长江江豚脐带永生化成纤维细胞系建立及细胞生长特性研究

取长江江豚(Neophocaena phocaenoids asiaeorientalis)产后胎盘脐静脉,经组织块培养,差异贴壁法纯化,构建长江江豚的原代细胞系;经外源癌基因SV40 T antigens(猿猴病毒T抗原)转染构建稳定脐带细胞系,并对长江江豚的永生化后的成纤维细胞的细胞形态、转染效率、生长曲线和活率等...     

Population Genetic Structure of the Yangtze Finless Porpoise (Neophocaena phocaenoides asiaeorientalis): Implications for Management and Conservation

Understanding the population genetic structure is a prerequisite for conservation of a species. The degree of genetic variability characteristic of the mitochondrial DNA control region has been widely exploited in studies of population genetic structure and can be useful in identifying meaningful population subdivisions. To estimate the genetic profile of the Yangtze finless porpoise (Neophocaena phocaenoides asiaeorientalis), an endangered freshwater population endemic to China, the complete mtDNA control region was examined in 39 individuals belonging to seven different stocks inhabiting the middle and lower reaches of the Yangtze River. Very low genetic diversity was found (nucleotide diversity 0.0011± 0.0002 and haplotypic diversity 0.65± 0.05). The mtDNA genetic pattern of the Yangtze population appears to indicate a founder event in its evolutionary history and to support the marine origin for this population. Analyses by F_st and Φ_st yielded statistically significant population genetic structure (F_st = 0.44, P < 0.05; Φ_st = 0.36, P < 0.05). These results may have significant implications for the management and conservation of the Yangtze finless porpoise in the future.     

固化河岸对长江江豚栖息活动的影响

长江江豚主要栖居在近岸浅水水域,喜好泥沙质类型的河岸。但在长江干流,有很多自然河岸被固化,河岸固化给长江江豚分布和栖息活动造的影响仍不甚清楚。2016年3月—2017年1月,对长江干流2个自然河段江豚的数量和分布做了12次考察,并收集了这2个河段岸型的相关数据,来分析固化河岸对江豚栖息活动的影响。12次考察累计发现江豚215头次,平均每次考察观察到江豚(17.92±7.09)头次。研究区域的固化河岸约占岸线总长的59%,分析发现,仅约13.9%的江豚分布在固化河岸水域,86.1%的江豚均分布在自然河岸水域。江豚在单位河岸长度的分布数量与该段固化河岸长度所占的比例呈显著负相关(r=-0.639,P0.01)。在自然河岸,分布在近岸50 m水域的江豚占31.8%,而在固化河岸,仅观察到2头江豚活动在近岸50 m水域内。由此可见,固化河岸对江豚的近岸分布有明显影响。调查还发现,安庆城区建设带约10 km江段12次考察均未发现江豚分布,城区建设带可能对江豚栖息已造成严重影响。长江干流的固化河岸所占比例非常高,研究结果提示这可能导致长江干流江豚栖息地的丧失和破碎化加剧,在制定长江江豚保护措施必需慎重考虑此因素的影响,并据此提出相应的栖息地保护和恢复方案。     

Development and characterization of polymorphic microsatellite loci in the endangered Yangtze finless porpoise (Neophocaena phocaenoides asiaeorientalis)

The finless porpoise (Neophocaena phocaenoides) is one of the smallest cetacean species widely distributed in the shallow coastal waters of the Indo-Pacific Oceans. The population size of the Yangtze subspecies (N. p. asiaeorientalis) has sharply decreased in the last two decades and access to objective data on its population structure and genetic diversity would be of great assistance for their proper management. Here we report on the isolation of nine polymorphic microsatellite using the “Fast Isolation by AFLP of Sequences Containing repeats” (FIASCO) protocol. Polymorphism was assessed using 30 porpoise individuals randomly sampled in the Yangtze River. The number of alleles per locus varies from 2 to 9, with an average value of 5.56, whereas the ranges of observed and expected heterozygosities were 0.300–0.633 (mean 0.496) and 0.473–0.804 (mean 0.659), respectively.     

长江安庆段航道整治对长江江豚数量和分布的时空影响

栖息地丧失和破碎化是威胁长江江豚(Neophocaena asiaeorientalis)种群长期发展的主要因素之一。航道整治工程是导致江豚栖息地丧失和破碎化的一个主要原因,研究工程施工和运行对江豚数量和分布的影响可为江豚栖息地管护提供有益信息。通过对湖口-吉阳矶水域江豚数量和分布的持续调查,分析了安庆段航道整治工程施工和运行对江豚的影响。12次目视调查共在研究水域发现江豚258群次613头次,工程施工前、施工期和初步运行期单次考察分别平均观察到江豚(67.0±24.3)头次、(35.6±22.2)头次和(50.0±2.8)头次。在主要施工区共发现江豚79群次185头次,施工前、施工期和初步运行期单次考察分别平均观察到江豚(18.60±12.03)头次、(10.4±7.3)头次和(20.0±4.2)头次。工程不同时期主要施工区江豚的数量及占比在统计学上没有显著差异。主要施工区S2和S3工程段施工期江豚平均分布密度((0.18±0.17)头/km、(0.04±0.08)头/km)较施工前((0.62±0.16)头/km、(0.40±0.29)头/km)显著下降,S5和S6工程段施工期平均...     

Establishment and characterization of two embryonic cell lines of <Emphasis Type="Italic">Bombyx mori</Emphasis>

Two cell lines, i.e., BmE-SWU1 and BmE-SWU2, were established from silkworm embryonic tissues of the reversion phase through primary culture in Grace’s medium supplemented with 20% fetal bovine serum. The BmE-SWU1 cell line mainly included diploid spindle cells and round cells, which were large and had severe heteroploidy karyotypes. The population doubling time of the 30th passage of the cell line was 58.7 hr. BmE-SWU2 cells were oblong or round, and small. The population doubling time for the 30th passage of the cell line was 46.6 hr. Of BmE-SWU2 cells 89.9% were diploid (2n = 56). Both strains were attached to epithelial-like cell lines and were susceptible to Bombyx mori nucleopolyhedroviruse (BmNPV). Inter simple sequence repeat (ISSR) fingerprinting of silkworm embryonic cell line was obtained.     

长江安庆段江豚主要栖息地鱼类群落结构

长江皖河交汇处(皖河口)是安庆江段江豚主要的栖息地之一。为了解江豚主要栖息地鱼类群落结构,于2013年9月—2014年8月对皖河口及其附近4个断面逐月进行了鱼类调查。研究结果表明,共采集鱼类35种,与历史资料相比,鱼类物种多样性已经严重下降,并且小型化趋势明显。5个断面当中,皖河口鱼类种数最多,为27种,且以中上层鱼类餐和油餐为主要优势种;其余4个断面分别为16、20、19和14种,且多数断面以底层鱼类黄颡鱼和长须黄颡鱼为主要优势种。方差分析结果表明,皖河口渔获物的总数量和总重量与其他断面无显著差异,但总种数却显著高于断面1和5。对中上层、中下层和底层鱼类的进一步分析发现,5个断面的显著差异仅存在于中上层鱼类,且皖河口中上层渔获物的数量和重量均显著高于其他断面。由此推断,江豚选择皖河口作为主要栖息地可能主要由于其更加丰富的中上层鱼类资源。研究结果不仅有助于了解鱼类资源的变化,还对江豚的原地和迁地保护具有重要意义。     

Analysis of the bacterial diversity in the fecal material of the endangered Yangtze finless porpoise, <Emphasis Type="Italic">Neophocaena phocaenoides asiaeorientalis</Emphasis>

The aim of this study was to determine the bacteria present in the fecal material of the endangered Yangtze finless porpoise, Neophocaena phocaenoides asiaeorientalis. Fecal samples were collected from 12 Yangtze finless porpoises living in the wild at Poyang Lake, located in Jiangxi Province, China. To determine the bacterial diversity, a 16S rRNA gene clone library using the bacterial PCR primers fD1 and rP2, was prepared. A total of 138 near-full-length sequences were analyzed and 39 operational taxonomic units (OTUs) were identified. Sequences showing ≥97% similarity were grouped together as an OTU. Six different phyla were identified in which 38 OTUs were classified. Most of the OTUs contained sequences belonged to the phylum Firmicutes (51.3%), followed by Tenericutes (17.9%), Proteobacteria (15.4%), Actinobacteria (7.7%), Deinococcus-Thermus (2.6%) and Cyanobacteria (2.6%). A phylum could not be assigned for one clone within one OTU (2.6%). It appears that the Yangtze finless porpoise has a more diverse range of bacteria compared to other aquatic mammals, such as seals.     

Seasonal changes in the local distribution of Yangtze finless porpoises related to fish presence

The Yangtze finless porpoise (Neophocaena asiaeorientalis asiaeorientalis) is an endangered freshwater porpoise subspecies unique to the Yangtze River basin. Seasonal variations in local distribution of the animal, as well as fish presence, sand dredging, ship navigation, and bridges were examined as potential factors affecting the occurrence of the animals. Passive acoustic surveys were performed regularly from May 2007 to August 2010, near the conjunction of the Yangtze River and Poyang Lake. The distribution of the porpoises was seasonally site‐specific. In May and August, the animals were detected more often at river junctions than in the lake, but vice versa from November to February. The rate of the porpoise detection was significantly higher in areas of fish presence than in areas of absence. The number of porpoises detected did not differ significantly between the sand dredging operation and the prohibition period (in 2008), although the number of vessels obviously declined in 2008. Ship traffic and bridges also did not appear to affect the presence of porpoises. These results showed the relative importance of the various environmental factors, which is important for conservation of not only Yangtze finless porpoise but also endangered isolated cetaceans.     

Reproductive seasonality and maturity of male Neophocaena phocaenoides asiaeorientalis in captivity: A case study based on the hormone evidence

To provide hormone evidence on reproductive seasonality and maturity of the Yangtze finless porpoise, the authors monitored the monthly variations of serum reproductive hormones of a male Yangtze finless porpoise in captivity via radioimmunoassay from 1997 to 2003. It was demonstrated that the immature animal had a serum T level of 28–101?ng?dL^?1 (65?±?52?ng?dL^?1), reached puberty with the serum T concentration ranging between 250 and 590?ng?dL^?1 (390?±?130?ng?dL^?1), and attained reproductive maturity when the serum T level went beyond 1120?±?180?ng?dL^?1 in the breeding season. The body length growth of the animal showed a pattern similar to the serum T variations during its adolescent period from the age of 4 to 6 years. Moreover, the serum T concentration of the male porpoise exhibited significant seasonal variations, indicating that its breeding season may start as early as March and end as late as September.     

Development of tetranucleotide microsatellite loci for the finless porpoise (<Emphasis Type="Italic">Neophocaena phocaenoides</Emphasis>)

The finless porpoise, Neophocaena phocaenoides, is endemic to the coastal waters of the Indo-Pacific, ranging from the Persian Gulf to Japan. Nine tetranucleotide microsatellite loci were isolated from the finless porpoise (Neophocaena phocaenoides). Polymorphism of each locus was assessed in 39 unrelated individuals from the Yellow Sea and the South China Sea of the Chinese waters. The number of alleles per locus varied from 2 to 11. The ranges of observed and expected heterozygosity were 0.154–0.795 and 0.146–0.839, respectively. Cross-species amplification of these loci was tested in other cetacean species. These microsatellite markers described here will be suitable for population genetic studies of finless porpoises and other cetacean species.     

Auditory temporal resolution and evoked responses to pulsed sounds for the Yangtze finless porpoises (<Emphasis Type="Italic">Neophocaena phocaenoides asiaeorientalis</Emphasis>)

Temporal cues are important for some forms of auditory processing, such as echolocation. Among odontocetes (toothed whales, dolphins, and porpoises), it has been suggested that porpoises may have temporal processing abilities which differ from other odontocetes because of their relatively narrow auditory filters and longer duration echolocation signals. This study examined auditory temporal resolution in two Yangtze finless porpoises (Neophocaena phocaenoides asiaeorientalis) using auditory evoked potentials (AEPs) to measure: (a) rate following responses and modulation rate transfer function for 100 kHz centered pulse sounds and (b) hearing thresholds and response amplitudes generated by individual pulses of different durations. The animals followed pulses well at modulation rates up to 1,250 Hz, after which response amplitudes declined until extinguished beyond 2,500 Hz. The subjects had significantly better hearing thresholds for longer, narrower-band pulses similar to porpoise echolocation signals compared to brief, broadband sounds resembling dolphin clicks. Results indicate that the Yangtze finless porpoise follows individual acoustic signals at rates similar to other odontocetes tested. Relatively good sensitivity for longer duration, narrow-band signals suggests that finless porpoise hearing is well suited to detect their unique echolocation signals.     

Serum-free culture of an embryonic cell line from <Emphasis Type="Italic">Bombyx mori</Emphasis> and reinforcement of susceptibility of a recombinant BmNPV by cooling

We established the first continuous cell line that uses a serum-free culture from the embryo of Bombyx mori (Lepidoptera: Bombycidae), designated as NIAS-Bm-Ke17. This cell line was serially subcultured in the SH-Ke-117 medium. The cells adhere weakly to the culture flask, and most cells have an oval shape. The cell line was subcultured 154 times, and the population doubling time is 83.67 ± 5.22 h. Random amplification of polymorphic DNA-polymerase chain reaction with a tenmar single primer for discrimination of insect cell lines recognized the NIAS-Bm-Ke1 cell line as B. mori. This cell line does not support the growth of the B. mori nuclear polyhedrosis virus (BmNPV) in the absence of the heat-inactivated hemolymph of B. mori. However, the heat-inactivated hemolymph in 1% volume of the medium supported a high level of susceptibility to BmNPV. In addition, the cooling treatment of the cells at 2.5°C also enhanced the susceptibility. We report a new serum-free culture system of the B. mori cell line for the baculovirus expression vector system.     

长江东流河道整治对长江江豚种群数量的影响

长江江豚是生活在长江中下游的濒危水生哺乳动物,通常活动在江心洲和浅水缓滩附近。航道整治改变了鱼类栖息活动的水文环境,也给豚类生存带来负面影响。本文根据长江东流河道整治施工前和施工后各3 次野外考察资料,分析了3 种流态中江豚的数量与群结构。结果表明,该工程实施前江豚主要栖息活动在边滩的分离区,其次在洲头的分流区。工程实施后,整治江段的江豚种群数量年下降率达8. 9% 。江豚在分流区活动已消失,在分离区集群规模较小,且在流态之间移动增大。本文研究结果也提示长江江豚就地保护难度越来越大,从长江干流中把长江江豚迁入故道中是一件刻不容缓的工作。     

长江江豚自然保护区建设管理存在的问题及调整建议

长江江豚(Neophocaena asiaeorientalis)是生活在中国长江的珍稀濒危鲸类动物, 自然栖息地破坏是造成其种群衰退的重要原因。目前中国已经在长江中下游流域建立了8个长江江豚自然保护区。然而, 早期保护区的功能区划随经济发展和生态保护的现实矛盾日渐突出, 按照现行法规对保护区难以实行有效管理, 迫切需要进行优化调整。文章基于国家政策、长江江豚生物学特点及分布规律, 兼顾航运等重大经济发展需求, 提出了长江豚类自然保护区调整的原则和建议: (1)将长江江豚喜好的近岸水域及一定范围的重要江滩和洲滩湿地, 及江心洲汊江一侧水域划入核心保护区; (2)将中间主航道水域以及长江大堤至核心区边界之间的水域划入一般控制区; (3)兼顾历史遗留问题, 细化不同区域的管控措施。该调整方案一方面使保护区功能区划更加符合长江江豚的分布特征和偏好生境, 有利于对其自然种群和重要栖息地的有效保护。另一方面也有助于理顺自然保护区管理上存在的法律障碍, 为长江江豚自然保护区建设、规划和管理提供新的思路和模式。     

Mitochondrial control region variability of baiji and the Yangtze finless porpoises,two sympatric small cetaceans in the Yangtze river

BaijiLipotes vexillifer (Miller, 1918) and the Yangtze finless porpoiseNeophocaena phocaenoides asiaeorientalis (Pilleri and Gihr, 1972) are two sympatric small cetaceans inhabiting the middle and lower reaches of the Yangtze River. In this study, a fragment (420–428 bp) of the mitochondrial control region was sequenced to provide the first comparative survey of genetic variability and population structure in these two endangered species, with samples of finless porpoises from the Yellow/Bohai Sea, East China Sea, and South China Sea also included. Low values of haplotype diversity and nucleotide diversity were found for both species, especially for the baiji and the Yangtze River and South China Sea populations of finless porpoises. The analysis of molecular variance (AMOVA) supported a high level of overall genetic structure among three porpoise populations in Chinese waters, with greatest differences found between either the Yangtze River population or the Yellow Sea population and the South China Sea population. The differentiation between the Yangtze and Yellow Sea populations was not significant, and the males have higher genetic differentiation than the females, suggesting a significant female-biased dispersal between these two populations. This study showed that the Yangtze finless porpoise, unlike the sympatric baiji, was not a genetically isolated population. The Yangtze and Yellow Sea porpoises should be included in the same management unit, but further studies using more samples and especially based on more molecular markers are urgently needed to confirm this.     

Estimating the rate of increase for the finless porpoise with special attention to predictions for the Inland Sea population in Japan

The finless porpoise Neophocaena asiaeorientalis inhabits coastal waters and rivers in East Asia and is exposed to various human activities. This species is listed on the IUCN Red List of Threatened Species due to a reduction in abundance. Although human-induced mortality can be a threat to porpoise populations, future anthropogenic impacts have not been quantitatively evaluated due to lack of demographic information. Adequate future population projections are needed to form the basis for conservation measures before the population declines to critical levels. We conducted a population viability analysis for the population of finless porpoise in the Inland Sea, Japan using a Leslie matrix model composed of age-specific survival and fertility rates. We described the uncertainty in the annual rate of increase (λ) for the finless porpoise using randomly sampled estimates of survival rate for other cetaceans with similar life histories. Plausible median estimates of λ ranged from 1.041 (age at first reproduction [AFR] = 7) to 1.056 (AFR = 5). Future population changes and extinction probabilities were predicted after combining these estimates with a predicted human-induced mortality rate (M) and available abundance estimates. The extinction probability after 100 years was 0 %. However, the probability of the quasi-extinction (<100 individuals) was as high as 79.0 % after 100 years. The results also suggest that the persistence of the finless porpoise population could be achieved with a small effort to reduce anthropogenic mortality.     

A new continuous cell line from larval ovaries of silkworm, <Emphasis Type="Italic">Bombyx mori</Emphasis>

A new continuous cell line from ovarian tissue of commercial variety “Kolar Gold” of silkworm, Bombyx mori, was established and designated as DZNU-Bm-12. The tissue was grown in MGM-448 insect cell culture medium supplemented with 10% fetal bovine serum (FBS) and 3% heat-inactivated B. mori hemolymph at 25 ± 1°C. The migration of partially attached small round refractive cells from the fragments of ovarioles began from the beginning of explantation. The cells multiplied partially attached in the primary culture initially, and some of them become freely suspended after 20 passages. The cells were adapted to MGM-448 and TNM-FH media each with 10% FBS and the population doubling time of cell line was about 36 and 24 hr, respectively. The chromosome number was near diploid at initial passages and slightly increased at 176th passage, but a few tetraploids and hexaploids were also observed. DNA profiles using simple sequence repeat loci established the differences between DZNU-Bm-12 and DZNU-Bm-1 and most widely used Bm-5 and BmN cell lines. The cell line was found susceptible to B. mori nucleopolyhedrovirus (BmNPV) with 85–90% of the cells harboring BmNPV and having an average of 3–17 OBs/infected cell. We suggest the usefulness of this cell line in BmNPV-based baculoviral expression system and also for studying in vitro virus replication.     

长江江豚细菌性疾病的诊治

人工饲养或易地保护长江江豚(Neophocaena asiaeorientalis asiaeorientalis), 其水体中常含有病原菌且易引起江豚发病, 探讨长江江豚的细菌性疾病诊疗方法十分必要。文章利用细菌的分离培养与鉴定, 结合血液检测结果, 对安庆西江围网内一头患病江豚进行了病原确诊, 结果发现患病江豚的致病原为金黄色葡萄球菌(Staphylococcus aureus)和魔氏摩根菌(Morganella morganii)。依据致病菌的药敏试验结果, 综合患病江豚的整体状况制定了治疗方案并对其进行系统治疗, 结果预后良好。研究的成功开展为江豚、海豚等鲸类动物细菌性疾病的诊断与防治提供了良好借鉴。     

鲸类微卫星引物对长江江豚的适用性研究

微卫星在长江江豚 (Neophocaenaphocaenoidesasiaeorientalis)中的应用研究还未见报道。本研究采用已发表的来自 6个鲸种的 2 3对微卫星引物对一个长江江豚群体DNA样本进行了微卫星扩增。结果表明其中有 7对引物在此群体中的扩增产物是稳定且多态的 ,序列分析结果表明这 7对引物的扩增产物都具有AC或GT两碱基重复单元 ,从而证明了扩增的有效性。研究结果表明用从其他鲸类分离出的微卫星引物可以快速筛选到适用于长江江豚指纹分析的引物