中华绒螯蟹幼体消化酶活力与氨基酸组成的研究

本文对中华绒螯蟹（Ｅｒｉｏｃｈｅｉｒｓｉｎｅｎｓｉｓ）各期幼体的消化酶活力与氨基酸组成进行了分析。实验结果表明，在中华缄螯蟹幼体发育过程中，五种消化酶活力表现出三种变化模式。胃蛋白酶、类胰蛋白酶和淀粉酶活力在食性转换过程中出现较明显变化，其中胃蛋白酶和类胰蛋白酶活力在状幼体期达到最大值；淀粉酶活力在状幼体期达最大值；纤维素酶和脂肪酶活力极微。氨基酸含量随着幼体发育逐渐增加，在必需氨基酸中以亮氧酸含量最高，色氨酸含量最低；非必需氨基酸中含量最高者为谷氨酸，最低者为胱氨酸。同时单个必需氨基酸含量与必需氨基酸总量的比值（Ａ／Ｅ）在幼体不同发育阶段略有差异，但基本趋于一致。     

克氏原螯虾幼体发育时期消化酶活力及氨基酸含量研究

采用生物化学方法对克氏原螯虾各期幼体消化酶活力与氨基酸组成进行了分析。实验结果显示,在克氏原螯虾幼体发育过程中,五种消化酶活力(胃蛋白酶、类胰蛋白酶、淀粉酶、纤维素酶和脂肪酶)表现出两种变化模式:胃蛋白酶和类胰蛋白酶在幼体发育Ⅱ龄幼体期和Ⅳ龄幼体期活力较高,其中Ⅳ龄幼体期该两种酶活力最高;淀粉酶、纤维素酶和脂肪酶活力为先增高后降低,在Ⅲ龄幼体期,三种酶的活力达到最高,但与Ⅳ龄幼体期的酶活力比较无显著差异。总氨基酸含量在幼体发育早期逐渐降低,幼体发育至Ⅲ龄幼体期,其氨基酸含量最低,Ⅳ龄幼体期又有所增加。在测定的所有氨基酸中谷氨酸含量最高,必需氨基酸中赖氨酸含量最高,单个必需氨基酸含量与必需氨基酸总量的比值(A/E)在整个胚胎发育过程中的变化趋于一致。     

中华绒螯蟹胚胎发育过程中的消化酶活力及氨基酸组成

采用生物化学方法测定了中华绒螯蟹(Eriocheir sinensis)胚胎发育过程中7个发育期的5种水解酶(类胰蛋白酶、胃蛋白酶、淀粉酶、脂酶和纤维素酶)活力和氨基酸的组成。结果表明，胚胎发育各阶段消化酶活力及氨基酸组成的变化与其发育关系密切。在整个胚胎发育过程中，类胰蛋白酶、胃蛋白酶和纤维素酶的活力呈“高-低-高”的变化趋势，脂酶则呈现逐渐上升的趋势，而淀粉酶的变化趋势不明显。但在原蚤状幼体期，5种酶的活力均显著升高，这与孵化后幼体开口摄食的自身调控机理有关。可溶性蛋白质的含量呈“低-高-低”的变化趋势，明显与相关酶的活力有关。氨基酸含量在前6期变化不大，在原蚤状幼体期显著下降。所有氨基酸中，谷氨酸的含量最高；必需氨基酸中以亮氨酸的含量最高，并且单个必需氨基酸含量与必需氨基酸总量的比值(A／E)在胚胎发育不同阶段略有差异，但基本趋于一致。     

温度对中国对虾幼体生长发育与消化酶活力的影响↑（*）

本文通过计数、称重和酶学分析的实验方法研究温度对中国对虾幼体生长发育与消化酶活力的影响。实验表明，温度对中国对虾各期幼体的存活率、变态率和增重量及三种消化酶活力有明显的影响，呈一个峰值变化。在实验温度范围内随着幼体发育，温度对存活率的影响逐渐减弱，对增重量影响逐渐增大。还表明温度对其各期幼体消化酶活力的影响与食性有关，各期幼体的三种消化酶活力对环境温度有各自的适应性，适温范围为２２－２８℃，而且在幼体发育过程中，其适宜温度逐渐上升。     

中国对虾幼体消化酶活力的实验研究

以酶学分析方法测定了中国对虾各期幼体几种消化酶活力，实验结果表明，在中国对虾幼体发育过程中，五种消化酶活力表现出四种变化模式，其中胃蛋白酶和类胰蛋白酶活力逐渐增大，淀粉酶活力呈下降趋势，纤维素酶和脂肪酶活力极微，在食性转换过程中，胃蛋白酶、类胰蛋白酶和淀粉酶出现较明显的变化。中国对虾幼体消化酶活力对饵料中的营养物质有着明显的适应性，而且饥饿实验表明消化酶活力受个体发育的影响。作者认为中国对虾幼体消     

中国对虾（Penaeus chinensis）幼体发育各阶段脂肪酸组成的研究↑（*）

通过对中国对虾幼体的五个发育阶段的脂肪酸组成的测定，分析比较了幼体脂肪中１１种主要脂肪酸的含量变化。在卵脂肪中占主要比例的脂肪酸为棕榈酸，棕榈油酸和ＥＰＡ，油酸；在仔虾脂肪中占主要比例的脂肪酸为棕榈酸，油酸，亚油酸，ＥＰＡ及ＤＨＡ。从卵到仔虾整个发育过程中，幼体脂肪中的豆蔻酸，棕榈酸，棕榈油酸的含量逐渐减少，而硬脂酸，亚油酸，亚麻酸和ＤＨＡ的含量逐渐增加。     

金乌贼(Sepia esculenta)早期发育阶段相关酶活性的变化

采用生化方法测定了金乌贼(Sepia esculenta)不同发育阶段的受精卵以及0–10日龄幼体的胰蛋白酶、胃蛋白酶、碱性磷酸酶、脂肪酶和谷丙转氨酶5种酶比活力的变化。结果显示,可溶性蛋白浓度总体呈先上升后下降再上升的变化趋势,其中,在囊胚和原肠期(Ⅱ)最高,在初孵幼体(Ⅶ)中最低,含量分别为81.50 mg/g和4.24 mg/g。胰蛋白酶、胃蛋白酶和谷丙转氨酶比活力的变化趋势相近,总体呈先上升后下降再上升的变化趋势,其中,在胚胎发育阶段均能检测出这3种酶的活性,说明主要自母体获得,但活力相对较低;在初孵幼体中,这3种酶活性均显著升高,比活力分别为0.51 U/g、1.68 U/mg和37.84 U/g;5日龄幼体(Ⅷ)中,3种酶比活力均显著下降至出膜前水平;10日龄幼体(Ⅸ)中,3种酶比活力均有小幅度上升。碱性磷酸酶和脂肪酶比活力的变化趋势相近,总体呈上升趋势,其中,在胚胎发育阶段仅检测到这2种酶的极低活性,幼体孵出后2种酶的比活力才显著上升,标志着器官发育的逐步完善和消化能力的逐步增强。     

金乌贼(Sepia esculenta)早期发育阶段相关酶活性的变化

采用生化方法测定了金乌贼(Sepia esculenta)不同发育阶段的受精卵以及0-10日龄幼体的胰蛋白酶、胃蛋白酶、碱性磷酸酶、脂肪酶和谷丙转氨酶5种酶比活力的变化.结果显示,可溶性蛋白浓度总体呈先上升后下降再上升的变化趋势,其中,在囊胚和原肠期(Ⅱ)最高,在初孵幼体(Ⅶ)中最低,含量分别为81.50 mg/g和4.24 mg/g.胰蛋白酶、胃蛋白酶和谷丙转氨酶比活力的变化趋势相近,总体呈先上升后下降再上升的变化趋势,其中,在胚胎发育阶段均能检测出这3种酶的活性,说明主要自母体获得,但活力相对较低;在初孵幼体中,这3种酶活性均显著升高,比活力分别为0.51 U/g、1.68 U/mg和37.84 U/g;5日龄幼体(Ⅷ)中,3种酶比活力均显著下降至出膜前水平;10日龄幼体(Ⅸ)中,3种酶比活力均有小幅度上升.碱性磷酸酶和脂肪酶比活力的变化趋势相近,总体呈上升趋势,其中,在胚胎发育阶段仅检测到这2种酶的极低活性,幼体孵出后2种酶的比活力才显著上升,标志着器官发育的逐步完善和消化能力的逐步增强.     

不同发育期的幼体和不同规格刺参消化道中四种消化酶的活性

测定了刺参耳状幼体、樽形幼体、五触手幼体、稚参、3种规格幼参和成参[体质量分别为(82.349±1.532)g、(147.639±2.031)g和(179.866±1.609)g]消化道中蛋白酶、淀粉酶、褐藻酸酶和纤维素酶的活性。试验结果表明,耳状幼体至樽形幼体蛋白酶活力下降,之后,快速上升。稚参蛋白酶活性显著高于幼体。相反,从耳状幼体至稚参,淀粉酶的活力逐渐升高,至稚参达最大值。幼体期褐藻酸酶逐渐上升,至五触手幼体达峰值,然后下降,稚参降至最小值。幼体纤维素酶活性一直很低,变化不大,但稚参的纤维素酶活性增强。幼参和成参的蛋白酶和淀粉酶活力随着个体发育而逐渐上升,而褐藻酸酶的活性却随着个体的生长而缓慢下降,稚参达最小值。成体的纤维素酶活性低而稳。4种消化酶中,蛋白酶活力最高,纤维素酶最低。     

ω_3HUFA对中华绒螯蟹幼体存活率及体脂肪酸组成的影响

分别以经海水小球藻、牟氏角毛藻和５０ＤＥ－Ｇ强化剂强化的轮虫投喂中华绒螯蟹早期幼体,至Ｚａｏ状Ⅲ期后改喂卤虫无节幼体,探讨了饵料ω－３系列高度不饱和脂肪酸对幼体发育和存活的影响。结果表明：小球藻轮虫组的幼体发育至第Ⅰ期幼蟹的存活率最高,为１２．００％,强化剂轮虫组和角毛藻轮虫组次之,分别为１０．６７％和９．６７％,而酵母轮虫组最低,仅为３．６７％;结合分析Ｚａｏ状Ⅴ期幼体脂肪酸组成可知,幼体发育至第Ⅰ期幼蟹的存活率与其总脂含量和ω－３ＨＵＦＡ含量密切相关,而Ｚａｏ状Ⅴ期幼体总脂含量与ω－３ＨＵＦＡ含量随饵料轮虫总脂和ω－３ＨＵＦＡ含量的增加而提高,表明早期幼体所摄取的ω－３ＨＵＦＡ对后期幼体发育和存活有明显的促进作用。     

The amino acid profiles in developing eggs and larvae of the freshwater Percichthyid fishes,trout cod,Maccullochella macquariensis and Murray cod,Maccullochella peelii peelii

Results on changes in the total amino acids (protein bound + free) and the free amino acids (FAA) in relation to development, from egg (unfertilised and/or fertilised) to yolk-sac resorbed larva, before first feeding, in two Percichthyid fish, trout cod, Maccullochella macquariensis and Murray cod, Maccullochella peelii peelii, which lay demersal, adhesive eggs, are presented. Throughout development, the FAA accounted for only a small proportion (0.19 % in fertilised eggs of both species) of the total amino acid pool. Nine essential amino acids (EAA) and eight non-essential amino acids (NEAA) were quantified in the amino acid pool at all stages of development. In both species, the total amino acid content decreased during the transformation (at 20 ± 1 °C) from newly hatched larva to yolk-sac resorbed larva. Overall, the changes in the TEAA and TNEAA reflected that of the amino acid pool. In trout cod, all but one EAA (lysine) and two NEAA (cysteine and glycine) decreased with ontogeny, from fertilised egg to yolk-sac resorbed larva. In Murray cod, however, the exceptions to the general decline were two NEAA (aspartic acid and glycine). In contrast, the FAA increased with development, the changes being reflected in both FEAA and FNEAA. Qualitatively, the predominant free amino acids in trout cod and Murray cod eggs were alanine, lysine, leucine and serine. Because the egg protein and the total amino acid contents declined with development, it is concluded that the rate of breakdown of yolk protein was higher than the anabolic and catabolic processes during embryogenesis. Data also suggest that in freshwater fish FAA are an unlikely primary energy substrate during embryogenesis.     

The changes in the biochemical compositions and enzymatic activities of rotifer (<Emphasis Type="Italic">Brachionus plicatilis</Emphasis>, Müller) and <Emphasis Type="Italic">Artemia</Emphasis> during the enrichment and starvation periods

The changes in the biochemical compositions and enzymatic activities of rotifer (Brachionus plicatilis) and Artemia, enriched and stored at 4°C temperature, were determined. The total starvation period was 16 h and samples were taken at the end of the 8th and 16th hours. In present study, the rotifer and nauplii catabolized a large proportion of the protein during the enrichment period. Lipid contents of both live preys increased during the enrichment period and decreased in nauplii and metanauplii throughout the starvation period but lipid content of the rotifer remained relatively constant during the starvation period. The changes observed in the amino acid compositions of Artemia and the rotifer were statistically significant (P < 0.05). The conspicuous decline the essential amino acid (EAA) and nonessential amino acid (NEAA) content of the rotifer was observed during the enrichment period. However, the essential amino acid (EAA) and nonessential amino acid (NEAA) contents of Artemia nauplii increased during the enrichment period. The unenriched and enriched rotifers contained more monounsaturated fatty acid (MUFAs) than polyunsaturated fatty acid (PUFAs) and saturated fatty acids (SFA). However, Artemia contained more PUFAs than MUFAs and SFA during the experimental period. A sharp increase in the amounts of docosahexaenoic acid (DHA) during the enrichment of the rotifer and Artemia nauplii was observed. However, the amount of DHA throughout the starvation period decreased in Artemia metanauplii but not in Artemia nauplii. Significant differences in tryptic, leucine aminopeptidase N (LAP), and alkaline phosphatase (AP) enzyme activities of Artemia and rotifer were observed during the enrichment and starvation period (P < 0.05). The digestive enzymes derived from live food to fish larvae provided the highest contribution at the end of the enrichment period. In conclusion, the results of the study provide important contributions to determine the most suitable live food offering time for marine fish larvae. Rotifer should be offered to fish larvae at the end of the enrichment period, Artemia nauplii just after hatching and before being stored at 4°C, and Artemia metanauplii at the end of the enrichment and throughout the starvation period.     

斑节对虾Chitinase-2基因的克隆及其在蜕皮和幼体发育过程中的表达分析

研究以斑节对虾(Penaeus monodon)转录组获得的几丁质酶基因(Pm Chi)片段,运用RACE技术克隆了PmChi基因c DNA全长,命名为PmChi-2。PmChi-2基因c DNA全长2 050 bp,其中5'-非编码区(5'-UTR)144 bp、3'-UTR 319 bp和开放阅读框(ORF)1 587 bp,编码528个氨基酸。生物信息学分析显示,PmChi-2与其他甲壳动物Chi-2的相似性为78%~97%。实时荧光定量PCR结果显示,PmChi-2在蜕皮前期(D期)表皮中表达水平最高,在胃、鳃、腹神经节和眼柄中表达水平依次降低,在其他组织(肝胰腺、肠、肌肉、心脏)中几乎不表达。不同蜕皮阶段,PmChi-2在鳃、胃和表皮3种组织中的表达变化模式基本一致,均在蜕皮期(E期)表达量最低,而最高表达量在鳃中出现在蜕皮后期(A期),在胃和表皮中为D期。幼体不同发育阶段分析揭示PmChi-2 mRNA在幼体发育阶段的无节幼体到糠虾幼体第二期表达量维持在一个低水平,在糠虾幼体第三期PmChi-2 mRNA表达水平显著升高,在仔虾期又显著下降,推测PmChi-2 mRNA可能与斑节对虾幼体发育密切相关。研究结果表明PmChi-2基因可能在斑节对虾蜕皮以及幼体的变态发育中发挥重要作用,为深入研究斑节对虾几丁质酶发育调控提供了重要信息。     

Amino Acid Composition of Developing Larval Freshwater Prawn Macrobrachium rosenbergii

Abstract.— Amino acid profiles of freshwater prawn Macrobrachium rosenbergii were determined during early larval stages (I-IX) to provide some baseline biochemical information of the growing larvae. The larvae obtained from several females were stocked into three 250-L tanks at a density of 30 larvae/L. The feeding regime consisted of newly hatched Artemia nauplii and egg custard containing 5% cod liver oil. For each developmental stage, larval samples from each experimental tank were pooled, freeze dried and after acid hydrolysis, the quantification of amino acids was done by a reverse phase high performance liquid chromatography ( N = 2). The highest represented of the total amino acids were for glutamic acid and phenylalanine (with cystine) with ranges from 13.4–16.6 and 9.7–11.5%, respectively, whereas tryptophan (1.4–1.6%). methionine (1.4–2.7%) and histidine (2.9–4.2%) were relatively lower. The levels of the following essential amino acids did not significantly change during larval development: arginine, leucine, phenylalanine (plus cystine), threonine, tryptophan and valine. Despite statistically significant changes that were observed in levels of some amino acids, amino acid composition appeared to be relatively unchanged during the larval growth. The results may suggest that the amino acid requirements of the freshwater prawn is relatively constant during larval life and can be satistied by a suitable protein source that resembles the larval amino acid profile.     

Comparative digestive enzyme ontogeny in two marine larval fishes: Pacific threadfin (Polydactylus sexfilis) and bluefin trevally (Caranx melampygus)

The specific activity of digestive enzymes; aspartic and serine protease, collagenase, lipase, acid and alkaline amylase, acid and alkaline phosphatase, and chitinase was assayed throughout early development in two species of marine fishes: the Pacific threadfin (Polydactylus sexfilis) and bluefin trevally (Caranx melampygus). Specific enzyme activities were determined on whole larval extracts sampled at selected stages of development, from day 0 to day 30 post-hatching. Similar developmental patterns of enzyme specific activity were observed in the two species, although differences in timing, amplitude and effects of first feeding were noted. Amylase activity increased prior to first feeding, peaking at the middle of the larval period, and becoming nearly undetectable by the time of larval-to-juvenile metamorphosis. Serine protease, collagenase, lipase and alkaline (and acid for threadfin) phosphatase activities increased gradually, followed by sharp increases to a plateau during the second half of larval development. Aspartic protease and chitinase activities, in addition to acid phosphatase (for trevally), were low to undetectable in the first half of development, increasing through metamorphosis. In the trevally only, this group of enzymes exhibited high activity levels at the time of hatching, followed by a precipitous drop. Species-dependent differences in enzyme specific activity at first feeding may have been a result of differences in yolk utilization. These results, taken in the context of earlier reports, lead us to conclude that carbohydrate utilization may play a significant role in the earlier phases of development among some marine larvae, followed by a shift to protein and lipid utilization.     

盐度胁迫对三疣梭子蟹(Portunus trituberculatus)鳃中游离氨基酸含量的影响

本研究分析了不同盐度环境下三疣梭子蟹(Portunus trituberculatus)鳃组织中游离氨基酸(FAAs)的含量及浓度变化规律,明确 FAAs 的组成及其在盐度适应中发挥的功能。结果显示,在正常海水中,三疣梭子蟹鳃组织中牛磺酸(Tau)的浓度最高(2.54 mg/g),其次分别为丙氨酸(Ala,0.65 mg/g)、谷氨酸(Glu,0.64 mg/g)、精氨酸(Arg,0.58 mg/g)、甘氨酸(Gly,0.48 mg/g)和脯氨酸(Pro,0.25 mg/g);在盐度10–50范围内,梭子蟹鳃中总游离氨基酸(TOFAA)的含量随盐度的增加而显著升高(P<0.05),盐度50时,TOFAA 的含量为盐度10时的1.48倍。进一步分析发现,非必需氨基酸(NEAA)(Gly、Glu、Ala 和 Pro)的浓度随盐度的升高而上升,而必需氨基酸(EAA)的浓度变化不显著(P>0.05)。研究表明,FAAs 在三疣梭子蟹盐度适应中具有重要功能,其中发挥渗透调节功能的主要为 NEAA。     

Heat treatment affects protein quality and protease activity in decapsulated cysts of Artemia when used as starter food for larvae of African catfish Clarias gariepinus (Burchell)

Decapsulated cysts of Artemia subjected to different heat treatments (40, 60, 80 and 96 °C) were fed to African catfish Clarias gariepinus larvae. Heated cysts, untreated cysts and live Artemia nauplii as control constituted the experimental diets. Protein denaturation and solubility, total alkaline protease and specific trypsin activities in the cyst diets were evaluated. The growth of catfish larvae and the proteolytic activity of larval samples during development were also determined. Heat treatment of cysts increased protein denaturation and decreased protein solubility. The protease activity in the cyst diets decreased with higher heating temperatures. The growth of catfish larvae differed according to the diet; higher fish growth was achieved with nauplii and cysts heated at 40 °C. The digestive enzyme activity in larval samples remained similar in all dietary treatments during larval development. The quality of food protein and the way this protein is processed might be more important for successful larval growth than exogenous enzyme supply.