Pathogenicity of Plesiomonas shigelloides and Citrobacter freundii isolated from the endangered Chinese sturgeon (Acipenser sinensis).

Chinese sturgeon (Acipenser sinensis) is an indigenous species of China and is listed as a critically endangered species. Recently, second filial generations of Chinese sturgeon in the Yangtze River Fisheries Research Institute suffered from a severe disease. In this study, two kinds of pathogenic bacteria were isolated from diseased sturgeon and identified as Plesiomonas shigelloides and Citrobacter freundii, based on 16S rDNA gene sequence alignment analysis. Antimicrobial susceptibility testing showed that P. shigelloides was resistant to ampicillin, penicillin, midecamycin, oxacillin, and clindamycin; and sensitive to tocefatriaxone, piperacillin, cefoperazone, cefazolin, and ciprofloxacin. C. freundii was resistant to ampicillin, penicillin, midecamycin, oxacillin, and clindamycin; and sensitive to chloramphenicol, cefuroxime, norfloxacin, ciprofloxacin, and ceftazidime. The median lethal dose (LD50) values of P. shigelloides and C. freundii were 4.50 × 103 colony forming units (CFU)/g and 3.20 × 103 CFU/g, respectively. Clinical symptoms of challenged sturgeons were the same as those of naturally infected sturgeons. Histopathological examination disclosed severe damage in the viscera of P. shigelloides and C. freundii-infected sturgeons. This is the first report suggesting that P. shigelloides infection is associated with mortality of Chinese sturgeon. The results of this study revealed the pathogenesis and severe pathogenicity of P. shigelloides and C. freundii in cultured Chinese sturgeon, and offer insights into the prevention and treatment of bacterial infection caused by P. shigelloides and C. freundii in cultured sturgeons.

Comparation of the structural characteristics and biological activities of chondroitin sulfates extracted from notochord and backbone of Chinese sturgeon (Acipenser sinensis).

To compare the structural properties and biological activities of chondroitin sulfate (CS) in two different tissues of Chinese sturgeon (Acipenser sinensis) and Russian sturgeon (Acipenser gueldenstaedti), we extracted their backbone cartilage CS (Cart-CS) and notochord CS (Noto-CS), and analyzed the CS structural properties using chromatographic and spectroscopic methods. The molecular weights of Chinese sturgeon Cart-CS and Noto-CS were 54.7 and 25.4 kDa, respectively, and the molecular weights of Russian sturgeon were 50.0 and 38.4 kDa, respectively. The disaccharide composition results showed that Cart-CS was mainly composed of CS-C, while Noto-CS was almost composed of pure CS-A. The antioxidant activity of sturgeon CS and its effect on collagen fibril formation were discussed. Sturgeon CS exhibited higher antioxidant activity than shark and bovine CSs. Sturgeon CS inhibited the self-assemble of type I collagen into fibrils. The inhibition effect of Cart-CS was higher than that of Noto-CS. The high value-added utilization of Cart-CS and Noto-CS will increase the value of sturgeon by-products. Furthermore, the disaccharide composition of CS in sturgeon depends on tissues of origin, but not on species. It means that the CS of Chinese sturgeon can be substituted by the CS of other commercial sturgeon. That will contribute to the protection of endangered species of Chinese sturgeon from illegal fishing and increase the value of commercial sturgeon by-products.

The last giants of the Yangtze River: A multidisciplinary picture of what remains of the endemic Chinese sturgeon.

The Chinese sturgeon, an important endemism of the Yangtze River, belongs to 'the most critically endangered group of species' worldwide, with overfishing and habitat destruction being the main drivers towards extinction. Newly obtained microchemical comparisons between animals and water from different river locations revealed a probable shifting of the spawning ground few kilometers downstream compared to the only previously known site, located under the Gezhouba Dam. This offers a glimmer of hope for an adaptive response to habitat perturbation caused by the recently built Three Gorges dam on the Yangtze River. On the other hand, genetic data provide an estimate of about 20 breeders participating in the only significant breeding event of the past 10 years. This warns of a near species extinction forecast if no in situ and ex situ conservation efforts occur promptly. Given these results we propose a list of priority conservation actions that urgently need to be promoted, supported, and put into practice.

Spermatogonia From Cryopreserved Testes of Critically Endangered Chinese Sturgeon Efficiently Colonized and Preferentially Proliferated in the Recipient Gonads of Yangtze Sturgeon.

The critically endangered Chinese sturgeon, Acipenser sinensis, presents late sexual maturity and has a large body size. Germ cell transplantation is a powerful technique for the production of gametes from large-bodied species in closely related recipients with a smaller body size and shorter generation time. To accelerate reproduction of Chinese sturgeon, donor spermatogonia collected from the cryopreserved testes of 3-year-old Chinese sturgeon were intraperitoneally transplanted into 7-8 days post-hatch larvae of Yangtze sturgeon (Acipenser dabryanus) with shorter generation interval. At 2 months post-transplantation (mpt), donor spermatogonia had colonized in the 81.25% of recipient gonads, with average numbers about two times those of endogenous primordial germ cells. Within the next 2 months, the rate of endogenous germ cell division in females (2-3 times) was faster than that in males (once), whereas colonized donor-derived spermatogonia divided about 2-3 times and twice in recipient females and males, respectively. Furthermore, the expression of germ cell-related genes, dazl, dead end, and vasa, in transplanted fish was higher than that in non-transplanted fish, suggesting the incorporation and proliferation donor spermatogonia in recipient. At 18 mpt, donor-derived spermatogonia survived in the 75.00% of recipient gonads. These results showed that the somatic microenvironment of Yangtze sturgeon gonad can support the long-term colonization, proliferation, and survival of xenogeneic germ cells. Thus, this study suggested that small-bodied Yangtze sturgeon is promising recipient as surrogate for Chinese sturgeon gamete production.

Effectiveness assessment of using riverine water eDNA to simultaneously monitor the riverine and riparian biodiversity information.

Both aquatic and terrestrial biodiversity information can be detected in riverine water environmental DNA (eDNA). However, the effectiveness of using riverine water eDNA to simultaneously monitor the riverine and terrestrial biodiversity information remains unidentified. Here, we proposed that the monitoring effectiveness could be approximated by the transportation effectiveness of land-to-river and upstream-to-downstream biodiversity information flows and described by three new indicators. Subsequently, we conducted a case study in a watershed on the Qinghai-Tibet Plateau. The results demonstrated that there was higher monitoring effectiveness on summer or autumn rainy days than in other seasons and weather conditions. The monitoring of the bacterial biodiversity information was more efficient than the monitoring of the eukaryotic biodiversity information. On summer rainy days, 43-76% of species information in riparian sites could be detected in adjacent riverine water eDNA samples, 92-99% of species information in riverine sites could be detected in a 1-km downstream eDNA sample, and half of dead bioinformation (the bioinformation labeling the biological material that lacked life activity and fertility) could be monitored 4-6 km downstream for eukaryotes and 13-19 km downstream for bacteria. The current study provided reference method and data for future monitoring projects design and for future monitoring results evaluation.

Screening and identification of female-specific DNA sequences in octaploid sturgeon using comparative genomics with high-throughput sequencing.

In this study, six candidate female-specific DNA sequences of octaploid Amur sturgeon (Acipenser schrenckii) were identified using comparative genomic approaches with high-throughput sequencing data. Their specificity was confirmed by traditional PCR. Two of these sex-specific sequences were also validated as female-specific in other eight sturgeon species and two hybrid sturgeons. The identified female-specific DNA fragments suggest that the family Acipenseridae has a ZZ/ZW sex-determining system. However, one of the two DNA sequences has been deleted in some sturgeons such as Sterlet sturgeon (Acipenser ruthenus), Beluga (Huso huso) and Kaluga (H. dauricus). The difference of sex-specific sequences among sturgeons indicates that there are different sex-specific regions among species of sturgeon. This study not only provided the sex-specific DNA sequences for management, conservation and studies of sex-determination mechanisms in sturgeons, but also confirmed the capability of the workflow to identify sex-specific DNA sequences in the polyploid species with complex genomes.

Characterization of underutilized root starches from eight varieties of ramie (Boehmeria nivea) grown in China.

Ramie root is an underutilized starch source. In this study, eight ramie varieties were investigated for starch properties. Starch content ranged from 18.6% to 50.1% in dry root. Starches from different varieties showed similar morphology including ellipsoidal, spherical and truncated granules with size D[4,3] from 10.1 to 14.1 µm. Starch had amylose content from 20.8% to 28.5%. All ramie varieties had B-type starches with relative crystallinity from 24.8% to 27.1%, ordered degree from 0.724 to 0.897 and lamellar thickness from 9.1 to 9.6 nm. Starches had gelatinization peak temperature from 70.5 to 73.8 °C and enthalpy from 14.9 to 15.8 J/g. Starches had swelling power and water solubility from 27.9 to 31.9 g/g and from 11.7% to 15.5%, respectively, at 95 °C, and exhibited different pasting properties with breakdown viscosity from 36 to 377 mPa s and setback viscosities from 1295 to 1863 mPa s. Starch pastes exhibited pseudoplastic behavior and different rheological properties. Native, gelatinized and retrograded starches had resistant starch from 81.7% to 83.9%, from 1.7% to 5.1% and from 5.6% to 13.3%, respectively. The eight varieties were divided into 3 groups according to starch properties. This study is helpful for selecting suitable ramie variety as starch source.

Physicochemical properties of a new starch from ramie (Boehmeria nivea) root.

A new starch was isolated from ramie root, and its physicochemical properties were investigated. Ramie dry root contained 45.9% starch. Starch had truncated, ellipsoidal, and spherical granule shapes with size from 7 to 30 µm and D[4,3] about 14.1 µm. Starch contained 38.9% apparent amylose content and 22.4% true amylose content, exhibited B-type crystallinity, and had 26.6% relative crystallinity, 0.82 ordered degree, and 9.2 nm lamellar thickness. Starch had 71.8 °C gelatinization peak temperature and 15.6 J/g gelatinization enthalpy, and exhibited 31.4 g/g swelling power and 17.1% water solubility at 95 °C. Starch had peak, hot, breakdown, final, and setback viscosities at 3048, 2768, 279, 4165, and 1397 mPa s, respectively, and showed peak time at 4.36 min and pasting temperature at 75.0 °C. The native, gelatinized, and retrograded starches contained 15.1%, 94.0%, and 86.5% rapidly digestible starch and 83.3%, 4.0%, and 10.7% resistant starch, respectively. Compared with potato and rice starches, ramie starch was somewhat similar to potato starch but significantly different from rice starch in starch component, crystalline structure, and functional properties. Therefore, ramie starch exhibited the potential to be used as a thickening agent, resistant-digesting food additive, and alternative to potato starch in food and nonfood industries.

Tracing the genetic footprints of vertebrate landing in non-teleost ray-finned fishes.

Rich fossil evidence suggests that many traits and functions related to terrestrial evolution were present long before the ancestor of lobe- and ray-finned fishes. Here, we present genome sequences of the bichir, paddlefish, bowfin, and alligator gar, covering all major early divergent lineages of ray-finned fishes. Our analyses show that these species exhibit many mosaic genomic features of lobe- and ray-finned fishes. In particular, many regulatory elements for limb development are present in these fishes, supporting the hypothesis that the relevant ancestral regulation networks emerged before the origin of tetrapods. Transcriptome analyses confirm the homology between the lung and swim bladder and reveal the presence of functional lung-related genes in early ray-finned fishes. Furthermore, we functionally validate the essential role of a jawed vertebrate highly conserved element for cardiovascular development. Our results imply the ancestors of jawed vertebrates already had the potential gene networks for cardio-respiratory systems supporting air breathing.

Molecular characterization and expression profiles of six genes involved in vitellogenic deposition and hydrolysis of Chinese sturgeon (Acipenser sinensis) suggesting their transcriptional regulation on ovarian development.

Ovary development of Chinese sturgeon (Acipenser sinensis) in controlled breeding has been reported to respond to dietary lipid levels. However, the corresponding molecular regulatory mechanism about ovary development of Chinese sturgeon is still unclear. To elucidate the molecular mechanism of vitellogenic deposition and hydrolysis, six key genes, namely, vtgr (vitellogenin receptor), atp6v1c1 (Vacuolar H+-ATPase subunit c1), atp6v1h (Vacuolar H+-ATPase subunit h), ctsb (cathepsin B), ctsd (cathepsin D) and ctsl (cathepsin L) involved in vitellogenic deposition and hydrolysis of Chinese sturgeon were cloned and characterized, and their spatio-temporal mRNA expression profiles as well as transcriptional responses to dietary lipid level were investigated. The full-length cDNA sequences of these six genes showed similar domain structure to their respective orthologous genes from other vertebrates. Tissue-specific expression patterns of these genes were observed in ovary, liver, muscle, spleen, brain, gill, intestine, heart, stomach and kidney. Ovarian expression level of vtgr was the highest in stage II, and ctsl expression was the highest in stage IV, while the mRNA expressions of other 4 genes were the highest in stage III. The increase of dietary lipid level promoted ovary development and elevated the expressions of vtgr, atp6v1c1, atp6v1h, ctsb and ctsd in the ovary. The results of the present study indicated that these genes are crucial for vitellogenic deposition, and provided a preliminary understanding on the molecular regulation of vitellogenic deposition and hydrolysis during ovary development of Chinese sturgeon.

The American Paddlefish Genome Provides Novel Insights into Chromosomal Evolution and Bone Mineralization in Early Vertebrates.

Sturgeons and paddlefishes (Acipenseriformes) occupy the basal position of ray-finned fishes, although they have cartilaginous skeletons as in Chondrichthyes. This evolutionary status and their morphological specializations make them a research focus, but their complex genomes (polyploidy and the presence of microchromosomes) bring obstacles and challenges to molecular studies. Here, we generated the first high-quality genome assembly of the American paddlefish (Polyodon spathula) at a chromosome level. Comparative genomic analyses revealed a recent species-specific whole-genome duplication event, and extensive chromosomal changes, including head-to-head fusions of pairs of intact, large ancestral chromosomes within the paddlefish. We also provide an overview of the paddlefish SCPP (secretory calcium-binding phosphoprotein) repertoire that is responsible for tissue mineralization, demonstrating that the earliest flourishing of SCPP members occurred at least before the split between Acipenseriformes and teleosts. In summary, this genome assembly provides a genetic resource for understanding chromosomal evolution in polyploid nonteleost fishes and bone mineralization in early vertebrates.

Quantifying the Colonization of Environmental Microbes in the Fish Gut: A Case Study of Wild Fish Populations in the Yangtze River.

In aquatic animals, gut microbial communities shift with host development and living environments. Understanding the mechanism by which the environment impacts the gut microbial communities of aquatic animals is crucial for assessing and managing aquatic ecosystem health. Here, we proposed a simplified framework for the colonization and dynamics of gut microbial communities. Then, to quantify the colonization of environmental microbes in the wild fish gut, the current study used 16S rRNA gene amplicon sequencing to obtain the structure of the water environmental microbial community and the gut microbial community in 10 wild fish populations (Leiocassis crassilabris, Leiocassis longirostris, Pelteobagrus vachelli, Silurus asotus, Siniperca chuatsi, Coilia brachygnathus, Aristichthys nobilis, Hypophthalmichthys molitrix, Coreius heterodon, and Xenocypris argentea) from the Wuhan section of the Yangtze River, and the relationship of these microbial communities was analyzed. The results identified that in most individuals, approximately 80% of gut microbes [at the operational taxonomic unit (OTU) level] were shared with the water environmental microbial community (except for individuals of Siniperca chuatsi and Coilia brachygnathus, approximately 74%). In approximately 80% of individuals, more than 95% of microbial species (OTUs) in the gut were transient. For fish species, more than 99% of microbial species (OTUs) that were introduced into the gut were transient. Nearly 79% of OTUs and 89% of species of water environmental microbes could be introduced into the fish gut. Driven by the introduction of transient microbes, fishes with similar feeding habits had similar gut microbial communities. The results indicated that for adult wild fishes, most gut microbiota were transient from the environmental microbiota that were related to fish feeding habits. We therefore encourage future research to focus on environmental microbiota monitoring and management to promote the better conservation of aquatic animals. It was important to note that, because of various influence factors, interspecific differences and individual variations on gut microbial community characteristics, the quantification of gut microbes in the current work was approximate rather than accurate. We hope that more comparable research could be conducted to outline the quantitative characteristics of the relationship between gut microbial community and aquatic environment microbial community as soon as possible.

Cryopreservation of germline stem cells in American paddlefish (Polyodon spathula).

Most sturgeon and paddlefish are critically endangered; therefore, effective measures to conserve these genetic resources are required. Cryopreservation of gonad tissues containing germline stem cells could be an effective strategy for long term preservation and restoration of fish species using germ cell transplantation procedure. The aim of this study was to develop an optimal procedure for long-term cryopreservation of American paddlefish gonads using a slow-freezing method. Through optimization of permeating cryoprotectants, nonpermeating cryoprotectants, and supplementation of proteins, gonad tissues were frozen with a cryomedium containing 1.3 M dimethyl sulfoxide, 0.1 M trehalose, and 10 % fetal bovine serum at a cooling rate of -1 °C/min. This method was also successfully utilized for the cryopreservation of Yangtze sturgeon testes. Viability of gonadal cells isolated from frozen gonads was not different from cells isolated from fresh gonadal tissues, while the number of gonadal cells dissociated from frozen gonads was less. Germline stem cells dissociated from long-term (1 year) cryopreserved gonads were labeled with PKH26 fluorescent dye and intraperitoneally transplanted into larvae of Yangtze sturgeon. The colonization of transplanted germline stem cells was confirmed by the presence of PKH26-labeled donor germline stem cells and donor-derived mtDNA sequence in the recipient gonads, providing evidence that germline stem cells from sturgeon and paddlefish gonads that had been preserved for a long period maintained their functions. The results of present study indicate the procedures used are effective for long-term preservation of critically endangered species within the Acipenseriformes order which can later be regenerated using surrogate broodstock technology.

Assessment of Yangtze sturgeon as recipient for the production of American paddlefish gametes through spermatogonia transplantation.

The Chinese paddlefish (Psephurus gladius), one of the world's largest freshwater fish, was last seen alive in 2003; they are presumed now to be extinct. In fish, germ cell transplantation is currently known as one of the most powerful assisted reproductive technologies for the conservation of endangered species. In the event that a Chinese paddlefish is unexpectedly caught in the near future, we aimed to develop an experimental strategy to produce paddlefish gametes in the gonads of surrogate sturgeon. Spermatogonia were collected from the testes of 2.5-year-old immature male American paddlefish (Polyodon spathula), the species most closely related to the Chinese paddlefish, by Percoll gradient centrifugation, and transplanted into the peritoneal cavity of Yangtze sturgeon (Acipenser dabryanus) larvae at 7-8 days post-hatch. At two months post-transplantation, donor-derived spermatogonia had efficiently colonized in the recipient gonads and proliferated. A PCR analysis developed to detect xenogenic donor-derived mtDNA sequences in recipient gonads revealed that American paddlefish germ cells survived for at least seven months after transplantation in the gonads of Yangtze sturgeon recipients. These results show that the somatic microenvironment of Yangtze sturgeon gonads was able to support the colonization, proliferation, and survival of xenogeneic germ cells from a different taxonomic family. This study provides key information that could lead to future restoration of Chinese paddlefish using germ cell transplantation.

Transcriptome analysis of ovarian maturation in a chondrostei Chinese sturgeon Acipenser sinensis.

Chinese sturgeon (Acipenser sinensis) with an evolutionary history of over 200 million years, has a long lifespan, and an extremely late and asynchronous sexual maturation (8-18 years for males and 14-26 years for females), resulting in the difficulty of mature adult culture. However, little is known about the regulatory mechanisms of the transition among ovarian maturation stages in the Chinese sturgeon. We performed de novo transcriptome sequencing of the Chinese sturgeon at different ovarian maturation stages (FII, FIII, and FIV). The number of differentially expressed genes (DEGs) between FII and FIII/FIV (33,517/34,217) was more than that between FIII and FIV (22,123), suggesting that the transition from FII to FIII/FIV is more important than that from FIII to FIV for ovarian maturation. The number of upregulated genes was more than that of the downregulated genes, suggesting that increased gene expression was involved in the transition from FII to FIII/FIV. The representative pathways of DEGs were steroid biosynthesis, fatty acid biosynthesis, fatty acid elongation, glycerolipid metabolism, biosynthesis of unsaturated fatty acid, and α-linolenic acid metabolism. The differential expressions from the transcriptome sequencing were validated with real-time reverse-transcription polymerase chain reaction. We also found 13 genes in sexual development, female sex determination, gonadal development, ovarian maturation, ovarian follicle development, and oocyte development pathways, which were differently expressed among fish at FII, FIII, and FIV. We suggest that enhanced synthesis of steroid, unsaturated fatty acid, and α-linolenic acid may contribute to ovarian maturation of the Chinese sturgeon. These potential determinants provide a glimpse of the molecular architecture of ovary development in sturgeons.

Metabolomics and gene expressions revealed the metabolic changes of lipid and amino acids and the related energetic mechanism in response to ovary development of Chinese sturgeon (Acipenser sinensis).

Captive breeding has been explored in Chinese sturgeon (Acipenser sinensis) for species protection. However, gonad development from stage II to IV of cultured female broodstocks is a handicap. This study aimed to explore the physiological and metabolic changes during the ovary development from stage II to IV of female Chinese sturgeon and the related energy regulatory mechanism, which may be helpful to address the developmental obstacle. The results showed that the oocyte volume increased and the muscle lipid content decreased with the ovary development. Ovarian RNA levels of most genes related to lipid and amino acid metabolism were higher in stage II and III than in stage IV. Serum contents of differential metabolites in arginine, cysteine, methionine, purine, tyrosine, lysine, valine, leucine and isoleucine metabolism pathways peaked at stage III, while the contents of sarcosine, alanine and histidine, as well as most oxylipins derived from fatty acids peaked at stage IV. These results indicated the more active amino acids, lipid metabolism, and energy dynamics of fish body in response to the high energy input of ovary developing from stage II to III, and the importance of alanine, histidine, taurine, folate and oxylipins for fish with ovary at stage IV.

Adjusting the Prerelease Gut Microbial Community by Diet Training to Improve the Postrelease Fitness of Captive-Bred Acipenser dabryanus.

As one of the most important tool for biodiversity restoration and endangered species conservation, reintroduction has been implemented worldwide. In reintroduction projects, prerelease conditioning could effectively increase postrelease fitness and survival by improving animals' adaptation to transformation from artificial to natural environments. However, how early-life diet training affects individuals' adaptation, fitness, and survival after release remains largely unknown. We hypothesized that early-life diet training would adjust the host's gut microbial community, the gut microbial community would influence the host's diet preference, and the host's diet preference would impact its adaptation to diet provision transformation and then determine postrelease fitness and survival. To verify this hypothesis, we investigated the growth characteristics and gut microbes of Yangtze sturgeon (Acipenser dabryanus) trained with natural and formula diets at both the prerelease and postrelease stages. The results showed that (1) the gut microbial communities of the individuals trained with a natural diet (i.e., natural diet group) and formula diet (i.e., formula diet group) evolved to the optimal status for their corresponding diet provisions, (2) the individuals in the natural diet group paid a lower cost (i.e., changed their gut microbial communities less) during diet transformation and release into the natural environment than did the individuals in the formula diet group, and (3) the gut microbes in the natural diet group better supported postrelease fitness and survival than did the gut microbes in the formula diet group. The results indicated that better prerelease diet training with more appropriate training diets and times could improve the reintroduction of Yangtze sturgeon by adjusting the prerelease gut microbial community. Because a relationship between diet (preference) and gut microbes is common in animals from insects (such as Drosophila melanogaster) to mammals (such as Homo sapiens), our hypothesis verified by the case study on Yangtze sturgeon applies to other animals. We therefore encourage future studies to identify optimal training diets and times for each species to best adjust its prerelease gut microbial community and then improve its postrelease fitness and survival in reintroduction projects.

Extinction of one of the world's largest freshwater fishes: Lessons for conserving the endangered Yangtze fauna.

The mega river ecosystem of the Yangtze River was once home to diverse aquatic megafauna but is increasingly affected by various anthropogenic stressors that have resulted in continuous loss of biodiversity, such as the probable extinction of Yangtze River Dolphin. The Chinese paddlefish, Psephurus gladius, was one of only two extant members of a relict lineage that was most diverse and widespread 34-75 million years ago. It is also one of the largest freshwater fish species, reaching up to 7 m in length. The Chinese paddlefish was once common in the Yangtze River, with c.25 t being harvested per annum during the 1970s. Populations have, however, declined drastically since the late 1970s as a result of overfishing and habitat fragmentation. Here, a basin-wide capture survey during 2017-2018 found 332 fish species, but did not find a single specimen of Chinese paddlefish. Furthermore, 140 historically reported fish species have not been found and most of them are considered highly endangered. Based on 210 sightings of Chinese paddlefish during the period 1981-2003, we estimated the timing of extinction to be by 2005, and no later than by 2010. In addition, the paddlefish probably became functionally extinct (i.e. it was unable to reproduce) by 1993, before it went extinct. It is likely that the lack of reproduction was among the major causes of extinction. As no individuals exist in captivity, and no living tissues are conserved for potential resurrection, the fish should be considered extinct according to the IUCN Red List criteria. The delayed extinction of Chinese paddlefish resulted from multiple threats, suggesting that optimizing conservation efforts on endangered Yangtze fauna is urgently needed.

Molecular characterization, expression pattern and evolution of nine suppressors of cytokine signaling (SOCS) gene in the swamp eel (Monopterus albus).

Suppressors of cytokine signaling (SOCS) family members have negative effects on cytokine signaling pathways involved in immunity, growth and development. Owing to their typical feature, they have been extensively studied in mammalians, but they have not offered systematic studies among teleosts. In the present study, nine SOCS family genes were identified in the swamp eel genome and analyzed regulation mechanisms of SOCS family members in swamp eels. The open reading frames of MaSOCS1a, MaSOCS1b, MaSOCS2, MaSOCS3a, MaSOCS3b, MaSOCS4, MaSOCS5, MaSOCS6 and MaSOCS7 were 663 bp, 603 bp, 717 bp, 618 bp, 645 bp, 1188 bp, 1488 bp, 1611 bp and 1998 bp and encoded 220, 238, 200, 205, 214, 395, 496, 536 and 655 amino acids, respectively. All SOCS proteins have no signal peptides. Multiple alignment revealed that MaSOCS family members possessed a typical conserved SOCS box and SH2 region. Phylogenetic analyses showed that all SOCS proteins were divided into two main clusters. Taken together with the similarity and identity of SOCS protein amino acids, these results indicated that MaSOCS family members shared conserved with other homologous genes, in which MaSOCS7 was more conserved. Further syntenic analysis confirmed the phylogenetic analysis results and annotation of SOCS protein, suggesting that MaSOCS5 shared a common ancestor gene with that of fish and humans. MaSOCS family members were constitutively expressed in a wide range of tissues with different levels. In particular, spleen and head kidneys play an important role in immune-related pathways. After Aeromonas veronii and polyinosinic-polycytidylic acid (poly I:C) challenge in the spleen and head kidney, MaSOCS family members exhibit different expression profiles. These expression patterns indicated that MaSOCS family members could make acute responses after pathogen invasion. Taken together, these results indicate that MaSOCS family members participate in the immune response against pathogens and offer a solid foundation for future studies of SOCS function.