Molecular Cloning and Tissue-specific Expression of Cu/Zn and Mn-superoxide Dismutase in the Three-keeled Pond Turtle, Chinemys reevesii

Both copper/zinc superoxide dismutase （SOD; Cu/Zn-SOD, SOD1） cDNA and manganese SOD （Mn-SOD, SOD2） cDNA were cloned for the first time from the three-keeled pond turtle, Chinemys reevesii, using RT-PCR and RACE methods in this work. The SOD1 cDNA was 749 bp long and consisted of a 32-bp 5＇-untranslated region （UTR）, a 249-bp 3＇-UTR, and a 468-bp open reading frame （ORF） encoding a 155-amino-acid protein with 16.0 kDa predicted molecular mass and 5.95 theoretical isoelectric point （p/）. The SOD2 cDNA was 1687 bp long and comprised 94-bp of 5＇-UTR, 912-bp 3＇-UTR and 681-bp ORF encoding a 226-amino-acid protein with 25.0 kDa predicted molecular mass and 8.83 pI. The deduced amino acid sequence of SOD1 showed relatively high similarity （77.4%-87.1%） and identity （65.4%-74.4%） with the published sequences of SOD1 from other vertebrate species, whereas SOD2 protein shared slightly higher similarity （83.6%-95.6%） and identity （76.1%-88.9%） with other reported vertebrates SOD2s. Phylogenetic analysis revealed that the C. reevesii SOD1 and SOD2 were separately clustered together, and were highly conserved during evolution. Both SOD mRNA expression was detected widely in the brain, liver, muscle, kidney, gut, spleen, lung and heart at variable levels. The highest expression of the two SODs was observed in muscle, and followed in brain, liver, kidney, gut and heart, whereas low transcriptional levels were found in spleen and lung. Meanwhile, high activity of SOD 1 was kept in brain, liver, muscle, kidney and heart, and followed in gut, spleen and lung. The activities of SOD2 in brain, liver, muscle, kidney, gut and heart were significantly higher than those in spleen and lung.

Differentiating Trachemys scripta elegans Shell Glue from Chinemys reevesii Shell Glue by UPLC-QTOF/MS Coupled with Binary Compare Tool of UNIFI

Objective This study was conducted to develop an method for identification of Trachemys scripta elegans(Ba Xi Gui,巴西龟)shell glue from Chinemys reevesii(Zhong Hua Cao Gui,中华草龟)shell glue.Methods In this research,an ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry(UPLC-QTOF/MS)method coupled with Binary Compare tool of UNIFI software was validated to differentiate Trachemys scripta elegans(Ba Xi Gui,巴西龟)shell glue from Chinemys reevesii(Zhong Hua Cao Gui,中华草龟)shell glue.The gelatins were digested enzymatically into peptides using trypsin.The UPLC-QTOF/MS data of the trypsin digested samples was subjected to Binary Compare of UNIFI software in order to screen out the unique peptides.PEAKS■Studio software was utilized to identify the sequence of the marker peptides.Results It was found that m/z 641.3 was the marker peptide in samples of Trachemys scripta elegans(Ba Xi Gui,巴西龟)shell glue,and m/z 649.3 was the marker peptide in samples of Chinemys reevesii(Zhong Hua Cao Gui,中华草龟)shell glue,with possible amino acid sequence of GEAGPSGPAGPTGAR and GESGPSGPAGPTGAR respectively.Conclusions The results will be helpful for the differentiating between these 2 gelatins,and it can be also used for quality control of Tortoise shell glue(Gui Jia Jiao,龟甲胶).

Effect of Dietary Vitamin C on the Antioxidant Defense System of Hibernating Juvenile Three-keeled Pond Turtles (Chinemys reevesii)

Juvenile three-keeled pond turtles(Chinemys reevesii) were fed diets supplemented with vitamin C(Vc) at doses of 0(basal diet, Vc0), 100(Vc100), 200(Vc200), 500(Vc500) and 2500(Vc2500) mg/kg diets at 28°C for 4 weeks, respectively. Then, the water temperature was gradually reduced to 10°C, and the turtles were induced into hibernation. Liver tissue samples were collected at three time points: start of hibernation(T1), 4 and 6 weeks’ hibernation(T2 and T3). A control group fed with the basal diet was set to parallel the whole treatment process, but reared at 28°C constantly. The results showed that hibernation mildly affected the antioxidant system and the influence varied with hibernating time. Hepatic malondialdehyde content of the Vc100 group was significantly lower than that of the other groups at T1. At T2, hepatic MDA in the groups of Vc500 and Vc2500 decreased significantly, while no clear differences were found among all groups at T3. The activities of antioxidant enzymes showed a positive correlation with dietary Vc dose before hibernation. After hibernation, total antioxidant capability was not affected by Vc. Superoxide dismutase activity became similar in different groups at T2, but decreased in higher Vc groups(≥ 200 mg/kg) at T3. Glutathione peroxidase and glutathione-S-transferase activities decreased significantly with dietary Vc supplementation(≥ 100 mg/kg) at T2, but recovered at T3. The result indicates that under normal rearing condition, low dietary Vc supplementation(< 100 mg/kg) might be beneficial to the antioxidant defense system. The effect of dietary Vc on the antioxidant defense system differed during hibernation.

孵化温度对乌龟性别及质量的影响

为评估不同孵化温度对乌龟(Chinemys reevesii)性别及质量的影响,探求更好的稚龟孵化条件,本研究将乌龟受精卵置于25、27、29、31、33℃、转温温度(33℃转29℃)条件下孵化,统计孵化率、测量稚龟形态特征以及运动性能和生理生化指标等。结果显示:27℃孵化率最高,为42.67%,且稚龟的尾长、背甲长、背甲宽、体高、腹甲长最大;27℃孵出的稚龟翻身速度最快,与31、33℃存在显著性差异;29℃孵出的稚龟游泳速度最快,与25、33℃呈现显著性差异;33℃孵化,孵化期最短和雌性比例最高,与25、27、29℃孵化差异显著,而33℃孵化的雌性比例与转温温度孵化没有显著性差异;33℃转29℃孵化温度下腹甲宽、体重最大,乌龟生理生化指标中仅总抗氧化能力存在显著性差异,33℃的总抗氧化能力最低,与29℃有显著性差异,转温温度组处于两者之间,与两者没有显著性差异。综上所述,27℃、29℃与转温温度处理能产生高质量的稚龟,而转温温度与恒定的27℃、29℃相比,其孵化周期更短,稚龟雌性比率更高,与31、33℃相比,则稚龟质量更好。综上结果表明本研究的转温温度方式可用来做为规模化扩繁全雌乌龟苗种的生产方式。

Effects of Pesticide Exposure on Embryonic Development and Hatchling Traits of Turtles

Deltamethrin is a widespread environmental hormone with endocrine-disrupting properties, but its effect on embryonic development of reptiles is largely unexplored. We investigated the effects of deltamethrin on embryonic development and offspring traits in two turtle species, one with parchment-shelled eggs and the other with rigidshelled eggs. Deltamethrin exposure during egg incubation did not affect hatching success and hatchling body size in either species. However, embryonic exposure to deltamethrin resulted in reduced hatchling locomotor performance in the red-eared slider turtle（Trachemys scripta） with parchment-shelled eggs, but not in the Chinese three-keeled pond turtle（Chinemys reevesii） with rigid-shelled eggs. These results suggest that parchment-shelled eggs are likely more vulnerable to deltamethrin than rigid-shelled eggs.