EFFECT OF DENSITY ON LARVAL DEVELOPMENT AND FEMALE PRODUCTIVITY OF TISBE-HOLOTHURIAE (COPEPODA, HARPACTICOIDA) UNDER LABORATORY CONDITIONS

POPULATION-DYNAMICS; FOOD; FISH

Changes in RNA, DNA, protein contents and growth of turbot Scophthalmus maximus larvae and juveniles

The growth potential of turbot Scophthalmus maximus larvae and juveniles was studied using nucleic acid-based indices and protein variables. The experiment was carried out from 4 to 60 days post hatching (dph). A significant increase in instantaneous growth rate during metamorphosis and retarded growth rate during post-metamorphic phase were observed. Ontogenetic patterns of DNA, RNA and protein all showed developmental stage-specific traits. The RNA:DNA ratio decreased up to 12 dph, then increased rapidly till 19 dph and fluctuated until 35 dph followed by a decline to the end. The RNA:DNA ratio was positively correlated with growth rate of juveniles during the post-metamorphic phase, whereas this ratio was not a sensitive indicator of growth during the pre-metamorphic phase and metamorphosis. The protein:DNA ratio showed a similar tendency to the RNA:DNA ratio. Changes of DNA content and protein:DNA ratio revealed that growth of S. maximus performed mainly by hyperplasia from 4 to 12 dph and hypertrophy until 21 dph during the pre-metamorphic larval phase. Growth was dominantly hypertrophical from the early- to mid-metamorphosing phase and hyperplastic thereafter. The results show that the DNA content and protein:DNA ratio can evaluate growth rates of larval and juvenile S. maximus on a cellular level.

Cadmium toxicity to embryonic-larval development and survival in red sea bream Pagrus major

At 18 degrees C and 33 psu, 24 and 48 h LC50 values of cadmium (Cd) for red sea bream Pagrus major embryos were 9.8 and 6.6 mg l(-1), respectively, while 24,48, 72, and 96 h LC50 values for larvae were 18.9,16.2, 8.0, and 5.6 mg l(-1), respectively, indicating that embryos were more sensitive to Cd toxicity than larvae. Cd concentrations at >= 0.8 mg l(-1) led to low hatchability (0-90% in >= 0.8 mg l(-1) solutions vs. 97-100% in lower ones), delay in time to hatch, high mortality (38-100% vs. 1-10%), morphological abnormality (42-100% vs. 1-10%), reduced length (3.55-3.60 vs. 3.71-3.72 mm) in the embryos and larvae. They were Cd concentration dependent and potential biological significant endpoints for assessing the risk of Cd to aquatic organisms. Heart beat and yolk absorption of the larvae were significantly inhibited at some high concentrations but they were not as sensitive as other endpoints to Cd exposure. (C) 2008 Elsevier Inc. All rights reserved.

Molecular cloning and functional analysis of cathepsin B in nutrient metabolism during larval development in Meretrix meretrix

Seed rearing is an important part in large scale clam culture industry. Since the nutritional history affects early development in bivalve, the condition of larval nutrition plays a key role in successful seed rearing. So far, the molecular mechanism of nutrient uptake in bivalve larvae is unclear. As one of the important proteolytic enzymes, cathepsin B of several organisms has been reported to be involved in digestion. We intended to analyze whether cathepsin B is involved in larval nutrient metabolism in the economic bivalve, clam Meretrix meretrix. The full length of M. meretrix cathepsin B (MmeCB) cDNA was cloned, which is 1647 bp with an open reading frame of 1014 bp. The deduced amino acid sequence encoded a preproenzyme of 337 residues with Cys-114, His-282 and Asn-302 composing cathepsin B activity center. The temporal and spatial expressions of MmeCB mRNA were examined from trochophore to post larva stages by whole mount in situ hybridization. In trochophore stage, no detectable signal was found. In the later three stages, MmeCB mRNA was detected in the digestive gland, suggesting a possible role of MmeCB in digestion. Moreover, MmeCB mRNA was also observed in the epidermal cells in D-veligers. Cathepsin B specific inhibitor (CA074 methyl ester) was applied to block the activity of cathepsin B in unfed larvae. The average shell lengths of treated larvae were smaller than that in control groups. The results of mRNA epidermal distribution and inhibitor treatment in D-veligers indicated that MmeCB may be also associated with other pathway of nutrient metabolism in larval epidermis. The overall results in this paper revealed that MmeCB might play a role in larval nutrient metabolism. (C) 2008 Elsevier B.V. All rights reserved.

A Dicer-1 gene from white shrimp Litopenaeus vannamei: Expression pattern in the processes of immune response and larval development

Dicer is a member of the RNAase III family which catalyzes the cleavage of double-stranded RNA to small interfering RNAs and micro RNAs, and then directs sequence-specific gene silencing. In this paper, the full-length cDNA of Dicer-1 was cloned from white shrimp Litopenaeus vannamei (designated as LvDcr1). It was of 7636 bp, including a poly A tail, a 5' UTR of 136 bp, a 3' UTR of 78 bp, and an open reading frame (ORF) of 7422 bp encoding a putative protein of 2473 amino acids. The predicted amino acid sequence comprised all recognized functional domains found in other Dicer-1 homologues and showed the highest (97.7%) similarity to the Dicer-1 from tiger shrimp Penaeus mondon. Quantitative real-time PCR was employed to investigate the tissue distribution of LvDcr1 mRNA, and its expression in shrimps under virus challenge and larvae at different developmental stages. The LvDcr1 mRNA could be detected in all examined tissues with the highest expression level in hemocyte, and was up-regulated in hemocytes and gills after virus injection. These results indicated that LvDcr1 was involved in antiviral defense in adult shrimp. During the developmental stages from fertilized egg to postlarva VII, LvDcr1 was constitutively expressed at all examined development stages, but the expression level varied significantly. The highest expression level was observed in fertilized eggs and followed a decrease from fertilized egg to nauplius I stage. Then, the higher levels of expression were detected at nauplius V and postlarva stages. LvDcr1 expression regularly increased at the upper phase of nauplius, zoea and mysis stages than their prophase. The different expression of LvDcr1 in the larval stages could provide clues for understanding the early innate immunity in the process of shrimp larval development. (C) 2010 Elsevier Ltd. All rights reserved.

三种海水经济鱼类早期发育生物学的研究

海水经济鱼类的养殖在我国已经形成第四次海水养殖浪潮，经济效益显著，有力地推动了我国海水养殖的产业结构调整和可持续发展。然而在海水养殖发展过程中也存在着诸多问题，尤其是早期发育阶段的高死亡率，严重制约了我国海水养殖产业的稳定和健康发展。 海水鱼类养殖的关键为高质量，高存活率苗种的生产和培育，由于鱼类种类繁多，生物多样性丰富，对应实际的繁育技术，尤其是新品种的开发，必须要做出相应的调整。这就要求我们必须对每一种鱼类早期发育有所了解，并将形态和组织上的数据用于指导生产。 本文通过显微观察和组织学研究，主要描述和研究了我国北方三种重要的海水经济鱼类(条斑星鲽、杂交鲆、条石鲷)的早期发育生物学，并结合实际生产进一步阐明关键期的产生原因，机理以及采用相应的对策。具体结果如下： 1.条斑星鲽：作为冷温性鲆鲽鱼类，条斑星鲽早期发育过程的特征主要有： ① 条斑星鲽受精卵无油球，卵子呈半浮性；不同步卵裂现象提前，发生在第三次卵裂；卵裂期裂球大小差异大。孵化过程较长，在水温8 ± 0.3℃，盐度33的条件下，经9 d孵化。条斑星鲽胚胎发育的不同时期对温度的敏感性不同，其中原肠期对温度比较敏感。 ②在8-10℃，盐度33的条件下，8-9 dph开口摄食。且开口时，其吻前端出现有一点状黑褐色素，构成了条斑星鲽仔鱼“开口期”的重要标志。卵黄囊于消失。在后期仔鱼末期，背鳍和臀鳍上形成特有的黑褐色条斑带。 ③杯状细胞首先出现在咽腔后部和食道前段，胃腺和幽门盲囊出现于29 dph，变态期始于30dph。在条斑星鲽早期发育过程中，观察到其直肠粘膜层细胞质出现大量嗜伊红颗粒，为仔鱼肠道上皮吸收的蛋白质。 ④首先淋巴化的免疫器官是头肾，然后是胸腺和脾脏，这与大部分硬骨鱼类不同。条斑星鲽除头肾和脾脏外，胸腺实质也形成MMCs。其中以脾脏形成MMCs最为丰富，形态多样。 2. 杂交鲆：为同属的牙鲆和夏鲆间的远缘杂交种，其发育过程的特点为： ① 在温度为15.4～16.0℃，杂交鲆胚胎从受精到孵化所需的时间为76 h左右，胚孔关闭前期，胚胎先出现视囊及克氏囊，而后形成体节。孵出前胚体在卵膜内环绕不到1周。 ② 孵化后消失。杂交鲆群体变态间隔长(34-60 dph)，且变态高峰期出现的冠状幼鳍不明显(与母本牙鲆相比)，数量为7-8根。 ③组织学观察发现，其消化系统中胃腺出现较晚，且胃腺发育过程缓慢(与母本牙鲆相比)。甲状腺滤泡增生不明显，颜色较浅，数量较少。杂交鲆在早期发育过程中，并没有出现鳔原基。 3. 条石鲷作为岩礁性的暖水性鱼类，早期发育过程也较为特殊，包括外形以及内部的器官结构。主要特点有： ① 受精卵：受精卵卵黄上具有龟裂结构，为鱼卵的分类特征之一。 ② 初孵仔鱼：初孵仔鱼背鳍膜上的黑色素，从体背面向背鳍膜边缘移动，到3dph仔鱼基本消失，此为本种仔鱼发育所特有的特点。 ③ 后期仔鱼和稚鱼：肠道肌肉层加厚明显，仔稚鱼胃肠排空率急剧上升，死亡率增加，通过改善常规的投饵方式部分解决了这个死亡高峰的问题。在幼鱼初期，牙齿融合为骨喙，为石鲷科鱼类的特征。 ④胸腺上皮分泌细胞：类似的现象同样在虹鳟鱼中发现，但是虹鳟鱼胸腺上皮分泌细胞不如条石鲷的丰富，同样也不如条石鲷的排列整齐，而是零星分布在胸腺上皮与咽腔接触的表面。除了正常的造血器官—脾脏和头肾外，肝脏、胰腺和鳔等多种组织等也出现MMCs，此现象在硬骨鱼类不多见，一般发生在软骨鱼类。

Toxicity of lead, cadmium and mercury on embryogenesis, survival, growth and metamorphosis of Meretrix meretrix larvae

In order to assess the toxicity of heavy metals on the early development of Meretrix meretrix, the effects of mercury (Hg), cadmium (Cd) and lead (Pb) on embryogenesis, survival, growth and metamorphosis of larvae were investigated. The EC50 for embryogenesis was 5.4 mu g l(-1) for Hg, 1014 mu g l(-1) for Cd and 297 mu g l(-1) for Pb, respectively. The 96 h LC50 for D-shaped larvae was 14.0 mu g l(-1) for Hg, 68 mu g l(-1) for Cd and 353 mu g l(-1) for Pb, respectively. Growth was significantly retarded at 18.5 mu g l(-1) (0.1 mu M) for Hg, 104 mu g l(-1) (1 mu M) for Cd and 197 mu g l(-1) (1 mu M) for Pb, respectively. The EC50 for metamorphosis, similar to 48 h LC50, was higher than 96 h LC50. Our results indicate that the early development of M. meretrix is highly sensitive to heavy metals and can be used as a test organism for ecotoxicology bioassays in temperate and subtropical regions.

Effects of rearing temperature and density on growth, survival and development of sea cucumber larvae, Apostichopus japonicus (Selenka)

In laboratory conditions, effects of rearing temperature and stocking density were examined on hatching of fertilized egg and growth of auricularia larvae of Apostichopus japonicus respectively. Data series like larval length and density, metamorphic time, and survival rate of the larvae were recorded. Statistics showed that for A. japonicus, survival rate (from fertilized egg to late auricularia) decreased significantly with the increasing rearing temperature (P < 0.05). At different temperatures SGR was statistically significant as well (P < 0.05) from day 1, and maximal SGR was found on day 9 at 24A degrees C (159.26 +/- 3.28). This study clearly indicated that at low temperature (< 24A degrees C), metamorphic rate was remarkably higher than at higher temperature (> 26A degrees C). Hatching rate was significantly different between 0.2-5 ind./ml groups and 20-50 ind./ml groups. Rearing larvae at the higher density had the smaller maximal-length, whereas needed longer time to complete metamorphosis. This study suggested that 21A degrees C and 0.4 ind./ml can be used as the most suitable rearing temperature and stocking density for large -scale artificial breeding of A. japonicus's larvae.