Xiaofu Pan

The Sinocyclocheilus cavefish genome provides insights into cave adaptation

BackgroundAn emerging cavefish model, the cyprinid genus Sinocyclocheilus, is endemic to the massive southwestern karst area adjacent to the Qinghai-Tibetan Plateau of China. In order to understand whether orogeny influenced the evolution of these species, and how genomes change under isolation, especially in subterranean habitats, we performed whole-genome sequencing and comparative analyses of three species in this genus, S. grahami, S. rhinocerous and S. anshuiensis. These species are surface-dwelling, semi-cave-dwelling and cave-restricted, respectively.ResultsThe assembled genome sizes of S. grahami, S. rhinocerous and S. anshuiensis are 1.75 Gb, 1.73 Gb and 1.68 Gb, respectively. Divergence time and population history analyses of these species reveal that their speciation and population dynamics are correlated with the different stages of uplifting of the Qinghai-Tibetan Plateau. We carried out comparative analyses of these genomes and found that many genetic changes, such as gene loss (e.g. opsin genes), pseudogenes (e.g. crystallin genes), mutations (e.g. melanogenesis-related genes), deletions (e.g. scale-related genes) and down-regulation (e.g. circadian rhythm pathway genes), are possibly associated with the regressive features (such as eye degeneration, albinism, rudimentary scales and lack of circadian rhythms), and that some gene expansion (e.g. taste-related transcription factor gene) may point to the constructive features (such as enhanced taste buds) which evolved in these cave fishes.ConclusionAs the first report on cavefish genomes among distinct species in Sinocyclocheilus, our work provides not only insights into genetic mechanisms of cave adaptation, but also represents a fundamental resource for a better understanding of cavefish biology.

A new cave-dwelling loach, Triplophysa xichouensis sp. nov. (Teleostei Nemacheilidae) from Yunnan, China

A new cave-dwelling loach of the genus Triplophysa, T. xichouensis, is described from an outlet of a subterranean river in Xisa Town, Xichou County, Yunnan Province, China. It can be distinguished from its congeners by the following characters: dorsal-fin rays iii, 8; anal-fin rays ii, 6; pectoral-fin rays i, 9 or 10; pelvic-fin rays i, 5 or 6; branched caudal-fin rays 16(8+8); eyes highly degenerated to a very tiny black dot; dorsal-fin origin closer to snout tip than to caudal-fin base and anterior to vertical line of pelvic-fin origin; pectoral fin length about two-thirds the distance between pectoral-fin origin to pelvic-fin origin; caudal peduncle slender, its length about three times its depth; caudal fin emarginate; body smooth and scaleless; lateral line complete and straight; anterior chamber of air bladder wrapped in dumbbell-shaped bony capsule and the posterior one well developed, long, oval; intestine short, bending in zigzag shape behind stomach. A key for the cave-dwelling species of Triplophysa is provided. urn:lsid:zoobank.org:pub:9162FFB1-7911-47C3-AE50-6A00E9590327.

Threatened fishes of the world: Ptychobarbus chungtienensis Tsao 1964 (Cyprinidae)

Common name: Chungtien schizothoracin (English). Local name: Zhongdian Chongchunyu (Chinese). Conservation status: EN—China Species Red List, Vol. 1 Red List (Wang and Xie 2004). Identification: D III 7-8; A II 5; LL 100–110; pharyngeal teeth two rows, 3,4–4,3; one pair of barbels. Sides of body above lateral line is brown with a golden shine and densely covered with black speckles, sides below lateral line are yellowish and densely covered with black speckles, and the abdomen is silver. Illustration by Bao-Rong Wu. Distribution: Endemic to Zhongdian Plateau of Jinsha River drainage including Lake Bita, Lake Napa, Lake Shudu, Nayahe River, Xiaozhongdian River and Gezan River (Huang and Chen 1986; Chen 2010). Abundance: Since 1986, populations have declined or disappeared in most areas. Species is now extirpated from Lake Napa. Most remaining individuals are in Lake Bita with a few in Xiaozhongdian River and Naya River (Wang and Xie 2004). Habitat and ecology: Juveniles occur in the littoral zone of the lake and streams; the adults inhabit the middle and lower water layers of the lake. Reproduction: Individuals return from the lake to streams to spawn in June. Males dig pits with anal fin rays and lays eggs in the pit. Parental fishes spawn several times from July to September every year. Threats: Habitat degradation, water pollution, over-exploitation and exotic species. Most notably, Micropercops swinhonis invasion is a critical threat. Conservation actions: Lake Bita and primary forest around the lake have been well protected since 1984 by establishing Bitahai Provincial Natural Reserve. A population status monitoring of Chungtien Environ Biol Fish (2010) 89:1–2 DOI 10.1007/s10641-010-9654-5

Cryopreservation of sperm from Neolissochilus benasi

Cryopreservation of sperm from Neolissochilus benasi was studied in 2011. The effects of various cryoprotectants of different concentrations, dilution ratios of milt to extender, storage volume and thawing temperature on motility of post-thawing of spermatozoa were examined to optimize cryopreservation procedures. Semen was stored in liquid nitrogen in 1.8 mL cryovial for 24 h, and the intensity of sperm motility was measured before and after cryopreservation. Post-thawing motility of frozen sperm obtained with cryoprotectants 10% MeOH or 15% EG were higher than for others. The most effective dilution ratio of milt to extender is 1:7. The maximal storage volume is 60 μL of 1.8 mL cryovial and the optimal sperm equilibration period in the extender D-15+10% MeOH was between 10-60 min. Thawing was optimal in a 37 Degrees Celsius water bath. When fresh sperm motility is (62.33±2.05)%, this cryopreservation protocol resulted in frozen-thawed semen with 20%-30% motile. The overall effect is not ideal, and cannot achieve extensive application. Different breeding management of different ground protection may have contributed to this result. Therefore, it is necessary to reduce stress capture induced in management of parent fish and provide suitable forming conditions. In the ex situ conservation of rare fish the broodstocks management of males is as important as that for females and the key to obtaining high quality larval fish.

Establishment and characterization of a fibroblast-like cell line from Anabarilius grahami (Cypriniformes: Cyprinidae).

Though Yunnan province contains some 562 known species of fish, no cell lines from any of these have been made available to date. To protect germplasm resources and provide an effective tool in solving problems at cellular level of Anabarilius grahami, a fish endemic to Fuxian Lake, Yunnan, China, we established and characterized the major features of a continuous cell line (AGF II) from the caudal fin tissue of A. grahami. This AGF II cell line consists of fibroblast-like cells and has been subcultured more than 60 times over the course of a year. The cell line was maintained in DMEM/F12 supplemented with 10% FBS, with a cellular doubling time of 51.1 h. We continued with more experiments to optimize the culture and storage conditions, and found a variety of interesting results: cells could grow at temperature between 24 °C and 28 °C, with the optimal temperature of 28 °C. Likewise, the growth rate of A. grahami fin cells increased when the FBS proportion increased from 5% to 20%, with the optimal growth at the concentrations of 20% FBS; cells were able to grow in L-15 and DMEM/F12 with optimal growth at L-15; DMSO is a better cryoprotectant than Glycerol, EG and MeOH for AGFII cells with optimal concentration of 5% DMSO. Chromosome analysis also showed that the distribution of chromosome number varies from 38 to 52, with a modal peak at 48 chromosomes, accounting for 39.8% of all cells. Using the same primer pairs specific to mtDNA, the AGF II cell sequences obtained by PCR were identical to those from muscle tissues of A. grahami. Both chromosome analysis and PCR amplification confirmed the AGF II cells were from A. grahami, also indicating that that current long-term artificial propagation of A. grahami has been successful. Finally, we noted that when cells were transfected with pEYFP-N1 and pECFP-N1 plasmid, bright fluorescent signals were observed, suggesting that this cell line may be suitable for use in transfection and future gene expression studies.Though Yunnan province contains some 562 known species of fish, no cell lines from any of these have been made available to date. To protect germplasm resources and provide an effective tool in solving problems at cellular level of Anabarilius grahami, a fish endemic to Fuxian Lake, Yunnan, China, we established and characterized the major features of a continuous cell line (AGF II) from the caudal fin tissue of A. grahami. This AGF II cell line consists of fibroblast-like cells and has been subcultured more than 60 times over the course of a year. The cell line was maintained in DMEM/F12 supplemented with 10% FBS, with a cellular doubling time of 51.1 h. We continued with more experiments to optimize the culture and storage conditions, and found a variety of interesting results: cells could grow at temperature between 24 Degrees Celsius and 28 Degrees Celsius, with the optimal temperature of 28 Degrees Celsius. Likewise, the growth rate of A. grahami fin cells increased when the FBS proportion increased from 5% to 20%, with the optimal growth at the concentrations of 20% FBS; cells were able to grow in L-15 and DMEM/F12 with optimal growth at L-15; DMSO is a better cryoprotectant than Glycerol, EG and MeOH for AGFII cells with optimal concentration of 5% DMSO. Chromosome analysis also showed that the distribution of chromosome number varies from 38 to 52, with a modal peak at 48 chromosomes, accounting for 39.8% of all cells. Using the same primer pairs specific to mtDNA, the AGF II cell sequences obtained by PCR were identical to those from muscle tissues of A. grahami. Both chromosome analysis and PCR amplification confirmed the AGF II cells were from A. grahami, also indicating that that current long-term artificial propagation of A. grahami has been successful. Finally, we noted that when cells were transfected with pEYFP-N1 and pECFP-N1 plasmid, bright fluorescent signals were observed, suggesting that this cell line may be suitable for use in transfection and future gene expression studies.

Broodstocks management, fecundity and the relationship between egg size and embryo survival ability of Sinocyclocheilus grahami

Broodstock management, fecundity and egg size of the golden-line barbel Sinocyclocheilus grahami were studied from 2007 to 2010. The induced spawning success of female S. grahami was 25.2% in 2007 and dramatically increased to 91.3% in 2010. The nucleus deviation rate and hatching success were 61.5% and 30.4%, respectively, in 2007 and increased to 85.2% and 44.5%, respectively, in 2010. Providing nutritious food for broodstocks of S. grahami can ensure optimum breeding conditions as well as high-quality eggs and fingerlings. There also seems to be a relationship between absolute fecundity (F) and standard length (SL), as described by the power-exponent function F=0.0004826SL(3.166) (R(2)=0.6424,P<0.05). The average of number of spawn egg was 2118.4+/-899.1 from 2007 to 2010, the average of absolute fecundity was 2402.9+/-881.9 from 2007 to 2010, and the average of relative fecundity was 70.4+/-20.8 from 2007 to 2010. The number of spawn egg, absolute fecundity and relative fecundity increased in individuals with a longer body length. Additionally, egg size contributed to the survival rate of embryos. The different batches reached an asymptotic, low or stable embryonic mortality during the first two days; the balance was broken in the subsequent seven days, as high embryonic mortality was observed in smaller eggs. The mortality of embryos from eggs larger than 2.0 mm was, contrastingly, rather stable. Embryos from bigger eggs have stronger survival potential, as bigger eggs can provide more energy and thus, a more favorable environment for early development.

Artificial Fertilization and Embryonic Development of Sinocyclocheilus grahami(Regan) from Dianchi Lake, China

Sinocyclocheilus grahami, a troglophile species, is a cyprinidae fish endemic to Dianchi Lake, Yunnan, China. We have attempted the artificial propagation of S. grahami for several years and recorded the success of S. grahami artificial fertilization for six years and found that egg quality improved with enhanced broodstock management, with negligible effects on S. grahami artificial fertilization resulting from the injection of LHRH-A2. In this study, we also provide a comprehensive staging series of this species. The eggs have relatively large yolks (1.8–2.2 mm) and are strongly adhesive. The egg envelope is thick and transparent. The embryonic development of S. grahami can be divided into thirty stages based on morphological features visible using light microscopy. Stages can be further grouped into six periods: zygote, cleavage, blastula, gastrula, segmentation and hatching. Embryos hatched 144.0–168.0 hours after fertilization (HPF). S. grahami embryonic development is generally similar to that of zebrafish (Danio rerio) and Anabarilius grahami but is relatively slower.

滇池金线鲃( Sinocyclocheilus grahami )与抚仙金线鲃( S. tingi )肌肉营养成分初步分析

In this study, we investigated the nutritive composition in the muscle of Sinocyclocheilus grahami and S. tingi . The contents of crude protein in the fresh muscle of S. grahamiand S. tingican be described as being 21.7% and 20.6%, crude fat were 3.43% and 2.66%, and total amino acid were 19.23% and 17.67%, respectively. Essential amino acids accounted for 44.08% and 43.69% of total amino acids of S. grahami and S. tingi, respectively. The values of essential amino acid index (EAAI) of the two species were 70.00% and 65.99%, respectively, with S. grahami being better than S. tingi. Ultimately, the amino acid composition of S. grahamiand S. tingi muscle tissue met the standards of the Food and Agriculture Organization of the United Nations as well as those of the World Health Organization (FAO/WHO). According to the nutritive evaluation in amino acid score (AAS), the first limited amino acids of both fish were cystine+methionine. Compared with other commercial fish, the nutrition value of the muscle of Sinocyclocheilusi> fish can generally be considered as being better.

[Sinocyclocheilus xichouensis, a new species of golden-line fish from the Red River drainage in Yunnan, China (Teleostei: Cypriniformes)].

In the present study, a new species of the genus Sinocyclocheilus Fang 1936, Sinocyclocheilus xichouensis, was described from the Ganhaizi tributary of Chouyang River, Red River drainage, located in southeast Yunnan, China. This species has normal eyes and a strong dorsal spine with serrations on the lower 3/5 part. In general, this species is similar in morphology to S. macrophthalmus, S. guishanensis, S. angustiporus, S. lateristritus, S. qiubeiensis, S. grahami, S. qujingensis, S. maculatus and S. purpureus distributed in the Nanpanjiang River, and S. qiubeiensis distributed in the Red River. It is distinguished from S. macrophthalmus by possessing fewer than 9 gill rakers. However, S. xichouensis can be distinguished from S. guishanensis by its interorbital width/SL of 8.1~9.9%, rostral barbels extended to posterior margin of eye and maxillary barbels extended to posterior preopercular. It is distinct from S. lateristritus by dorsal-fin origin opposite of pelvic fin origin, with no a black stripe along the lateral line. It is distinguished from S. grahami by 74~88 lateral-line scales, 20 scale rows above the lateral line, and 16 scale rows below the lateral line. It is distinguished from S. qujingensis by a curved lateral line, 74-88 lateral-line scales, 48 circumpeduncular scales. It can be distinguished from S. yimenensis by the ratio of predorsal length, dorsal-fin base length, preanal length, anal fin length, prepectoral length, caudal-peduncle length and lower jaw length to SL, 47.1%-53.7%, 12.8%-15.8%, 66.0%-71.0%, 13.7%-17.1%, 26.0%-29.5%, 19.3%-24.7%, 4.7%-7.0%, respectively. It is distinguished from S. maculatus and S. purpureus by possession of lateral line and scaled body and distinguished from S. angustiporus and S. qiubeiensis by 35~39 predorsal scales, 6 gill rakers, and interorbital width/SL of 8.1%-9.9%.

Selection of Reliable Reference Genes for Quantitative Real-Time PCR in Golden-Line Barbell (Sinocyclocheilus grahami) During Juvenile and Adult Stages

In order to obtain reliable results of gene expression, appropriate internal control gene(s) were required for normalization before real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Here, we selected eight candidate housekeeping genes as the potential reference genes in normalizing qRT-PCR data in ten tissues of the juvenile and adult stages of a naturally tetraploid cyprinid fish Sinocyclocheilus grahami. Before qRT-PCR, appropriate primers were designed and compared for distinguishing different copies in some duplicate genes. Candidate reference genes were evaluated for their stability using online software RefFinder, which is integrated by four normal software of the comparative delta-CT, BestKeeper, NormFinder and GeNorm. According to the rankings produced by these analyses, Eef2, ACTB and G6PD were the most stable reference genes as internal controls for qRT-PCR in juvenile, while B2M, Eef2 and RPL17 were shown to be most appropriate in adult fish. All the analyses show that GAPDH was the least stable expression gene which would be unsuitable as reference gene at both stages. This is the first report about reference gene selection in Sinocyclocheilus and we think that is beneficial for the future gene expression studies in golden-line barbel.