Telomere dynamics in the Sydney rock oyster (Saccostrea glomerata): an investigation into the effects of age, tissue type, location and time of sampling.

Telomere length has been purported as a biomarker for age and could offer a non-lethal method for determining the age of wild-caught individuals. Molluscs, including oysters and abalone, are the basis of important fisheries globally and have been problematic to accurately age. To determine whether telomere length could provide an alternative means of ageing molluscs, we evaluated the relationship between telomere length and age using the commercially important Sydney rock oyster (Saccostrea glomerata). Telomere lengths were estimated from tissues of known age individuals from different age classes, locations and at different sampling times. Telomere length tended to decrease with age only in young oysters less than 18 months old, but no decrease was observed in older oysters aged 2-4 years. Regional and temporal differences in telomere attrition rates were also observed. The relationship between telomere length and age was weak, however, with individuals of identical age varying significantly in their telomere length making it an imprecise age biomarker in oysters.

Effect Of A Negative Poisson Ratio In The Tension Of Ceramics

The effect of a negative Poisson ratio is experimentally revealed in the tension deformation of a natural layered ceramic. This effect can increase the volume strain energy per unit volume by 1100% and, simultaneously, decrease the deformation strain energy per unit volume by about 44%, so that it effectively enhances the deformation capacity by about 1 order of magnitude in the tension of the material. The present study also shows that the physical mechanisms producing the effect are attributed to the climbing on one another of the nanostructures in the natural material, which provides a guide to the design of synthetic toughening composites.

The History, Present Condition, and Future of the Molluscan Fisheries of North and Central America and Europe: Volume 2, Pacific Coast and Supplemental Topics

Over 100 molluscan species are landed in Mexico. About 30% are harvested on the Pacific coast and 70% on the Atlantic coast. Clams, scallops, and squid predominate on the Pacific coast (abalone, limpets, and mussels are landed there exclusively). Conchs and oysters predominate on the Atlantic coast. In 1988, some 95,000 metric tons (t) of mollusks were landed, with a value of $33 million. Mollusks were used extensively in prehispanic Mexico as food, tools, and jewelry. Their use as food and jewelry continues. Except in the States of Baja California and Baja California Sur, where abalone, clams, and scallops provide fishermen with year-round employment, mollusk fishing is done part time. On both the Pacific and Atlantic coasts, many fishermen are nomads, harvesting mollusks wherever they find abundant stocks. Upon finding such beds, they build camps, begin harvesting, and continue until the mollusks become so scarce that it no longer pays to continue. They then look for productive beds in other areas and rebuild their camps. Fishermen harvest abalones, mussels, scallops, and clams by free-diving and using scuba and hooka. Landings of clams and cockles have been growing, and 22,000 t were landed in 1988. Fishermen harvest intertidal clams by hand at wading depths, finding them with their feet. In waters up to 5 m, they harvest them by free-diving. In deeper water, they use scuba and hooka. Many species of gastropods have commercial importance on both coasts. All species with a large detachable muscle are sold as scallops. On the Pacific coast, hatchery culture of oysters prevails. Oyster culture in Atlantic coast lagoons began in the 1950's, when beds were enhanced by spreading shells as cultch for spat. (PDF file contains 228 pages.)

Stocking, Enhancement, and Mariculture of Penaeus orientalis and Other Species in Shanghai and Zhejiang Provinces, China

China's marine aquaculture landings provide only 18% of its combined freshwater and amrine capture and culture landings, at a per-capita consumption of only 3.2 kg/yr out of a total of 18.1 kg/yr. We described development and some of the results of long-term mariculture and stocking/enhancement projects that have been underway for up to 20 years in the Hangzhou Bay area. Penaeus orientalis (also referred to as P. chinensis) stocking provided up to 400 t/yr, at a total cost-benefit ratio of up to 8 Yuan of landed shrimp per Yuan invested in shrimp stocking. Over 40 t of Penaeus orientalis were produced commercially in 1993, with proceeds being used to fund mariculture and fisheries research. Large scale edible jellyfish restocking is also underway, while semicommercial culture of abalone, Haliotis diversicolor, has been successful. Technical problems limitig mariculture have been solved successfully for some species.

Biographic Memoir of Ernest Ingersoll: Naturalist, Shellfish Scientist, and Author

The name Ernest Ingersoll is well-known to many shellfishery biologists as the author of two outstanding monographs on the shellfisheries of the United States and Canada in the 1880's. The first (Ingersoll, 1881a), entitled "A Report on the Oyster-Industry of the United States," was a 252-page description of historical and contemporary oyster fishing' marketing methods, and statistical data in the eastern provinces of Canada and the coastal states of the United States. The second (Ingersoll, 1887), entitled "The Oyster, Scallop, Clam, Mussel, and Abalone Industries," was a l20-page summary of the first monograph about oysters as well as a history and description of contemporary methods and statistical data of the other shellfisheries. Although Ingersoll was, by profession, a naturalist and author but only briefly a shellfish scientist, these monographs are regarded as benchmarks, providing the principal descriptions of shellfisheries in North America in the 1700's and 1800's.

Molecular determination of oxytetracycline-resistant bacteria and their resistance genes from mariculture environments of China

Aims: To assess the diversity of antibiotic-resistant bacteria and their resistance genes in typical maricultural environments. Methods nand Results: Multidrug-resistant bacteria and resistance genes from a mariculture farm of China were analysed via cultivation and polymerase chain reaction (PCR) methods. Oxytetracycline (OTC)-resistant bacteria were abundant in both abalone and turbot rearing waters, accounting for 3.7% and 9.9% of the culturable microbes. Multidrug resistance was common, with simultaneous resistance to OTC, chloramphenicol and ampicillin the most common resistance phenotype. 16S rDNA sequence analyses indicate that the typical resistant isolates belonged to marine Vibrio, Pseudoalteromonas or Alteromonas species, with resistance most common in Vibrio splendidus isolates. For OTC resistance, tet(A), tet(B) and tet(M) genes were detected in some multidrug-resistant isolates, with tet(D) being the most common molecular determinant. For chloramphenicol resistance, cat II was common, and floR was also detected, especially in marine Pseudoalteromonas strains. Conclusions: There is the risk of multidrug-resistant bacteria contamination in mariculture environments and marine Vibrio and Pseudoalteromonas species serve as reservoirs of specific antibiotic resistance determinants. Significance and Impact of the Study: This paper and similar findings from Korea and Japan indicate the potential for widespread distribution of antibiotic resistance genes in mariculture environments from the East Asian region of the world.

A review of advances in research on marine molluscan antimicrobial peptides and their potential application in aquaculture

Antimicrobial peptides (AMPs) are important components of the host innate immune response against microbial invasion. They are usually characterized by their small-size, heat-stability and broad range of antimicrobial activity. This review covers research advances on marine mollusc AMPs, specifically those isolated from mussels, scallops, oysters, venerid clams and abalone, which mainly include MGD, mytilin, myticin, mytimycin, big defensin, and RPD-1. Their structural characteristics, antibacterial activity, and expression pattern as well as peptide distribution and their release following microbial challenge are also discussed. In addition, the prospect of the application of AMPs as food additives or their use in immunostimulation to prevent diseases of aquatic animals, as well as their potential hazards, are also discussed.

cDNA cloning, characterization and expression analysis of the antioxidant enzyme gene, catalase, of Chinese shrimp Fenneropenaeus chinensis

Catalase is an important antioxidant protein that protects organisms against various oxidative stresses by eliminating hydrogen peroxide. The full-length catalase cDNA of Chinese shrimp Fenneropenaeus chinensis was cloned from the hepatopancreas using degenerate primers by the method of 3' and 5' rapid amplification of cDNA ends PCR. The cDNA sequence consists of 1892 bp with a 1560 bp open reading frame, encoding 520 amino acids with high identity to invertebrate, vertebrate and even bacterial catalases. The sequence includes the catalytic residues His71, Asn144, and Tyr354. The molecular mass of the predicted protein is 58824.04 Da with an estimated pl of 6.63. Sequence comparison showed that the deduced amino acid sequence of F. chinensis catalase shares 96%, 73%, 71% and 70% identity with that of Pacific white shrimp Litopenaeus vannamei, Abalone Haliotis discus hannai, Zhikong scallop Chlamys farreri and Human Homo sapiens, respectively. Catalase transcripts were detected in hepatopancreas, hemocytes, lymphoid organ, intestine, ovary, muscle and gill. by real-time PCR. The variation of catalase mRNA transcripts in hemocytes and hepatopancreas was also quantified by real-time PCR and the result indicated that the catalase showed up-regulated expression trends in hemocytes at 14 h and in hepatopancreas at 37 h after injection with white spot syndrome virus (WSSV). (c) 2008 Elsevier Ltd. All rights reserved.

Transformation of Platymonas (Tetraselmis) subcordiformis (Prasinophyceae, Chlorophyta) by agitation with glass beads

A transient transformation system for the unicellular marine green alga, Platymonas subcordiformis, was established in this study. We introduced the pEGFP-N1 vector into P. subcordiformis with a glass bead method. P. subcordiformis was incubated in cell wall lytic enzymes (abalone acetone powder and cellulase solutions) to degrade the cell wall. The applicable conditions for production of viable protoplasts were pH 6.5, 25 degrees C, and 3 h of enzyme treatment. The protoplast yield was 61.2% when P. subcordiformis cells were added to the enzyme solution at a concentration of 10(7) cell ml(-1). The protoplasts were immediately transformed with the pEGFP-N1 vector using glass-bead method. The transformation frequency was about 10(-5), and there was no GFP activity observed in either the negative or the blank controls. This study indicated that GFP was a sensitively transgenic reporter for P. subcordiformis, and the method of cell wall enzymolysis followed by glass bead agitation was applicable for the transformation of P. subcordiformis.

Molecular characterizations of chloramphenicol- and oxytetracycline-resistant bacteria and resistance genes in mariculture waters of China

In order to gain an understanding of the diversity and distribution of antimicrobial-resistant bacteria and their resistance genes in maricultural environments, multidrug-resistant bacteria were screened for the rearing waters from a mariculture farm of China. Both abalone Haliotis discus hannai and turbot Scophthalmus maximus rearing waters were populated with abundant chloramphenicol-resistant bacteria. These bacteria were also multidrug resistant, with Vibrio splendidus and Vibrio tasmaniensis being the most predominant species. The chloramphenicol-resistance gene cat II, cat IV or floR could be detected in most of the multidrug-resistant isolates, and the oxytetracycline-resistance gene tet(B), tet(D), tet(E) or tet(M) could also be detected for most of the isolates. Coexistence of chloramphenicol- and oxytetracycline-resistance genes partially explains the molecular mechanism of multidrug resistance in the studied maricultural environments. Comparative studies with different antimicrobial agents as the starting isolation reagents may help detect a wider diversity of the antimicrobial-resistant bacteria and their resistance genes. (C) 2009 Elsevier Ltd. All rights reserved.

皱纹盘鲍的遗传育种与养殖技术研究

优良的种质是产业发展的重要保证，品种更新和养殖技术的发展已经给世界农业带来了令人瞩目的成就，然而我国水产生物的育种工作刚处于起步阶段，而育种技术的研究则更是滞后。借鉴陆生生物中发展起来的相对成熟的研究方法，可以帮助加快海洋生物遗传育种相关研究的进度。本研究以我国北方海区重要的海洋经济动物－皱纹盘鲍为研究对象，从表型遗传、数量性状遗传等2个方面开展了皱纹盘鲍的遗传育种研究，同时从幼鲍培育密度与分选效应等方面研究了皱纹盘鲍的中间培育技术。 主要结果如下： 1. 皱纹盘鲍的贝壳颜色遗传、食物对贝壳颜色表现型的影响，贝壳颜色与生长速度间的关系 将贝壳颜色为橘红色（O表型）的突变型皱纹盘鲍与贝壳颜色为绿色（G表型）的野生型皱纹盘鲍进行了连续2代的交配实验。结果表明：皱纹盘鲍橘红色的贝壳颜色相对于绿色的贝壳颜色为隐性性状，皱纹盘鲍的贝壳颜色表型受单位点、2个等位基因遗传控制，其中基因型为oo的个体，贝壳颜色的表现型为橘红色（O表型），而基因型为GG或Go的个体，贝壳颜色的表现型为野生型（G表型）。 为探讨食物类型对不同基因型皱纹盘鲍贝壳颜色表现型的影响，对不同贝壳颜色表型的个体投喂不同种类的食物，结果表明，除遗传因素外，皱纹盘鲍的贝壳颜色表现型显著地受食物类型的调控。其中oo基因型的个体，在摄食底栖硅藻（Navicula sp.）和红藻时，贝壳颜色的表型为橘红色；而在摄食褐藻、绿藻和以海带粉为唯一海藻源的人工配合饵料时，贝壳颜色的表型为黄色。GG和Go基因型的个体，在摄食底栖硅藻、红藻时，贝壳颜色的表型为褐红色；在摄食褐藻、绿藻和以海带粉为唯一海藻源的人工配合饵料时，贝壳颜色的表型为绿色。该结果表明，相同基因型的皱纹盘鲍在摄食不同类型的食物时，贝壳表现型不同，即不同类型的食物可以导致2种基因型皱纹盘鲍的贝壳颜色表现型在一定范围内发生转换：oo基因型的个体，贝壳的颜色可以表现为橘红色或者黄色，不会出现野生型皱纹盘鲍的褐红色或绿色；而GG与Go基因型的个体，相应的贝壳颜色表型只能是褐红色或者绿色，不会出现oo基因型可能表现的橘红色或黄色。特定基因型的皱纹盘鲍，在摄食特定类型的食物时贝壳的相应部位可表现出特定的颜色。皱纹盘鲍的这种“食物－贝壳颜色”的相关性可作为一种形态标记，用于标识皱纹盘鲍的个体和群体，该标记技术可用于皱纹盘鲍的养殖技术和遗传学研究。 此外，选用了贝壳颜色遗传学实验中建立的贝壳颜色发生分离的家系为实验材料，以壳长为指标，分析比较了来自相同家系的O表型与G表型个体之间的生长速度。结果表明，在幼鲍发育至412天止的3-5个统计时段内，没有在同一家系来源的2种贝壳颜色表型个体之间检验到生长速度的显著差异。 2. 皱纹盘鲍不同选育群体及杂交群体的贝壳形态参数分析 在皱纹盘鲍的7个群体中（包括已经对生长速度为指标进行了多代人工选育的群体4个、野生群体之间直接杂交繁育的杂交F1群体3个），测量了4-6龄成体样本的壳长（L）、壳宽（W）、壳高（H）和壳重（Sw），并计算了L/（L+W+H）、W/（L+W+H）、H/（L+W+H）和Sw/（L×W×H）等4个壳形态学参数。用方差分析方法（MANOVA、ANOVA）统计并比较了这些壳形态参数在皱纹盘鲍群体间的遗传变异。结果表明，4个壳形态参数在不同群体间变异系数分别为0.34、0.74、2.62和6.54，其中，H/（L+W+H）与Sw/（L×W×H）在各供试群体间均具有较高的多态性且差异达显著水平，表明这2个参数在不同群体间存在较高的遗传变异。由于在活体情况下无法测量壳重（Sw）性状，建议以参数H/（L+W+H）为指标对皱纹盘鲍贝壳形态（如壳型）等进行人工选择。 3. 皱纹盘鲍成体阶段生长性状的遗传参数估计 采用巢式设计，分析了成体阶段不同发育期皱纹盘鲍的壳长与生长速率的遗传力、不同发育期的壳长性状之间的遗传相关、以及不同发育期的生长速率之间的遗传相关，结果表明：（1）壳长遗传力在受精后第70 、130、320、320、380、490与550天的雄性组分估计值分别为0.161 ± 0.075、0.312 ± 0.131、0.326 ± 0.331、0.135 ± 0.228、0.153 ± 0.185和0.180 ± 0.106；雌亲组分估计分别为0.312 ± 0.172、0.699 ± 0.168、0.695 ± 0.168、0.977 ± 0.407、0.427 ± 0.195和0.449 ± 0.027。（2）生长速率遗传力在受精后第320~380天、490 ~ 550天，雄、雌组分估计值分别为0.080 ± 0.120（雄）、 0.210 ± 0.191（雌）以及0.299 ± 0.146（雄）、0.306± 0.148（雌）。雌亲组分的壳长遗传力和生长速率遗传力估计值较大且均达显著水平，表明皱纹盘鲍在成体阶段依然受母性效应的影响。成体阶段生长性状遗传力水平的估计对制定科学的皱纹盘鲍育种方案有指导意义。（3）雄亲组分估计的不同发育期（第390 ~ 550天）壳长间遗传相关为0.597 ~ 1.000，雌亲组分估计为0.589 ~ 1.177。由雄亲、雌亲组分估计，受精后第320~380天与第490 ~ 550天两个发育阶段生长速率间遗传相关均接近于0。雌亲组分估计不同发育期壳长间遗传相关均达显著水平（t0.05, d.f.=13 = 4.33 ~ 11.69，P<0.01），表明壳长性状早期选择有效，即在皱纹盘鲍早期阶段依据壳长性状对个体进行择优或去劣可在后期阶段获得壳长较大的个体。由于使用的雄亲数目少（8个父系半同胞），实验中以雄亲组分估计的遗传参数误差较大。 4. 皱纹盘鲍选育系间的群体杂交 进行了皱纹盘鲍4个人工选育系之间的完全双列杂交实验，以群体交配的方式共建立了16个组合；此外，以大连“98”选群与汕头“S”选群为亲本，以群体交配的方式建立了4个交配组合。对不同方向的杂交组合进行了中亲杂种优势、超亲杂种优势以及配合力等方面的评价。 （1）测量了4个选育群体（R、97、S和J）及其各杂交组合在受精后第9、20和30天时的壳长，统计分析了不同选育系间壳长性状的差异、评价了不同方向杂交组合的中亲与超亲杂种优势、以及配合力。结果如下： 选育系群体内交配繁育的4个组合，在受精后第9、20和30天的壳长均有显著差异，其中，97  97组合在早期发育各阶段均为最小，分别为0.462 ± 0.023mm、0.698 ± 0.057mm和1.476 ± 0.234mm；S  S组合的3次测量值均为最大，分别为0.522 ± 0.023mm、0.824 ± 0.084mm和1.798 ± 0.229mm。 两个方向杂交组合与选育系亲本群体内交配组合的平均值和高亲值比较，得到如下结果：（A）受精后第9天壳长表现正向中亲杂种优势的组合有6个、表现负向中亲杂种优势的组合6个，其中J  97组合的中亲优势率最高，为9.05%；R  S组合最低，为-6.61%。正向高亲杂种优势组合有4个、负向高亲杂种优势组合有8个，其中S  J组合的高亲优势率最高，为5.77%；R  S组合最低，为-7.96%。（B）受精后第20天壳长表现正向中亲杂种优势的组合有7个、表现负向中亲杂种优势的组合5个，其中J  97组合的中亲优势率最高，为12.60%；J  R组合最低，为-8.72%。正向高亲杂种优势组合有3个、负向高亲杂种优势组合有11个，其中J  97组合的高亲优势率最高，为12.20%；J  R组合最低，为-12.67%。（C）受精后第30天壳长表现正向中亲杂种优势的组合有7个、负向中亲杂种优势的组合5个，其中97  S组合的中亲优势率最高，为24.08%；S  97组合最低，为-12.69%。正向高亲杂种优势组合有6个、负向高亲杂种优势组合有6个，其中97  S组合的高亲优势率最高，为15.95%；S  J组合最低，为-19.44%。上述结果表明，皱纹盘鲍不同选育系之间的交配组合，杂种优势率差异很大，因此，通过组配实验，将杂种优势率高的交配组合选择出来应用于生产，可望显著提高目标性状的产量。 对早期发育阶段各生长期壳长性状，亲本一般配合力（GCA）、各杂交组合间特殊配合力（SCA）以及正反交（REC）效应值进行方差分析，结果表明：各亲本GCA差异显著，说明各选育群体存在显著的遗传差异，其中汕头选群“S”在测量的各个生长期均为正值且显著大于其它各亲本；特殊配合力（SCA）以及正反交（REC）效应值较大在各杂交组合间存在显著差异，说明在早期生长发育阶段非加性遗传效应（显性和上位效应）占主导地位。综合各个生长期亲本GCA和杂交组特殊配合力（SCA）以及正反交（REC）效应值，杂交组合97×S在早期生长阶段不仅有较高SCA值而且两个亲本也具有较大的GCA值，表明选育系97和S较适宜作为杂交亲本使用。 （2）大连“98”选群与汕头“S”选群进行2×2因子设计的群体杂交实验，比较了各交配组合早期存活相关性状如受精率、孵化率、变态率以及壳长性状，评价了两个方向杂交组合平均以及不同方向杂交组合的中亲杂种优势率。结果表明早期发育阶段各组合间的受精率无显著差异，而孵化率、变态率等两个杂交方向平均的中亲杂种优势率为5.49%与12.53%，高于壳长性状的优势率（0.936-1.534%）。方差分析结果表明不同方向的杂交组合在早期发育阶段存活相关性状以及壳长性状存在显著差异。孵化率、变态率性状，S×98的中亲杂种优势率分别为13.21%与21.10%，均高于98×S的-3.84%与3.85%；而第10和25d壳长性状，S×98的中亲杂种优势率为1.14%与-2.52%，低于98×S的1.93%与4.41%。 为进一步评价“98”选群与“S”选群不同交配组合在不同温度条件下的生长，进行了基因型与环境的互作研究。从“98”选群与“S”选群的4个交配组合中分别取5月龄幼鲍100头，各组合随机分成3组，每组1个重复，分别于12°C、16°C和 22°C温度条件下进行培育，比较各交配组合基因型与温度对幼鲍生长的影响。不同温度条件下，各组合壳长生长的方差分析结果表明，基因型和温度都能够对幼鲍生长以及最终壳长产生极显著的影响（P < 0. 01），它们的交互作用也达到显著水平（P < 0.05）。杂交子代的幼鲍壳长在12°C、16°C和 22°C温度条件下均表现出杂种优势，双向杂交的中亲杂种优势率分别为5.32%、5.55%和0.03%，表明低温条件（12°C），比高温条件（22°C）下有更强的杂种优势。汕头“S”选群的早期孵化率、变态率、生长性状以及低温条件下幼鲍生长性状的单亲杂种优势率分别为16.64%、42.49%、3.42~5.79%和5.73~9.15%，单亲杂种优势率较大，表明可通过杂交手段，显著地改良汕头“S”选群在早期发育阶段的生长速度、存活率以及幼鲍期的生长性状。本研究的结果支持了Lerner（1954）杂种优势的基因与环境互作学说。 5. 皱纹盘鲍幼鲍的中间培育技术研究 （1）对南方越冬方式的评价 目前，每年的11月前后，将6-7月龄幼鲍运往南方的闽东、闽中、闽南沿海越冬，翌年4月至6月再运回到北方（大连、山东半岛）的养殖模式已经普遍应用于皱纹盘鲍的实际生产，为评价南方越冬的幼鲍培育方式，本研究分别以不同幼鲍材料在闽东三都海湾进行了越冬培育实验。 选择生产上壳长分别为18.37 ± 1.28 mm、15.89 ± 1.10 mm、14.55 ± 1.10 mm与10.59 ± 0.84 mm的幼鲍进行了为期6.5个月的越冬培育，实验结束时，存活率分别为95.56 ± 2.21%、90.55 ± 1.96%、83.97 ± 1.63%与63.30 ± 2.79%。回归分析表明，供试幼鲍在实验起始时的壳长与越冬阶段的存活率成正相关（P = 0.018 < 0.05）。该结果表明，提高幼鲍的规格可显著提高皱纹盘鲍的越冬成活率，因此对于实际生产而言，采取适当措施提高皱纹盘鲍越冬苗种的规格将大幅增加生产的收益，而采用生长速率快的品种、品系或提早采苗均可实现该目标。综合各规格组幼鲍，幼鲍在南方开放性水域进行越冬培育的平均存活率较高，可达到91.38±0.01%，从幼鲍南方越冬的存活曲线可以看出，幼鲍的死亡主要集中在从大连运至福建某地后的15天内，出现死亡高峰的原因可能是由于运输过程的胁迫。此外，2月及4月中下旬水温出现显著降低或回升时也有较明显的死亡出现。该部分结果，对皱纹盘鲍幼鲍的养成管理有指导意义，可以通过合理安排越冬时间、避开死亡的敏感期等措施减少苗种越冬阶段的死亡量。 以中国大连野生群体繁育的子一代为亲本（10♀，10♂），以群体交配的方式繁育F2代个体为实验材料，分别于南方海区以及北方室内升温水方式下进行生长、存活比较，结果表明南方越冬培育方式下，幼鲍壳长的日增长率为81.37-108.89 µm•day-1，与北方室内升温培育条件相比，壳长生长提高了1.08 ~ 1.68倍；而存活率无显著差异。皱纹盘鲍幼鲍南方越冬方式的优势主要体现在鲍鱼幼鲍的生长速度加快，同时节约养殖场的能耗 （2）幼鲍培育过程中的养殖密度与分选效应评价 以3种规格皱纹盘鲍幼鲍为材料比较幼鲍在4个培育密度以及分选或混养条件下壳长的平均日生长及特定生长率。在南方越冬培育方式下实验进行106天，多因素方差分析结果表明实验初始幼鲍的壳长以及培育密度对壳长的生长有显著影响，而且密度效应在不同幼鲍起始规格组中有不同表现；分选没有能够提高不同规格组的生长。本研究的结果对皱纹盘鲍幼鲍的越冬培育有一定的指导作用。

皱纹盘鲍生长和抗逆相关新基因的初步研究

在刘晓研究员已构建的皱纹盘鲍全长cDNA文库及克隆随机测序的结果基础上，分析发现了皱纹盘鲍胸腺肽β（Tβ）cDNA全长，该序列全长487bp，生物信息学分析显示其含有一个135bp的开放阅读框（ORF），编码一个含44个氨基酸的蛋白质分子，另外其5'和3'非翻译区长度分别是69bp和283bp，3'端非翻译区具有AATAAA mRNA加尾信号及PolyA寡聚腺苷酸尾巴。采用荧光定量PCR的方法研究皱纹盘鲍Tβ的组织特异性发现，Tβ在各组织中均有分布，但在外套膜中高表达，推测和其组织结构中富含参与免疫分泌的颗粒有关。利用荧光定量PCR检测感染鳗弧菌后皱纹盘鲍腹足肌中Tβ在不同时间点的转录表达发现，Tβ基因表现出具有显著差异的时间依赖性，12h后Tβ基因的表达量明显升高，且在处理后24h达到最高值，然后表达量开始回落，至处理后72h，Tβ表达量基本降至初始值。推测皱纹盘鲍在受到病原菌侵染后Tβ基因表达量上调可能是由于Tβ基因参与了皱纹盘鲍的免疫反应，它可能在皱纹盘鲍的抗逆机制中发挥着重要作用。 发现了一个在皱纹盘鲍大小个体中差异表达的长1555bp含有完整3’端的新基因片断，暂命名为TH。利用荧光定量PCR检测皱纹盘鲍RwRh、J1Rh、J1Jm、RwJm 4个家系大小个体中TH基因的差异表达，发现其在大个体中的表达量显著高于小个体，推测其可能与皱纹盘鲍生长速度有某种关联。荧光定量PCR对皱纹盘鲍TH基因时空差异表达的研究发现，其在发育过程中的表达呈现出一定的规律性，即在围口壳幼虫期前始终保持较低水平的表达，当围口壳形成后表达量急剧增加，至幼鲍时达到最高值，在成鲍中也维持较高水平的表达，提示该基因可能调控壳的发育。同时，皱纹盘鲍TH基因具有组织特异性表达，在外套膜中的表达量显著高于其他组织，推测和其组织结构中富含参与壳形成的颗粒有关。另外还比较了皱纹盘鲍RwRh、J1Rh、J1Jm、RwJm 4个家系中TH基因的差异表达，结果发现在这4个家系中的相对表达呈现出一定的规律，即J1Rh＞RwRh＞RwJm＞J1Jm，可能有助于解释家系之间生物学性状上的差异。

皱纹盘鲍基因组单核苷酸多态标记开发与应用

本研究利用皱纹盘鲍的EST序列进行单核苷酸多态（SNP）标记开发；对等位基因特异性PCR（AS-PCR）方法进行了优化，使之适合SNP基因型分析；对一个作图家系开发基因相关SNP标记，并对标记在子代个体中的分离情况进行了分析，探讨了借助SNP在遗传连锁图谱上定位功能基因的方法。 对大约5800条EST序列进行拼接，共获得含有4条以上序列的contig 150个，在86个含有单碱基突变位点的contigs中发现SNP 302个，碱基置换类型A/G（C/T）、A/C（G/T）、A/T、C/G的位点数目分别为147、90、21、16个。50个contigs在BLASTx分析后具有匹配（E值小于1E-5），其中的220个SNPs全部为同义cSNPs，位于遗传密码子的第三简并位。粗略估算，皱纹盘鲍核基因组中SNP的平均分布密度不低于1%。 通过扩增DNA pool后产物直接测序共验证了28个SNP。PCR直接测序法操作简单，结果可靠，一次测序可能验证多个位点，有时还可以发现新的突变位点。通过重点研究引物3’端不同错配对PCR扩增的影响，对AS-PCR的引物设计原则及反应条件进行了探讨及优化，发现在AS引物的3’端倒数第二位引入额外的强错配后，AS-PCR检测SNP的特异性会得到很大的提高，从而使AS-PCR可以满足小规模的SNP基因型分析。 根据EST-contig或者单一的EST序列设计PCR引物74组，其中43组可以特异扩增皱纹盘鲍基因组DNA。用这些引物扩增一个作图家系的父母本，并对PCR产物纯化后分别进行双向直接测序，在18个基因片段中发现了86个SNP，其中82个是新SNP，并且绝大多数SNP位于内含子序列中。这些SNP在父母本中的基因型，在多数情形下，是一方为纯合（AA），而另一方为杂合（AB）；父母本均为杂合和均为纯合的形式则较少。在父母本中杂合形式的SNP位点，理论上可以在子代中发生分离。 在每个基因的内含子中选择父母本基因型为AA×AB或者AB×AA的SNP位点，设计AS-PCR分型引物并检测123个子代个体的基因型。在对9个基因中的11个SNP位点（包括5个母本标记和6个父本标记）进行子代基因型分析后发现，2个位点不符合孟德尔1：1分离（P < 0.05），符合预期分离比率的9个位点存在较大可能定位于遗传连锁图谱。通过分析两组父母本标记（F142和M142，F459和M459）发现，在根据父母本序列设计SNP分型引物时，可以设计指向同一基因的成对的父母本标记，从而使两个单亲标记可作为一个共同标记使用。

The manganese superoxide dismutase gene in bay scallop Argopecten irradians: Cloning, 3D modelling and mRNA expression

A novel manganese superoxide dismutase (MnSOD) was cloned from bay scallop Argopecten irradians by 3' and 5' rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA of MnSOD was of 1207 bp with a 678 bp open reading frame encoding 226 amino acids. The deduced amino acid sequence contained a putative signal peptide of 26 amino acids. Sequence comparison showed that the MnSOD of A. irradians shared high identity with MnSOD in invertebrates and vertebrates, such as MnSOD from abalone Haliotis discus discus (ABG88843) and frog Xenopus laevis (AAQ63483). Furthermore, the 3D structure of bay scallop MnSOD was predicted by SWISS-MODEL Protein Modelling Server and compared with those of other MnSODs. The overall structure of bay scallop MnSOD was similar to those of zebrafish Danio rerio, fruit fly Drosophila melanogaster, Chinese shrimp Fenneropenaeus chinensis, human Homo sapiens, and had the highest similarity to scallop Mizuhopecten yessoensis and abalone H. discus discus. A quantitative real-time PCR (qRT-PCR) assay was developed to detect the mRNA expression of MnSOD in different tissues and the temporal expression in haemocytes following challenge with the bacterium Vibrio anguillarum. A higher-level of mRNA expression of MnSOD was detected in gill and mantle. The expression of MnSOD reached the highest level at 3 h post-injection with V. anguillarum and then slightly recovered from 6 to 48 h. The results indicated that bay scallop MnSOD was a constitutive and inducible protein and thus could play an important role in the immune responses against V anguillarum infection. (c) 2008 Elsevier Ltd. All rights reserved.

Otimização das condições de cultivo de Ulva lactuca (Chlorophyta, Chlorophyceae) para alimentação de Haliotis tuberculata

Dissertação de Mestrado, Estudos Integrados dos Oceanos, 26 Fevereiro de 2016, Universidade dos Açores.