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.

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.

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.

Response of Invasive Plant Alligatorweed to Salt Stress at Early Growth Stage

[Objective] The study aimed to investigate the early growth stage＇s response of invasive plant alligatorweed（Alternanthera philoxeroides） under salt stress.[Method] The alligatorweed was cultivated with four different NaCl concentrations 0（control）, 100, 200, 300 mmol/L diluted in 1/2 Hoagland＇s solution at early stage.During the period, we measured the number of nodes and leaves, length, fresh weight, dry weight and water content of stems to determine the effect of salt on alligatorweed growth. [Result] Compared with control, the node number, leaf number,length, fresh weight and dry weight of the stems significantly changed in NaCl solution, and the difference was positively related to NaCl concentration. We concluded that middle and high concentrations of NaCl significantly inhibited the growth of alligatorweed, by suppressing the node number, leaf number, length, fresh weight and dry weight of the stems. Among them, the stem length and fresh weight were more sensitive to NaCl stress. [Conclusion] The present result is helpful to understand the alligatorweed＇s adaptability to saline environment, and also helpful to prevent its invasion in such environment.

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.

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.

Biochemical composition of the alligator pipefish, Syngnathoides biaculeatus (Bloch, 1785)

Considering the economic importance in traditional Chinese medicine(TCM) and lack of baseline information, we evaluated the proximate composition, fatty acid and amino acid profiles, trace element content and C:N ratio in the alligator pipefish, Syngnathoides biaculeatus. Amongst proximate principals, a crude protein formed the major biochemical component((58.9±2.2)% dry weight). Mean percent concentrations(dry weight) of other components such as a total lipid(TL), ash and nitrogen-free extract measured were,(1.8±0.2)%,(19.2±2.2)% and(20.1±0.45)%, respectively. The fatty acid profile revealed the presence of 27 saturated fatty acids(SFA) with 13 straight-chained and 14 branched-chained, 28 unsaturated fatty acids(UFA) with 14 monounsaturated and 14 polyunsaturated and nine other minor fatty acids. Mean percent contributions of total SFAs and UFAs to TL were found to be(55.41±0.24)% and(44.05±0.25)%, respectively. Altogether, 16 dif ferent amino acids with an equal number of essential(EAA) and non-essential(NAA) ones were identified. Percent contributions by EAA and NAA to the total amino acid content were 38.11% and 61.89%, respectively. Trace metal concentrations in S. biaculeatus were generally low and their distribution followed the order, Mg>Fe>Zn>Mn>Cu>Cr>Ni>Hg>Co. The C:N ratio was(4.37±0.04)%. The profile of major biochemical constituents in alligator pipefish, S. biaculeatus revealed its potential use in TCM as well as a nutritional diet for human consumption. The results of the study would also form the basis for formulation and optimization of diets for the culture of S. biaculeatus.

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.

Effect of Alligator Pepper on Litter Weight of Rats Fed on High Glycemic Index Diet

This study was carried out to find out if alligator pepper can prevent the development of fetal macrosomia in Sprague-Dawley rats fed with high glycemic index diet. Fifteen adult female Sprague-Dawley rats of proven fertility were acclimatized for a period of two weeks in a well-ventilated laboratory and fed with grower’s mash and water ad libitum. Thereafter, they were randomly placed in separate cages after which a male Sprague-Dawley rat of proven fertility was introduced into each of the cages containing the female rats. They were left in the cages for three days during, which mating was confirmed to have occurred. The male rats were then withdrawn from the female rats, which were placed in separate maternity cages. The female rats were randomly allocated to three groups A, B and C so there were 5 pregnant rats in each group. The rats in group A were fed with 50 g of grower’s mash daily while the rats in groups B and C were fed with 50 g of cooked fermented cassava respectively. All rats were given water ad libitum throughout the duration of the experiment. The rats in groups A and B were intraperitoneally injected with 13.3 ml per Kg body weight of distilled water while the rats in group C were intraperitoneally injected with 13.3 mg/Kg body weight of aqueous extract of alligator pepper respectively. They were observed for 18 - 25 days. The litters of the rats in group B had a significantly higher mean weight than the rats in group A while the litters of the rats in group C had a significantly lower mean weight than the rats in groups B. It was concluded that intraperitoneally injected aqueous extract of alligator pepper, in the first trimester of pregnancy, can prevent the development of fetal macrosomia in Sprague-Dawley rats.

Breeding the Yangtze Alligator for Profit

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Peasants and Alligators: A Love Story in China's Countryside

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The Origin of Alligator sinensis

Alligator sinensis and Alligator mississippiensis are put under the Alligator family and Alligator genus as two different species and have been thought that they are closely related in taxonomy. The results of our DNA-DNA hybridization experiments showed that A. sinensis is a relatively young species originated in Asia,whereas A. mississippiensis is the older.It seems that the alligator and crocodilian could evolve from each other.The evolutionary rate of the crocodilian is slower than those of birds and higher vertebrates.The phylogeny of four crocodilians have been obtained and the hypothesis that the ancestor of Crocodylus porosus is a transitional form between A.mississippiensis and A.sinensis is proposed.

Analysis of PFAAs in American alligators part 1： Concentrations in alligators harvested for consumption during South Carolina public hunts

Environmental contamination resulting from the production or release of harmful chemicals can lead to negative consequences for wildlife and human health. Perfluorinated alkyl acids（PFAAs） were historically produced as protective coatings for many household items and currently persist in the environment, wildlife, and humans. PFAAs have been linked to immune suppression, endocrine disruption, and developmental toxicity in wildlife and laboratory studies. This study examines the American alligator, Alligator mississippiensis,as an important indicator of ecosystem contamination and a potential pathway for PFAA exposure in humans. Alligator meat harvested in the 2015 South Carolina（SC） public hunt season and prepared for human consumption was collected and analyzed for PFAAs to determine meat concentrations and relationships with animal body size（total length）, sex, and location of harvest. Of the 15 PFAAs analyzed, perfluorooctane sulfonate（PFOS） was found in all alligator meat samples and at the highest concentrations（median 6.73 ng/g）. No relationship was found between PFAA concentrations and total length or sex.Concentrations of one or all compounds varied significantly across sampling locations, with alligators harvested in the Middle Coastal hunt unit having the highest PFOS concentrations（median 16.0 ng/g; p = 0.0001）. Alligators harvested specifically from Berkley County, SC（located in the Middle Coastal hunt unit） had the highest PFOS concentrations and the greatest number of PFAAs detected（p 〈 0.0001）. The site-specific nature of PFAA concentrations in alligator meat observed in this study suggests a source of PFAA contamination in Berkley County, SC.

Analysis of PFAAs in American alligators part 2： Potential dietary exposure of South Carolina hunters from recreationally harvested alligator meat

Exposure to perfluorinated alkyl acids（PFAAs） has been linked to many harmful health effects including reproductive disorders, developmental delays, and altered liver and kidney function. Most human exposure to environmental contaminants, including PFAAs,occurs through consumption of contaminated food or drinking water. This study uses PFAA data from meat samples collected from recreationally harvested American alligators（Alligator mississippiensis） in South Carolina to assess potential dietary exposure of hunters and their families to PFAAs. Consumption patterns were investigated using intercept surveys of 23 hunters at a wild game meat processor. An exposure scenario using the average consumption frequency, portion size, and median perfluorooctane sulfonic acid（PFOS） concentration in alligator meat from all hunt units found the daily dietary exposure to be 2.11 ng/kg body weight per day for an adult human. Dietary PFOS exposure scenarios based on location of harvest suggested the highest daily exposure occurs with alligator meat from the Middle Coastal hunt unit in South Carolina. Although no samples were found to exceed the recommended threshold for no consumption of PFOS found in Minnesota state guidelines, exposure to a mixture of PFAAs found in alligator meat and site-specific exposures based on harvest location should be considered in determining an appropriate guideline for vulnerable populations potentially exposed to PFAAs through consumption of wild alligator meat.

空心莲子草的质量标准研究

[目的]为空心莲子草(ALLIGATOR ALTERNANTHERA HERB)的质量控制提供依据,全面剖析空心莲子草的生药学特征。[方法]通过性状鉴别、显微鉴别、理化鉴别、常规成分检查、薄层色谱鉴定及含量测定,来全面剖析空心莲子草的生药学特征。[结果]根据试验结果,建议空心莲子草的质量标准为:空心莲子草的水分含量不超过10%,灰分含量不超过26%,酸不溶物不超过6.5%,水溶性浸出物不低于40%,醇溶性浸出物不低于25%,总黄酮的含量不得低于2.0%。[结论]该研究可以为空心莲子草的质量标准提供科学依据。

Compressive Biomechanics of the Reptilian Intervertebral Joint

This study compared the pre-sacral intervertebral joints of the American alligator(Alligator mississippiensis)with those from specimens of Varanus.These two taxa were chosen because they have similar number of pre-sacral vertebrae and similar body weights;however,Varanus can move bipedally and has diarthrotic intervertebral joints,whereas Alligator has intervertebral discs and cannot move bipedally.This study consisted of three objectives:(1)to document the anatomy of the intervertebral joint,(2)to quantify the compressive biomechanics of the intervertebral joints and explore which features contributed to compression resistance,and(3)to quantify the impact of compression on the intervertebral foramen and spinal nerves in these two taxa.The experimental results revealed that the diarthrotic intervertebral joints of Varanus were significantly(4x)stiffer than the intervertebral disc of Alligator,and that a significant component of this increased stiffness arose from the facet joints.Compressing the intervertebral joints of the two taxa caused a reduction in foraminal area,but the magnitude of this reduction was not significantly different.We hypothesize that the main factor preventing spinal nerve impingement in Varanus during gravitational compression is the relatively small size of the spinal ganglion/nerve relative to the foraminal area.

Effects of physiological integration on defense strategies against herbivory by the clonal plant Alternanthera philoxeroides

Aims The plant-herbivore interaction is one of the most fundamental inter-actions in nature.Plants are sessile organisms,and consequently rely on particular strategies to avoid or reduce the negative impact of herbivory.Here,we aimed to determine the defense strategies against insect herbivores in the creeping invasive plant Alternanthera philoxeroides.Methods We tested the defense response of A.philoxeroides to herbivory by a leaf-feeding specialist insect Agasicles hygrophila and a pol-yphagous sap-feeding insect Planococcus minor.We also tested the mechanisms triggering defense responses of A.philoxeroides by including treatments of artificial leaf removal and jasmonic acid application.Furthermore,we examined the effect of physiological integration on these defense strategies.Important Findings The combination of artificial leaf removal and jasmonic acid appli-cation produced a similar effect to that of leaf-feeding by the real herbivore.Physiological integration influenced the defense strat-egies of A.philoxeroides against herbivores,and increased biomass allocation to aboveground parts in its apical ramets damaged by real herbivores.Our study highlights the importance of physio-logical integration and modular plasticity for understanding the consequences of herbivory in clonal plants.

Trans-generational effects in the clonal invader Alternanthera philoxeroides

Aims Recent studies have revealed heritable phenotypic plasticity through vegetative generations.In this sense,changes in gene regulation induced by the environment,such as DNA methylation(i.e.epigenetic changes),can result in reversible plastic responses being transferred to the offspring generations.This trans-generational plasticity is expected to be especially relevant in clonal plants,since reduction of sexual reproduction can decrease the potential for adaptation through genetic variation.Many of the most aggressive plant invaders are clonal,and clonality has been suggested as key to explain plant invasiveness.Here we aim to determine whether trans-generational effects occur in the clonal invader Alternanthera philoxeroides,and whether such effects differ between populations from native and non-native ranges.Methods In a common garden experiment,parent plants of A.philoxeroides from populations collected in Brazil(native range)and Iberian Peninsula(non-native range)were grown in high and low soil nutrient conditions,and offspring plants were transplanted to control conditions with high nutrients.To test the potential role of DNA methylation on trans-generational plasticity,half of the parent plants were treated with the demethylating agent,5-azacytidine.Important Findings Trans-generational effects were observed both in populations from the native and the non-native ranges.Interestingly,trans-generational effects occurred on growth variables(number of ramets,stem mass,root mass and total mass)in the population from the native range,but on biomass partitioning in the population from the non-native range.Trans-generational effects of the population from the native range may be explained by a‘silver-spoon’effect,whereas those of the population from the non-native range could be explained by epigenetic transmission due to DNA methylation.Our study highlights the importance of trans-generational effects on the growth of a clonal plant,which could help to understand the mechanisms underlying expansion success of many clonal plants.

Effects of soil substrate heterogeneity and moisture on interspecific competition between Alternanthera philoxeroides and four native species

Aims Clonal plant species have the potential for high relative performance in heterogeneous environments,and this might increase the com-petitive ability and invasiveness of introduced clonal plant species.It was hypothesized that clonal species whose performance responds more to heterogeneity of a resource have higher competitive ability in habitats where this resource is more heterogeneous and that this relationship is stronger when other resources are less limiting.Methods To test these hypotheses,the perennial clonal herb Alternanthera philoxeroides,which is invasive in China,was grown alone or with each of four clonal perennial,co-occurring herbs native to China,i.e.Alternanthera sessilis,Cynodon dactylon,Hemarthria altissima and Wedelia chinensis.Plants were given homogeneous or het-erogeneous soil substrate crossed with low and high levels of soil moisture.Important Findings Effects of heterogeneity on the accumulation of mass and ramets and on competitive effect and response of A.philoxeroides differed between native species and interacted with effects of soil moisture.A.philoxeroides reduced the final total mass or ramet number of the native species except A.sessilis,and the negative competitive effects on H.altissima and C.dactylon were more pronounced in heterogeneous than in homogeneous soil.Competitive response of A.philoxeroides was more negative to A.sessilis than to the other native species.Across native species,the competitive response of A.philoxeroides was more negative in heterogeneous than in homo-geneous soils at low moisture level,but the reverse was true at high moisture level.Results do not consistently support either hypoth-esis,but do suggest that competitive ability can be partly explained by individual species traits such as size,and that some competi-tive effects and responses are emergent properties of interspecific interactions.