白斑综合征病毒对红螯光壳螯虾幼虾肝胰腺免疫酶活性及其超微结构的影响

利用生物化学方法和电镜技术,研究人工注射免疫多糖及WSSV对红螯光壳螯虾幼虾肝胰腺POD、LSZ、SOD、PO的活性变化及肝胰腺超微结构的影响。试验分对照组、实验组Ⅰ(注射WSSV)、实验组Ⅱ(注射免疫多糖)、实验组Ⅲ(注射免疫多糖48 h后注射WSSV)4组,结果显示,随着处理时间的增加,实验组Ⅰ与对照组相比POD、PO、LSZ活性均呈现明显降低趋势(P<0.01),而SOD活性呈现先升高后降低的趋势(P<0.01);实验组Ⅱ的螯虾4种酶活性呈现先升后降,与对照组相比酶活性增加(P<0.05);实验组Ⅲ4种酶活性均高于实验组Ⅰ(P<0.05),但与对照组相比SOD、POD、LSZ活性降低(P<0.01)。红螯光壳螯虾幼虾肝胰腺组织由肝小管组成,肝小管由基膜和上皮细胞组成。超微结构显示,对照组螯虾幼虾肝胰腺单层柱状上皮细胞的表面微绒毛排列整齐,各细胞器结构完整;实验组Ⅰ上皮细胞微绒毛受损、断裂,核膜解体,细胞核破裂,粗面内质网断裂;实验组Ⅱ上皮细胞粗面内质网核糖体增多;实验组Ⅲ与实验组Ⅰ相比,细胞核结构完整,粗面内质网肿胀,线粒体部分畸变。结果说明感染WSSV的幼虾肝胰腺形态结构受损,并进一步影响其生物学功能。人工注射免疫多糖能提高幼虾免疫相关酶活性,在一定程度上能抵抗WSSV的侵染。     

氨氮急性胁迫及其毒后恢复对红螯光壳螯虾幼虾相关免疫和代谢指标的影响

应用实时荧光定量PCR技术,结合生物酶和代谢产物测定,研究了氨氮急性胁迫对红螯光壳螯虾幼虾代谢及免疫系统的毒性影响及其毒后恢复情况。实验首先进行3 d的氨氮胁迫,取样后剩余虾移入曝气自来水进行7 d的毒后恢复实验。结果表明,3 d氨氮胁迫后,肌肉ACP、AKP、SOD活性表达均受到显著影响,随着氨氮浓度的升高酶活性分别降低,最高浓度组(16 mg/L)降低为对照组的76%、68%和62%。线粒体MnSOD、胞外Cu/ZnSOD的mRNA表达量也随着氨氮浓度增加而下降,最高浓度组降低至对照组的69%和68%。CAT、GPX活性以及GPX和GST的mRNA表达量变化不显著。肝胰腺中可溶性蛋白和甘油三酯含量随着氨氮浓度升高而降低,最高浓度组分别降低至对照组的72%和59%,AST活性在12 mg/L浓度组显著升高至对照组的134%。7 d恢复期过后,ACP和AKP活性以及各代谢指标恢复到正常水平;而SOD和GPX活性高于对照组。各抗氧化基因的表达量都不同程度高于对照组。实验表明,高浓度氨氮胁迫能抑制部分免疫相关酶的活性及基因表达,对免疫系统造成损害。氨氮胁迫下,红螯光壳螯虾动员蛋白质和脂肪来供能应对胁迫。7 d的恢复时间不足以让红螯光壳螯虾从胁迫中完全恢复,其肌肉仍处于轻度氧化应激状态。     

饲料中大豆磷脂对红螯光壳螯虾性腺发育期营养物质积累的影响

基础饲料中添加5种不同水平的大豆磷脂(SL):0%(Diet 1),1%(Diet 2),2%(Diet 3),4%(Diet 4)和6%(Diet 5)。饲喂初始体质量为(25.64±1.53)g的红螯光壳螯虾(Cherax quadricarinatus)雌虾8周,观察其卵巢发育期个体生长、性腺指数等参数的变化,分析卵巢和肝胰腺营养物质积累的快慢以及与营养物质积累相关基因脂肪酸结合蛋白(FABP)mRNA表达的差异。结果表明:饲料中不同水平的SL对卵巢发育期红螯光壳螯虾的成活率和相对增重率(WG)影响不显著(P>0.05),但饲喂≥2%(SL)饲料的雌虾性腺指数(GSI)显著升高(P<0.05),雌虾的肝胰腺指数(HSI)则随饲料中SL的增加而呈下降趋势(P>0.05);随着饲料中SL的增加,虾肝胰腺中脂滴的体积增大,数量增多,结构更加完整,质地更加均匀,卵巢中脂滴和卵黄颗粒的数量增多,体积增大;同时,饲料中SL的增加促进了FABP在肝胰腺和卵巢中的表达量。综上所述,饲料中的SL对红螯光壳螯虾雌虾卵巢发育具有促进作用,在培育红螯光壳螯虾雌虾亲体过程中,含6.5%鱼油的基础饲料至少补充2%的SL有利于雌虾卵巢的快速发育。本研究旨在为深入研究虾蟹生殖生理学和规模化红螯光壳螯虾早繁苗种生产等提供基础资料。     

不同脂肪源对红螯光壳螯虾幼虾生长、消化酶活性及其肌肉生化组成的影响

通过在人工配合饲料中分别添加5%的花生油、猪油、鱼油和豆油作为主要脂肪源,以商业饲料为对照,进行8周饲喂实验,研究不同脂肪源对红螯光壳螯虾(Cherax quadricarinatus)幼虾生长、消化酶活性及其肌肉生化组成的影响。结果显示,不同脂肪源对红螯光壳螯虾幼虾的体长增长率和特定增长率影响不显著(P0.05);但增重率各组间存在显著性差异(P0.05),以豆油组最高,达到2332.93%,花生油组最低,为1839.50%;豆油组肝胰腺指数显著高于其他各组(P0.05),为0.75%。幼虾存活率以豆油组最高(P0.05),达到83.3%,鱼油组较低,仅为56.7%。不同脂肪源饲喂组的肝胰腺胃蛋白酶活力无显著差异(P0.05);脂肪酶活力花生油组显著高于其他实验组(P0.05),为1177.23U/g(prot);淀粉酶活力各组间差异显著(P0.05),由高到低依次为鱼油组、对照组、豆油组、猪油组、花生油组;纤维素酶活力以花生油组较高,为61.14U/(gprot()P0.05)。幼虾腹部肌肉中各种脂肪酸的含量明显受到饲料中脂肪酸种类和含量的影响,饱和脂肪酸含量猪油组显著高于其他组(P0.05);单不饱和脂肪酸以花生油组含量最高,豆油组最低;多不饱和脂肪酸则以豆油组含量最高(P0.05)。在各实验组中,豆油组红螯光壳螯虾幼体具有最高的体质量增长率和存活率,较高的肝胰腺指数和肝胰腺消化酶活力,豆油组幼虾腹部肌肉中多不饱和脂肪酸尤其是亚油酸和亚麻酸含量也较高。因此,以豆油作为主要脂肪源能够满足红螯光壳螯虾幼体的生长需要,获得较好的饲养效果,并降低饲料成本。     

Percoll不连续密度梯度离心法分离红螯光壳螯虾血细胞

建立了利用Percoll不连续密度梯度离心分离红螯光壳螯虾血细胞的方法,并对配制分离体系的缓冲液以及分离体系的密度组成进行了优化。结果显示,由20%、65%和100%的Percoll组成的分离体系分离效果最优。在转速为1 810 r/min的条件下离心20 min之后,可将红螯光壳螯虾血细胞分为SGC与GC 2个细胞层。经流式细胞术分析,发现细胞层纯度均在95%以上,细胞死亡比率低于1.5%,可用于后续的细胞功能分析。此方法简单有效,为后续研究螯虾的免疫防御机制及病害防治方法奠定了基础。     

眼柄摘除对红螯光壳螯虾生长、性腺发育及体色的影响

为探究眼柄摘除对红螯光壳螯虾(Cherax quadriarinatus)生长、性腺发育及体色的影响,对6月龄红螯光壳螯虾[体质量(47.47±3.48)g]进行了摘除眼柄处理实验。结果显示,4周后摘除双侧眼柄组红螯光壳螯虾体质量为(84.40±13.41)g,显著高于不摘除眼柄组[(49.63±6.47)g]和摘除单侧眼柄组[(51.47±4.08)g](P<0.05);摘除双侧眼柄组红螯光壳螯虾存活率为60.0%±4.8%,显著低于不摘除眼柄组和摘除单侧眼柄组(分别为94.4%±2.3%和97.2%±2.1%);摘除双侧眼柄组红螯光壳螯虾性腺指数为1.23±0.50,显著高于不摘除眼柄组(0.20±0.06)和摘除单侧眼柄组(0.35±0.08)。利用ImageJ软件对红螯光壳螯虾体色RGB值分析表明,摘除双眼柄组红螯光壳螯虾体色G值和B值(分别为99.26±5.23和98.40±3.58)显著高于摘除单侧眼柄组(59.02±3.85和44.07±4.57)(P<0.05)。结果表明,眼柄摘除能促进红螯光壳螯虾生长和性腺发育,影响体色变化。     

红螯光壳螯虾组织蛋白酶L基因的克隆及维生素C对其表达的影响

为深入了解红螯光壳螯虾组织蛋白酶L基因的表达特性及维生素C对其表达的影响,实验利用RACE-PCR技术及荧光定量PCR技术,从红螯光壳螯虾肝胰腺中克隆得到组织蛋白酶L基因cDNA全长序列,命名为CqCatL(GenBank登录号:KJ913663),同时检测了该基因在红螯光壳螯虾各个组织及添加了不同浓度维生素C的组别中的表达。结果显示,该基因全长1 810 bp,开放阅读框长度为1 026 bp,编码341个氨基酸残基,预测的分子量和等电点(pI)分别为37.63 ku和5.17。同源性分析结果显示,该基因编码的蛋白与其他虾蟹类有较高的相似性,说明组织蛋白酶L基因在甲壳动物具有较高的保守性。组织荧光定量PCR结果显示,CqCatL基因在红螯光壳螯虾的多个组织中均有表达,其中肝胰腺中表达量最高,其次为血细胞,在肠及触角腺中也有一定量的表达。在基础饲料中添加不同水平的维生素C后,CqCatL基因的表达量也存在明显差异,其中维生素C添加量为400 mg/kg的组别中该基因的表达量最高。研究表明,组织蛋白酶L基因在红螯光壳螯虾的生长发育过程中有重要的作用,且其表达量受维生素C的影响。     

饲料中非蛋白能源物质对红螯光壳螯虾幼虾生长、生理、生化指标的影响

在等蛋白质、等能量基础上,研究碳水化合物与脂类比例(CHO∶L)为10.75∶1、4.81∶1、2.66∶1、1.52∶1和0.87∶1的5组试验饲料对红螯光壳螯虾[初始体质量(1.72±0.01)g]相关生长、生理、生化指标的影响。8周试验结果表明,CHO∶L比例为2.66∶1时,红螯光壳螯虾的增重率、特定生长率和饲料利用率达到最高。高比例的CHO∶L(10.75∶1)和低比例的CHO∶L(0.87∶1)都会显著地抑制(P<0.05)红螯光壳螯虾的生长和饲料的利用。饲料脂肪水平为40～145 g/kg时,虾的脂肪酶和碱性磷酸酶活力显著升高(P<0.05),己糖激酶和丙酮酸激酶活力则呈显著降低趋势(P<0.05)。CHO∶L对虾胃蛋白酶活力影响显著(P<0.01),CHO∶L为2.66∶1和1.52∶1表现出比较高的活力,显著高于(P<0.05)其它试验组。碳水化合物为156.3～360.4 g/kg范围内,虾淀粉酶活力随饲料中碳水化合物的升高而显著升高(P<0.01)。红螯光壳螯虾增重率分别与饲料中碳水化合物和脂肪水平进行二次回归分析得出,红螯光壳螯虾对配合饲料中碳水化合物和脂肪的最适需求量分别为268.28和120.22 g/kg,相对应的CHO∶L为2.20∶1,且红螯光壳螯虾对碳水化合物的利用能力要高于对脂肪的利用。     

不同温度对红螯螯虾虾苗生长的影响

以体质量0.02 g红螯螯虾为研究对象,开展了不同温度(22、25、28、31、34℃)对红螯螯虾虾苗生长影响的试验。试验结果表明随着温度的升高,红螯螯虾虾苗的成活率不断降低,22℃时成活率最高,为48.21%±8.50%,34℃时成活率最低,为21.55%±3.09%,31℃时虾苗特定生长率最高,为7.50%±0.41%。     

红螯光壳螯虾响应长期水体盐度胁迫的基因和代谢通路

为探究长期盐度胁迫条件下红螯光壳螯虾肠道基因表达和代谢通路的变化。实验采用Illumina Hiseq 2 500平台对0、5、10和15这4个盐度下养殖5周的红螯光壳螯虾的肠道组织进行了高通量测序。研究共获76 534个unigenes,通过与NR、NT、KO、Swissprot、PFAM、GO和KOG数据库进行比对,成功注释37 378个unigenes。通过引入FPKM(Fragments Per Kilo bases per Million fragments)来估算基因表达水平,基于NR和Pfam两个数据库的蛋白注释结果,448 362个unigenes在GO数据库得到注释。根据KO功能注释与Pathway的联系,9 483个unigenes在KEGG数据库被注释分类到34条通路途径。通过DEGseq进行基因表达差异分析,以盐度0为对照组,显著差异基因筛选条件设置为Q value<0.05且差异倍数|Fold Change|>2,盐度5、10和15组分别获得差异基因2 733、91和236个,其中盐度5组共有2 068个基因显著上调,665个基因显著下调。将所有差...     

Intensification of redclaw crayfish Cherax quadricarinatus culture: II. Growout in a separate cell system

In the process of exploring ways to intensify crayfish culture, a growout system of individual cages (cells) was designed to determine the effects of gender and cell size on the growth of the red claw crayfish Cherax quadricarinatus. Cells of three different diameters—large (25 cm), medium (20 cm) and small (16 cm)—were used. When crayfish were stocked at a mean weight of approximately 10 g, growth rate of males was significantly higher than that of females. The growth rate of the males in the large cells was 0.31±0.14 g/day, while that of the females was 0.18±0.09 g/day. The size of the cell had significant influence on the weight of males. Male crayfish in the large and medium cells grew better than those in the small cells. When males were stocked at a higher mean weight (about 23 g), their mean weight after 206 days was higher in the large cells (69.28±15.72 g) than in the small cells (58.11±12.66 g), suggesting that the growth of large males was also affected by cell size. Regardless of cell size, male animals of this species grew faster than females under conditions of individual cells. This intensive culture method appears to present a powerful improvement in yields, by as much as two orders of magnitude, in comparison with communal cultures.     

Intensification of redclaw crayfish Cherax quadricarinatus culture: I. Hatchery and nursery system

Intensification of an indoor hatchery and nursery system for the Australian redclaw crayfish Cherax quadricarinatus (von Martens) (Decapoda: Parastacidae) was obtained by increasing the surface area available for the crayfish juveniles and by synchronizing the age of the hatchlings held in each tank. The former improvement was facilitated by distributing an artificial seaweed-like material throughout almost the entire volume of small (275 l) hatching tanks. As the number of egg-bearing females was increased from 3 to 8 per hatching tank, the number of juveniles per liter also increased to as many as 6.5 juveniles/l, without reaching an apparent upper limit. The hatchlings were kept in the tanks for 75 days from the day females were found to be gravid and then harvested and graded according to size. The average juvenile weight at harvest was 0.34±0.04 g. The weight distribution of the juvenile males was not significantly different from that of the juvenile females on the day of harvest, and in both the distribution was positively skewed.     

In vivo dry matter and protein digestibility of three plant-derived and four animal-derived feedstuffs and diets for juvenile Australian redclaw, Cherax quadricarinatus

Dry matter and protein digestibility of three plant-derived and four animal-derived feedstuffs and diets in which they were included were evaluated for juvenile Australian redclaw. The ingredients evaluated were: soy paste, textured wheat, sorghum meal, two sardine meals (67% and 58% crude protein), squid meal, and red crab meal. A reference diet was formulated and produced in the CIBNOR nutrition laboratory. Seven experimental diets were then made including 15% of each ingredient in the reference diet. The experiment consists of a single-factor, completely randomized design with five replicates per treatment. Digestibility was measured indirectly, using chromic oxide as a marker. Plant-derived ingredients and the corresponding diets had, in general, a higher digestibility than animal ingredients. Soy paste and sorghum meals, and the diets in which they were included, showed an excellent dry matter (over 87%) and protein (approximately 90%) digestibility. Some of the animal ingredients such as sardine meal 67% CP and squid meal had a good dry matter digestibility (over 80%), but were significantly lower than plant-derived ingredients. The lowest dry matter and protein digestibility was recorded for sardine meal 58% CP and red crab meal. It is concluded that juvenile redclaw are omnivorous and able to efficiently consume diets containing plant- and animal-derived ingredients, but they can digest plant-derived ingredients more efficiently.     

红螯螯虾XO-SG神经细胞的原代培养

本研究采用酶解的方法获得红螯螯虾眼柄XO-SG复合体的单个神经细胞,并依据显微观察对XO-SG神经细胞进行分类,同时利用Leibovitz's L-15等作为基础培养基离体培养解离的神经细胞。目的是建立虾类XO-SG神经细胞的分类标准并确定合适的培养条件,便于在体内外开展神经内分泌系统的调控研究。结果显示,根据神经细胞形态特点,红螯螯虾XO-SG神经内分泌细胞分为6种类型,不同类型的细胞在细胞大小以及显微结构上存在明显差异;建立了红螯螯虾眼柄XO-SG神经元的体外原代培养方法,细胞在改良的L-15培养基中存活状态良好,原代培养的神经细胞可以在体外存活14 d。离体培养过程中,不同类型神经细胞的再生速率存在差异,部分细胞在第2天就出现再生的轴突或树突。再生过程基本可以持续7 d,随后细胞开始萎缩凋亡。本研究为红螯螯虾眼柄神经细胞的分类以及利用神经细胞在体外开展虾类的内分泌代谢和调节机制研究提供了基础依据和研究平台。     

Shifting the natural spring–summer breeding season of the Australian freshwater crayfish Cherax quadricarinatus into the winter by environmental manipulations

Ninety sexually mature Cherax quadricarinatus females were exposed to various combinations of photoperiod and temperature for 2 months during the summer. Females were randomly assigned to either “winter” “semi-winter” or “summer” simulation treatments. In the “winter” treatment, crayfish were exposed to a simulated winter photoperiod (gradual decrease from 14L:10D to 10L:14D, 4 weeks at short day length followed by gradual increase to 14L:10D) and temperature (gradual decrease from 27 to 15 °C, held for 4 weeks, and then gradual increase to 27 °C). In the “semi-winter” treatment, crayfish were exposed to a simulated winter photoperiod and a summer temperature (27–29 °C). In the “summer” treatment, the crayfish were exposed to summer water temperatures and a photoperiod of 14L:10D. Following the 2 months of conditioning, the females were stocked for 7 months in small groups with males under environmental conditions similar to those of the “summer” treatment. All females were individually tagged and molting and spawning events were monitored. Females exposed to “semi-winter” conditioning in the summer, demonstrated a threefold increase in the rate of first spawning during the winter (December–February) compared with the other females. Crayfish breeders can easily implement these findings since shifting the breeding season into the winter only requires shortening of the photoperiod in the summer. The stocking of ponds in the spring with large nursed juveniles that hatched from eggs spawned in the winter, would allow the attainment of market size at the end of the limited growout season in temperate zones.     

Characterisation of cellulase activity in the digestive system of the redclaw crayfish (Cherax quadricarinatus)

Endogenous cellulase activity was identified in the gastric fluid and digestive gland of the redclaw crayfish. Cellulase showed maximal activity from pH 4 to 5 and was stable for up to 2 h at 40°C. Cellulase activity in the digestive gland was unaffected by antibiotic treatment. Taken together these findings suggest a significant endogenous component for redclaw cellulase activity. Partial purification of cellulase activity was performed using anion exchange and gel filtration chromatography. One major and one minor band of activity were identified subsequently by SDS-PAGE and zymography. The molecular weight of the major band was estimated at 40 kDa while the minor band was estimated at 30 kDa. Redclaw cellulase enzymes demonstrated broad substrate specificity, hydrolysing polysaccharides containing β-1,4 and mixed β-1,4 and β-1,3 glycosidic bonds but showed a preference for soluble substrates. Hydrolysis products of cellodextrins of various lengths also showed that the enzymes liberated free glucose. Exposure of redclaw to antibiotics resulted in a dramatic decline in bacterial populations in the gastric contents (>90%) but only a 40% decline in cellulase activity.     

Evaluation of practical diets containing different protein levels on gonad development of female redclaw crayfish Cherax quadricarinatus

The effect of five experimental diets with different crude protein content (220, 270, 330, 390 and 450 g kg⁻¹) on gonad development of female Cherax quadricarinatus was tested under laboratory conditions. After 70 days, a significant linear relationship indicated that higher concentrations of protein and carbohydrates in the hepatopancreas were produced as the dietary crude protein increased ( P  < 0.05). There were significant responses of the gonadosomatic index, hepatosomatic index, biochemical composition of the gonad (protein, lipids, carbohydrates and energy) and frequency of secondary vitellogenic oocytes to dietary protein level, as indicated by significant fits of the quadratic equation to the observed experimental data. The optimal response of the criteria parameters corresponded to levels of crude protein in the range 284–355 g kg⁻¹. Overall, 330 g kg⁻¹ crude protein with a protein : energy ratio of 15.6 mg kJ^–1 was considered the most adequate concentration of dietary protein for gonad development and biochemical composition in female redclaw crayfish.