White-spot disease of Chinese soft-shelled turtles (Trionyx sinens) caused by Paecilomyces lilacinus

Chinese soft-shelled turtles （Trionyx sinens） in culture farms using an artificial warming system in Zhejiang, China, often show typical signs of white-spot disease such as white spots on their bodies, skin lesions, anorexia and eventually death. The sick turtles were mostly 5-80 g in weight. A suspected fungal pathogen was isolated from the sick turtles and verified as Paecilomyces lilacinus by sequence analysis of the internal transcribed spacer （ITS） of its ribosomal DNA （rDNA）. Detailed morphological examinations were also conducted to confirm the white-spot disease.

Effects of Brown Fish Meal Replacement with Fermented Soybean Meal on Growth Performance, Feed Efficiency and Enzyme Activities of Chinese Soft-shelled Turtle, Pelodiscus sinensis

A 120-day feeding experiment was conducted to investigate the effects of partial replacement of brown fish meal (BFM) by fermented soybean meal (FSBM) in diets of Chinese soft-shelled turtle (Pelodiscus sinensis). The turtles (initial mean body weight, (115.52 ± 1.05) g) were fed with three experimental diets, in which 0%, 4.72% and 9.44% BFM protein was replaced by 0%, 3% and 6% FSBM, respectively. Results showed that the feeding rate (FR), specific growth rate (SGR) and feed efficiency ratio (FER) of turtles fed with the diet containing 3% FSBM were not significantly different from the control group (0% FSBM) (P > 0.05). However, FR, SGR and FER of turtles fed with the diet containing 6% FSBM were significantly lower than those of the control group (P < 0.05). No significant differences were observed in the activities of serum glutamic-oxalacetic transaminase and glutamicpyruvic transaminase among dietary treatments (P > 0.05). However, the uric acid concentration in turtles fed with the diet containing 3% or 6% FSBM was significantly lower than that in the control group (P < 0.05). There were no significant differences in the activities of lysozyme, alkaline phosphatase and total superoxide dismutase among dietary treatments (P > 0.05). The results suggested that FSBM could replace 4.72% BFM protein in turtle diets without exerting adverse effects on turtle growth, feed utilization and measured immune parameters.

Physiological responses in vitamin C system during hibernation in juvenile Chinese soft-shelled turtle Pelodiscus sinensis

Vitamin C (Vc) is an important antioxidant that helps turtles tolerating stressful environment. This work quantified changes in tissue Vc levels during winter hibernation in the soft-shelled turtle Pelodiscus sinensis , to reveal the stress response pattern of tissue Vc during hibernation and contribute basic data for turtle culture. We sampled juvenile soft-shelled turtles at pre-hibernation (17.0°C mud temperature;MT), during hibernation (5.8°C MT) and after arousal (20.1°C MT) in the field. The transcript levels of the gene encoding L-gulonolactone oxidase (GLO), the key enzyme for Vc synthesis, decreased significantly during hibernation. However, GLO activity did not match the GLO transcription patterns and remained stable during hibernation, and showed temperature-dependent kinetic characteristics. Vitamin C levels in the brain, liver, kidney, and spleen (but not muscle) all decreased significantly during hibernation, but recovered to pre-hibernation levels or even higher levels after arousal. The soft-shelled turtle endured 5 months of hibernation with no significant oxidative damage in most tissues, except in the spleen. Splenic Vc was nearly exhausted during hibernation, accompanied by a significantly elevated malonaldehyde (MDA) level. Although the high level of oxidative damage quickly decreased after arousal, the potential tissue damage in the spleen during hibernation might account for the weakened immune capacity of turtles after hibernation.

Effects of ration level and feeding frequency on digestibility in juvenile soft-shelled turtle, Pelodiscus sinensis

The effects of ration level and feeding frequency on digestibility in juvenile soft-shelled turtle, Pelodiscus sinensis, were investigated. Four ration levels 1.5%, 2.5%, 4.0% and satiation （6.0% BW/d） were used. Apparent digestibility （AD） of dry matter （DMAD）, protein （PAD） and protein real digestibility （PRD） were significantly affected by ration level, but not by feeding frequency when the ration level was similar. However, the feeding frequency affected the AD, DMAD, PAD and PRD significantly when the turtles were fed to satiation. The relationship between fecal protein content （Y） and protein intake （At） can be expressed as a quadric equation： Y=-0.1742＋0.1476X-0.0003X^2 （r^2=0.876, n=27, F=93.92, P〈0.01）.

Effects of Dietary Soy Protein Concentrate on Growth, Digestive Enzymes Activities and Target of Rapamycin Signaling Pathway Regulation in Juvenile Soft-Shelled Turtle, <i>Pelodiscus sinensis</i>

Soft-shelled turtle, Pelodiscus sinensis is important aquatic species in China, and searching for alternatives protein resources to fish meal (FM)-based feeds in feed has become urgent and important for its sustainability development. The present study was conducted to assess the effects of dietary soy protein concentrate (SPC) on growth, digestive enzymes and target of rapamycin (TOR) signaling pathway of juvenile P. sinensis (4.56 ± 0.09 g). SPC was applied to replace FM protein at 0%, 15%, 30% and 60% (designated as T0, T15, T30 and T60, respectively), and each diet was fed to triplicate groups. The results showed that there was no significant difference in growth performance and feed utilization except of the turtles fed with T60 diet, of which showed poorer daily weight gain and feed conversion rate. The pepsin/trypsin and Na+-K+ ATP-ase activities decreased dramatically when SPC level increased, and lipase activities in liver and intestinal tract also showed decline tendency. However, amylase activities were unaffected. No significant differences were observed in TOR, S6K1 and 4E-BP1 genes mRNA expression level of TOR signaling pathway among the treatments. However, the relative phosphorylated level of these proteins decreased significantly when SPC level increased. The present study indicated that high SPC substitution level would suppress digestive enzymes and TOR signaling pathway proteins phosphorylated level and eventually result in growth reduction of P. sinensis.

Discovery of virus pathogen from soft-shelled turtle Trionyx sinesis

THE soft-shelled turtle Trionyx sinesis, because of its nutritional and medicinal values,

Effects of acute cold exposure on oxidative balance and total antioxidant capacity in juvenile Chinese soft-shelled turtle,Pelodiscus sinensis

Acute cold exposure may disturb the physiological homeostasis of the body in ectotherms.To date,there has been no information on the effects of cold exposure on homeostasis of reactive oxygen species(ROS)or antioxidant defense response in the Chinese soft-shelled turtle,Pelodiscus sinensis.In this study,P.sinensis juveniles were acclimated at 28℃,transferred to 8℃ as cold exposure for 12 h,then moved back to 28℃ rewarming for 24 h.We measured the ROS level and total antioxidant capacity(TAC)in the brain,liver,kidney and spleen at 2 and 12 h cold exposure,and at the end of the rewarming period.Malonaldehyde(MDA)and carbonyl protein were used as markers of oxidative damage.Turtles being maintained simultaneously at 28℃ were used as the control group.Cold exposure did not disturb the ROS balance in all 4 tissues,while rewarming raised the ROS level in the brain and kidney of P.sinensis.Cold exposure and rewarming decreased the TAC in the brain,liver and spleen but did not change the TAC in the kidney.MDA and carbonyl protein levels did not increase during the treatment,indicating no oxidative damage in all 4 tissues of P.sinensis.Our results indicated that extreme cold exposure did not impact the inner oxidative balance of P.sinensis,but more ROS was produced during rewarming.P.sinensis showed good tolerance to the harsh temperature change through effective protection of its antioxidant defense system to oxidative damage.This study provides basic data on the stress biology of P.sinensis.

Supplementation with turmeric residue increased survival of the Chinese soft-shelled turtle (Pelodiscus sinensis) under high ambient temperatures

Turmeric residue（TR）, containing residual levels of curcumin, is a solid by-product waste generated after the extraction and separation of curcumin from turmeric root. A feeding trial was conducted to evaluate the effects of TR on the survival of Chinese soft-shelled turtles（SSTs）, Pelodiscus sinensis, under a high ambient temperature. A total of 320 female SSTs were assigned randomly to two diets： basal diet（the control group, n=160） and an interventional diet supplemented with 10% TR（the TR group, n=160）. Our results demonstrated that supplementation of TR increased the SST survival rate by 135.5%, and superoxide dismutase（SOD） activity of SST liver by 112.8%, and decreased the malondialdehyde（MDA） content of SST liver by 36.4%, compared to the control group. The skin of the SST fed TR showed a golden color. High-performance liquid chromatography（HPLC） analysis indicated that the concentrations of curcumin in TR and the skin of the SST fed TR were（1.69±0.30） and（0.14±0.03） μg/g, respectively. Our observation suggests that supplementation of TR increased the survival rate of SST under high ambient temperatures. We speculated that the increased survival rate and tolerance at the high ambient temperature were associated with the anti-oxidation activity of curcumin from TR. Moreover, curcumin in TR could be deposited in SST skin, which made it more favored in the market of China. Our findings provide new knowledge and evidence to effectively reuse TR as a feed additive in animal and aquatic farming.

Division of Chinese soft-shelled turtle intestine with molecular markers is slightly different from the morphological and histological observation

The Chinese soft-shelled turtle(Pelodiscus sinensis)is a commercially important species in Asian countries.Knowledge of its nutritional requirements and physiology is essential for determining the appropriate content of the feed for this animal.However,the lack of functional characterization of the intestine of this turtle limits the understanding of its absorption and utilization of nutritional materials.To solve this problem,this work utilized anatomical and histological methods to characterize 9 segments sampled along the anterior-posterior axis of the intestine.Furthermore,9 genes,which have been well documented in the intestine division of mammals and fish,were employed to functionally characterize the 9 sampled segments.Our results suggest that regions covering from the starting site to S3(position at 29.9%of the total length from the starting of the intestine)are the equivalent of mammalian dedumonen,and those covering S4(40.2%)and S5(65.4%),posterior to S8(92.7%),are the equivalent of the mammalian ileum and the large intestine,respectively.As to the region spaning S6(81.3%)and S7(87.3%),its functional equivalent(small intestine or large intestine)may be variable and depends on the functional genes.This molecular characterization in relation to the division of the intestine of Chinese soft-shelled turtle may contribute to the understanding of the nutritional physiology of the turtle,and promote Chinese soft-shelled turtle production.

Medicinal Soft-Shelled Turtle

Ingredients:Whole soft-shelled turtle; Chinese wolf-berry fruit(Lycium Chinese), milk vetch membrane root(Astragalus membranaceus), Chinese angelica andAmerican ginseng--about 25g of

黄沙鳖致病性蜡样芽孢杆菌的分离鉴定及药敏试验

【目的】对广西武宣县某养殖场患病黄沙鳖(Pelodiscus sinensis)进行病原菌分离鉴定及药敏试验,为防治黄沙鳖蜡样芽孢杆菌病提供参考依据。【方法】采用常规方法对患病黄沙鳖进行细菌分离,通过人工感染试验确定其致病性。采用API 50CH生化鉴定和16S rRNA序列分析进行细菌鉴定,并以K-B纸片扩散法及二倍稀释法进行药敏试验。【结果】从患病黄沙鳖肝脏中分离获得1株具有芽孢的革兰氏阳性杆菌GXWXAA,对健康黄沙鳖致死率达100.0%。经鉴定,菌株GXWXAA属于蜡样芽孢杆菌(Bacillus cereus),与蜡样芽孢杆菌JY5(HQ833024)、JY7(HQ833025)、JY9(HQ833026)和VBE16(MG027671)的亲缘关系最近,相似性均达99.9%。菌株GXWXAA对新霉素、恩诺沙星和多西环素等15种抗生素敏感,对头孢曲松、环丙沙星和依诺沙星中介,对青霉素G、氨苄青霉素、复达欣、复方新诺明和多粘菌素B耐药。新霉素、恩诺沙星和多西环素对菌株GXWXAA的最小抑菌浓度分别为0.488、0.122和0.244μg/mL。【结论】引起广西武宣县某养殖场黄沙鳖发病的病原菌属于蜡样芽孢杆菌,建议选择新霉素、恩诺沙星和多西环素等抗生素对患病黄沙鳖进行治疗。

黄沙鳖抗菌肽cathelicidin基因克隆及表达特征分析

【目的】克隆黄沙鳖cathelicidin基因并分析其组织分布特征及在攻毒后的表达变化,为深入研究cathelicidin功能及在黄沙鳖先天免疫中的潜在作用提供基础数据。【方法】采用RT-PCR结合RACE克隆黄沙鳖cathelicidin基因cDNA全长序列,利用生物信息学分析软件对黄沙鳖cathelicidin基因及其推导氨基酸序列进行结构特征分析,并采用实时荧光定量PCR检测黄沙鳖cathelicidin基因在不同组织中的表达特征及攻毒后的表达变化。【结果】黄沙鳖cathelicidin基因cDNA序列全长1026 bp(GenBank登录号MK250818),包括483 bp的开放阅读框(ORF)、315 bp的5'非编码区(5'UTR)和228 bp的3'非编码区(3'UTR)。黄沙鳖cathelicidin基因cDNA序列编码160个氨基酸,包括氨基端的信号肽区域、含有4个保守半胱氨酸(Cys)残基的cathelin区域及羧基端的成熟肽区域。黄沙鳖cathelicidin基因推导氨基酸序列与中华鳖cathelicidin基因推导氨基酸序列(KY948708.1)的相似性最高,为97.5%;基于cathelicidin基因推导氨基酸序列相似性构建的系统发育进化树也显示黄沙鳖与中华鳖的亲缘关系最近。黄沙鳖cathelicidin基因在脾脏和肝脏中的相对表达量相对较高,其次是心脏,在脑组织、肾脏、肠道、肌肉、表皮和背甲的相对表达量较低。以温和气单胞菌攻毒后,黄沙鳖脾脏cathelicidin基因相对表达量呈升—降—升—降的波动变化趋势,出现2个峰值(攻毒后第3和36 h),对应的cathelicidin基因相对表达量分别是对照(注射等量无菌生理盐水)的23.1和3.5倍。【结论】黄沙鳖cathelicidin基因在脾脏和肝脏中高表达量,且可被温和气单胞菌感染诱导,说明cathelicidin基因在黄沙鳖抵抗病原感染过程中发挥重要作用。

黄沙鳖β-防御素基因克隆及其表达分析

【目的】明确β-防御素在黄沙鳖先天免疫中的潜在作用,为开展黄沙鳖绿色病害防治及促进其养殖业健康发展打下基础。【方法】运用RACE克隆黄沙鳖β-防御素基因(Hs-BD1)cDNA序列,采用SignalP-5.0、PredictProtein、PSIPRED、Robetta和Clustal X等在线软件进行生物信息学分析,并通过实时荧光定量PCR检测Hs-BD1基因在黄沙鳖不同组织中的表达特征及细菌感染前后的表达变化。【结果】Hs-BD1基因c DNA序列全长493 bp,包括72 bp的5’非编码区(5’-UTR)、201 bp的开放阅读框(ORF)及220 bp的3’非编码区(3’-UTR)。Hs-BD1基因编码66个氨基酸残基,包括22个氨基酸残基组成的信号肽区、3个氨基酸残基组成的前肽区和41个氨基酸组成的成熟肽区。其中,成熟肽区具有6个保守的半胱氨酸残基(31Cys、58Cys、38Cys、52Cys、42Cys和59Cys)及位于C1和C2间的甘氨酸残基(Gly),即Hs-BD1基因属于β-防御素家族。Hs-BD1氨基酸序列与中华鳖β-防御素16的相似性最高(93.9%),基于β-防御素序列相似性构建的系统发育进化树也显示黄沙鳖与中华鳖和西部锦龟聚为一支,其亲缘关系相对较近。6个保守的半胱氨酸残基分别以C1-C5、C2-C4和C3-C6的连接方式形成3个二硫键;Hs-BD1成熟蛋白三级结构是由α-螺旋、β-折叠和无规则卷曲组成。Hs-BD1基因在黄沙鳖肝脏、脾脏、肺脏和肾脏中的相对表达量较高,在心脏、肠道、肌肉、脑组织和表皮中的相对表达量均较低;以温和气单胞菌攻毒后,Hs-BD1基因在黄沙鳖脾脏中的相对表达量呈上升—下降—上升—下降的变化趋势,在攻毒后第3和36 h共出现2个表达峰值,对应的相对表达量分别是攻毒前(0 h)的20.8和10.6倍,差异均极显著(P<0.01)。【结论】Hs-BD1基因在黄沙鳖的多个组织中均有表达,尤其在肝脏、脾脏、肺脏和肾脏中的相对表达量较高,且可被温和气单胞菌感染诱导表达,说明Hs-BD1基因在黄沙鳖抵抗病原感染的过程中发挥调控作用。

中草药防治黄沙鳖病害试验

通过在水泥池中对病鳖用药情况进行认真观察,对比分析,研究中草药防治黄沙鳖病害有效方法,为进一步研究中草药防治黄沙鳖病害提供科学依据。黄沙鳖主要病害发生在6-8月,捕捉患病的鳖,筛选9组中草药在水泥池进行鳖病防治试验,每天用药1次,连续用药7d,仔细观察、记录全过程,从中得出最佳防治效果的3组中草药配伍。

黄沙鳖β-防御素Hs-BD1的原核表达及抗菌活性检测

【目的】构建黄沙鳖β-防御素1(Hs-BD1)原核表达并分析其抗菌活性,为深入分析Hs-BD1的生理功能及开发新型黄沙鳖抗菌药物提供理论依据。【方法】构建Hs-BD1成熟肽序列的重组表达载体pCold-TF-Hs-BD1,采用异丙基硫代半乳糖苷(IPTG)诱导重组蛋白rHs-BD1表达,以十二硫酸酯钠—聚丙烯酰胺凝胶电泳(SDS-PAGE)和免疫印迹(Western blotting)检测重组蛋白rHs-BD1的表达情况。采用Ni柱亲和层析法对重组蛋白rHs-BD1进行纯化,分析其对几种革兰氏阳性和阴性菌的抑菌活性、溶血性和抗氧化能力。【结果】SDS-PAGE检测结果显示,重组蛋白rHs-BD1在电泳凝胶约58.1 kDa处出现特异性条带,其分子量大小与预测值相符。经Western blotting检测,重组蛋白rHs-BD1可与Anti-6×His Tag抗体发生特异性反应,表明Hs-BD1基因在原核表达系统成功表达,经纯化的重组蛋白rHs-BD1含量为400.0μg/mL。对已纯化的重组蛋白rHs-BD1进行体外抗菌活性检测,结果显示其对无乳链球菌(Streptococcus agalactiae) HPG1和金黄色葡萄球菌(Staphylococcus aureus)HPN1的最小抑菌浓度(MIC)为40.00μg/mL,对大肠杆菌(Escherichia coli)YLG1、类志贺邻单胞菌(Plesiomons shigelloides)DAL1和迟钝爱德华氏菌(Edwardsiella tarda)FCH1的MIC为20.00μg/mL,表明重组蛋白rHs-BD1对一些革兰氏阳性和阴性菌具有较强的抗菌活性。20.0、40.0、60.0和80.0μg/mL重组蛋白rHs-BD1对新鲜黄沙鳖血细胞的溶血率分别为1.5%、2.7%、3.3%和4.2%,20.0、40.0、80.0和120.0μg/mL重组蛋白rHs-BD1对1,1-二苯基-2-苦肼基(DPPH)自由基的清除率分别为2.6%、5.2%、11.6%和17.0%。【结论】经原核表达获得的Hs-BD1重组蛋白rHs-BD1具有一定的广谱抗菌活性和抗氧化能力,且溶血作用较弱,对黄沙鳖机体毒副作用较低,可用于新型黄沙鳖抗菌药物开发。

中草药防治黄沙鳖病害技术

利用中草药成份中含有的清热、解毒、抑菌、杀菌、抗病毒等功效,组成中草药配方,经试验,筛选出几组中草药配方防治黄沙鳖腐皮病、红脖子病、腮腺病等主要疾病,并在生产实践中取得了明显的治疗效果。