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. of the beach(Ali et al., 2005; Kamel and Mrosovsky, 2005; Turkozan et al., 2012). Other species such as snakes and lizards select nest sites with more moist substrates, which significantly increases their body size at hatching(Brown and Shine, 2004; Reedy et al., 2013). Nest site preference may be maintained for one or more breeding seasons(Janzen and Morjan, 2001; Kamel et al., 2006). When female chose the same site or area during different years, it is called nest site fidelity(Switzer, 1993). This nest choice behavior may influence offspring survival(Lindeman, 1992) and nest site fidelity has been documented in numerous species such as birds(Lindberg and Sedinger, 1997), turtles(Mitrus, 2006; Walde et al., 2007), tuatara(Refsnider et al., 2010), and crocodilians(Elsey et al., 2008).Chinese alligator(Alligator sinensis) is a critically endangered freshwater crocodilian endemic to China(Thorbjarnarson and Wang, 1999). Recent investigations show that there are no more than 130 Chinese alligators     

自然环境下影响扬子鳄繁殖的环境因子

生态因子对生物的生长、发育、生殖和分布有着直接和间接的影响.在1998～2005年对扬子鳄繁殖研究中心内自然环境下鳄卵及其繁殖地环境因子进行调查,包括窝卵数、产卵时间、出壳时间、孵化期内温度、湿度的变化情况、雏鳄孵出后的存活数量以及巢区的植被盖度.调查发现天气状况直接影响母鳄产卵时间、卵的孵化质量与雏鳄的出壳数量.盖度相对大于0.5的窝巢中多数卵基本能正常发育,盖度小于0.5的窝巢中可能只有少数几枚卵发育正常.     

Eleven novel microsatellite markers for the Chinese alligator (Alligator sinensis)

Chinese alligator (Alligator sinensis) is a critically endangered species endemic to China. In this study, we developed 11 novel microsatellite loci for this rare species and applied them to examine genetic variation of indigenous alligators from Changxing Nature Reserve and America-born Chinese alligators. The 11 polymorphic microsatellites presented a total of 31 alleles among 57 individuals scored, yielding an average of 2.82 alleles per locus. One allele was unique to the American population but four private alleles were detected in the Changxing population. The average expected and observed heterozygosities were 0.400 and 0.482 for the Changxing alligators and 0.520 and 0.621 for the America-born individuals, respectively. These microsatellite markers would be useful tools in the genetic examination of this endangered species.     

A bacterial artificial chromosome library for the Chinese alligator (Alligator sinensis)

Chinese alligator (Alligator sinensis) is a rare and endangered species endemic to China. To better understand genetic details of the Chinese alligator genomic structure, a highly redundant bacterial artificial chromosome (BAC) library was constructed. This library consists of 216,238 clones with an average insert size of about 90kb, indicating that the library contains 6.8-fold genome equivalents. Subsequently, we constructed a 516kb contig map for the Chinese alligator olfactory receptor (OR) genes, which spans nine BAC clones, and subjected the BACs to full sequencing. The sequence analysis revealed that this contig contained 16 OR functional genes and meanwhile demonstrated that the nine BACs, which constituted the contig, overlapped correctly, proving the usability of this genome library. As a result, this BAC library could provide a useful platform for physical mapping, genome sequencing or complex analysis of targeted genomic regions for this rare species.     

Isolation of polymorphic microsatellite loci from the Chinese alligator (Alligator sinensis)

Ten microsatellite loci were isolated from the Chinese alligator (Alligator sinensis), using a partial genomic DNA library and an enrichment method. Fifty microsatellite repeats were screened from the library of 888 positive clones, 10 of which were polymorphic. These microsatellite loci developed here are another set of novel molecular markers for A. sinensis, and they will be suitable for population genetic studies as well as for kinship analysis.     

The sex ratio of wild Chinese alligators Alligator sinensis

The Chinese alligator Alligator sinensis is one of the most endangered crocodilian species, and typically exhibits temperature-dependent sex determination. It is extremely important to clarify the sex structure of Chinese alligators to implement recovery projects successfully. However, the sex ratio of wild Chinese alligators remains unknown. In this study, we collected 28 years of sex ratio data from Chinese alligators residing in the natural and artificial habitats of Changxing Nature Reserve, China, and examined the differences in the sex ratio dynamics between these two populations. We observed that the sex ratio of wild Chinese alligators is 1 male to 4.507 females, which was significantly lower compared to that of the captive population （1 to 2.040; P 〈 0.001）, and is significantly different to previously documented sex ratios for this species （all P 〈 0.01）. Furthermore, we documented an annually stable （P = 1.000） female-biased sex ratio for wild alligators at hatching [1 male to 4.747 females; 0.174 （0.167~）.182）], in contrast to a dramatically fluctuating sex ratio （P 〈 0.001） in captivity [1 male to 1.674 females; 0.374 （0.246-0.593）], showing a potential mechanism for adjusting the sex structure. Finally, we found that the hatchling sex ratios were similar to that of the population sex ratio （P = 0.748）, with little correlation to air temperature values in the 60-70 day incubation period during the breeding season （July and August; both P 〉 0.05）. Overall, this study indicates that the stabilized female-biased sex ratio of Changxing Chinese alligators might result from selection pressure caused by local mate competition and major inbreeding .     

Social Displays of the American Alligator (Alligator mississippiensis)

Adult alligators perform two conspicuous social displays, bellowsand headslaps. Both of these behaviors are performed from ahead oblique tail arched (HOTA) posture. Bellow displays,by both males and females, involve the production of a loud,roaring vocalization. Male alligators also produce a infrasonicsignal, termed subaudible vibrations, just prior to the audiblebellow. Bellowing occurs throughout the year but is most frequentduring the courting season, when alligators bellow daily inchoruses. Chorus length appears to be correlated with the sizeof the adult population. Chorusing is frequently initiated byfemales but seems to be perpetuated by male alligators. Bellowingmay serve to attract alligators of the opposite sex and possiblyto space out animals of the same sex. The headslap display is an assertion display consisting of eightcomponent behavioral acts: the elevated posture, HOTA posture,subaudible vibrations, headslap, jawclap, growl, inflated posture,and tail wag. Each act component is variable in presence andintensity producing a highly variable, graded signal. The alligatortypically remains motionless in the HOTA posture for about 16sec before executing the headslap/jawclap acts. The headslapdisplay involves a rapid clapping shut of the jaws as the undersurfaceof the head is slapped against the water surface. Headslappingis most common in early morning and afternoon hours. Analysisof 1,050 headslap displays by 91 known individuals indicatesthat 94.5% of the observed displays were performed by males.Headslap displays are generally performed from typical displaysites which the alligator seeks out prior to the display. Responsesto headslap displays include headslapping by others, lunges,approaches, and bellow growling. The headslap display functionsas a declaration of presence. A musky odor is commonly detected in association with both ofthese social displays, suggesting a possibly important, butlittle understood, pheromonal component of these behaviors. The displays both involve complex signals including visual,auditory, olfactory and possibly tactile channels of communication.Most of the behaviors described are shared with many other speciesof crocodilians.     

Genetic diversity of Chinese alligator (Alligator sinensis) revealed by AFLP analysis: an implication on the management of captive conservation

Chinese alligator (Alligator sinensis) is one of the most critically endangered species among 23 extant crocodiles in the world. To prevent the extinction of the species, a captive propagation started at early 1980s, and the total number of alligator was brought up to 10 thousands from dozens of founder in 2000. But several genetic investigations showed those alligators were under an extremely low genetic diversity status with few detectible polymorphic loci. To get more insight into its genetic diversity for the management of captive Chinese alligator, AFLP was adopted to characterize variations in the population. Total of 347 bands were generated from 47 individuals using 3 primer combinations, of which 203 (58.50%) were polymorphic, and 35 AFLP phenotypes were revealed from those individuals. Comparing the results between RAPD and AFLP analysis on almost same sample set clearly indicated that AFLP is more efficient in revealing polymorphic loci, especially in those populations with extremely low genetic diversity. In present three assays, electrophoresis profile also displayed 3 individuals possessing very highly polymorphic AFLP phenotypes that were never been found by RAPD and mtDNA D-loop sequencing, implicating that we should offer these individuals more breeding opportunities to maintain the genetic diversity in the population and restrict those carrying few polymorphic loci from reproduction.     

使用SSR和mtVNTR分子标记识别扬子鳄个体

扬子鳄是中国特有的濒危物种,为有效避免种群衰退,最大限度地保持该物种现有的遗传多样性,有必要对种群的个体进行个体识别研究,以便重建遗传谱系,指导现有繁育工作。应用SSR(Simple sequence repeats)与mtVNTR(Variable number tandem repeats on mitochondrial DNA)两种分子标记对扬子鳄39个个体进行了个体识别分析,结果显示：8个SSR座位的累计个体识别率与累计父权排除率分别达0．9968、0．7697,mtVNTR的个体识别率为0．9146,联合SSR与mtVNTR两种分子标记的累计个体识别率理论值达0．9997,并在实际分析中将39个扬子鳄个体完全区分开,其区分能力较RAPD(Random amplified polymorphic DNA)、AFLP(Amplified fragment length polymorphism)及mtDNA控制区5’端序列分析等分子标记要高。此外,SSR和mtvNTR还可对某些低频等位基因及其携带个体做有效的筛查,这对今后进行大量扬子鳄个体的分子标记识别和群体的遗传谱系建立等工作将具有一定实际意义[动物学报51(3)：501—506,2005]。     

扬子鳄的保护遗传学研究进展

保护遗传学是主要研究与灭绝风险相关的遗传因素以及如何利用遗传学管理方法降低物种灭绝风险的科学,是保护生物学和分子遗传学的交叉学科.近几十年来,遗传学研究在生物多样性保护的理论和实践中发挥着越来越重要的作用.本文回顾了AFLP、mtDNA D-loop、RAPD、微卫星DNA、MHC等DNA分子标记技术在扬子鳄的样品采集、生物多样性、个体鉴定、繁殖管理、野外放归等保护遗传学方面研究所取得的一些进展.对扬子鳄保护的工作提出了建议:重建扬子鳄的谱系;加大对扬子鳄的放归力度;加强饲养种群之间的基因交流;借鉴密河鳄的管理经验.     

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 ,可进行扬子鳄皮质用品等的分子鉴定及部分序列的扩增和测序研究     

人工养殖的扬子鳄适应自然环境的能力

为了复壮扬子鳄野生种群数量,实施养殖扬子鳄放归自然是重要途径之一.通过对人工养殖扬子鳄进行野外放归的试验研究,以探明其在野外环境下活动情况和适应能力.2007年6～11月,采用无线电遥测跟踪、望远镜观察和夜间辅助灯光望远镜观察等方法,对健康的4条5.5年龄和3条2.5年龄的人工养殖扬子鳄在自然环境下活动情况进行观察;在2008和2009年的5月,将这些鳄重捕后测量它们的大小并察看生长情况.结果显示:扬子鳄的释放初期经历了过渡期和争夺领域期2个阶段,20天后处于不同的区域内,具领域特征和营建洞穴行为,能够安全越冬;逐渐恢复野生习性,自我保护的警惕性提高,逃避威胁的能力加强;小龄鳄生长明显,大龄鳄没有明显生长,这与它们所处环境的食物种类有关;鳄性成熟后,能够成功繁殖.总之,人工养殖扬子鳄能够适应相应的自然环境,能够正常生活、生存和繁衍.     

Febrile response to infection in the American alligator (Alligator mississippiensis)

Temperature probes were inserted into the stomachs of juvenile American alligators (Alligator mississippiensis) maintained outdoors at ambient fluctuating temperatures. Internal body temperatures (T_b) were measured every 15 min for two days, and then the alligators were injected with bacterial lipopolysaccharide (LPS), pyrogen-free saline, or left untreated. Alligators injected intraperitoneally with LPS exhibited maximum T_bs 2.6 ± 1.1 °C and 3.5 ± 1.2 °C higher than untreated control animals on days one and two after treatment, respectively. T_bs for these animals fell to within control ranges by day three postinjection. Similarly, mean preferred body temperatures (MPBTs) were significantly higher for LPS-injected alligators on days one (4.2 ± 1.8 °C) and two (3.5 ± 1.6 °C) after treatment. Intraperitoneal injection of heat-killed Aeromonas hydrophila, a gram-negative bacterium known to infect crocodilians, resulted in a fever while injection of Staphylococcus aureus (gram positive) did not elicit a febrile response. Injection of LPS in alligators maintained indoors in a constant temperature environment resulted in no increase in internal T_b. These results indicate that alligators did not exhibit a febrile response in the absence of a thermal gradient, and suggest that febrile responses observed are probably behavioral in nature.     

扬子鳄小胃黏膜的组织化学和细胞学

扬子鳄（Alligator sinensis）的小胃是在胃幽门部与十二指肠的交接处由胃幽门括约肌和十二指肠括约肌突入管腔形成的一个小腔,其生理功能一直不清楚。本文采用组织化学、电子显微镜和免疫组织化学技术对扬子鳄小胃黏膜的组织化学成分、细胞超微结构及细胞类型进行了较全面的研究。小胃黏膜上皮PAS反应呈强阳性,AB染色呈弱阳性,主要分泌中性糖蛋白和少量的含硫酸性糖蛋白。电子显微镜下,扬子鳄小胃黏膜上皮主要由表面黏液细胞组成,偶见内分泌细胞。小胃腺部则70％-90％为内分泌细胞,其余为少量的腺黏液细胞和泌酸胃酶细胞。应用7种胃肠激素的抗血清在小胃黏膜中检测出了5-羟色胺（5-HT）、胃泌素（Gas）、胰高血糖素（Glu）、生长抑素（SS）、P-物质（SP）和血管活性肠肽（VIP）细胞,以Glu细胞密度最高,VIP细胞密度最低。未检测到胰多肽（PP）细胞。本研究结果表明,从组织化学成分和细胞类型看,扬子鳄的小胃与胃同源;从细胞超微结构和内分泌细胞所占百分比例看,扬子鳄的小胃已出现了明显的特化,泌酸胃酶细胞中未见泌酸小管,可能没有泌酸功能。内分泌细胞含量丰富,可能在调节胃肠道功能中发挥重要作用[动物学报54（6）：1044—1050,2008]。     

Vocalization of the tree frogPolypedates maculatus (Rhacophoridae)

The period of calling activity ofPolypedates maculatus lies between April and October. Males possess an indistinct subgular vocal sac which turns yellow during the breeding season. Mating calls type I, type II and distress calls have been identified. Mating calls type I and type II consist of a single pulse group. Type I call comprises of 7–22 pulses, whereas type II call consists of 4–6 pulses. Pulses are short. The frequency spectrum is broad and continuous. Distress calls, with 6 hormonics, are given by the females with their mouth open.     

扬子鳄与密河鳄基因组DNA的复性动力学和组织结构的研究

通过DNA复性动力学的分析,测定了扬子鳄和密河鳄基因组的组织结构,两者的复性曲线非常相象,高度重复DNA含量很少,而中度重复DNA序列各占基因组的30％左右。中度重复序列部分能再分成三个组分,虽然每个组分的序列复杂性和拷贝数有差别,但每部分的总复杂性是相似的。我们已测得扬子鳄基因组的单拷贝序列为3.68×10’bp,基因组为5.65×10’bp;密河鳄基因组的单拷贝序列为3.67×10’bp,基因组为5.24×10’bp。根据基因组的组织结构的分析,在分子水平上表明两种鳄鱼是亲缘关系十分相近的种。