Insights into Body Size Evolution: A Comparative Transcriptome Study on Three Species of Asian Sisoridae Catfish.

Body size is one of the most important attributes of a species, but the basic question of why and how each species reaches a different "right size" is still largely unknown. Herein, three phylogenetically closely related catfishes from Sisoridae, including one extraordinarily large-sized Bagarius yarrelli and two average-sized Glyptothorax macromaculatus and Oreoglanis setiger, were comparatively studied using RNA-Seq. Approximately 17,000 protein-coding genes were annotated for each of the three fishes, and 9509 genes were identified as high-confidence orthologous gene pairs. Comparative expressions uncovered a similar functional cluster about ribosome biogenesis was enriched in different tissues of the upregulated genes of Bagarius yarrelli. Moreover, differentially expressed genes and positively selected genes revealed that the glycolysis/pyruvate metabolism and cell cycle pathways have also greatly enhanced in this large-sized species. In total, 20 size-related candidate genes (including two growth modulators: the serine/threonine-protein kinases 3 (AKT3) and adaptor protein 1 (SH2B1), and a crucial pyruvate kinase (PKM2A)) were identified by multiplying comparative analyses along with gene functional screening, which would play major roles in enabling the large body size associated with Bagarius yarrelli and provide new insights into body size evolution. In conjunction with field observations and morphological comparisons, we hypothesize that habitat preferences promote size divergence of sisorids.

Mycobacterium tuberculosis infection depressed cytotoxic T cells activity owing to decreasing NKG2C and increasing NKG2A expression.

Cytotoxic T lymphocytes (CTLs) play protective roles in immunity against tuberculosis (TB) infection by strongly inhibiting intracellular mycobacterial growth. In TB infection, the impairing mechanism of CTLs function remains unclear. In this study, we identified that the cytotoxic granule molecules expression levels of perforin (PRF) and granulysin (GNLY) in CD3+ and CD8+ CTL cells were significantly depressed in TB patients compared to those in healthy donors. The frequencies of T-CTLs, co-expressing granzyme B (GZMB), PRF and GNLY, were obviously decreased in TB patients. Moreover, NKG2C highly expressed in T-CTLs, was an effective activator of cytotoxic activity of CD3+ T cells. And, NKG2C+CD3+ T cells potently inhibited intracellular mycobacterial growth. The proportions of NKG2C+ cells in CD3+ and CD8+ T cells were dramatically decreased in TB patients. Contrarily, NKG2A, an inhibitor of T cells cytotoxic activities, was highly expressed in T-CTLs of CD3+ and CD8+ T cells in TB patients. Here, we successfully discovered that depressed CTLs activities in TB patients were attributed to low expression of cytotoxic granule molecules and high expression of inhibitory NKG2A receptor, suppression of agonist receptor NKG2C. Thus, NKG2 receptors were potential targets for immunotherapy of tuberculosis, especially for multidrug-resistant tuberculosis.

Evolution of the intermuscular bones in the Cyprinidae (Pisces) from a phylogenetic perspective.

Intermuscular bones (IBs) are widely present in morphologically generalized teleost fishes and are commonly found in the Cyprinidae. Intermuscular bones are small, hard spicules of bone that are formed by ossification in the myosepta between neighboring myomeres. Why fish have IBs, and whether there is any evolutionary pattern to their occurrence, has been poorly understood. However, the presence of IBs does substantially affect the meat quality and commercial values of many cyprinid fishes in aquaculture. In this study, we sampled 592 individuals of cyprinid fishes to systematically investigate the evolution of IBs from a phylogenetic point of view. We found that the total number of IBs in the Cyprinidae ranged from 73 to 169, and we clarified that only two categories of IBs (epineural and epipleural) were present in all examined cyprinids. Most of the IBs were distributed in the posterior region of the fish, which might be an optimal target for selecting fewer IB strains in aquaculture. There was a positive correlation between IBs and the number of vertebrae, thus making it possible to predict the approximate number of IBs by counting the number of vertebrae. Although the IBs displayed some correlation with phylogenetic relationships in some lineages and to ecological factors such as diet (especially carnivore), in an overall view the variations of IBs in cyprinids were extremely diverse. The number and patterns of IBs in these fishes may reflect their phylogenetic history, but have been shaped by multiple environment factors. In this study, we also confirmed that X-ray photography remains an optimal and reliable method for the study of IBs.

Whole-Genome Sequencing of the Giant Devil Catfish, Bagarius yarrelli.

As one economically important fish in the southeastern Himalayas, the giant devil catfish (Bagarius yarrelli) has been known for its extraordinarily large body size. It can grow up to 2 m, whereas the non-Bagarius sisorids only reach 10-30 cm. Another outstanding characteristic of Bagarius species is the salmonids-like reddish flesh color. Both body size and flesh color are interesting questions in science and also valuable features in aquaculture that worth of deep investigations. Bagarius species therefore are ideal materials for studying body size evolution and color depositions in fish muscles, and also potential organisms for extensive utilization in Asian freshwater aquaculture. In a combination of Illumina and PacBio sequencing technologies, we de novo assembled a 571-Mb genome for the giant devil catfish from a total of 153.4-Gb clean reads. The scaffold and contig N50 values are 3.1 and 1.6 Mb, respectively. This genome assembly was evaluated with 93.4% of Benchmarking Universal Single-Copy Orthologs completeness, 98% of transcripts coverage, and highly homologous with a chromosome-level-based genome of channel catfish (Ictalurus punctatus). We detected that 35.26% of the genome assembly is composed of repetitive elements. Employing homology, de novo, and transcriptome-based annotations, we annotated a total of 19,027 protein-coding genes for further use. In summary, we generated the first high-quality genome assembly of the giant devil catfish, which provides an important genomic resource for its future studies such as the body size and flesh color issues, and also for facilitating the conservation and utilization of this valuable catfish.

A novel immortalization vector for the establishment of penaeid shrimp cell lines.

Cell immortalization technology based on gene transfer has been successfully used to generate cell lines from a wide variety of cell types. The inability to stably introduce and express foreign genes has hampered application of this strategy in shrimp cells. We report here the use of replication-defective pantropic retrovirus to achieve a novel immortalization vector in which simian virus 40 large T antigen (SV40T) gene is expressed from Moloney murine leukemia virus (MoMLV) promoter. Data confirmed the presence of transferred SV40T gene and its stable mRNA expression in transduced lymphoid cells of Penaeus chinensis. The transduced cells showed a higher growth rate and a longer replication life-span compared with their untransduced counterparts. These results indicate the pantropic retrovirus-based immortalization-inducing gene delivery system is a potential tool for establishing cell lines from shrimp.

Genome Assembly for a Yunnan-Guizhou Plateau "3E" Fish, Anabarilius grahami (Regan), and Its Evolutionary and Genetic Applications.

A Yunnan-Guizhou Plateau fish, the Kanglang white minnow (Anabarilius grahami), is a typical "3E" (Endangered, Endemic, and Economic) species in China. Its distribution is limited to Fuxian Lake, the nation's second deepest lake, with a significant local economic value but a drastically declining wild population. This species has been evaluated as VU (Vulnerable) in the China Species Red List. As one of the "Four Famous Fish" in Yunnan province, the artificial breeding has been achieved since 2003. It has not only re-established its wild natural populations by reintroduction of the artificial breeding stocks, but also brought a wide and popular utilization of this species to the local fish farms. A. grahami has become one of the main native aquaculture species in Yunnan province, and the artificial production has been emerging in steady growth each year. To promote the conservation and sustainable utilization of this fish, we initiated its whole genome sequencing project using an Illumina Hiseq2500 platform. The assembled genome size of A. grahami is 1.006 Gb, accounting for 98.63% of the estimated genome size (1.020 Gb), with contig N50 and scaffold N50 values of 26.4 kb and 4.41 Mb, respectively. Approximately about 50.38% of the genome was repetitive. A total of 25,520 protein-coding genes were subsequently predicted. A phylogenetic tree based on 4,580 single-copy genes from A. grahami and 18 other cyprinids revealed three well-supported subclades within the Cyprinidae. This is the first inter-subfamily relationship of cyprinids at genome level, providing a simple yet useful framework for understanding the traditional but popular subfamily classification systems. Interestingly, a further population demography of A. grahami uncovered a historical relationship between this fish and Fuxian Lake, suggesting that range expansion or shrinkage of the habitat has had a remarkable impact on the population size of endemic plateau fishes. Additionally, a total of 33,836 simple sequence repeats (SSR) markers were identified, and 11 loci were evaluated for a preliminary genetic diversity analysis in this study, thus providing another useful genetic resource for studying this "3E" species.

An integrative approach to reappraising species validity in Pseudexostoma (Teleostei: Sisoridae).

The catfish genus Pseudexostoma (Sisoridae) currently contains three species: P. yunnanensis from the upper Irrawaddy River (Dayingjiang), and P. brachysoma and P. longipterus from the upper Salween River (Nujiang). We adopted an integrative approach combining both morphology-based (traditional measurements, truss-based and geometric morphometrics, fin skeleton examination and X-ray photography) and molecular approaches (genetic distances, haplotype network, phylogenetic relationships) to reappraise species validity in Pseudexostoma. Our results showed that P. longipterus cannot be distinguished from P. brachysoma from either morphological or molecular analyses, and that the former name should thus be considered a junior synonym of the latter. The two remaining geographically isolated species, P. yunnanensis and P. brachysoma, showed a distinct genetic distance equated to the typical 2% sequence divergence threshold delineating different species of fishes, and with a divergence time dated to approximately 1.22-2.14 million years ago. Although P. yunnanensis and P. brachysoma share some morphological characters that appear gradually diverging (body depth at anus and interorbital distance), some degree of overlap still exists, we suggest the two species are distinct based on the total evidence.

[Progress on gene targeting].

Gene targeting is a rising technology in molecular biology,which is defined as the introduction of exogeneous DNA to specific site in genome by homologous recombination,and consequently change the hereditary character of the cell. This technology provides a new and powerful means for research in developmental biology,molecular genetics,immunology and medicine. Progresses have been made in exploring gene structure and function,gene expression and regulation,transgene and gene therapy with the application of gene targeting. But there are some problems in gene targeting,especially for the low efficiency. This article just provided a review of the principle and program of gene targeting,and discussed the possible approaches to increase the efficiency of gene targeting.

Monocrotophos pesticide modulates the expression of sexual differentiation genes and causes phenotypic feminization in zebrafish (Danio rerio).

Monocrotophos (MCP) is an organophosphorus pesticide moderately toxic to fish, and it has significant estrogenic properties in vivo. In this study, zebrafish (Danio rerio) were exposed to 0.001, 0.010, and 0.100 mg/L 40% MCP pesticide in a semi-static manner from fertilization to 40 days post-hatching. Histological analyses were performed to determine whether sex differentiation in zebrafish was affected by MCP, and the mRNA expression levels of genes involved in sexual differentiation were quantified by real-time PCR to clarify the possible mechanism(s) of action. The results revealed a prominent increase in the proportion of females (71%) in the 0.100 mg/L MCP pesticide treatment as well as the presence of one intersex individual in each of the groups exposed to 0.001 and 0.100 mg/L MCP. MCP exposure stimulated forkhead transcription factor gene L2 (foxl2) expression and suppressed doublesex/mab-3 related transcription factor 1 (dmrt1) expression, indirectly leading to elevated gonadal aromatase (cyp19a1a) gene expression, which should promote phenotypic feminization. In addition, MCP treatment increased the transcription of brain aromatase (cyp19a1b), resulting in an indirect impact on sexual differentiation. The results from this investigation can be used for risk and hazard assessment of MCP pesticide.