Carbonic anhydrase and Na/K-ATPase activities during the molt cycle of low salinity-reared white shrimp <Emphasis Type="Italic">Litopenaeus vannamei</Emphasis>

Changes in hemolymph osmolality, ion concentrations, and enzymatic activities of carbonic anhydrase (CA) in the gills and epidermal tissue, and Na/K-ATPase in the gills during the molt cycle were investigated in the white shrimp Litopenaeus vannamei. Hemolymph osmolality was high in the intermolt and early premolt stages, but started to decrease prior to ecdysis through to postmolt stages A and B. Changes in Na⁺ and Cl⁻ ion concentrations paralleled those in hemolymph osmolality. CA activity levels in the anterior and posterior gills were low at intermolt stage C₀ and premolt stage D₀, and maximum at premolt stage D₃. In the epidermal tissue, activity was relatively high at intermolt stage C₀ and premolt stage D₀, but fluctuated towards premolt stage D₃ and postmolt stage A. On the other hand, Na/K-ATPase activity in the gills decreased between intermolt stage C₀ and premolt stage D₂, but increased at premolt stage D₃ and postmolt stage A. The changes in patterns of CA activity during the molt cycle suggest that CA may be involved in supplying counter-ions for Na⁺ and Cl⁻ uptake during molting. Branchial Na/K-ATPase appears to be involved in producing local osmotic gradients in order to support water influx across the epithelium.     

中华绒螯蟹蜕皮周期及蜕皮过程中肝胰腺消化酶活性的变化

采用Darch蜕皮周期分期方法,对中华绒螯蟹(Eriocheir sinensis)的蜕皮周期进行了划分,并初步探讨了中华绒螯蟹蜕皮过程中肝胰腺消化酶活性变化。根据第三颚足末端刚毛和表皮的形态学特征,可将中华绒螯蟹的蜕皮周期分为蜕皮后期(A-B期)、蜕皮间期(C期)、蜕皮前期(D期)和蜕皮期(E期),其中D期可进一步分为D0、D1、D3-43个亚期。在蜕皮过程中,肝胰腺淀粉酶和胰蛋白酶活性在蜕皮后A-B期开始升高,于蜕皮前早期D0阶段达最大(P<0.05),随后又逐渐降低,至蜕皮前晚期D3-4阶段降至最低(P<0.05);胃蛋白酶活力在蜕皮周期中于蜕皮前晚期D3-4阶段显著降低(P<0.05),其他阶段变化不明显(P>0.05)。结论认为,中华绒螯蟹蜕皮过程可分为C、D0、D1、D3-4、A-B 5个时期,蜕皮过程中肝胰腺淀粉酶、胰蛋白酶和胃蛋白酶活性发生周期性变化。     

日本沼虾蜕皮过程中头胸甲外骨骼超微结构的改变

采用HE染色及石蜡切片的扫描电镜观察方法,描述了日本沼虾(Macrobrachium nipponense)蜕皮间期(C期,intermolt stage)及新旧表皮更替变化剧烈的蜕皮前晚期(D4期,late premolt stage)和蜕皮后期(postmolt stage)A-B期头胸甲外骨骼的形态结构变化特点。结果显示,日本沼虾外骨骼分为上表皮、外表皮、内表皮三层,D4期出现新的上表皮和外表皮,B期出现新内表皮,外表皮强嗜碱性而内表皮弱嗜酸性。扫描电镜观察发现,C期头胸甲内、外表皮均为几丁质-蛋白质纤维构成的平行板层结构且板层内有发达的孔道系统(pore canals,pc),但内外表皮板层间排列紧密程度不同,外表皮板层切面边缘较整齐,结构致密,板层内pc大小均一、近似圆形,内表皮板层切面边缘粗糙,结构较疏松,板层内pc大小不等、多为梭形。在蜕皮前后,新外表皮结构变化显著,与C期相比,D4期新外表皮超微结构与旧内表皮结构相似,而在A期,外表皮超微结构由疏松变为致密,这可能与蜕皮后表皮的钙化相关。本研究旨为阐明沼虾的蜕皮机制提供基础资料。     

中华绒螯蟹蜕皮周期中肝胰腺细胞组成的变化

取一年生未成熟中华绒螯蟹 (Eriocheir sinensis)幼蟹, 甲壳宽度在15~40 mm, 暂养在75 cm×50 cm×45 cm的玻璃缸内, 24 h充气, 自然光照, 每天换水1/3, 每次换水后投喂土豆、杂鱼等食物, 动物适应实验室条件1周后进行实验。为得到蜕皮后的样本, 将处于蜕皮前晚期的个体在相同条件下单独饲养。根据已报道方法, 将中华绒螯蟹的蜕皮周期分为蜕皮间期C期、蜕皮前D₀、D₁和D_3–4期、蜕皮后A-B期等5个时期。采用细胞学和组织学方法观察中华绒螯蟹蜕皮过程中肝胰腺细胞组成的变化, 结果显示, R细胞数量在蜕皮间期和蜕皮前期都占绝对优势, 虽然在蜕皮前早期D₀期数量显著下降(P<0.05), 但从D₁期开始上升, 于蜕皮前晚期D_3–4期达到最高(62.06 ± 3.92)%, 而在蜕皮后A-B期又显著降低(P<0.05)。F细胞的数量除在D₁期显著增加外(P<0.05), 其余时期的变化无统计学差异(P>0.05)。B细胞数量在蜕皮前早期D₀期显著增高(P<0.05), 随后开始下降, 直到蜕皮前晚期D_3–4期恢复到间期水平, 而蜕皮后A-B期数量又显著增高(P<0.05)。E细胞数量除在蜕皮前早期D₀期稍微上升外, 其余时期基本稳定(P>0.05)。饥饿对处于不同蜕皮时期的中华绒螯蟹肝胰腺细胞组成的影响不同, 饥饿48 h后, 处于D₀时期的中华绒螯蟹与正常组相比, 肝胰腺R细胞数量无显著变化, B细胞和E细胞数量显著下降, F细胞数量上升。处于D₁时期的中华绒螯蟹与正常组相比, 肝胰腺R细胞、B细胞、F细胞和E细胞数量均无显著变化。说明中华绒螯蟹蜕皮过程中肝胰腺细胞组成的变化和蜕皮周期密切相关。本研究通过探讨中华绒螯蟹蜕皮过程中肝胰腺组织结构及细胞组成变化, 了解甲壳动物蜕皮过程的基础生物学现象, 并为其健康养殖提供理论基础。

     

Vitamin D3 and the renal handling of phosphate in American eels

Cholecalciferol (Vitamin D₃) increased plasma inorganic phosphate concentration in American eels,Anguilla rostrata, in a dose-dependent fashion. This response was more marked in phosphate loaded fish. In control as well as phosphate loaded eels the hyperphosphatemic response to D₃ was associated with a sharp reduction in renal phosphate clearance relative to¹⁴C-polyethelene glycol (PEG) clearance. Glomerular filtration and urine flow rates were not affected by D₃. As renal phosphate clearance, even in phosphate loaded eels, never significantly exceeded that of PEG, it is suggested that D₃ reduced the relative clearance rate of phosphate by increasing renal phosphate reabsorption rather than by reducing the tubular secretion of phosphate.     

In vitro methyl farnesoate secretion by mandibular organs isolated from different molt and reproductive stages of the crab Oziotelphusa senex senex

ABSTRACT:   Considerable evidences indicate that mandibular organ (MO) of crustaceans secrete terpenoid hormone (methyl farnesoate [MF]), which regulates molting and reproduction. In the freshwater south Indian rice field crab Oziotelphusa senex senex , MO isolated during premolt and vitellogenesis, secreted greater amounts of MF, which indicates that the regulation of the crustacean molt and reproduction is complex and also involves MF, besides steroid (ecdysteroid) and peptide (sinus gland peptide) hormones.     

日本沼虾N-乙酰-β-D-氨基葡萄糖苷酶基因克隆及KK-42对其表达的影响

为进一步探究KK-42缩短蜕皮周期的分子机制,本研究以日本沼虾幼虾为材料,通过RACE技术克隆了几丁质降解途径中的限速酶基因NAGase c DNA全长序列,定量测定了KK-42处理不同时间对头胸甲表皮组织中NAGase m RNA相对表达量和对应酶活力的影响。序列分析结果显示,NAGase c DNA全长2 536 bp,编码617个氨基酸。同源性分析显示,NAGase基因保守性较低,与凡纳滨对虾的相似度最高,仅为68%。系统进化分析显示,日本沼虾、三疣梭子蟹、凡纳滨对虾、中国明对虾聚为一个大类,凡纳滨对虾和中国明对虾亲缘关系更为接近,日本沼虾单列一个分支。Real-time PCR分析表明,NAGase相对表达量在蜕皮前D_0期达到峰值;KK-42处理后3 h,D_4期的相对表达量是对照组的253%,处理后6 h,C期和D_0期较对照组分别提高了226%和187%。NAGase酶活力从C期到D_4期逐渐提高,KK-42处理能明显提高C和D_0期NAGase酶活力,尤其对C期的影响最为显著,在处理后3、6和12 h分别提高了11.26、5.99、7.15倍。结果提示,KK-42对日本沼虾表皮NAGase的诱导效应可能是其缩短蜕皮周期的分子机制之一。     

Mixed feeding schedule of low and high protein in the diet of Labeo rohita (Hamilton) fingerlings: effect on growth performance,haemato‐immunological and stress responses

Four experimental diets D₁, D₂, D₃ and D₄ having 100, 300, 350 and 400 g protein kg^?1 diet, respectively, were prepared. Fingerlings of Labeo rohita (5.45 ± 0.03 g) were fed with different diets following a mixed feeding schedule with the feeding cycle of 28 days that consisted of either 28 days feeding of diet D₁ or D₂ or 21 days feeding with D₁ diet and 7 days with either D₃ or D₄ diet. Different treatments were T₁ (D₁ for 28 days), T₂ (D₂ for 28 days), T₃ (D₁ for 21 days and D₂ for 7 days), T₄ (D₁ for 21 days and D₃ for 7 days) or T₅ (D₁ for 21 days and D₄ for 7 days). Fingerlings were sampled after three feeding cycles (84 days). Higher (P < 0.05) growth performance and protein utilization efficiency were found in the T₃ group, which was similar to the T₂ group. The blood count, NBT, lysozyme activity, serum parameters and stress indicator parameters also complement for the better immunity in the T₂ and T₃ groups. Therefore, it could be concluded that fingerlings feeding alternately a low‐protein diet (D₁) for 21 days followed by a normal‐protein diet (D₂) for 7 days in a cycle of 28 days for 84 days maintained better growth and health status.     

罗氏沼虾细胞色素P450家族CYP302a1基因克隆及其在蜕皮周期中的表达

蜕皮是甲壳动物重要的生理活动,与其蜕皮激素的合成密切相关,细胞色素P450(CYP)302a1是甲壳动物蜕皮激素合成通路中的关键酶之一。本研究克隆了罗氏沼虾CYP302a1基因(Mr-CYP302a1),cDNA全长1859 bp,开放阅读框(ORF)为1629 bp,编码543个氨基酸(aa),分子量大小为61.09 ku,等电点为8.42。氨基酸序列分析显示CYP302a1基因的保守结构域含有5个P450基因家族特征保守区域:heme-binding、helix-K、helixC、helix-I及PERF。系统进化分析结果显示Mr-CYP302a1首先与绿虾CYP302a1聚为一支,然后与凡纳滨对虾及三疣梭子蟹等十足目甲壳动物的CYP302a1聚为一支,与甲壳动物的亲缘关系最近。实时荧光定量PCR(qRT-PCR)检测表明Mr-CYP302a1在罗氏沼虾的多个组织中均有表达,其中在Y器官中的表达量最高,性腺中次之。同时研究发现,MrCYP302a1基因在罗氏沼虾的蜕皮后期(A期和B期)表达量很低,蜕皮间期(C期)表达量开始上升,在蜕皮前期D1亚期达到峰值。对Mr-CYP302a1进行蛋白表达及多克隆抗体制备,蛋白印迹法(Western blot,WB)检测表明Mr-CYP302a1蛋白在罗氏沼虾Y器官中的表达量最高,在蜕皮过程中的蜕皮前期D1亚期达到峰值。综上所述,该基因在罗氏沼虾的蜕皮过程中扮演着十分重要的角色。     

In vivo calcitropic effect of some vitamin D compounds in the marine Antarctic teleost,Pagothenia bernacchii

The Antarctic notothenioid, Pagothenia bernacchii, were found to have plasma total and free calcium levels, plasma inorganic phosphate and whole body calcium efflux rates which were similar to those seen in other teleosts. But total bone calcium was lower than reported for other teleosts. A single injection of vitamin D₃ (5 ng g^–1 fish) increased plasma total and plasma free calcium and these increases were associated with an increase in whole body calcium efflux and bone calcification. Conversely, the same treatment with 1,25-(OH)₂-D₃ reduced plasma free calcium. This seco-steroid also increased the specific activity of ⁴⁵Ca in bone at 40h post-injection but did not significantly effect total bone calcium, plasma total calcium or whole body calcium efflux. 25-OH-D₃ at the same dose had no effect on any of the parameters tested and none of the seco-steroids tested had any effect on plasma total inorganic phosphate. These data show that both D₃ and 1,25-(OH)₃-D₃ can have calcitropic effects in this marine teleost and that these two forms of vitamin D can exert different effects within the same species.     

A quantitative determination of deformities in barramundi (Lates calcarifer; Bloch) fed a vitamin deficient diet

Cultured barramundi, Lates calcarifer, suffer from abnormalities affecting the jaw, opercula and spine. The aim of this study was to quantify for the first time the effects of supplemented dietary vitamin C, vitamin D₃ and ultraviolet (UV) light on the development of jaw, opercula and spinal deformities. Four diets were formulated to contain (i) no vitamin C or vitamin D₃, (ii) only vitamin D₃, (iii) only vitamin C and (iv) both vitamin C and vitamin D₃. In addition, two commercial diets (diets 5 and 6) were also tested. These diets were replicated in the presence, and in the absence, of ultraviolet (UV) light as this may also affect skeletal development. Diets formulated with 170 ± 1 mg kg^?1 and 195 ± 0.5 mg kg^?1 of vitamin C (diets 3 and 4, respectively) and the commercial diets (diets 5 and 6) had significantly lower incidences of spinal deformities (0–2.5%; P < 0.01) and opercula deformities (nil detected). Spinal deformities were ‘broken back’ syndrome, which was found only in the precaudal vertebrae, and lordosis which was only in the caudal vertebrae. UV light and vitamin D₃ did not affect spinal or opercula deformities. There was no change in the occurrence of jaw deformities in vitamin C, vitamin D₃ or UV light treatments.     

蜕壳周期内中华绒螯蟹钙含量、组织结构及相关基因表达变化

运用生化分析、组织切片和分子生物学等技术,测定蜕壳期(蜕下旧壳后0.5 h内)和蜕壳后期(蜕下旧壳0.5 h后至新壳未完全硬化之前)平均体质量(5.63±2.35)g中华绒螯蟹肝胰腺和鳃中的钙含量,观察肝胰腺和鳃组织结构变化,用荧光定量PCR技术分析肝胰腺和鳃中的钙网蛋白(CRT)基因的表达情况.试验结果表明,蜕壳后期...     

Effect of high inclusion of rendered animal by‐product ingredients on growth,digestibility and economic performances in climbing perch Anabas testudineus

A 70‐day growth trail was conducted to investigate the effects of inclusion of high levels of meat and bone meal (MBM) and protein concentrate (PC) on growth, digestibility and economic performances of climbing perch, Anabas testudineus. Four isonitrogenous diets were formulated by lowering the level of dietary fishmeal protein at 0 (D₁, control), 70 (D₂), 85 (D₃) and 100% (D₄) with a mixture of MBM and PC protein (1:1). Triplicate groups of 300 fish (mean weight of 0.80 g) stocked in each 40 m² pond and fed the respective test diets. A digestibility trial was conducted after the growth trial in indoor glass aquarium. The result showed that growth parameters were significantly decreased (P < 0.05) with fishmeal replacement levels. However, significant differences were not found in feed conversion ratio and survival of fish. No difference was also found in protein efficiency ratio among D1, D2 and D3. Similar to growth parameters, total fish production was highest in D1, intermediate in D2 and D3; and lowest in D4. Apparent digestibility coefficients of dry matter, protein and lipid were highest (P < 0.05) in D1 and lowest in D4. The economic analysis revealed that the benefit cost ratio was ranked by D₃ (1.81), D₂ (1.71), D₁ (1.66) and D₄ (1.46) respectively. Upon considering the overall performances and unavailability of finite protein sources, it can be concluded that 70–85% fishmeal could be replaced with a mixture of MBM and PC (1:1) in practical diets for climbing perch.     

莱州湾鱼类群落的关键种

关键种对群落结构稳定性起着决定作用,它的筛选对于整个生态系统的研究都具有重要的理论和实际意义。本研究基于2011年5月对莱州湾渔业底拖网数据,以摄食关系为基础构建了莱州湾鱼类群落种间相互作用关系网,运用网络分析法计算了该关系网的13种重要性指数及Key Player Problem参数(F、DF和DR)。根据13种指数的排序结果、聚类信息和3个Key Player Problem参数,对莱州湾鱼类群落的关键种进行了筛选。结果显示,13种重要性指数可划分为4个信息组:a(D、CC、IC、TI~1和TI~7)即基本信息组、b(D_(in)、H_(in)和K_t)即信息输入组、c(D_(out)、H_(out)和K_b)即信息输出组和d(BC和K)即信息控制组。细纹狮子鱼(D、D_(in)、BC、CC、IC、H_(in)、TI~1、TI~7、K、K_t、F、~DF和~DR)和六丝矛尾虾虎鱼(D_(out)、H_(out)和K_b)在莱州湾鱼类群落的网络分析结果中处于最高地位,密切联系着群落的其他种群,控制着群落的结构和能流,属于群落的关键种,其中细纹狮子鱼是关键捕食者,控制着群落中其他重要食物竞争者和捕食者密度,六丝矛尾虾虎鱼是关键被捕食者,通过维持捕食者的密度来限制其他被捕食者的密度。     

Development of 25 Novel Microsatellite Loci and Genetic Variation Analysis in Breeding Populations of the Pearl Oyster,Pinctada fucata

In this study, we screened 103,050 gene sequences of Pinctada fucata and identified 1450 simple sequence repeats (SSRs) distributing in 1355 sequences, in which 97 have sufficient flanking sequences for primer design, and 25 novel microsatellite loci were stable and polymorphic with N_a which varied from 2 to 6. Six microsatellite loci were found in the open reading frame of their corresponding sequences. With the 25 SSR loci, five breeding populations and a wild population, a total of 240 individuals from wild population (W), three consecutive selected populations (G₁, G_2, and G₃), and two backcross populations (D_o, D_r) (40 individuals per population) were assayed. In these populations, we detected 108 alleles of 111 bp to 362 bp. Each population had mean H_o of 0.5718–0.7366 and mean H_e of 0.5830–0.6954. Except the F_st = 0.0697 (W vs. G₃), pairwise F_st values ranged from 0.0131 to 0.0473, the genetic identity coefficients varied from 0.8688 to 0.9663, and genetic distance ranged from 0.0343 to 0.1307, all of these suggested that the breeding populations had genetic differentiation but at a low level generally. Besides, all the genetic index values, backcross populations (D_o, D_r) were higher than corresponding G₃ values, indicating that backcross could make the offspring incline to the recurrent parents.     

Pectin plays a role in restricting the utilization of rapeseed meal by Chinese mitten crab,Eriocheir sinensis

The present study aims to assess the anti‐nutritional effect of pectin and to better understand rapeseed meal (RSM) as a feed ingredient for Chinese mitten crab, Eriocheir sinensis. A semipurified diet containing 280 g/kg fish meal was formulated as a control diet (D_F). A second diet (D_P) was supplemented with 26.7 g/kg pectin; the amount was estimated to be equal to that in the third diet (D_R), which was formulated to include 400 g/kg RSM. Another diet (D_RPase) was supplemented with 12 g/kg pectinase based on D_R in which pectin was degraded by pectinase. The diets were randomly fed to twelve tanks of the crab for 8 weeks. Compared with the crabs fed D_F, the specific growth rate, protein efficiency ratio, apparent digestibility coefficients (ADC) of dry matter and protein, activity of aspartate transaminase (AST) and trypsin (TP), and high‐density lipoprotein (HDL) in the serum decreased significantly in the crabs fed D_R; the ADC of dry matter, activity of AST and serum HDL decreased significantly in the crabs fed D_p, but they were higher than in the crabs fed D_R. Compared with the crabs fed D_R, the ADC of dry matter, activity of AST and TP, and serum HDL increased significantly in the crabs fed D_RPase. The mRNA expression activity of both Es‐pl44 and Es‐pp1 was D_F < D_P < D_RPase < D_R. These results indicate that pectin contributed to low utilization of RSM in Chinese mitten crab.     

Substitution of fish meal with sunflower seed meal in diets for Nile tilapia (Oreochromis niloticus L.) reared in earthen ponds

This study investigated sunflower seed meal (SSM) as dietary protein replacement of fish meal (FM) for Nile tilapia (Oreochromis niloticus) juveniles (initial mean weight of 19.8 ± 6.3 g) reared in earthen ponds for 210 days. SSM replaced 25%, 50%, 75%, and 100% (i.e., D₂₅, D₅₀, D₇₅, and D₁₀₀, respectively) of FM in an isonitrogenous and isocaloric diet. The optimum FM replacement level of D₂₅ as predicted by the equation y = 177.5 + 26.5x – 7.9x² yielded an optimum weight of about 184 g within 100 days. Thus D₂₅ was the most effective SSM level for Nile tilapia growth in earthen ponds. Fish growth declined as SSM exceeded D₂₅. The length-weight relationship (r ≥ 90%) depicted an isometric fish growth. Nutrient utilization parameters were similar in all the diets except for D₁₀₀. The protein content of fish carcass was highest in the control and D₂₅, while fiber level was highest in D₁₀₀. Amino acids imbalance and high fiber content could have reduced the fish growth at D₅₀, D₇₅, and D₁₀₀.     

Carbonic anhydrase and Na/K-ATPase activities at different molting stages of the giant freshwater prawn <Emphasis Type="Italic">Macrobrachium rosenbergii</Emphasis>

ABSTRACT:   In the present study, the role of carbonic anhydrase (CA) and Na/K-ATPase in the gill and epidermal tissues in the giant freshwater prawn Macrobrachium rosenbergii was examined as a function of molting stage. CA activity levels in the front and back gills were low at the intermolt stage C₀, but increased significantly at premolt stage D₃, and then decreased after molting. In the epidermal tissue, activity levels decreased gradually towards premolt to a minimum level at stage D₃, but became elevated at postmolt stages A and B. Na/K-ATPase levels in the front and back gills did not change significantly during the molt cycle. CA in the gill is possibly involved in supplying counter-ions for ion uptake, while CA in the epidermal tissue may play a role in mineralizing the exoskeleton after ecdysis. Na/K-ATPase in the gills may function in ion uptake from the ambient medium; however, since its activity was not influenced by the molt cycle, it probably does not have a major role in osmoregulation in the freshwater environment.