Polymorphism of Exon 3 of MHC Class Ⅱ B Gene in Chinese Alligator(Alligator sinensis)

The polymorphism of MHC class II B gene in 14 Chinese alligators was analyzed, which came from three different areas： a wild population from Xuancheng, Anhui, a captive population from Changxing, Zhejiang, and a captive population from Anhui Research Center for Reproduction of Chinese Alligators. The gene fragment was amplified using a pair of specific primers designed from the MHC gene sequence of the spectacled caiman. A total of 34 sequence haplotypes of exon 3 were detected in the sampled Chinese alligators. The numbers of haplotypes of the 3 Chinese alligator populations were 15, 10, and 9, respectively. The overall estimation of the MHC polymorphism in the Chinese alligator population was higher than those in mammals and in cypdnid fish, The rates of nonsynonymous substitutions （dN） occurred at a significantly lower frequency than that of synonymous substitutions （ds）, which were not consistent with the common rule. This result might suggest that the polymorphism of exon 3 seemed not to be maintained by the balancing selection. The neutrality test of Tajima excluded the null hypothesis that the polymorphism of exon 3 was generated by a random drift, and the fact that D = -0.401 indicated an excess of rare mutations in the Chinese alligator. The nucleotide diversity of the sequences and the phylogenetic relations were also analyzed, and the results suggested that there was no significant difference in genetic diversity among the 3 populations of Chinese alligator.

Ecological Networks of Allometric Growth,Feeding Behavior and Gut Microbiota in Chinese Alligator Hatchlings

Chinese alligator hatchlings in captivity are fragile and have a high mortality rate within first year.The body mass gain of reptile animals is closely related to their feeding behavior and gut microbiota.This study analyzed the intrinsic factors influencing the allometric growth of Chinese alligator hatchlings based on their body mass gain,feeding behavior,and gut microbiota.This information would enhance the health management of Chinese alligator hatchlings.There was a significant correlation between the total distance moved,the average number of conspecifics nearby,and body mass gain.Chinese alligator hatchlings with a greater growth rate showed greater activity and more independent behavior during feeding than those with a lower growth rate.Moreover,after feeding started,some functions of the gut microbiota showed significant relationships with growth rate and feeding activity.Chinese alligator hatchlings with a greater growth rate showed greater levels of heme biosynthesis than those with a lower growth rate,and feeding activity was inhibited by long-chain fatty acid biosynthesis.These results elucidate the relationships between health,feeding behavior,and the gut microbiota of Chinese alligator hatchlings.Understanding the intrinsic factors of their health and feeding behavior can improve the health management of Chinese alligator hatchlings in captivity for conservation.

Nest-site Use by the Chinese Alligator (Alligator sinensis) in the Gaojingmiao Breeding Farm,Anhui,China

Nest-site and nesting material used by the Chinese alligator （Alligator sinensis） was studied at the Gaojingmiao Breeding Farm, Langxi County, Anhui, China from May to September 2009. In this study, artificial nesting materials were placed in 43 potential nesting sites before the nesting season, 11 of which were used. Additionally, eight nests were built at natural sites without artificial nesting materials provided. Seven environmental variables were measured at each nest site： distance from water, height from water surface, sunlight duration, nearest bank slope, nest site slope, vegetation coverage and concealment. Statistical analyses indicated that concealment was significantly different between used and unused nest sites, with concealment being significantly correlated to the use of materials- placed sites. In comparing the nests at artificial vs. natural sites, only the nearest bank slope differed significantly. Further, principal component analysis of natural nests indicated that the duration of nest exposure to sunlight and vegetation coverage were more influential than the other factors studied.

Nest Site Preference and Fidelity of Chinese Alligator (Alligator sinensis)

Oviparous female reptiles select nesting sites with optimal ecological factors that contribute to egg development. Chinese alligator （Alligator sinensis）, an oviparous reptile, is a critically endangered crocodilian with temperature-dependent sex determination. Research on its nesting behavior may facilitate the protection of this species. In this study, we monitored nesting behavior over eight years. We compared selected frequency of nest sites, distance from nest site to water, height from nest site top to the water surface, distance from nest site to human activity region, and canopy density between nest sites on the island and bank. The results showed that 45 nest sites were used by female alligators over eight years and each site was selected from one to 10 times. The selected frequency of nest site occurrence on the island was higher than that on the bank （P 〈 0.001）. We observed that 88% of the individual alligators （15/17） showed different degrees of nest site fidelity. However, Chinese alligators might not always be loyal to only one nest site because of environmental changes or interspecific competition at nest sites. Our findings suggest that female alligators prefer to nest at island, which might be because of the nests on the island had a higher canopy density （P = 0.010） and were further from the human activity region （P 〈 0.001） than those on the bank did. It would be beneficial to reduce human activities during the breeding season and protect the vegetation of Chinese alligator habitats in the future.

ISSR Markers as a Tool for Assessing Genetic Diversity in the Chinese Alligator (Alligator sinensis)

Eight different inter simple sequence repeat(ISSR) markers were used as tools to investigate genetic variability and population differentiation in the Chinese alligator,Alligator sinensis,in this study. Eleven polymorphic bands(17.2%) out of a total of 64 were generated from 110 individuals in three populations. Analysis of molecular variation showed that most of the genetic variation(98.0%) occurred within the populations. Dendrogram relationship based on Nei’s unbiased genetic diversity illustrated that two breeding populations were genetically closely related. The Nm value of the study was 4.520,suggesting that high levels of gene flow existed and no differentiation appeared in the populations. In a reconstructed Neighbor-Joining tree,the haplotypes coming from the same populations did not gather as a class,suggesting the three populations had no apparent geographic pattern. This study shows that ISSR markers could be well applied as a feasible tool to assess genetic diversity in Chinese alligator individuals.

扬子鳄（Alligator sinensis）肾的超微结构

用透射电镜观察扬子鳄肾的超微结构。它的近曲小管和收集管上皮细胞无质膜内褶，远曲小管上皮细胞有较少的质膜内褶。扬子鳄肾小管的这些结构特征与它们在淡水生活有关。本文还就扬子鳄的栖息环境及其肾的超微结构与半咸水生活的美洲鳄进行了比较。

扬子鳄(Alligator sinensis)生态学研究进展

简要介绍了半个多世纪以来 ,扬子鳄自然种群的变化、种群衰落的原因、生活史对策、栖息地选择、行为生态、生理生态等方面的主要研究成果 ,并对将来的研究进行了展望。

扬子鳄(Alligator sinensis)眼球形态学参数的测量

本文对11条扬子鳄的眼球的各种形态学参数进行了测量。结果表明,扬子鳄眼球形状呈稍扁圆球形;眼球大小与其它鳄类相似,且随年龄增长而增长;角膜在眼球的覆盖面比昼行哺乳类大,比水生哺乳类小;晶状体与角膜比值较小,这表明扬子鳄眼球聚焦能力较差;另外,角膜、巩膜和晶状体的生长关系表明,角膜轴径比巩膜轴径生长快,角膜屈光度随年龄增长而加大,因而成鳄较幼鳄近视。

Identification of Cloaca Bacteria from Candidate Releasing Chinese Alligators

The Chinese alligator （Alligator sinensis） is a critically endangered species in China. Wild populations of Chinese alligator are on the edge of extinction. Through a release program, some captive-bred alligators will be selected and released into the wild to supplement and renew natural populations. The purpose of this study was to provide data to select healthy individuals for release. Through bacteriological and molecular identification, six different genera, eight species and an unclassified bacterium were identified in 13 bacterial strains, which were isolated from the cloaca of 25 Chinese alligators. One genus and four species were identified in eight bacterial strains, which were isolated from the water where the alligators live. According to the analysis, except for the unclassified bacterium, the other bacteria from the cloaca were not pathogenic and were different from the bacteria isolated from the water. Thus, it was concluded that the 24 Chinese alligators were healthy, and could be selected to be released into the wild. As subject AS 12 was identified carrying an unclassified bacteria, of which the characteristics were unknown, it was suggested that the AS 12 individual not be released.

扬子鳄(Alligator sinensis)卵体积和重量估算及其对孵化成功率的影响

收集了安徽省扬子鳄国家级自然保护区2004、2006、2007年的800枚扬子鳄卵和2007年的188条幼鳄的相关数据。经统计分析,得到卵的体积、重量的回归方程(V=0.537LB2+1.151,R2=0.960;W=0.593LB2-0.541,R2=0.975)和估算公式(V=0.553LB2,F=0.267,P=0.606>0.05;W=0.585LB2,F=0.003,P=0.960>0.05)。公式V=0.553LB2估算出的卵的体积与实际体积相比,误差小于3%;公式W=0.585LB2估算的卵的重量与实际鲜重相比,误差小于2%。基于卵的体积和重量推导公式,还探讨卵的体积、重量与孵化成功率和繁殖成功率之间的关系,结果表明,扬子鳄卵的体积、重量对孵化成功率和繁殖成功率均有明显的影响,较大的卵孵化出幼鳄的几率及成活率都更高。

Current Status of Alligator sinensis

The status about the resources of Alligator sinensis was investigated by sampling methods from August to September in 1994.In the National Nature Reserve for Chinese Alligator (ANNRCA) in Anhui,156 marking caves of alligators were found,77 alligators were seen by lighting-counting method and 253 by visiting.Analysing the results above,there were probably 667～740 alligators in ANNRCA and the population age pyramid was wide at the top and narrow at the base,which indicated that the survival situation of the population was serious.In the Chinese Alligator Reproduction Research Center of Anhui Province (CARRCAP),there were 4376 alligators by the end of the year 1994,of which 248 were breeding alligators,1542 recently hatched and 2586 of different ages.The population with an age pyramid that is narrow at the top and wide at the base shows that it has fast growing potential.

Host Genetic Background Impacts Microbiome Composition in Newborn Alligator

Genetic factors play a key role in determination of the structure of the cloacal flora for newborn Chinese Alligators.We collected the cloacal microbiomes for 24 newborn Chinese Alligators from three different genetic backgrounds for 16S gene amplicon sequencing.The number of cloacal flora for the Chinese Alligators from different groups was comparable but differed structurally.There were variations in proportions of floral compositions at the phylum and family levels;however,the main difference was at the genus level.There were two significant differences in richness and evenness among the three groups.Non-metric multidimensional scaling NMDS analysis revealed that the 24 samples could be clearly divided into three categories based on their genetic backgrounds(stress=0.0244).Thus,we postulated that newborn Chinese Alligators with different genetic backgrounds have different immune strengths,which affects individual responses to environmental microorganisms.In summary,newborn Chinese Alligators from different genetic backgrounds exhibit variations in cloacal microbiome.

Establishment and cryopreservation of liver, heart and muscle cell lines derived from the Chinese alligator (Alligator sinensis)

The Chinese alligator, Alligator sinensis, is a critically endangered species. A conservation project of gene resources for an endangered species first involves the preservation of organs, tissues, gametes, genomic DNA libraries and cell lines. The present study is the first to establish and cryopreserve cell lines of liver, heart and muscle tissues from the Chinese alligator. The study revealed that there was a large discrepancy in cell migration time in primary cultures among liver (11-12 d), heart (13-14 d) and muscle (17-18 d) tissue pieces. The differences in time in primary cell culture suggested that it was relatively easy to build visceral-derived cell lines for reptiles. Biological analysis showed that the population doubling time for thawed cells was approxi- mately 36 h. Karyotyping revealed that the frequency of Chinese alligator cells showing chromosome number as 2n=32 was 88.6%-93.4%. Chinese alligator cell lines established here provide a vital resource for research and are likely to be useful for protection of this rare and critically endangered species. Furthermore, the establishment of these methods may supply technical and theoretical support for preserving genetic resources at the cellular level for other reptile species.

Complete mitochondrial DNA sequence of Chinese alligator, Alligator sinensis, and phylogeny of crocodiles

The 16746-neucleotide (nt) sequence of mitochondrial DNA (mtDNA) of Chinese alligator, Alligator sinensis, was determined using the Long-PCR and primer walking methods. As is typical in vertebrates, the mtDNA encodes 13 proteins, 2 rRNA, 22 tRNA genes, and a noncoding control region. The composition of bases is respectively 29.43% A, 24.59% T, 14.86% G, 31.12% C. The gene arrangement differs from the common vertebrate gene arrangement, but is similar to that of other crocodiles. DNA sequence data from 12S rRNA, 16S rRNA, protein-coding genes and combined sequence data were used to reconstruct the phylogeny of reptiles with the MP and ML methods. With this large data set and an appropriate range of outgroup taxa, the authors demonstrate that Chinese alligator is most closely related to American alligator among three crocodilian species, which suppors the traditional viewpoint. According to the branch lengths of ML tree from the combined data set, the primary divergence between Alligator and Caiman genus was dated at about 74.9 Ma, the split between Chinese alligator and American alligator was dated at 50.9 Ma.

扬子鳄(Aligatorsinensis)卵壳的扫描电镜观察

为比较扬子鳄受精卵及未受精卵卵壳，用扫描电镜观察其超微结构。结果表明：其卵壳由外致密层、蜂窝层、乳突层、外壳膜和内壳膜５层组成。并就经孵化的卵壳外致密层表面出现的腐蚀坑和裂纹产生的原因及生理意义进行了讨论。

Testing reintroduction as a conservation strategy for the critically endangered Chinese alligator: Movements and home range of released captive individuals

The Chinese alligator (Alligator sinensis) is considered the most critically endangered crocodilian as a result of the near total loss of its habitat and its extremely small and fragmented wild populations. Plans for population recovery lie mostly with wetland res- toration and the reintroduction of captive-reared animals. We carried out a first-trial release of 3 adult Chinese alligators (1♂, 2♀) into a pond at the Hongxing conservation site, Xuancheng, southern Anhui Province; the animals were radio-tracked from May to October in 2003. We hypothesized that after a period of adaptation, the alligators would establish definable home ranges. Two (1♂, 1♀) of the 3 alligators were monitored for the whole of the tracking period. The male had an annual home-range size of 7.61 hm2, and the female 4.00 hm2. Water temperature and pond water level were two important factors influencing the alligators' distributions, and daily movements. The radio-tracked alligators had overlapping home ranges, which notably included the one substantial island in the pond; that island is the only known nesting site of the local native wild alligators. Aggressive interactions between the released alligators and native wild alligators were observed during the breeding season around this island. All the three reintroduced alligators survived the winter of 2003 and were alive in the same pond in 2008. We concluded that the Hongxing conservation site provided a suitable habitat for the reintroduced alligators. However, the low water level in the pond resulting from farmland irrigation in August and September can be a substantial threat to the alligators' survival. Therefore, regu- lations on irrigation in summer and autumn are needed to balance the water needs of the alligators and agriculture.

草鱼敏感的负趋音筛选

声学屏障是一种阻拦鱼类进入危险区域进而保护鱼类资源的重要手段,为筛选鱼类敏感的负趋音作为声学屏障,本研究采用6种单频音(500~3000 Hz)和1种宽频音(扬子鳄吼叫声)作为实验用音对草鱼幼鱼进行负趋音的筛选研究,通过在水槽两端交替播音来对比草鱼幼鱼对不同声音的行为反应。结果显示,大多数实验鱼对声音有反应,扬子鳄吼叫声与其他声音组的差异显著。单频音实验组中播放500 Hz单频音时平均反应次数最大,为(1.7±0.6)次,而播放扬子鳄吼叫声时平均反应次数高达(5.0±0.9)次,显著高于其他实验音。在总平均速度中,宽频音组中草鱼的总平均速度显著高于其他组,表明草鱼对扬子鳄吼叫声敏感,产生逃窜行为。研究表明,草鱼对扬子鳄吼叫声具有负趋音性,扬子鳄吼叫声是一种对草鱼具有驱赶、威慑作用的声音。本研究可为过鱼设施中辅助诱驱鱼手段及水利结构中避免鱼类的夹带提供参考依据。

雅鲁藏布江支流湘曲湘河鱼道声驱鱼效果初探

为探求野外场景下利用声驱鱼技术能否提高鱼道进口的通过数量,本研究在雅鲁藏布江支流湘曲湘河鱼道进口2~#上游的河道布置水下扬声器并24 h循环播放捕食者的声音(扬子鳄吼叫声),利用PIT遥测系统对湘河水利枢纽的过鱼对象:拉萨裸裂尻鱼、双须叶须鱼和斯氏高原鳅的负趋音行为进行监测。为考虑监测过程中水文情势和环境因子变化对负趋音行为的影响,实验通过构建耦合多因素的Logistic回归模型,采用AIC模型准则筛选最优模型,并识别影响鱼类负趋音行为的关键因素。结果显示:(1)实验组进入鱼道的数量显著高于对照组;(2)声音和鱼道流量是影响鱼道进口通过率的关键因素;(3)声音开启后,拉萨裸裂尻鱼和双须叶须鱼的鱼道进口通过率显著高于斯氏高原鳅;(4)监测期间2~#鱼道进口流量在0.81~1.32 m~3/s的区间内变化,在此区间内鱼道流量和鱼道进口通过率之间呈负相关趋势(P=0.007),当鱼道流量过高时,鱼道进口通过率会随鱼道流量的增加而降低。研究表明,声驱鱼技术在实际工程应用中能在一定程度上阻止鱼类进入电站尾水等危险区域并增加鱼类进入鱼道的概率。本研究可为声驱鱼技术在鱼道工程的应用和湘河水利枢纽过鱼设施的优化设计提供依据和参考。

扬子鳄源粘质沙雷氏菌XCYZE株的分离鉴定及其致病性分析

目的查明一例源于安徽省扬子鳄管理中心的幼鳄死因。方法从幼鳄脑部组织坏死灶采集病料,进行细菌分离与鉴定,并对其致病性进行分析。结果菌落在血琼脂平板上生长呈红色的革兰氏阴性菌;生理生化试验显示该菌在葡萄糖、甘露醇、乳糖、柠檬酸盐和硫化氢等中均呈阳性;PCR扩增其16S rDNA基因,其大小约1500 bp,经遗传进化树分析,与已报道的粘质沙雷氏菌GRD1株在同一分支;其致病性试验可导致实验鼠在36 h内全部死亡,出现严重肺出血、肝脏肿大、脾脏肿大、腹水现象等。结论从扬子鳄脑内分离的病原是粘质沙雷氏菌,具有较强的致病性,且对多种抗菌药具有一定的耐药性。本试验为野生动物或者人类感染粘质沙雷氏菌的诊断和治疗提供参考意见。

扬子鳄鞣制皮革和鳞片的DNA提取方法

运用一种改进的提取方法 ,作者从鞣制皮革中成功地提取了总DNA ,同时还对尾尖皮、鳞片、盐腌生皮等皮质进行了DNA提取 ;用 12SrRNA基因扩增的通用引物、扬子鳄鉴别引物、微卫星引物及RAPD引物进行PCR扩增 ,并对部分扩增结果进行测序 ,以检验提取效果。结果证明 ,几种皮质标本都可提取出DNA ,其中尾尖皮和鳞片的提取效果较好 ,用四种引物都可扩增出明显亮带 ;盐腌生皮和鞣制皮提取结果也很好 ,并且用12SrRNA通用引物、扬子鳄鉴别引物扩增的亮带较明显 。