Heritability and phenotypic correlations of gonad sweetness in the sea urchin Strongylocentrotus intermedius

Gonad is the only edible part of sea urchins. Thus, a number of studies have been focusing on how to improve both quantity and quality of their gonads. However, as far as our knowledge, the genetic basis of gonad flavor remains totally unknown in sea urchins. In the present study, we found that the heritability of gonad sweetness was at a high level of 0.56, clearly indicating that it is, to a large extent, under genetic control. Gonad sweetness was significantly positively correlated with gonad weight (P < 0.05), a* (P < 0.01) and b* (P < 0.01), while significantly negative correlated with L* (P < 0.01), ΔE ₁ (P < 0.01) and ΔE ₂ (P < 0.01). The present study provides valuable information into the genetic basis of gonad sweetness and evidences that gonad sweetness is potential to be improved in sea urchin genetic breeding programs.     

虾夷马粪海胆不同家系和性别间性腺性状的比较

对来自虾夷马粪海胆20个家系的600枚海胆的性腺湿重、指数、颜色、水分和脂肪酸组成进行了测量和分析,结果显示性腺湿重和性腺指数在家系间均有极显著差异,但在性别间差异不显著,两个性腺产量性状在整个群体内均具有较大的变异;虾夷马粪海胆性腺颜色介于黄色和橙红色之间,且色泽鲜艳,经CIEL*a*b*标准量化和方差分析,L*和a*在家系间无显著差异,但b*、亮黄色差(ΔE1)和亮橙黄色差(ΔE2)在家系间均具有极显著差异,在性别间,雌性海胆的L*、a*和b*值以及两个色差均极显著优于雄性;性腺水分在家系和性别间有极显著和显著差异;性腺总脂中含有较高的EPA(12.11±2.65)%和AA(10.27±3.40)%,而DHA含量较低(0.49±0.64)%,但其在群体内变异极大(130.77%),高不饱和脂肪酸在家系间具有极显著差异,而在性别间仅AA有显著差异(雄性较高)。研究结果表明,对性腺产量和品质进行家系或性别选择均可达到提升性腺产量和品质的目的。     

Estimates of heritabilities and genetic correlations for growth and gonad traits in the sea urchin Strongylocentrotus intermedius

The present study is the first estimate for the sea urchin, Strongylocentrotus intermedius, of genetic parameters for growth and gonad traits during the entire reproductive period. 2165–5764 sea urchins were sampled from 74 to 120 families at six different growth stages. Six hundred individuals were sampled from 20 randomly chosen families at harvest. Coefficients of variation for most growth and gonad traits were high (18.08–78.66%) suggesting a good variation foundation for breeding. Point estimate for heritabilities based on the sire components of variance were moderate to high for test height (0.24–0.39), test diameter (0.21–0.48) and body weight (0.16–0.49). Heritabilities of gonad wet weight, gonad index and gonad moisture at harvest were 0.17, 0.41 and 0.50 respectively (moderate or high). Gonad colour parameters, L^* (lightness) and a^* (redness) have a heritability of 0, while b^* (yellowness) and colour difference ΔE₁ and ΔE₂ have very similar heritabilities of 0.2, 0.18 and 0.21 respectively. Heritabilities of β‐carotene concentration and content were 0.89 and 0.44 respectively. The genetic correlations between the two measured traits ranged from ?0.984 to 0.999. Gonad wet weight, L^*, b^*, β‐carotene concentration and β‐carotene content had significant positive genetic correlations with all the growth traits. However, gonad index, gonad moisture and colour differences ΔE₁ and ΔE₂ had significant negative genetic correlations with growth traits. The present study indicates that growth, gonad yield and most gonad quality traits could be improved by selection with moderate to high potential. Indirect selection should be applied on gonad traits using growth traits as secondary traits. The present study provides important theoretical guidance for future selective breeding programmes for S. intermedius.     

虾夷马粪海胆体腔细胞的类型及功能

取平均壳径分别为1．9cm和4．4cm虾夷马粪海胆(Strongylocentrotus intermedius)进行实验观察。结果表明，海胆体腔有2种类型的细胞，即变形吞噬细胞和色素细胞。变形吞噬细胞形状不定，能伸出伪足，核较大，线粒体、溶酶体等细胞器丰富。色素细胞具突起，内有紫红色颗粒，颗粒溶于酒精等多种溶剂中，使得电镜下细胞内含有大量空泡，细胞核很少见，细胞器较少。变形细胞离体后可凝集，具吞噬酵母的能力，吞噬能力与温度成正相关。色素细胞具有辅助的免疫功能。     

虾夷马粪海胆表型性状对性腺性状的影响效果分析

从19月龄虾夷马粪海胆Strongylocentrotus intermedius养殖群体中随机选择600枚,测量了壳高(TH)、壳径(TD)、活体重(BW)、性腺湿重(GWW)、性腺颜色(L*a*b*)、性腺指数(GI)、性腺水分(GM)7个性状。采用相关分析和通径分析方法,计算了各性状间的相关系数,并定量分析了壳性状对活体重及表型性状(壳高、壳径、活体重)对性腺性状的影响效果。结果表明,壳性状与活体重极显著相关(P0.01);4个性腺性状除构成性腺颜色性状的a*(绿-红)、b*(蓝-黄)分别与壳径、活体重呈显著相关(P0.05),而与其他表型性状无显著相关外,L*(暗-亮)、性腺湿重、性腺指数和性腺水分与表型性状均达到极显著相关(P0.01)。壳径对活体重的直接影响最大(0.717),活体重对性腺湿重的直接影响最大(0.598),壳径和活体重可作为间接选择性腺湿重的主要指标。利用曲线回归建立了壳性状预测活体重及活体重预测性腺湿重的最优模型:BW=0.001TD2.822(R2=0.984),GWW=0.039BW1.331(R2=0.753)。     

中间球海胆生长分化相关的AFLP标记

从同一遗传背景、同环境下培养的1龄中间球海胆(Strongylocentrotus intermedius)中,分别选择大个体50只、小个体30只,分别组成大小2组海胆,两组之间壳径、壳高2个指标均存在显著差异。共选用6对AFLP引物组合对2组海胆进行PCR扩增,发现43个标记在大小2组海胆间存在显著的频率差异(P0.05),其中29个位点差异极显著(P0.01)。所有存在显著频率差异的位点中,2个位点在大海胆组中缺失,4个位点在小海胆组中缺失,14个位点在大海胆组中出现的频率显著高于小海胆组(P0.05),而23个位点在小海胆组中出现的频率高于大海胆组(P0.05)。这一系列谱带可能与中间球海胆生长性状之间存在一定的相关关系。遗传多样性分析结果表明,大海胆组的平均Shannon氏指数、Nei氏杂合度2个指标均显著高于小海胆组(P0.05),提示杂种优势可能与中间球海胆的快速生长存在一定联系。     

虾夷马粪海胆溶菌酶基因全长cDNA的克隆与表达分析

本实验采用RT-PCR和cDNA末端快速扩增(RACE)技术克隆得到了虾夷马粪海胆(Strongylocentrotus intermedius)溶菌酶(LYZ)基因的全长cDNA序列。结果表明, 虾夷马粪海胆LYZ基因全长为912 bp, 含有1个480 bp的开放阅读框(ORF), 编码159个氨基酸, 其中第1−20个氨基酸为信号肽, 蛋白计算分子量为17.69 kD, 等电点为7.75。氨基酸比对分析表明, 虾夷马粪海胆LYZ基因与紫球海胆(Strongylocentrotus purpuratus)和刺参(Apostichopus japonicus)的i型LYZ基因相似百分比分别为91.4%和59.3%, 并且含有i型LYZ基因的保守序列DVGSLSCGP (Y)Y(F)QIK, 所以推断本实验克隆的溶菌酶为i型。采用实时定量PCR方法, 以β-actin为内标, 对其在虾夷马粪海胆各组织中的表达进行研究, 发现LYZ基因在围口膜中表达量最高, 其次是齿间肌、管足、肠、体腔液、雄性性腺和雌性性腺。利用脂多糖(LPS)刺激虾夷马粪海胆, 取刺激后不同时间的海胆体腔液, 对该基因的表达差异进行分析。结果表明, 虾夷马粪海胆的LYZ基因在LPS刺激后8 h时表达量最高, 12 h时开始逐步回落, 至36 h时回落至对照组相近水平。本结果可为虾夷马粪海胆免疫学研究及抗病相关分子标记的开发提供参考依据。

     

Effects of biofilms as the main and as a supplementary food on the survival,somatic growth and gonad enhancement of sea urchin Strongylocentrotus intermedius

Cultured sea urchins of similar size (mean ± SE = 4.33 ± 0.48 g in body weight) were fed biofilms only, kelp (Laminaria japonica)+biofilms (biofilms as supplementary food) and a control diet of kelp only for 7 months in the laboratory. The somatic growth and the survival rate of the sea urchins were measured monthly, and the gonad wet weight and gonad color difference were determined at the end of the experiment. The results show that diet did not significantly affect survival rate (P > 0.05), but had highly significant effects on somatic growth from the first month to the end of the experiment (P < 0.01). Sea urchins fed biofilms only showed negligible or even negative somatic growth at the end of the experiment. Sea urchins on the kelp+biofilms grazed biofilms and consumed kelp during the experiment, and showed sustained greater increase in body weight than those of fed kelp only after the fourth month (P < 0.05). The biofilms may have supplied micronutrients. At the end of the experiment, gonad production of sea urchins fed biofilms was too little (0.11 ± 0.09 g) to identify sex and measure color. Gonad wet weights of males and females and gonad color fed kelp+biofilms did not differ significantly from those of fed kelp only (P > 0.05). However, sea urchins fed kelp+biofilms were more uniform in gonad color than those fed kelp only (P < 0.01), indicating biofilms supplementation could reduce the percentage of low-grade roe. This study therefore reveals the potential of biofilms as a supplementary food in the culture of sea urchins.     

Estimates of genetic parameters for growth traits of the sea urchin,Strongylocentrotus intermedius

Heritabilities and genetic and phenotypic correlations were estimated for body weight, test diameter, and test height of the sea urchin from measurements on progeny resulting from 11 sires and 33 dams by artificial fertilization of 3 females by single males, and measurements at 8, 10, and 12 months after metamorphism. Point estimate for heritabilities based on the sire components of variance were moderate to high for body weight (0.21–0.49), test diameter (0.21–0.47), and test height (0.22–0.37). Genetic correlations were significant for body weight with test diameter (0.30∼0.65) and test height (0.30∼0.54) and test diameter with test height (0.31∼0.65). Genetic correlation estimates, derived the nested design and half-sib correlation analysis used in this study, appear to provide reliable estimates. Significant phenotypic correlations were found for body weight with test diameter (0.82∼0.86) and test height (0.49∼0.83), and test diameter with test height (0.47∼0.84). The phenotypic correlations for test height with body weight (0.491) and test diameter (0.467) at 12 months' of age were smaller than those earlier sampling periods.     

Purification and biochemical characterization of a cellulase from the digestive organs of the short-spined sea urchin Strongylocentrotus intermedius

We isolated a cellulase from the digestive organs of the short-spined sea urchin Strogylocentrotus intermedius using a combination of ion-exchange chromatography and gel filtration together with an assay for carboxymethylcellulase activity. The isolated cellulase was stained as a single band by Congo red. The molecular weight of the isolated cellulase, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions, was 59?kDa. The isolated cellulase exhibited hydrolytic activity toward carboxymethyl cellulose, with an optimum temperature and pH of 30?°C and pH 8.0, respectively. The thermal stability of the enzyme was characterized by determining the temperature at which activity decreased by 50?% with treatment for 30?min at pH 7.0 and found to be 32?°C. Cellulase activity remained at a high level at 5?C20?°C, which is the growth temperature of the short-spined sea urchin. These results confirm that the short-spined sea urchin should preferably be reared at a water temperature of <20?°C.     

Isolation and characterization of bacteria associated with a syndrome disease of sea urchin Strongylocentrotus intermedius in North China

Sea urchin, Strongylocentrotus intermedius, transplanted from Japan in 1989, has been widely cultured along the coasts of Liaoning and Shandong Provinces and has become the dominant and most economically important maricultured species in North China. However, a lesion syndrome symptom of S. intermedius broke out frequently these years, showing lethargy in activities, blackish peristomial membrane and body well lesions, and brought about high mortality eventually. Six representative prominent bacterial strains were isolated from diseased sea urchin from September 2009 to January 2010. By means of API 20NE and 16S rRNA gene sequences analysis, isolates were identified as Shewanella aquimarina, Pseudoalteromonas tetraodonis, Vibrio shilonii, V. harveyi, V. fortis and V. splendidus. Bacterial challenge tests showed that their representative isolates were virulent to S. intermedius with LD₅₀ values ranging from 9.2 × 10⁴ to 3.4 × 10⁶ CFU/g body weight, among which S. aquimarina , V. fortis and P. tetraodonis were highly virulent, and the other three isolates showed moderate virulence. The results indicated that a variety of bacteria including Shewanella, Pseudoalteromonas and Vibrio were involved in the mortality of S. intermedius, and the six isolates were opportunistic pathogens of sea urchins. All isolates reported herein were sensitive to ampicillin, enrofloxacin, ofloxacin, doxycycline and florfenicol.     

Interactive effects of family and stocking density on survival and growth of the sea urchin Strongylocentrotus intermedius

To investigate the effects of family, stocking density, and their interaction on mortality, growth rate, and size dispersal in the sea urchin Strongylocentrotus intermedius, 1,440 juveniles from six full‐sib families were exposed to three stocking densities, that is, low stocking density (LD), middle stocking density (MD), and high stocking density (HD), and reared for 10 months in sea‐based suspended cages. The results demonstrated significant family effects on cumulative mortality rate (CMR); specific growth rate for test diameter (SGR for TD); specific growth rate for body weight (SGR for BW); coefficient of variation for body weight at the 10th month (CV for BW₁₀); and significant stocking density effects on SGR for TD, SGR for BW, and CV for BW₁₀. Statistically significant family by stocking density interaction was only found in SGR for TD. A certain degree of family‐ranking inversions occurred in SGR for TD. The present study provides evidence for the existence of family by stocking density interaction on the growth rate of test diameter in the family selection for S. intermedius. More attention should be paid to this interaction effect to select correct parents in S. intermedius.     

Effects of continuous and diel intermittent feeding regimes on food consumption, growth and gonad production of the sea urchin Strongylocentrotus intermedius of different size classes

We investigated effects of a continuous and three diel feeding regimes on food consumption, growth and gonad production of cultured Strongylocentrotus intermedius of three size classes. There was no significant difference of food consumption, body weight and gonad production between nocturnal and diurnal feeding regimes of S. intermedius of all size classes, greatly challenging the old paradigm on the diel feeding preference of cultured S. intermedius. The continuous and three diel intermittent feeding regimes did not significantly affect body weight of sea urchins of all size classes. However, sea urchins in the large and small size classes which were fed continuously had significantly higher gonad wet weight and gonad index than those fed intermittently. Gonad of sea urchins in the medium size class did not change with feeding regime. The present study increases our understanding of the feeding behavior of cultured S. intermedius, with a potential of direct application in aquaculture.     

Effect of feed deprivation and refeeding on the MYP gene expression of the sea urchin (Strongylocentrotus intermedius)

To determine the main expression site of major yolk protein (MYP) gene and the mechanisms for adaptation to starvation and refeeding in Strongylocentrotus intermedius, MYP mRNA expression amounts were analysed using a real‐time RT‐PCR. The results showed that MYP could be transcribed in the intestine, stomach, gonad and coelomocytes, and that the intestine was the main expression site of MYP gene in non‐starved urchins. The MYP synthesis in the intestine decreased during 15 days of starvation (67.70%, 52.58% and 71.35% of the control at 5, 10 and 15 days of fasting respectively) and then increased dramatically by different amounts (the peaks were 2.71‐, 12.16‐ and 7.89‐fold that of the control respectively) during the refeeding stages. Nevertheless, the expression amounts in the gonads did not decline, but increased continuously during all periods of fasting (2.66‐, 3.72‐ and 13.19‐fold that of the control at 5, 10 and 15 days of starvation respectively) and during the refeeding stages. At the end of the recovery feeding experiment, the levels reached 9.58‐, 17.48‐ and 100.69‐fold that of the control. These data suggested that the ‘priority’ strategy for the sea urchin is to reduce MYP expression amounts in the intestine if food is limited and to increase MYP gene expression in the gonad to protect reproductive function.     

虾夷马粪海胆早期生长发育的遗传力估计

以虾夷马粪海胆(Strongylocentrotus intermedius)为亲本，采用不平衡巢式设计方法和人工授精技术，每个雄性海胆配5个雌性海胆，每个雌性个体产生若干幼体，构成了11个父系半同胞家系和35个母系全同胞家系，分别测定了每个母系孵化后生长到3月龄和5月龄的全同胞幼海胆40-50个后代的体重和壳径，应用数量遗传学原理和半同胞组内相关分析法研究了虾夷马粪海胆早期生长发育性状的遗传力。结果表明，3月龄和5月龄的海胆体重的狭义遗传力估计值为0．339-0．523，壳径的狭义遗传力估计值为0．316-0．487。分析结果显示，雌性遗传方差组分均显著大于雄性遗传方差组分，雌性遗传方差组分存在显著的母性效应，表明由雄性遗传方差组分估计的遗传力准确可取，父系半同胞组内相关法计算的狭义遗传力是遗传力的无偏估计值。     

中间球海胆野生和养殖群体遗传结构的微卫星分析

利用28对微卫星DNA分子标记对中间球海胆的1个野生和1个养殖群体进行了遗传多样性分析。结果表明：在28个基因座中,共检测到91个等位基因,每个基因座位检测到的等位基因数为2～6个,2个群体的平均等位基因数均为3.071 4,平均有效等位基因数分别为2.231 9、2.227 1,平均观察杂合度分别为0.523 4、0.536 5,平均期望杂合度为0.486 8、0.499 3,平均多态信息含量为0.447 7、0.439 6,群体间的多态性差异不显著。2个群体间的遗传相似系数为0.758 7,遗传距离为0.276 2。经HardyWeinberg平衡的卡方检验,有50％的位点显著偏离HardyWeinberg平衡。通过F检验发现,两个群体均处于不同程度的杂合子过剩状态。群体间发生分化程度很弱,遗传变异主要来自群体内个体之间。