Expansion and contraction of lake basin shape the genetic structure of Sinocyclocheilus (Osteichthyes: Cypriniformes: Cyprinidae) populations in Central Yunnan, China.

Geological events can strongly affect the genetic structures and differentiation of fish populations. Especially, as an endemic fish of the genus Sinocyclocheilus in the Yunnan-Guizhou Plateau, the effects of key geological events on the distributions and genetic structures remain poorly understood. Examining the phylogeographic patterns of Sinocyclocheilus fishes can be useful for elucidating the spatio-temporal dynamics of their population size, dispersal history and extent of geographical isolation, thereby providing a theoretical basis for their protection. Here, we used single nucleotide polymorphisms (SNP) method to investigate the phylogeographic patterns of Sinocyclocheilus fishes. Our analysis supports the endemicity of Sinocyclocheilus, but the samples of different regions of Sinocyclocheilus contain multiple ancestral components, which displayed more admixed and diversified genetic components, this may be due to the polymorphism of the ancestors themselves, or gene infiltration caused by hybridization between adjacent species of Sinocyclocheilus. We estimate that the most recent common ancestor (MRCA) of Sinocyclocheilus fish in the Central Yunnan Basin at approximately 3.75~3.11 Ma, and infer that the evolution of Sinocyclocheilus in the central Yunnan Basin is closely related to the formation of plateau lakes (around 4.0~0.02 Ma), and identifies the formation of Dianchi Lake and Fuxian Lake as key geological events shaping Sinocyclocheilus population structure. It is also the first time to prove that the altitude change has a great influence on the genetic variation among the populations of Sinocyclocheilus.

Decreased COVID-related adaptive behavior and increased negative affect: A multivariate latent growth curve model.

Data from a longitudinal questionnaire investigation of three time waves were used to investigate affective and behavioral changes and their covariant relationship among Chinese general population during the COVID-19 pandemic from March to May 2020. 145 participants aging from 15 to 63 completed three waves of survey. Latent growth curve analyses found that negative affect gradually increased as the pandemic continued. A faster increase in negative affect was related to a greater decrease in adaptive behavior and faster increase in non-adaptive behavior. A higher initial level of negative affect was related to a slower increase in non-adaptive behavior.

Construction of a chromosome-level genome assembly for genome-wide identification of growth-related quantitative trait loci in Sinocyclocheilus grahami (Cypriniformes, Cyprinidae).

The Dianchi golden-line barbel, Sinocyclocheilus grahami (Regan, 1904), is one of the "Four Famous Fishes" of Yunnan Province, China. Given its economic value, this species has been artificially bred successfully since 2007, with a nationally selected breed (" S. grahami, Bayou No. 1") certified in 2018. For the future utilization of this species, its growth rate, disease resistance, and wild adaptability need to be improved, which could be achieved with the help of molecular marker-assisted selection (MAS). In the current study, we constructed the first chromosome-level genome of S. grahami, assembled 48 pseudo-chromosomes, and obtained a genome assembly of 1.49 Gb. We also performed QTL-seq analysis of S. grahami using the highest and lowest bulks (i.e., largest and smallest size) in both a sibling and random population. We screened two quantitative trait loci (QTLs) (Chr3, 14.9-39.1 Mb and Chr17, 4.1-27.4 Mb) as the major growth-related locations. Several candidate genes (e.g., map2k5, stat1, phf21a, sox6, and smad6) were also identified, with functions related to growth, such as cell differentiation, neuronal development, skeletal muscle development, chondrogenesis, and immunity. These results built a solid foundation for in-depth MAS studies on the growth traits of S. grahami.

Restocking of Anabarilius grahami in Lake Fuxian, Southwest China: morphological and genetic effects.

The restocking of the endangered Kanglang white minnow ( Anabarilius grahami) in Lake Fuxian, China, has been conducted for 13 years. However, few studies have reported on the effectiveness of the captive breeding and release of this species. Here, we investigated variations in morphology, including body shape and skeletal deformities, and genetic features among hatchery-born and recaptured A. grahami from Lake Fuxian. Results showed that current hatchery-reared fish displayed a stubbier body shape than their wild conspecifics from the 1980s. Furthermore, high skeletal deformity ratios were found in two aquafarms (Luchong, 50%; Haikou, 45.2%), and the release of malformed fish elevated the skeletal deformity rate of wild stocks found near the Lake Fuxian release sites (west coast, 19.0%; east coast, 12.5%). Based on variations in the cytochrome b (cyt b) gene, existing A. grahami populations showed relatively high haplotype diversity and low nucleotide diversity. Hatchery populations exhibited reduced genetic variations based on microsatellite markers and reintroduction led to markedly lower genetic diversity around the west coast release sites of Lake Fuxian. Analysis of molecular variance (AMOVA) of cyt b and microsatellite analysis showed that the greatest genetic variations were found within populations, and genetic distance and Bayesian clustering analysis showed that the 14 populations clustered into one group. Based on morphological and genetic tests, we discuss corresponding recommendations, including release size, feed formulations, breeding strategies, and release tags, to minimize potential risks and improve hatchery practices for better restocking of this species.

Effects of osmotic pressure, temperature and stocking density on survival and sexual reproduction of Craspedacusta sowerbii.

The effects of osmotic pressure, temperature and stocking density on medusae survival of Craspedacusta sowerbii were examined. The medusae were shown to be sensitive to the variations of osmotic pressure. And the survival time was <90 h at 34 mOsm/L and it declined rapidly with rising osmotic pressure. The peak survival time of >200 h was recorded at 0.2 mOsm/L. Comparing with 27℃ and 32℃ treatments, 23℃ treatment yielded lower activities at a range of 8-13/min. However, there was a longer survival time. A non-linear relationship existed between survival time and stocking density. Lower density resulted in larger body size. And sexual reproduction resumed after breeding for >22 days. Newly-formed polyps and medusae appeared subsequently but only in the higher-density groups of 10, 14 and 18 ind./L. It suggested that the number of newly-formed polyps and medusae was highly dependent on stocking density. That is, a higher stocking density produced more organisms. However, newly-formed medusae died within one month and none grew a diameter of >5 mm.

[Use of alizarin red S and alizarin complexone for immersion marking Sinocyclocheilus grahami].

In the present study, we examined the potential uses of Alizarin complexone and Alizarin red S to mark S. grahami larvae and juveniles. Individuals of different ages (6 days, 20 days, 90 days) were immersed in four concentrations of a solution of each chemical (50, 100, 150 and 200 mg/L) for different periods of time (4-36 h). Marked individuals were euthanized 4-27 days after immersion, and the lapillus was removed and used to determine mark quality. After marking with 50-150 mg/L ALC and ARS, violet marks were visible under normal light, and scarlet fluorescent marks were visible under 510-560 nm green light. The clear marks under normal light were recorded, and a 100% survival rate was guaranteed under the following conditions: 6 day old larvae immersed in 50 mg/L ALC or ARS for 8h; 20 day old larvae immersed in 50 mg/L ALC or 100 mg/L ARS for 24h; 90 day old juveniles immersed in 100mg/L ALC or 150 mg/L ARS for 24 h.

[Overview of the artificial enhancement and release of endemic freshwater fish in China].

Due to declining fishery resources and the growing development of conservation aquaculture, artificial freshwater fish enhancement and releasing have begun to replace traditional means of recovering endemic and rare fish populations. Artificial proliferation can be beneficial both to endemic fish conservation and technical bottleneck breakthroughs. This overview presents a review of the latest research and the underlying principles behind the conservation implementation processes, as well as the research status of artificial enhancement and release of endangered freshwater fish species in China, such as Mylopharyngodon piceus, Ctenopharyngodon idellus, Hypophthalmichthys molitrix, H. nobilis, Acipenser sinensis, Myxocyprinus asiaticus, and Sinocyclocheilus grahami. The overview also presents evolutionarily significant units, sperm and egg quality, and cryopreservation technologies and cell cultures used in artificial enhancement and release, which help standardize genetic management and minimize the genetic differences between hatched and wild populations. Monitoring fish from cultivation to release is essential to evaluating wild population recovery and adjusting recovery plans. Moreover, the remaining problems of artificial releases are discussed in-depth, touching on issues such as the limitations of domestic hatching, the base number of wild populations necessary to the environment, the proper size at which to release juveniles' into the environment, the geographic confusion of populations, the contradictions in commercial fish selection and fish conservation, and "exotic species" invasion.

[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%.

[Artificial propagation and embryonic development of Neolissochilus benasi].

From 2009 to 2011, luteinizing hormone releasing hormone analogue (LHRH-A2) mixed with domperidon (DOM) was successfully applied during the artificial propagation of Neolissochilus benasi. Totally, 60 females and 100 males were injected with the hormone mixture, resulting in 47 (78.3%) females and 92 (92.0%) males being successfully spawned. A total of 1,986-5 854 eggs were spawned per female with an egg diameter varying between 2.2-2.8 mm, and an average nucleus deviation rate of 73.2%. Sperm density, vitality and life span were 16.32±2.89×10(9)/mL, 60.6±3.2% and 70.2±5.3 s, respectively. On the whole, the embryonic development of N. benasi was similar to that of zebra fish-albeit relatively slower-lasting approximately 120 hours. The development itself can be divided into six discrete stages: zygote, cleavage, blastula, gastrula, segmentation and hatching. Results showed that the average hatching rate was 32.4%, with 86.5% of larvae surviving 45 days after hatching. During embryonic development, deformities commonly occurred on the mouth, chest, ocular region, especially in the spinal column. To try to attempt improving future breeding efforts, we provided a survey of the embryonic developmental difficulties of N. benasi using LHRH-A2 followed by several potential solutions, including providing suitable breeding conditions and minimizing capture stresses.

[Analysis of the nutritional components in muscle of Sinocyclocheilus grahami and S. tingi].

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. grahami and S. tingi can 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. grahami and 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 Sinocyclocheilus fish can generally be considered as being better.

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 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.

[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.

[Fishery resource protection by artificial propagation in hydroelectric development: Lixianjiang River drainage in Yunnan as an example].

Hydroelectric developments can result in a number of negative environmental consequences. Conservation aquaculture is a branch of science derived from conservation and population recovery studies on endangered fishes. Here we discuss the impacts on fishes caused by hydropower projects in Lixianjiang, and evaluate effects and problems on the propagation of Parazacco spilurus, Hemibagrus pluriradiatus, Neolissochilus benasi and Semilabeo obscurus. A successful propagation project includes foraging ecology in fields, pond cultivation, juvenile fish raising, prevention and curing on fish disease, genetic management, artificial releasing and population monitoring. Artificial propagation is the practicable act on genetic intercommunication, preventing population deterioration for fishes in upper and lower reaches of the dam. For long-term planning, fish stocks are not suitable for many kind of fishes, but can prevent fishes from going extinct in the wild. Basic data collection on fish ecology, parent fish hunting, prevention on fish disease are the most important factors on artificial propagation. Strengthening the genetic management of stock population for keeping a higher genetic diversity can increase the success of stock enhancement. The works on Lixianjiang provide a new model for river fish protection. To make sure the complicated project works well, project plans, commission contracts, base line monitoring and techniques on artificial reproduction must be considered early. Last, fishery conservation should be considered alongside location development.

[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.

[Association of the Pro12Ala polymorphism in peroxisome proliferators activated receptor-gamma gene with rheumatoid arthritis in Sichuan Province of China].

OBJECTIVE: To investigate the association of the Pro12Ala variant in peroxisome proliferators-activated receptor gamma (PPAR gamma) gene with rheumatoid arthritis. METHODS: The genotypes of the Pro12Ala variant in the PPAR gamma gene were determined by polymerase chain reaction-restriction fragment length polymorphism in 421 unrelated subjects of the Han population in the Sichuan Province of China, including 207 subjects with rheumatoid arthritis and 214 subjects without the disease. The clinical data were also collected and analyzed. RESULTS: The allele frequencies in the case and control groups were 98.79%, 95.79% for allele P and 1.21%, 4.21% for allele A; the genotype frequencies were 97.58% and 91.59% for PP, 2.42% and 8.41% for PA, and 0 for AA. The A allele frequency was much lower in the RA group than that in the control group. CONCLUSION: The above data showed that the Pro12Ala variant of the PPAR gamma was associated with rheumatoid arthritis. The A allele might be a protective factor for RA. The Pro12Ala polymorphism in the PPAR gamma gene in Sichuan Han population is similar to that in other populations in China, but different from that in European and American populations.