Development and characterization of 45 novel microsatellite markers for sea cucumber (Apostichopus japonicus)

Simple sequence repeat‐enriched library screening and expressed sequence tag database mining were adopted to develop microsatellite markers for sea cucumber (Apostichopus japonicus). Eighty‐three microsatellite loci were selected for polymorphism assessment using 48 individuals. The results showed that 45 novel loci were polymorphic. The number of alleles ranged from two to 16, and the values of observed and expected heterozygosities varied from 0 to 0.9375 and from 0.1135 to 0.9674, respectively. No significant linkage disequilibrium between pairs of loci was found and 26 loci conformed to the Hardy–Weinberg equilibrium. These markers are therefore a potential tool for studies in the population structure and linkage map construction for A. japonicus.     

夏眠对刺参(Apostichopus japonicus (Selenka))能量收支的影响

夏眠是刺参最重要的生理特征;水温升高是其夏眠的主要诱发因子,而夏眠的临界温度与刺参体重密切相关。为揭示刺参夏眠对其能量利用对策的影响,测定了2种体重规格(134.0±13.5)g和(73.6±2.2)g刺参在10、15、20、25℃和30℃5个温度梯度下的能量收支。结果表明,温度和体重及其交互作用对刺参能量的摄入均有显著影响;而温度是影响其摄食能分配的主要因素。研究发现,刺参在非夏眠期、夏眠临界期和完全夏眠期的能量利用对策有所不同:在非夏眠期,刺参摄食能支出的最大组分是粪便能,占摄食能的比例超过50%,其次为呼吸耗能,占19.8%～39.4%,而生长能和排泄能占的比例较小,分别为5.7%～10.7%和2.9%～3.7%;在夏眠临界温度下,呼吸和排泄耗能占摄食能的比例均显著增大(分别为88.3%和13.6%),而生长能所占比例降为负值(-55.3%),刺参表现为负生长;而在夏眠期,刺参的摄食能和排粪能为零,为维持其基本生理活动,不得不动用以往贮存于体内的能量,消耗于呼吸和排泄等生理过程,供维持生命之用。总之,从能量生物学的角度看,夏眠的主要生态学意义在于刺参长时间处于相对高温环境,进而导致摄食受阻条件下的一种能量节约方式。     

A genetic linkage map of the sea cucumber, Apostichopus japonicus (Selenka), based on AFLP and microsatellite markers

We present the first genetic maps of the sea cucumber ( Apostichopus japonicus ), constructed with an F₁ pseudo-testcross strategy. The 37 amplified fragment length polymorphism (AFLP) primer combinations chosen identified 484 polymorphic markers. Of the 21 microsatellite primer pairs tested, 16 identified heterozygous loci in one or other parent, and six were fully informative, as they segregated in both parents. The female map comprised 163 loci, spread over 20 linkage groups (which equals the haploid chromosome number), and spanned 1522.0 cM, with a mean marker density of 9.3 cM. The equivalent figures for the male map were 162 loci, 21 linkage groups, 1276.9 and 7.9 cM. About 2.5% of the AFLP markers displayed segregation distortion and were not used for map construction. The estimated coverage of the genome was 84.8% for the female map and 83.4% for the male map. The maps generated will serve as a basis for the construction of a high-resolution genetic map and mapping of the functional genes and quantitative trait loci, which will then open the way for the application of a marker-assisted selection breeding strategy in this species.     

Reproductive cycle and releasing time for increase of resource of adult sea cucumber Apostichopus japonicus released to seed breeding grounds

The reproductive cycle and releasing time for effective increase of resource of adult sea cucumber (Apostichopus japonicus) have been studied in the west coast of Korea. Adult sea cucumbers collected in Seocheon-gun April 2013 for first release and in the uncontaminated Taean-gun area in the west coast of Korea. Divers monthly collected the specimens in the released area from December 2013 to November 2014 in order to investigate the reproductive cycle of A. japonicas in Taean-gun. Random specimens were dissected to examine the gonadal developmental stages and discharge rates of the guts and gonads. The reproductive cycle of A. japonicus in both sexes are classified into the following six successive stages in the Taean-gun: (1) Stage I (recovery stage from December to February), (2) Stage II (early growing stage from February to March), (3) Stage III (late growing stage from March to April), (4) Stage IV (mature stage from April to July), (5) Stage V (partly spawned stage in July), and (6) Stage VI (spent stage from August to November). The estivation period of this species is from July to October in the Taean-gun region while surface water temperature is approximately 20–25.4°C. Thus, the optimum period to easily harvest them is from October to November in the uncontaminated Taean-gun area. It is also the best releasing time because the recovery stage starts from December to February in the Taean-gun region.     

刺参“腐皮综合症”致病菌LNUB415的分离及防治的初步研究

从大连海域患“腐皮综合症”的养殖刺参肠道中分离纯化到1株优势细菌LNUB415,经人工回接感染试验确定其对刺参具有较强的致病性.经形态学、生理生化和16S rDNA序列分析,该菌被初步鉴定为蜡样芽胞杆菌(Bacillus cereus).从多种具有免疫增强作用的中草药中筛选到抑菌效果最好的黄连,可使刺参死亡率降低30％.     

Polymorphic markers for the sea cucumber Parastichopus californicus

Five polymorphic markers were isolated from a Parastichopus californicus genomic DNA library. In addition, a method was developed to extract DNA from both muscle and skin tissue. The number of alleles at these loci ranged from three to 14. Observed heterozygosity ranged from 0.2 to 0.9. These markers, the first of their type for a sea cucumber species, will prove useful for exploration of the population structure of P. californicus.     

刺参病害现状及其生物技术检测的研究进展

随着人工养殖规模的扩大,刺参养殖的病害问题已严重阻碍刺参养殖业的发展,如刺参腐皮综合症、化板病、病毒性疾病等.综述了近年来刺参的养殖工艺和模式,以及常见疾病的症状和防治手段.并针对刺参防御系统的特点,从免疫学和分子生物学方面做了相应的探讨和展望.     

A comparison of the effects of light intensity on movement and growth of albino and normal sea cucumbers (Apostichopus japonicus Selenka)

Albinism in the sea cucumber Apostichopus japonicus (Selenka) may be a significant biomarker that can be used to obtain information about behaviors and growth of this species. The goal of this study was to compare the behaviors and growth rate between albino and normal A. japonicus. Total light intensity and specific growth rate were used as statistical indices. Diverse light intensities were tested in the laboratory and in the field to identify ecological characteristics of albino A. japonicus. Experimental results revealed that: (1) both albino and normal specimens had negative phototaxis; (2) normal A. japonicus were more responsive to intense light than albino species, and the latter could withstand longer periods of exposure to intense light; (3) normal A. japonicus grew faster than albino specimens; and (4) both normal and albino A. japonicus grew faster in natural light than in the other light intensities tested.     

海水酸化及升温对刺参稚参生长和变色的影响

海洋酸化和海洋变暖是当下及未来海洋生物及其依存生态系统面临的主要环境压力和生态问题。当前,海洋生物早期发育气候变化生物学的研究主要集中于海洋酸化的影响,为了更好地探究海洋气候变化对海洋生物的影响,有必要研究海洋酸化和变暖联合作用下海洋生物的生态响应。以受精后24天的刺参稚参为研究对象,通过模拟当前和本世纪末海洋环境,观察海水酸化和升温对刺参稚参在体色发育关键时期生长、发育及体色变化的影响。实验设置对照组(大连近海水温,pCO₂ 400 mg·kg^-1)、升温组(对照组水温+2℃,pCO₂ 400 mg·kg^-1)、酸化组(对照组水温,pCO₂ 1000 mg·kg^-1)、酸化升温组(对照组水温+2℃,pCO₂ 1000 mg·kg^-1)。结果表明:温度升高2℃能够显著提高稚参发育速率,体色变化加快; pH值降低0.23个单位显著延迟稚参生长,体色变化减缓,个体间体重差异变大;升温2℃能抵消pH降低0.23个单位对...     

Effects of aestivation on the energy budget of sea cucumber Apostichopus japonicus (Selenka) (Echinodermata: Holothuroidea)

Apostichopus japonicus is a kind of temperate sea cucumbers, known to aestivate when water temperature rises above 20°C to 24.5°C. In this study, we measured the effects of aestivation on the energy utilization (i.e., energy allocation in growth, feces discharge, respiration and excretion) of A. japonicus with two different body weights (134.0 g ± 3.5 g and 73.6 g ± 2.2 g) at water temperature from 10°C to 30°C with an interval of 5°C. Noticeable variation in the energy utilization of sea cucumbers was observed in this study. During the non-aestivation period, energy deposit in growth was lower and the energy loss in feces accounted for the majority of the feeding energy. Under the threshold temperature, the feeding energy reduced and the proportion of energy deposit in growth became negative. During aestivation, sea cucumbers discontinued feeding energy, resulting in weight loss. Our study suggested that the ecological implication of aestivation in this species could lead to a model of energy saving during the long-term hot period.     

复合微生态制剂对幼刺参营养成分和 酶活力的影响

目的研究复合微生态制剂对幼刺参体壁营养成分、几种消化酶和免疫酶活力的影响。方法在封闭式循环系统中投喂不同的微生态制剂进行30 d刺参养殖实验。结果投喂液态复合微生态制剂组和粉状复合微生态制剂组的刺参体壁的粗脂肪、总糖、粗蛋白含量最高,两组与对照组相比差异有统计学意义(Ps0.05)。添加微生态制剂的3个实验组刺参肠道蛋白酶、淀粉酶活力均比对照组高,且差异有统计学意义(Ps0.05)。实验组刺参的肠道、体壁、体液过氧化氢酶活力均比对照组高,其中投喂液态复合微生态制剂实验组活力最高。实验组刺参组织的酸性磷酸酶和碱性磷酸酶活力也明显高于对照组(与对照组相比差异有统计学意义,Ps0.05)。结论微生态制剂可以有效改善幼刺参体壁营养成分,促进机体消化活力,提高刺参免疫力。     

The syndrome of sea cucumber (Apostichopus japonicus) infected by virus and bacteria

A outbreak of disease with symptoms of evisceration and skin ulteration led to mass mortality in sea cucumber Apostichopus japonicus cultivated in indoor ponds near the Dalian coast from December 2004 to April 2005. Spherical virus particles with a diameter of 75–200 nm were found in the cytoplasm of cells in the water-system, the alimentary canal and in the respiratory trees of the diseased and dying sea cucumber individuals by electron microscopic observation of ultrathin sections. Examination by negative stained samples revealed that all the diseased sea cucumbers were infected by the virus, while the healthly ones cultivated outside the contagious area were not. Two bacterial strains were also isolated from the diseased animals. When exposed to a medium containing the virus particles, regardless of whether the bacterial suspension was added, healthy sea cucumbers exhibited identical disease symptoms as the ones in the indoor ponds, and had a mortality of 90%–100%. However, when exposed to a medium in which there was only one of the two bacterial strains, 30%–80% of the sea cucumbers were infected and nearly 20% died. Negative staining showed that the viral particles were detected only in the bodies of the tested animals that were exposed to the viral medium. Histopathologically, the diseased sea cucumbers are characterized by karyopycnosis, and disintegration of the endoplasmic reticula and mitochondria in the epithelial cells in the water-system, the respiratory tree and the alimentary canal. Fundation items: Supported by commonweal program of State Oceanic Administration of China (200705007), Science and Technology Project of Liaoning Province (2004203001) and Science and Technology Project of Dlian (2004 BINS030).     

刺参养殖池塘颗粒悬浮物结构及其沉积作用

对不投饵刺参养殖池塘的颗粒悬浮物结构及其沉积量和沉积速度进行了研究.结果表明:(1)各池塘总颗粒悬浮物重量(TS)为(73.1±20.5)mg/L,变动于46.0～132.0mg/L之间;颗粒无机物(IS)含量为(50.6±12.6)mg/L,变动于28.5～76.5mg/L之间,占总颗粒悬浮物的69.13%;浮游生物干重(PZ)为(0.675±0.706)mg/L,变动于0.064～2.814mg/L之间,其中浮游植物干重(DWP)为(0.541±0.622)mg/L,变动于0.062～2.582mg/L之间,浮游动物干重(DWZ) 为(0.135±0.200)mg/L,变动于0.002～1.160mg/L之间;颗粒腐质为(21.91±19.59)mg/L,变动于4.18～83.86mg/L之间,占总颗粒悬浮物的比例为29.95%.(2)总颗粒物沉积量为(19.44±16.34)g·m-2·d-1,变动于6.33～91.98g·m-2·d-1之间,沉降速度为(0.30±0.30)m·d-1,变动于0.08～1.63m·d-1之间,新生态颗粒有机物沉积量为(5.09±7.30)g·m-2·d-1,变化于0.14～31.27g·m-2·d-1之间,总颗粒物沉积量和新生态颗粒物沉积量均是底层高于表层.     

Metabolic characteristics of sea cucumber Apostichopus japonicus (Selenka) during aestivation

Metabolic characteristics of the sea cucumber Apostichopus japonicus (Selenka) during aestivation were studied in the laboratory. The effects of water temperature on oxygen consumption rate (OCR) and ammonia-N excretion rate (AER) in A. japonicus were determined by the Winkler and Hypobromite methods, respectively. Mature (large, 148.5 ± 15.4 g, medium 69.3 ± 6.9 g) and immature (small, 21.2 ± 4.7 g) individuals aestivated at water temperatures of 20 and 25 °C, respectively. The metabolic characteristics of mature individuals were different from immature individuals during this period. The OCR of mature sea cucumbers peaked at 20 °C, and then dropped significantly at higher temperatures, whereas the OCR of the immature animals continued to increase slightly, even beyond the aestivation temperature. The AER of mature individuals peaked at 20 °C, while that of the immature animals peaked at 25 °C. The relationships between dry weight (DW) and absolute oxygen consumption (R) and absolute ammonia-N excretion (N) could be described by the regression equation R or N = aW^b. With the exception of 15 °C, the O / N ratios (calculated in atomic equivalents) of large size sea cucumbers was close to 20 across the temperatures used in this study, indicating that their energy source was a combination of lipid and protein. On the other hand, apart from small individuals maintained at 10 °C, the O / N ratios of the medium and small sea cucumbers were close to 10, indicating that protein was their major energy source. The O / N ratios in all size groups remained unchanged after aestivation was initiated.     

Induction of larval metamorphosis in the sea cucumber Stichopus japonicus by periphitic diatoms

The mass production of juvenile seeds of the sea cucumber,Stichopus japonicus has recently developed by the SeaFarming Center of Saga Prefecture. Methods for the culture ofperiphicic diatoms have been improved. There are three importantsteps in propagating the diatoms. The first step is theenrichment, with the addition of the nutrient salts, undercontrolled light intensity. The second step is washing withhigh pressure seawater and reversal of the plates. The laststep is elimination of copepods, which feed on diatoms, usinga pesticide. Small periphitic diatoms such as Navicula,Amphora, Achnanthes, and Nitzschia are easily culturedat a density of more than one million cells cm^–2, andthese diatoms are able to induce larval metamorphosis andserve as a food source for juvenile sea cucumbers.     

Spatiotemporal expression pattern of gonad-stimulating substance-like peptide of the sea cucumber, Apostichopus japonicus

The spatiotemporal expression pattern of gonad-stimulating substance-like peptide-containing polypeptide (GSSLP) in the sea cucumber Apostichopus japonicus was examined using immunochemistry. The GSSLP was detected in the gonads from shortly before the empirical breeding season (May and June) to July. On the basis of immunoblotting analysis, GSSLP showed considerable polymorphism among the organs examined in this study, particularly in the gonads, in which the polymorphism was associated with N-glycosylation and the formation of intra-molecular disulfide bonds. In the ovary, GSSLP was expressed from March to June and corresponded to two bands at 113 and 100 kDa under reducing conditions. In July, only the larger band weakly remained. In testis, GSSLP was detected first in April as two bands of 245 and 190 kDa under reducing conditions. The number of bands increased to five in June but decreased to three smeared bands in July. In the radial nerve and circumoral nerve ring, GSSLP corresponded to a single peptide of 170 kDa with little N-glycosylation and its expression level hardly changed throughout a year with no correlation with the breeding season. GSSLP was detected mainly in the morula cells in all the organs examined. In addition, GSSLP was detected in the follicle cells of the ovary and, for a brief period, in the jelly space, but never in the ooplasm. In testis, the morula cells were localized close to the invaginated inner epithelium, but never in the male gametes. In July animals, gonadal morula cells were rarely observed.     

The Syndrome of Sea Cucumber (Apostichopus japonicus) Infected by Virus and Bacteria

A outbreak of disease with symptoms of evisceration and skin ulteration led to mass mortality in sea cucumber Apostichopus japonicus cultivated in indoor ponds near the Dalian coast from December 2004 to April 2005. Spherical virus particles with a diameter of 75-200 nm were found in the cytoplasm of cells in the water-system, the alimentary canal and in the respiratory trees of the diseased and dying sea cucumber individuals by electron microscopic observation of ultrathin sections. Examination by negative stained samples revealed that all the diseased sea cucumbers were infected by the virus, while the healthly ones cultivated outside the contagious area were not. Two bacterial strains were also isolated from the diseased animals. When exposed to a medium containing the virus particles, regardless of whether the bacterial suspension was added,healthy sea cucumbers exhibited identical disease symptoms as the ones in the indoor ponds, and had a mortality of 90%-100%. However, when exposed to a medium in which there was only one of the two bacterial strains, 30%-80% of the sea cucumbers were infected and nearly 20% died. Negative staining showed that the viral particles were detected only in the bodies of the tested animals that were exposed to the viral medium. Histopathologically, the diseased sea cucumbers are characterized by karyopycnosis, and disintegration of the endoplasmic reticula and mitochondria in the epithelial cells in the water-system, the respiratory tree and the alimentary canal.     

Identification and functional analysis of foxl2 and nodal in sea cucumber,Apostichopus japonicus

Sea cucumber (Apostichopus japonicus) is an important mariculture species in China. To date, the mechanisms of sex determination and differentiation in sea cucumber remain unclear. Identifying sex-specific molecular markers is an effective method for revealing the genetic basis of sex determination and sex differentiation. In this study, foxl2 and nodal homologous genes were identified in A. japonicus. Foxl2 exhibited dynamic and sexually dimorphic expression patterns in the gonads, with prominent expression in the ovaries and minimal expression in the testis according to real-time quantitative PCR (RT-qPCR) study. As nodal was specifically expressed in the ovary, it could serve as an ovary-specific marker in sea cucumber. Additionally, knockdown of foxl2 or nodal using RNA interference (RNAi) led to the down-regulation of piwi, germ cell-less, and dmrt1, suggesting that foxl2 and nodal may play important roles in gonad maintenance of sea cucumber. Overall, this study adds to our understanding of the roles of foxl2 and nodal in the gonadal development of A. japonicus, which provides further insight into the mechanisms of sea cucumber sex determination and differentiation.