the Combination of Direct Stimulation and an Intestinal Microbiota-Mediated Effect: Insights from a Germ-Free Zebrafish Model

BACKGROUND Essential oils (EOs) are commonly used as animal feed additives. Information is lacking on the mechanisms driving the beneficial effects of EOs in animals, especially the role played by the intestinal microbiota of the host. OBJECTIVE The purpose of this study was to clarify the relative contribution of direct effects of EOs on the physiology and immune system of tilapia and indirect effects mediated by the intestinal microbiota by using a germ-free zebrafish model. METHODS Juvenile hybrid tilapia were fed a control diet or 1 of 4 treatment diets containing 60-800 mg Next Enhance 150 (NE) (an EO product containing equal levels of thymol and carvacrol)/kg for 6 wk. The key humoral and cellular innate immune parameters were evaluated after the feeding period. In another experiment, the gut microbiota of tilapia fed a control or an NE diet (200 mg/kg) for 2 wk were transferred to 3-d postfertilization (dpf) germ-free (GF) zebrafish, and the expression of genes involved in innate immunity and tight junctions was evaluated in zebrafish at 6 dpf. Lastly, NE was directly applied to 3-dpf GF zebrafish at 3 doses ranging from 0.2 to 20 mg/L, and the direct effect of NE on zebrafish was evaluated after 1 and 3 d. RESULTS NE supplementation at 200 mg/kg enhanced phagocytosis activity of head kidney macrophages (×1.36) (P < 0.05) and plasma lysozyme activity (×1.69) of tilapia compared with the control (P < 0.001), indicating an immunostimulatory effect. Compared with those colonized with control microbiota, GF zebrafish colonized with NE microbiota showed attenuated induction of immune response marker genes serum amyloid a (Saa; ×0.62), interleukin 1β (Il1β; ×0.29), and interleukin 8 (Il8; ×0.62) (P < 0.05). NE treatment of GF zebrafish at 2 and 20 mg/L for 1 d upregulated the expression of Il1β (×2.44) and Claudin1 (×1.38), respectively (P < 0.05), whereas at day 3 the expression of Occludin2 was higher (×3.30) in the 0.2-mg NE/L group compared with the GF control (P < 0.05). CONCLUSION NE may affect the immunity of tilapia through a combination of factors, i.e., primarily through a direct effect on host tissue (immune-stimulating) but also an indirect effect mediated by microbial changes (immune-relieving).

The Growth-Promoting Effect of Dietary Nucleotides in Fish Is Associated with an Intestinal Microbiota-Mediated Reduction in Energy Expenditure

Background: Nucleotides have been used as functional nutrients to improve the growth and health of animals, including fish. The mechanism involved in the growth-promotion effect of nucleotides is still unclear.Objective: We investigated the bioenergetic mechanism underlying the growth-promotion effect of nucleotides in zebrafish and the associated roles played by the intestinal microbiota.Methods: Larval zebrafish were fed a control or a 0.1% mixed nucleotides-supplemented diet for 2 wk. Standard metabolic rate, the minimal rate of energy expenditure by animals at rest, was evaluated by oxygen consumption with the use of a respirometer. The expressions of fasting-induced adipose factor (Fiaf), inflammatory cytokines, and genes involved in fatty acid (FA) oxidation were tested by quantitative reverse transcriptase-polymerase chain reaction. The intestinal microbiota from the nucleotide-fed fish (NT fish) or control fish was transferred to 3-d postfertilization germ-free zebrafish in which oxygen consumption and expression of cytokines and fiaf were evaluated.Results: Compared with controls, nucleotide supplementation at 0.1% increased the weight and energy gains of zebrafish by 10% and 25%, respectively (P < 0.01). Standard metabolic rate was 28% lower in NT fish than in controls (P < 0.001). Nucleotide supplementation downregulated the inflammatory tone in the head kidney of the fish. Moreover, NT fish had a 51% lower intestinal expression of fiaf than did controls (P < 0.05), which was consistent with decreased expression of key genes involved in FA oxidation [carnitine:palmitoyl transferase 1a (cpt1a) and medium-chain acyl coenzyme A dehydrogenase (mcad)] in liver and muscle. Germ-free zebrafish colonized with microbiota from NT fish had a 25% lower standard metabolic rate than did those colonized by control microbiota (P < 0.01), whereas direct nucleotide feeding of germ-free zebrafish did not affect standard metabolic rate relative to germ-free controls that were not fed nucleotides. Furthermore, germ-free zebrafish colonized with nucleotide microbiota exhibited downregulated inflammatory tone and 33% lower fiaf expression compared with their control microbiota-colonized counterparts.Conclusions: The growth-promoting effect of dietary nucleotides in zebrafish involves 2 intestinal microbiota-mediated mechanisms that result in reduced standard metabolic rate: 1) lower inflammatory tone and 2) reduced FA oxidation associated with increased microbial suppression of intestinal fiaf.

A Chitinase from Aeromonas veronii CD3 with the Potential to Control Myxozoan Disease

Background The class Myxosporea encompasses about 2,400 species, most of which are parasites of fish and cause serious damage in aquaculture. Due to the concerns about food safety issues and limited knowledge of Myxozoa life cycle and fish immune system, no chemicals, antibiotics or immune modulators are available to control myxozoa infection. Therefore, little can be done once Myxozoa establishment has occurred. Methodology/Principal Findings In this paper we isolated Aeromonas veronii CD3 with significant myxospore shell valve-degrading ability from pond sediment. A 3,057-bp full-length chitinase gene was consequently cloned, and the corresponding mature, recombinant chitinase (ChiCD3) produced by Escherichia coli had substantial chitinase activity. The deduced sequence of ChiCD3 contained one catalytic domain, two chitin-binding domains, and one putative signal peptide. ChiCD3 had an optimal activity at 50°C and pH 6.0, and retained more than 50% of its optimal activity under warm water aquaculture conditions (∼30°C and pH ∼7.0). After incubation with ChiCD3, 38.0±4.8% of the myxospores had damaged shell valves, whereas myxospores incubated with commercially available chitinases remained intact. Conclusion/Significance This study reveals a new strategy to control myxozoan disease. ChiCD3 that has capacity to damage the shell valve of myxospores can be supplemented into fish feed and used to control Myxozoa-induced diseases specifically.