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.

Comparative Analysis of the Expression of Resistance-Related Genes Respond to the Diversity Foliar Pathogens of Pseudostellaria heterophylla.

The foliar disease, which is the primary complex disease of Pseudostellaria heterophylla, can be caused by multiple co-infecting pathogens, resulting in a significant reduction in yield. However, there is a lack of research on the relationship between co-infection of various pathogens and the response of resistance-related genes in P. heterophylla. Through the use of 18S rDNA sequencing and pathogenicity testing, it has been determined that Fusarium oxysporum, Alternaria alternata, Arcopilus aureus, Botrytis cinerea, Nemania diffusa, Whalleya microplaca, and Cladosporium cladosporioides are co-infecting pathogens responsible for foliar diseases in P. heterophylla. Furthermore, the qRT-PCR analysis revealed that F. oxysporum, A. alternata, B. cinerea, A. aureus, N. diffusa, Schizophyllum commune, C. cladosporioides, and Coprinellus xanthothrix upregulated ten, two, three, four, seven, thirteen, five, one, and six resistance-related genes, respectively. These findings suggest that a total of 22 resistance-related genes were implicated in the response to diverse fungi, and the magnitude and frequency of induction of resistance-related genes varied considerably among the different fungi. The aforementioned gene associated with resistance was found to be implicated in the response to multiple fungi, including PhPRP1, PhBDRN15, PhBDRN11, and PhBDRN3, which were found to be involved in the resistance response to nine, five, four, and four fungi, respectively. The findings indicate that the PhPRP1, PhBDRN15, PhBDRN11, and PhBDRN3 genes exhibit a broad-spectrum resistance to various fungi. Furthermore, the avirulence fungi C. xanthothrix, which is known to affect P. heterophylla, was found to prime a wide range of resistance responses in P. heterophylla, thereby enhancing its disease resistance. This study provided insight into the management strategies for foliar diseases of P. heterophylla and new genetic materials for disease-resistant breeding.

Epinephelus coioides PCSK9 affect the infection of SGIV by regulating the innate immune response.

Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) in mammals is a multifunctional protein. In this study, PCSK9 of marine fish Epinephelus coioides was characterized. The full-length cDNA of E. coioides PCSK9 was 2458 bp in length containing 185 bp 5' UTR, 263 bp 3' UTR and 2010 bp open reading frame (ORF) encoding 669 amino acids with the predicted molecular weight of 71 kDa and the theoretical PI of 6.6. Similar to other members of PCSK9 family, E. coioides PCSK9 has three conserved domains: Inhibitor_ I9 super family, Peptidases_ S8_ PCSK9_ Proteinase K_ like, and PCSK9_ C-CRD super family. E. coioides PCSK9 mRNA could be detected in all the tissues examined by real-time quantitative PCR, with the highest expression in the brain, followed by skin, trunk kidney, head kidney, intestine, blood, liver, spleen, gill, muscle and heart. E. coioides PCSK9 was distributed in both the cytoplasm and nucleus. The expression of E. coioides PCSK9 was significantly upregulated during Singapore grouper iridovirus (SGIV) infection. Upregulated PCSK9 could significantly affect the activities of nuclear factor kappaB (NF-κB) promoter, SGIV-induced apoptosis, and the expressions of the key SGIV genes (ICP18, LITAT, MCP, and VP19) and the E. coioides proinflammatory factors (IL-6, IL-1ß, IL-8, and TNF-α). The results illustrated that E. coioides PCSK9 might be involved in the pathogen infection by regulating the innate immune response.

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.

[Dissection of seed development of Gastrodia elata at different temperatures].

The study is aimed to create seed materials and dissect the molecular mechanism of sexual propagation of Gastrodia elata. In this research, thirteen characteristics of flowers, flower stem, fruits, seeds and embryo of G.elata f. glauca and G.elata f. elata after bolting at room temperature(RT) and constant temperature(CT, 22 ℃) were determined. It was found that the constant temperature condition could prolong the bolting duration of G.elata and increased the number of flowers, while the variety of G.elata only affected the bolting duration, but had no effect on the number of flowers, and the G.elata f. elata was more likely to bolting than the G.elata f. glauca. The variety of G.elata was the main factor affecting the time of dehiscent fruit of G.elata, the temperature was the main factor affecting the fruits number and fruits diameter, and the constant temperature was more conducive to the fruits shape of G.elata than the room temperature. There was no significant difference in seed phenotype of G.elata varieties, but the seed embryo of G.elata seeds cultivated at constant temperature was fuller than that of G.elata cultivated at room temperature, and temperature had less influence on the seed phenotype of G.elata. But it was interesting to find that temperature and varieties had greater influence on the seed embryo of G.elata, constant temperature cultivation was more conducive to the formation of seed embryo of G.elata, and more the seed embryo of G.elata f. elata was easier to form than the seed embryo of G.elata f. glauca. However, the development of seeds and embryos of G.elata was significantly affected, and the development of seeds and embryos of G.elata f. glauca was more sensitive to temperature than G.elata f. elata. The research suggested that it is advisable for G.elata to produce seed materials by bolting at constant temperature(22 ℃).

Alantolactone mitigates renal injury induced by diabetes via inhibition of high glucose-mediated inflammatory response and macrophage infiltration.

Background: Inflammatory response plays a crucial role in the occurrence and development of diabetic nephropathy (DN). Drugs that carry anti-inflammatory effects have the potential to treat diabetic nephropathy. It's reported that alantolactone (ALA), a natural product, has a variety of pharmacological effects against inflammation and oxidation. However, the specific effects of alantolactone on DN and the mechanisms underlying alantolactone remain elusive. Therefore, the present study aimed to probe whether ALA could mitigate inflammation as mediated by high glucose (HG) in NRK-52E cells and reduce renal injury caused by diabetic nephropathy.Materials and methods: The anti-inflammatory effect of ALA was evaluated in the present study using ELISA and RT-qPCR. Western blot and macrophage adhesion assay were then performed to confirm anti-macrophage adhesion and the protein expression of cell adhesion molecules. Finally, the effect of ALA and its underlying mechanism was evaluated in vivo.Results: Results showed that ALA curbed HG-stimulated expression of macrophage adhesion and pro-inflammatory cytokines in renal NRK-52E cells. In addition, both pro-inflammatory cytokines and NF-kappa B witnessed reduced expression or activity in oral administration with ALA in vivo, thus inhibiting the increase of serum creatinine and urea nitrogen (BUN) levels. This in consequence ameliorated fibrosis and stemmed pathological worsening of diabetic renal tissues.Conclusions: These findings suggest that ALA may hold promise in the treatment of DN, and importantly, the anti-inflammatory system may prove to be a new strategy to treat human DN.

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.

[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.