Breeding Habitat Selection of Reeves’s Pheasant (Syrmaticus reevesii) in Dongzhai National Nature Reserve, Henan Province, China

Reeves＇s Pheasant （Syrmaticus reevesii） is a threatened pheasant species endemic to China. The habitat use of territorial male birds was surveyed by the help of live decoys in a core area of Dongzhai NationaI Nature Reserve. The breeding habitat selection of this pheasant was examined at two scales （115 m and 250 m scale, i.e. 4.15 hm^2 and 19.63 hm^2, respectively）, including the characteristics at distance scale. Investigation was based on line transect, RS and GIS in Dongzhai National Natural Reserve from 2001 to 2003. Moreover, a range of habitat variables were compared between used and control points at each scale, and stepwise logistic regression was applied to select the key scale and the key habitat factors in relation to breeding habitat selection of this bird. Our results stated that the territorial males at Baiyun occurred mostly in mixed forests, followed by fir forests, pine forests, shrubs, and broadleaf forests. The area of conifer forests was the key factor influencing habitat selection of this bird in breeding period at the scales of 115 m and 250 m, and the proximity of farmland was important for habitat selection in breeding seasons. Furthermore, Reeves＇s Pheasants attached great importance to the scale of 115 m. When considering a range of habitat variables at all scales within a multivariate regression, the leading factors having effect on habitat selection in the breeding period were areas of conifer forests at 115 m scale and the distance to farmland. In addition, these above results suggested that strengthening the management of suitable habitat, and optimizing the habitat configuration are important in promoting conservation of this bird. However, it also highlighted the importance of initiating future researches on the conifer forests and their impact on the population of Reeves＇s Pheasants, which would be beneficial to promote the habitat conservation of this pheasant more effectively.

白冠长尾雉(Syrmaticus reevesii)的研究现状及保护建议

白冠长尾雉(Syrmaticus reevesii)为中国特有珍稀雉类和国家Ⅱ级重点保护动物,由于人类活动的扩张,白冠长尾雉的种群数量在急剧下降,分布面积在迅速退缩,面临着绝迹的危险。为了保护白冠长尾雉,从生态学和行为学领域对白冠长尾雉进行综述,分析了其致危因素并提出了合理的保护建议,以期为白冠长尾雉进一步的研究和保护提供参考。

白冠长尾雉(Syrmaticus reevesii)卵壳的超微结构及元素组成研究

对采自河南董寨国家级自然保护区的白冠长尾雉（Syrmaticus reevesii）卵壳样品进行了检测和研究，利用扫描电子显微镜观察了卵壳的超微结构，采用电感偶合等离子体光谱仪测定了卵壳中的化学元素．结果表明：白冠长尾雉卵壳的表面晶体层、栅栏锥体层和壳膜层的厚度分别为20．8，220．8和62．5μm，分别占卵壳总厚度的6．8％，72．6％和20．6％；栅栏层有很多蜂窝小孔，其直径为（0．32±0．08）μm（n=30）；卵壳表面有许多圆形或椭圆形的气孔，可能与胚胎发育过程中的气体交换有关，气孔横断面呈细漏斗状，在其上半部有颗粒状填充物．同时比较了白冠长尾雉野生和笼养个体未孵化卵壳中21种化学元素的含量，发现在组成卵壳的化学元素中，野生白冠长尾雉的Ca，Mg，P，S的质量分数叫〉1mg·g，其中Ca含量超过卵壳元素总含量的40％；Na，Si，S，K和A1次之，w=0．1～1mg·g^-1；Fe，Zn，Pb，Mn，Cu，V，Ti元素的w=1～100μg·g^-1；Ni，Cr，Co，Se，Cd的含量最低，w〈1μg·g^-1．与野生个体的卵壳相比，保护区人工饲养的白冠长尾雉的卵壳成分有显著差异，其中S，Cu，Fe，Al，Mn，Si，Sr，Se，Cr等元素野生个体与笼养个体相比，同种元素在不同个体上w的差异均达到20％以上．饲养个体对Fe，Mn，Si和Sr等4种元素摄入明显不足，而S，Cu，Al，Se，Cr等元素则显著偏高，这可能是造成董寨自然保护区白冠长尾雉卵受精率较低的主要原因．建议适当调整饲料中这些元素的配比，以保证饲养条件下白冠长尾雉拥有良好的营养供给，从而提高人工繁殖的成功率．

乌龟（Chimemys reevesii）精子的超微结构

用透射电镜观察了乌龟精子的形态。精子长度６５～７０μｍ，头部长１７～２０μｍ，尖而弯曲，并有以下特点：１．顶体复合体细而尖；２．核内有两条小管；３．连接段在细胞核末端形成连接领；４．中段的线粒体由９或１０层不规则的同心圆状嵴及致密中心构成。研究发现乌龟精子同海龟类、喙头类及眼镜鳄的精子构造相似；但同蜥蜴类，鸟类及哺乳类的差别较大。

幼乌龟Chinemys reevesii配合饲料蛋白质最适含量的研究

首次采用蛋白质梯度法,以白鱼粉为蛋白源,测定出幼乌龟配合饲料最适蛋白质含量。养殖42d后,以相对增重率作衡量指标,用直线回归及抛物线回归方程拟合,结果表明:幼乌龟配合饲料蛋白质最适含量为35.75%～40.38%。

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.

The Effect of the Cyp19a1 Gene Methylation Modification on Temperature-dependent Sex Determination of Reeves' Turtle(Mauremys reevesii)

Temperature-dependent sex determination（TSD） is a type of environmental sex determination in which the sex of the embryos depends on the ambient temperature; however,the molecular mechanisms governing this process remain unknown.Aromatase,encoded by the cyp19a1 gene,which converts androgens into estrogens in animals,was considered to be the key gene for TSD.In this study,the 5＇-flanking region of the cyp19a1 gene in Reeves＇ turtle（Mauremys reevesii） was cloned,and the promoter region was identified using the luciferase reporter assay.Then the eggs of Reeves＇ turtle were incubated at different temperatures（26°C： male-biased temperature; 29°C： non-sex-biased temperature and 32°C： female-biased temperature）.During the thermosensitive period,the adrenal kidney gonad complexes（AKG） were sampled.DNA methylation analysis of the AKG samples showed that the promoter region of the cyp19a1 gene was significantly de-methylated in the female-biased temperature regime（P＆lt;0.01）.Quantitative analysis of the cyp19a1 gene and estrogen by q PCR and Elisa assay showed that the expression level of the cyp19a1 gene and estrogen content were both upregulated significantly at the female-biased temperature（P＆lt;0.01）.These results indicated that the de-methylation response of the cyp19a1 gene to incubation temperature,especially at the female-biased temperature,could lead to temperature-specific expression of aromatase and increased estrogen levels,which may further determine gonadal development in Reeves＇ turtle.These findings provide insights into the genetic mechanisms underlying TSD.

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.

Behavioral and Physiological Responses of Sea Slug (Onchidium reevesii) to Low-Frequency Noise

A large amount of marine noise pollution from coastal industrial construction and the water transportation industry affects the growth and development of marine life,which is a big issue threatening marine organisms.However,most studies have focused on vertebrates,such as marine mammals and fishes,and little is known about the effects of noise on invertebrates.Therefore,the impacts of low-frequency noise(100,300,and 500 Hz)on behavioral and physiological responsesof the sea slug(Onchidium reevesii)were investigated.Under laboratory conditions,sea slugs were stimulated with low-frequency noise at 100,300,and 500 Hz for 1 h.Then,hemolymph enzyme activities(glucose,albumin,triglycerides,superoxide dismutase,catalase,and malondialdehyde)were measured,and mRNA expression of the hsp70 gene was detected in hemolymph and the nervous system by quantitative realtime polymerase chain reaction(qRT-PCR)analysis,while expression of the hsp70 protein was analyzed by immunohistochemistry.The results demonstrated that sea slugs were away from the stimulus source under the influence of low-frequency noise compared to the control group.Enzyme activities,as well as hsp70 gene mRNA and protein expression levels,were significantly higher in the noiseexposed groups than those in the control group.Overall,these changes indicate that low-frequency noise caused oxidative stress in sea slugs in vivo,and the oxidative damage gradually increased when the noise frequency was increased from 100 to 500 Hz.

韩国乌龟(Mauremys reevesii)的分布及季节性移动距离研究(英文)

为确定韩国乌龟(Mauremys reevesii)的地理分布及生境状况,作者采用活捕和直接观察法全面调查了韩国所有国家公园及其附近地区,同时,在全罗南道求礼郡利用无线电发射器跟踪法研究测定了乌龟的家域。研究结果显示,在韩国共有60处观察到乌龟的分布,且多集中在韩国的南半部;最大季节性移动距离达196 m,在统计学上未出现个体间差异,但实际测量距离仍可发现较大差异;在韩国乌龟的家域确定为2.8~6.1 hm^2之间。

Lineage diversification and niche evolution in the Reeves’Butterfly Lizard Leiolepis reevesii(Agamidae)

We used mitochondrial cytochrome b and ND4 genes and 9 microsatellite loci to determine genetic diversity,population structure,evolutionary history,and migration patterns within the Reeves’butterfly lizard Leiolepis reevesii(Agamidae).Considering molecular-based phylogeographical lineages,we then performed niche equivalency and similarity tests between divergent lineages.Phylogenetic analyses based on mitochondrial DNA(mtDNA)data revealed 2 lineages(A and B)diverging≈0.84 million years ago and,respectively,restricted to the northern and southern portions of the Wuzhishan and Yinggeling mountain ranges.Lineage B contains individuals from southern Hainan;Lineage A includes individuals from all other localities and can be further divided into 3 clusters according to microsatellite data.The null hypothesis that the 2 lineages shared identical niches was rejected in all niche equivalency tests,indicating niche shifts during genetic divergence.Similarity tests provided evidence of niche conservatism,suggesting that the 2 lineages share more characteristics of their niche spaces than randomly expected.The niche similarity and equivalency tests indicated a complex niche pattern in which both lineages share a main portion of their ecological spaces.The climatic niche of Lineage B represented a marginal and specialized fraction of the entire ecological space of the climatic niche of Lineage A,with warmer conditions.Isolation caused by orogenesis and subsequent niche divergence,together with local adaptation,may have led to genetic differentiation and further lineage sorting in L.reevesii.

Analyses of the differential expression of cloned Caspase-3 and Caspae-9 genes,of Onchidium reevesii,exposed to extreme hot and cold conditions

During low tide intertidal organisms face severe temperature fluctuations.This situation may be more pronounced in the context of climate change.To investigate the molecular response mechanism of Onchidium reevesii when stressed by hot and cold temperatures,we cloned its Caspase-3 and Caspase-9 genes and detected their expression characteristics using RT-qPCR.The results showed that the full-length of the cDNA of Caspase-3 consist of 1831 bp,and included an open reading frame(ORF)of 894 bp,a 50UTR of 155 bp and a 30 UTR of 300 bp,and encoding for 297 amino acids.The full-length of the cDNA of Caspase-9 consisted of 2425 bp,and included an ORF of 1373 bp,a 50 UTR of 290 bp,and a 30 UTR of 759 bp,and encoding for 457 amino acids.During the evolution of species the two genes have been greatly conserved.A homology analysis and phylogenetic tree reconstruction revealed that O.reevesii is most closely related to Aplysia californica.The RT-qPCR showed that both genes were expressed in all of the tissues and that expression was highest in the hepatopancreas.It is speculated that these genes play an essential role in the immunity of O.reevesii to certain diseases.Conditions of temperature stress(40�C and 10�C)showed that both heat and cold stresses could induce significant changes in both genes.Compared to the control group,under cold stress conditions the levels of expression of Caspase-3 and Caspase-9 were significantly upregulated(P<0.05)in each experimental group.When exposed to a constant temperature of 10�C,their expression peaked after 1 h(4.32-fold,P<0.05;and 5.58-fold,P<0.05,respectively)and was at its lowest after 12 h(1.93-fold,P<0.05;and 2.35-fold,P<0.05,respectively).Their expression was upregulated after 24 h(2.68-fold,P<0.05;and 4.21-fold,P<0.05,respectively).The expression of both genes showed similar trends.Under heat stress,the expressions of the two genes showed a wave pattern.When exposed to a constant temperature of 40�C,their expression was up-regulated after 1,6,and 24 h,and down-regulated after 3 and 12 h.Except at 24 h,the other experimental groups did not differ significantly(P>0.05)from the control group.When subjected to various stress conditions the two genes displayed different expression patterns,indicating that the species has different strategies for coping with temperature changes.It further indicates that the effect of temperature on the species may play a role through the Caspase-dependent pathway.These may help to better understand the response,at the molecular level,of O.reevesii to conditions of hot and cold stress.

Home range and habitat use of male Reeves’s Pheasant(Syrmaticus reevesii)during winter in Dongzhai National Nature Reserve,Henan Province,China

Home range and habitat use of male Reeves’s pheasant(Syrmaticus reevesii)were studied during winter of 2001~2002 and 2002~2003 in the Dongzhai National Nature Reserve,Henan Province.Results from five individuals of Reeves’s pheasant with over 30 relocations,indicated that the average size of home range was 10.03±1.17 hm^(2) by Minimum Convex Polygon method,8.60±0.35 hm^(2) by 90%Harmonic Mean Transformation method,and 9.50±1.90 hm^(2) by 95%Fixed Kernel method.It was observed that the winter range is smaller than that in the breeding season.The mean core area of the home range was found to be 1.88±0.37 hm^(2).Although the habitat composition of the core area varied greatly for individuals,a large part of the habitats used were composed of conifer and broadleaf mixed forests,masson pine forests,fir forests,and shrubs.Habitat use within the study area was non-random,while habitats within home ranges were randomly used.Habitat use was dictated by tree diameter at breast height,shrub height and coverage at 2.0 m.The proximity between forests and shrubs were also found to be important in providing refuge for the birds during winter.Recommendations for conservation management include protecting the existing habitats in Dongzhai National Nature Reserve,increasing suitable habitat for Reeves’s Pheasant through artificial plantations(e.g.firs),and restoring some parts of the large shrub area into forests.

Ultrastructure and elemental composition of the eggshell of Reeve’s Pheasant(Syrmaticus reevesii)

The eggshell of Reeve’s pheasant(Syrmaticus reevesii)collected from the Dongzhai National Nature Reserve in Henan Province,China was studied.By using scanning electron microscopy and inductively coupled plasma(ICP)spectrometry,the ultrastructure and elemental composition of the eggshell was determined.The study showed that the average thickness of the surface layer of crys-tals,the palisade and cone layer,and the eggshell membrane were 20.8,220.8 and 62.5μm,respectively,accounting for 6.8%,72.6%and 20.6%of the total thickness of the eggshell.There were many vesicular holes in the palisade layer with an average diameter of 0.32±0.08μm(n=30).The function of these holes might be significant to air exchange.The shape of the eggshell pore on the surface layer of crystals is round or elliptical.The fracture surface of the pore is funnel-shaped.Some granules filled the upper part of the eggshell pores.The content of 21 elements in the eggshell of wild and captive Reeve’s pheasants was compared and presented.It indicated that among the elements that made up the eggshell of the wild pheasant,the content of Ca,Mg,P and S was much higher,ω>1 mg/g,withω(Ca)being higher than 40%of the eggshell.The contents of Na,Si,Sr,K and Al wereω=0.1-1 mg/g,while Fe,Zn,Pb,Mn,Cu,V and Ti had lower concentrations(ω=1-100μg/g).Theωof Ni,Cr,Co,Se,Cd were lower than 1μg/g.The elemental composition in the eggshell of the captive Reeve’s pheasant kept in the Dongzhai National Natural Reserve was significantly differ-ent from that of the wild species,with a difference of over 20%on S,Cu,Fe,Al,Mn,Si,Sr,Se and Cr.The lower intake of Fe,Mn,Si and Sr on the one hand and the higher intake of S,Cu,Al and Cr on the other hand might be responsible for the low fertility of captive Reeve’s pheasants in the Dongzhai National Nature Reserve.In order to ensure that the pheasants are receiving the proper amount of nutrition and to improve their breeding success,the amount of certain elements in the food should be adjusted.

Expression of mapk1 and egr1 genes in Onchidium reevesii under tidal stimulation

Previous studies have speculated that tidal rhythm of Onchidium reevesii was linked to its memory ability, which has not been well explained. Mapk1 and egr1 genes are closely related to memory formation in the MAPK signaling pathway that have been extensively studied in vertebrates. These two genes are involved in Long-term potentiation (LTP), which is generally regarded as one of the main molecular mechanisms underlying learning and memory. To investigate the relationship between tidal rhythm of O. reevesii and its memory ability, we studied the molecular mechanism of mapk1 and egr1 genes cooperating with tidal rhythm. The full-length cDNA sequence of mapk1 gene was cloned by RACE method and analyzed by bioinformatics, then qRT-PCR was used to analyze its expression levels in each tissue. A homology analysis and phylogenetic tree reconstruction revealed that O. reevesii is most closely related to the Biomphalaria glabrata. The qRT-PCR showed that mapk1 was expressed in all the tissues, but the highest expression was in the ganglion. We speculated that mapk1 is related to memory formation of O. reevesii. Then we used qRT-PCR to detect the expression of mapk1 and egr1 in ganglion of O. reevesi under tidal stimulation. The expression of mapk1 and egr1 at the rising tide points were significantly higher (P < 0.05) than that at the previous one lowest tide points on May 15-21, except the rising tide point on May 20 and May 21 of mapk1. The expression of mapk1 and egr1 genes in the ganglion sampled for 7-day were basically the same as that of the tide change of the same day. The expression of the two genes were upregulated at raising tide and downregulated at the lowest tide, it is presumed that the O. reevesii was affected by the tide for a long time and formed the tide memory.

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。

低温对乌龟稚龟应激水平和天然免疫能力的影响

为探究乌龟(Mauremys reevesii)稚龟对低温胁迫的生理适应性,采用背甲下静脉窦采血技术,在6、17和26℃条件下分别处理第1、10和28天时,测定了其身体大小、应激水平和血浆杀菌能力的变化。结果显示:(1)温度和处理时间均不影响稚龟的体重、背甲长和背甲宽(P> 0.05)。(2)6、17和26℃组嗜异性粒细胞与淋巴细胞的比值均不随处理时间而变化(P> 0.05),只在第1天时,6℃组显著低于17℃组(P <0.05)。(3)随处理时间延长,血浆杀菌能力17℃组显著下降(P <0.05),26℃组显著升高(P <0.05);只在第28天时,6℃组和17℃组均显著低于26℃组(P <0.05)。低温对乌龟稚龟的生长无明显的抑制作用,短期低温胁迫导致其应激反应能力增强,天然免疫能力明显下降,不利于其适应入眠前和出眠后环境温度的大幅度波动。

低氧胁迫对瘤背石磺Toll样受体4、血细胞和免疫酶活性的影响

为探究瘤背石磺面临低氧环境时固有免疫的应激机制,实验以RACE法对瘤背石磺TLR4基因进行全长克隆,并进行生物信息学分析,测定了低氧胁迫下瘤背石磺TLR4基因的表达变化,以及分析了血细胞活力和溶菌酶(LSZ)活性、肝脏中超氧化物歧化酶(SOD)和碱性磷酸酶(ALP)活性。结果显示,瘤背石磺TLR4基因cDNA全长共3605 bp,包括编码956 aa氨基酸的2817 bp开放阅读框。系统进化树表明,瘤背石磺TLR4基因与光滑双脐螺TLR4基因的进化地位较为接近。qRT-PCR结果显示,TLR4基因在瘤背石磺7个组织中均有表达,其中表达量最高的是肝脏。低氧胁迫下,各组织中TLR4基因的表达量皆显著上升,其中神经节在4 h时率先达到峰值。此外,血细胞活力、LSZ活性、ALP活性都是呈先下降后上升的趋势,而SOD活性呈先上升再下降再上升的趋势,波动幅度最为明显。研究结果初步说明了TLR4的生理功能,为探究潮间带生物免疫系统的低氧应激机制提供理论参考。

瘤背石磺NR1基因的克隆和序列分析以及低频声音刺激对其表达的影响

长期栖息于潮间带的瘤背石磺能感知潮汐来临时产生的低频声音,本研究模拟潮汐的低频声音刺激瘤背石磺,探讨NR1型受体基因在瘤背石磺感应低频声音的行为中发挥的作用。首先通过c DNA末端扩增法(RACE)得到瘤背石磺NR1基因(OrNR1) cDNA全长,进行生物信息学分析,并通过实时荧光定量PCR (qRT-PCR)检测OrNR1基因在各组织以及不同低频声波刺激下神经节中的表达。结果显示,OrNR1基因全长2 434 bp,其中5'非编码区(UTR)长357 bp, 3'UTR长184 bp,1 893 bp开放阅读框共编码630氨基酸。瘤背石磺OrNR1蛋白分子质量约为70.29 ku,等电点为5.93,分子式为C3 153H4 911N833O924S32。OrNR1基因含有信号肽,一个预测的甘氨酸和谷氨酸结合位点,一个跨膜域,多序列比对结果表明各物种间NR1具有较高的保守性。系统进化树结果显示,瘤背石磺OrNR1基因与加州海兔亲缘关系最高,符合传统形态学分类。qRT-PCR结果显示,OrNR1在瘤背石磺不同组织均有表达,在神经节中相对表达量最高,其次是肝胰腺、腹足,而在口器和背部皮肤中表达量较低OrNR1在频率为200、160 Hz的低频声音刺激下表达量在不同时间组均显著高于对照组,40 Hz的低频声音刺激下2 h后表达量显著高于对照组;在120 Hz时OrNR1基因表达量受到抑制,不同组的表达量有显著差异。推测该基因在瘤背石磺低频声音感知中起到了重要作用。