Abiotic and Biotic Influences on the Movement of Reintroduced Chinese Giant Salamanders (Andrias davidianus) in Two Montane Rivers.

PDF HTML阅读 XML下载 导出引用 引用提醒 大鲵细胞与大鲵虹彩病毒的微载体规模化培养工艺及优化 DOI: 作者: 作者单位: 1. 华中农业大学水产学院 水生动物医学系, 湖北 武汉 430070;2. 中国水产科学研究院 长江水产研究所, 湖北 武汉 430223 作者简介: 贾路路(1991-),女,硕士,从事水产动物医学研究.E-mail:1525825924@qq.com 通讯作者: 中图分类号: S941 基金项目: 公益性行业（农业）科研专项（201203086-05）. Technologies for large-scale cultivation of giant salamander cells and iridovirus by the Cytodex 3 microcarrier Author: Affiliation: 1. Department of Aquatic Animal Medicine, College of Fisheries, Huazhong Agricultural University, Wuhan 430070, China;2. Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan 430223, China Fund Project: 摘要 | 图/表 | 访问统计 | 参考文献 | 相似文献 | 引证文献 | 资源附件 | 文章评论 摘要:利用Cytodex 3微载体悬浮培养系统规模化培养大鲵肌肉细胞（GSM）和大鲵虹彩病毒（GSIV），研究了微载体培养GSM细胞的形态和增殖特性，同时测定了病毒在培养系统中的增殖动态相关指标。结果显示，在Cytodex 3微载体培养系统中，将GSM细胞在贴壁期以转速30 r/min，每静置40 min搅拌2 min的方式间歇搅拌，10 h后贴壁率可达95%，培养基中最适血清浓度为10%，最适微载体浓度为2 g/L，最适细胞初始接种密度为1.2×105 cells/mL；增殖期以25 r/min的连续搅拌方式可以达到最佳的细胞生长效能。倒置显微镜与扫描电镜观察结果显示，GSM细胞呈长梭形，紧密贴附在Cytodex 3微载体上，生长良好。采用优化的工艺条件培养GSM细胞，以感染复数（MOI）为0.5的剂量接种GSIV至规模化培养的GSM细胞，48 h后GSM细胞出现典型的细胞病变效应，72 h病毒滴度达到最高TCID50=10-8.50±0.20/mL。本研究为大鲵虹彩病毒病疫苗的规模化生产工艺研究奠定了前期基础。 Abstract:The Chinese giant salamander , a member of the family Cryptobranchidae, is the largest extant amphibian species in the world, which is valued for food, medicine, and research on animal evolution and biodiversity because of its unique phylogenetic position and physiological features. Within the last decade, Chinese giant salamander farming has increased rapidly in China. However, with the rapid development of Chinese giant salamander aquaculture, a severe epizootic recently occurred in cultured Chinese giant salamanders in Hubei, Hunan, Sichuan, Shannxi, and Zhejiang Provinces of China, causing tremendous economic losses. The causative pathogen has been identified as the giant salamander iridovirus (GSIV). At present, no effective methods are available for the control of the disease. Immunoprophylaxis is considered the best measure in controlling viral diseases in aquatic animals, and the large-scale cultivation technology of giant salamander cells and GSIV are of significance for the immunoprophylaxis of the disease. In this study, by using Cytodex 3 microcarriers to culture giant salamander muscle cells (GSM) and GSIV at a large scale, the GSM cell morphology, proliferative characterization, and GSIV growth dynamics were investigated. The results showed that the attachment efficacy reached 95% after 10 h of cultivation with intermittent agitation of 30 r/min for 2 min followed by 40 min still culture during the cell attachment period in the Cytodex 3 microcarrier culture system. The optimal culture conditions are as follows:serum concentration 5%, microcarrier concentration 2 g/L, and initial inoculation cell density of 1.2×105 cells/mL. During the cell growth period, the continuous stirring speed was 25 r/min. Under inverted microscopy and electron microscopy, the GSM cells on the Cytodex 3 microcarriers were long, spindle-shaped, and well adhered. After infection with GSIV at a multiplicity of infection of 0.5, the GSM cells on Cytodex 3 microcarriers showed the typical cytopathic effect at 48 h post infection, and the highest virus titer (TCID50/mL) of 10-8.50±0.20/mL was reached at 72 h post infection. This study established a solid foundation for further investigation on the large-scale technologies of GSIV vaccine production in the future. 参考文献 相似文献 引证文献

The Chinese giant salamander (<i>Andrias davidianus</i>), the American giant salamander (<i>Cryptobranchus alleganiensis</i>), and the Japanese giant salamander (<i>A. japonicum</i>) are precious aquatic protected animals that currently exist in the world. The latter two cannot be traded locally, whether wild or cultivated, and can only be protected, so they have not formed an industry. However, China allows the second generation of artificially bred offspring giant salamander to be traded and has already formed a factory scale breeding. The Chinese giant salamander has high economic value in food, health, medicine, beauty and other fields. In recent years, its artificial breeding industry has been widely developed in China and has a certain industrial scale. However, there are also many problems in the development of the giant salamander industry. In order to promote the high-quality development of the giant salamander industry and improve its quality, this article conducts research on the giant salamander industry, identifies existing problems, and provides suggestions. Firstly, this article analyzes the problems in the development process of giant salamanders from multiple perspectives, including management, service, market, research and development, and brand awareness. Secondly, corresponding industrial development strategies are proposed, including increasing regulatory efforts, enhancing service awareness, building market order, expanding sales channels, increasing research and development investment, reducing disease occurrence, extending the industrial chain, and strengthening brand awareness. Finally, the prospects for the development of the giant salamander industry are discussed.

The purpose of this study was to determine whether limited occurrence data for highly threatened species can provide useful spatial information to inform conservation. The study was conducted across central and southern China. We developed a habitat suitability model for the Critically Endangered Chinese giant salamander (Andrias davidianus) based on one biotic and three abiotic parameters from single‐site locality records, which represent the only relevant environmental data available for this species. We then validated model quality by testing whether increased percentage of predicted suitable habitat at the county level correlated with independent data on giant salamander presence. We randomly selected 48 counties containing historical records which were distinct from, and independent of, the single‐site records used to develop the model, and 47 additional counties containing >50% predicted suitable habitat. We interviewed 2,812 respondents near potential giant salamander habitat across these counties and tested for differences in respondent giant salamander reports between counties selected using each method. Our model predicts that suitable giant salamander habitat is found widely across central and southern China, with counties containing ≥50% predicted suitable habitat distributed in 13 provinces. Counties with historical records contain significantly more predicted suitable habitat than counties without historical records. There are no statistical differences in any patterns of respondent giant salamander reports in surveyed counties selected from our model compared with the areas of known historical giant salamander distribution. A Chinese giant salamander habitat suitability model with strong predictive power can be derived from the restricted range of environmental variables associated with limited available presence‐only occurrence records, constituting a cost‐effective strategy to guide spatial allocation of conservation planning. Few reported sightings were recent, however, with most being over 20 years old, so that identification of areas of suitable habitat does not necessarily indicate continued survival of the species at these locations.

Protective immunity of a Pichia pastoris expressed recombinant iridovirus major capsid protein in the Chinese giant salamander, Andrias davidianus

Draft genome sequencing and annotation of a low-virulence Morganella morganii strain CQ-M7, a multidrug-resistant isolate from the giant salamander in China

Cloning, sequence analysis and expression profiles of Toll-like receptor 7 from Chinese giant salamander Andrias davidianus.

Immunological responses and protection in Chinese giant salamander Andrias davidianus immunized with inactivated iridovirus

Chinese giant salamander(Andrias davidianus) is a rare and endangered endemic amphibian caudata in China.Chinese giant salamander sperm was cryopreserved in liquid nitrogen or stored at 0—4℃ on a short-term basis.The objective was to establish reliable technologies for the conservation of this endangered species and for artificial assisted reproduction in Chinese giant salamander farms.The studies showed that the spermatozoa remained motility only 3-5h at the room temperature but 6d at 4℃ and 9d at 0℃.Besides,the viability of spermatozoa precipitous declined after adding a dilute solution.An improved cryopreservation method for Chinese giant salamander sperm was established according to the mature technologies for cryopreservation of fish.Cryopreseved sperm was diluted in buffered extenders(pH between 7 and 7.5) containing intracellular(DMSO).The concentration of intracellular cryoprotectants ranged from 5% to 25%.In this paper,ten to fifteen percents of the sperm were motile after thawing.The ultrastructures of Chinese giant salamander sperm before and after cryopreservation were observed by SEM and TEM.Many deformations were found in the cryopreserved sperm,such as swelling or breaking of the plasma membrane,disappearing of the nuclear envelope,fracture of the perforatorium and axoneme,degenerating of the undulating membrane and disappearing of the mitochondrial ridges.The results indicated that these damages might be the main reasons for the decline of the motility in the cryopreserved sperm.This study provided an important reference for the establishment of standardized procedures of the short-term storage and cryopresevation of Chinese giant salamander's sperm as well as the artificial breeding of Chinese giant salamander.

Human‐mediated hybridization between native and non‐native species is causing biodiversity loss worldwide. Hybridization has contributed to the extinction of many species through direct and indirect processes such as loss of reproductive opportunity and genetic introgression. Therefore, it is essential to manage hybrids to conserve biodiversity. However, specialized knowledge is required to identify the target species based on visual characteristics when two species have similar features. Although image recognition technology can be a powerful tool for identifying hybrids, studies have yet to utilize deep learning approaches. Hence, this study aimed to identify hybrids between the native Japanese giant salamander (Andrias japonicus) and the non‐native Chinese giant salamander (Andrias cf. davidianus) using EfficientNetV2 and smartphone images. We used smartphone images of 11 individuals of native A. japonicus (five training and six test images) and 20 individuals of hybrids between A. japonicus and A. cf. davidianus (five training and 15 test images). In our experimental environment, an AI model constructed with EfficientNetV2 exhibited 100% accuracy in identifying hybrids. In addition, gradient‐weighted class activation mapping revealed that the AI model was able to classify A. japonicus and hybrids between A. japonicus and A. cf. davidianus on the basis of the dorsal head spot patterning. Our approach thus enables the identification of hybrids against A. japonicus, which was previously considered difficult by non‐experts. Furthermore, since this study achieved reliable identification using smartphone images, it is expected to be applied to a wide range of citizen science projects.

Andrias davidianus ranavirus (ADRV) is an emerging viral pathogen that causes severe systemic hemorrhagic disease in Chinese giant salamanders. There is an urgent need for developing an effective vaccine against this fatal disease. In this study, DNA vaccines containing the ADRV 2L gene (pcDNA-2L) and the 58L gene (pcDNA-58L) were respectively constructed, and their immune protective effects were evaluated in Chinese giant salamanders. In vitro and in vivo expression of the vaccine plasmids were confirmed in transfected cells and muscle tissues of vaccinated Chinese giant salamanders by using immunoblot analysis or RT-PCR. Following ADRV challenge, the Chinese giant salamanders vaccinated with pcDNA-2L showed a relative percent survival (RPS) of 66.7%, which was significant higher than that in Chinese giant salamanders immunized with pcDNA-58L (RPS of 3.3%). Moreover, the specific antibody against ADRV was detected in Chinese giant salamanders vaccinated with pcDNA-2L at 14 and 21 days post-vaccination by indirect enzyme-linked immunosorbent assay (ELISA). Transcriptional analysis revealed that the expression levels of immune-related genes including type I interferon (IFN), myxovirus resistance (Mx), major histocompatibility complex class IA (MHC IA), and immunoglobulin M (IgM) were strongly up-regulated after vaccination with pcDNA-2L. Furthermore, vaccination with pcDNA-2L significantly suppressed the virus replication, which was seen by a low viral load in the spleen of Chinese giant salamander survivals after ADRV challenge. These results indicated that pcDNA-2L could induce a significant innate immune response and an adaptive immune response involving both humoral and cell-mediated immunity that conferred effective protection against ADRV infection, and might be a potential vaccine candidate for controlling ADRV disease in Chinese giant salamanders.

Iridovirus Infection in Chinese Giant Salamanders, China, 2010

Simple SummaryThe molecular mechanisms of skin pigmentation in Andrias davidianus are not clear. In this study, we identified two albino individuals and found that Andrias davidianus had distinct regulatory mechanisms of skin pigmentation that differed from other vertebrates. The key signaling pathway and transcription factors related to melanin synthesis in other vertebrates did not play a significant role in Andrias davidianus. MITF mRNA in Andrias davidianus had a unique splicing form that was not reported in other vertebrates and a unique mutation existed in the SLC24A5 gene in albino Andrias davidianus. The results contributed to understanding the molecular mechanism of skin pigmentation in Andrias davidianus and accelerating the acquisition process of Andrias davidianus species with specific body colors by genetic means, which will help to enrich the aquaculture market of Andrias davidianus.The Chinese giant salamander (Andrias davidianus) has been increasingly popular in the aquaculture market in China in recent years. In the breeding process of Andrias davidianus, we found that some albino individuals were extremely rare and could not be inherited stably, which severely limits their commercialization in the aquaculture market. In this study, we performed transcriptome and small RNA (sRNA) sequencing analyses in the skin samples of wild-type (WT) and albino (AL) Andrias davidianus. In total, among 5517 differentially expressed genes (DEGs), 2911 DEGs were down-regulated in AL, including almost all the key genes involved in melanin formation. A total of 25 miRNAs were differentially expressed in AL compared to WT, of which 17 were up-regulated. Through the integrated analysis, no intersection was found between the target genes of the differentially expressed miRNAs and the key genes for melanin formation. Gene Ontology (GO) and KEGG pathway analyses on DEGs showed that these genes involved multiple processes relevant to melanin synthesis and the key signal pathway MAPK. Interestingly, the transcription factors SOX10 and PAX3 and the Wnt signaling pathway that play a key role in other species were not included, while the other two transcription factors in the SOX family, SOX21 and SOX7, were included. After analyzing the key genes for melanin formation, it was interesting to note an alternative splicing form of the MITF in WT and a critical mutation of the SLC24A5 gene in AL, which might be the main reason for the skin color change of Andrias davidianus. The results contributed to understanding the molecular mechanism of skin pigmentation in Andrias davidianus and accelerating the acquisition process of individuals with specific body colors by genetic means.

The Chinese giant salamander iridovirus (CGSIV), belonging to the genus Ranavirus in the family Iridoviridae, is the causative agent of an emerging infectious disease causing high mortality of more than 90% and economic losses in Chinese giant salamanders in China. In this study, a recombinant baculovirus-based vaccine expressing the CGSIV major capsid protein (MCP) was developed and its protective immunity in Chinese giant salamanders was evaluated. The recombinant Autographa californica nucleopolyhedrosis virus (AcNPV), expressing CGSIV MCP, designated as AcNPV-MCP, was generated with the highest titers of 1 × 108 plaque forming units/mL (PFU/mL) and confirmed by Western blot and indirect immunofluorescence (IIF) assays. Western blot analysis revealed that the expressed MCP reacted with mouse anti-MCP monoclonal antibodies at the band of about 53 kDa. The results of IIF indicated that the MCP was expressed in the infected Spodoptera frugiperda 9 (Sf9) cells with the recombinant baculovirus, and the Chinese giant salamander muscle cells also transduced with the AcNPV-MCP. Immunization with the recombinant baculovirus of AcNPV-MCP elicited robust specific humoral immune responses detected by ELISA and neutralization assays and potent cellular immune responses in Chinese giant salamanders. Importantly, the effective immunization conferred highly protective immunity for Chinese giant salamanders against CGSIV challenge and produced a relative percent of survival rate of 84%. Thus, the recombinant baculovirus expressing CGSIV MCP can induce significant immune responses involving both humoral and cell-mediated immunity in Chinese giant salamanders and might represent a potential baculovirus based vaccine candidate for Chinese giant salamanders against CGSIV.

IntroductionPerilla oil is a natural vegetable oil rich in alpha-linolenic acid extracted from Perilla seeds. Fish oil is a commonly used animal fat containing a large amount of n-3 polyunsaturated fatty acids. Soybean oil is a commonly used vegetable oil rich in linoleic acid. This study aimed to determine whether perilla, fish, or soybean oil can be used as a lipid source in the diet of the Chinese giant salamander (Andrias davidianus).MethodsThree isonitrogenous and isolipidic diets were created by adding 3.0% each of soybean oil (SO), fish oil (FO), and perilla seed oil (PSO). Eight adolescent giant salamanders were fed in rectangular tanks for 98 days with a total of 96 giant salamanders (mean body weight = 75.20g) and were fed an experimental diet until satiation.Results and DiscussionThe viscerosomatic index and crude lipid content of muscle were found to be considerably lower and the muscle crude protein was substantially higher in the PSO group than in the other groups (P<0.05). Linoleic acid levels were highest in SO, eicosapentaenoic acid and docosahexaenoic acid levels were highest in FO, and α-linolenic acid levels were highest in PSO in A. davidianus muscle tissue (P<0.05). The results of study indicate that the PSO diet can significantly increase the total antioxidative capabilities in the liver and intestine, by elevating the activities of total superoxide dismutase, carnitine transferase-1, and acetyl-CoA carboxylase in the liver. Meanwhile, the immunoglobulin M and high-density lipoprotein cholesterol levels were higher in the blood (P<0.05). Furthermore, it significantly reduced hepatic malondialdehyde, plasma endotoxin, D-lactic acid, and total cholesterol levels, and plasma alkaline phosphatase, diamine oxidase, aspartate transaminase, and alanine transaminase activities (P<0.05). Therefore, perilla, fish, and soybean oils can be used as single lipid sources for A. davidianus with respect to growth performance. On the other hand, perilla oil can enhance crude protein content of muscle, increase Immunoglobulin M(IgM) content and Alkaline phosphatase(AKP) enzyme activity, promote lipid metabolism, and maintain the health of the liver and intestine in A. davidianus.

Collagens made from giant salamander (Andrias davidianus) skin and their odorants