Spontaneous captive breeding and larval development in the green and red variants of the Japanese sea cucumber Apostichopus japonicus (Selenka 1867)

We report the spontaneous spawning, larval development, survival rate and larval growth rate patterns observed in the green and red variants of the Japanese sea cucumber Apostichopus japonicus. The green variant adapted well to the captive conditions in the Sasebo City Fisheries Center and spontaneously spawned without any induction or stimulation. One hundred individual green variants spawned nine times and produced approximately 155 million eggs. In contrast, 50 individual red variants showed poor adaptation to captivity and spawned spontaneously only three times, producing about 12 million eggs. Larval development and growth rate pattern was almost identical between the two variants of A. japonicus. In contrast, the larval survival rate for the green variant was over 90% up to the auricularia stage (10 days), but much low (less than 30%) for the red variant. We demonstrated that the green variant of A. japonicus was easier to rear in captivity. This provides a useful method for maintaining sustainable harvests and eventually contributing to restocking and sea ranching of the existing population. Further studies about optimal ecological conditions and behaviour are needed to improve egg production and survival rate of the red variant of A. japonicus.     

Effect of salinity on growth and energy budget of red and green colour variant sea cucumber Apostichopus japonicus (Selenca)

Sea cucumber, Apostichopus japonicus (Selenca), tolerates salinity fluctuations inhabiting intertide zone. This study deals with growth, food intake, food conversion and the bioenergetic responses of the red variant (wet weight of 2.60 ± 0.11g) and green variant (wet weight of 2.56 ± 0.08 g) A. japonicus to different salinities of 22, 26, 30, 34, and 38 psu at 16.5 ± 0.5°C. The results showed that salinity had a significant effect on specific growth rate (SGR) of both green and red variants A. japonicus (P < 0.05). Both colour variants of sea cucumber had highest SGR at 30 psu, and then decreased when salinity below or above this point. Maximum SGR (the green 1.07 ± 0.08% day^?1, the red 1.14 ± 0.09% day^?1 respectively) is related with maximum food intake (FI) and highest food conversion efficiency (FCE) (P < 0.05) occurring at 30 psu. Only under 22 psu, the green variant grew faster than the red variant (P < 0.05), and under other four salinity treatments there was no significant difference between SGR of two colour variant holothruians (P > 0.05). Values of adaptable salinity scope for green and red variants sea cucumber survival are 18.5~39 psu and 20.9~38.6 psu respectively. The average energy budget formula of sea cucumber at 30 psu was: 100C = 6G +42F +3U+49R (C, energy ingested; G, energy for growth; F, energy loss as faeces; U, energy used for ammonia excretion; R, energy loss for respiration). The sea cucumber had maximum energy ingested (C) and highest proportion of energy for growth (G) at 30 psu, and then decreased when salinity is above or below this salinity. Both red and green variants of A. japonicus deposited for growth were very low, and the energy loss in faeces and energy for respiration accounted for the majority of assimilation energy. The result clearly showed that the optimum condition for farming green and red variants A. japonicus, both with respect growth and energy allocation, is the salinity scope of 26 ~ 30 psu.     

A preliminary study on the potential value of a novel integrated aquaculture system on water purification

Intensive and semi-intensive sea cucumber aquaculture produces effluents that impact the quality of the local aquatic environment and sea cucumber growth; such effluents are an important cause of coastal water eutrophication. Integrated aquaculture has been shown to efficiently reduce the release of nutrients during aquaculture and to provide economic benefits. In this study, we report our investigation of a polyculture system utilizing Styela clava (stalked sea squirt) and Stichopus japonicus (Japanese sea cucumber) under two feeding modes. To examine purification of the water in the system, nutrients in the water and the sediment, attached heterotrophic bacteria, and the survival rates and growth rates of S. clava and S. japonicus were determined. Tank cultivation trials showed that, under the same feeding modes, the concentration of nutrients in the polyculture system was approximately 50–60 % of the sum of the nutrients in the S. japonicus mixture and S. clava monoculture systems. The abundance of attached heterotrophic bacteria in the polyculture system was remarkably lower than that of the mixed culture system under the same feeding mode. S. japonicus in the polyculture system grew better than those in the mixed culture system under the same feeding mode. These results demonstrate that the S. clava–S. japonicus integrated culture system offers a means of constraining or reversing the pollutive impacts of coastal sea cucumber aquaculture.     

Microsatellite genetic variation in wild and hatchery populations of the sea cucumber (Apostichopus japonicus Selenka) from northern China

Farming of the sea cucumber Apostichopus japonicus (Selenka) started 20 years ago and is still in rapid expansion in China. In order to assess the genetic status of both wild and cultivated stocks of this species, we used eight microsatellite markers to estimate the level of genetic diversity within five hatchery stocks and two wild populations of A. japonicus, and compared the degree of genetic differentiation between them. High levels of polymorphism were observed over all loci. The mean alleles and expected heterozygosities over the seven stocks were 10.4–12.3 and 0.735–0.783 respectively. The results of the microsatellite survey provide no evidence to show that hatchery practice of the sea cucumber in China to date has significantly affected the genetic variability of the cultured stocks. Significant differentiation was found between most pairs of the hatchery stocks and wild populations (F_st range: 0.008–0.036), and no obvious difference was detected between the wild populations (F_st=0.008). The information on the genetic variation and differentiation obtained in this study can be applied for future genetic monitoring of A. japonicus aquaculture stocks and will be useful for future genetic improvement by selective breeding, and for designing suitable management guidelines for these genetic materials.     

Stage-specific distribution of Japanese sea cucumber <Emphasis Type="Italic">Apostichopus japonicus</Emphasis> in Maizuru Bay,Sea of Japan,in relation to environmental factors

The Japanese sea cucumber Apostichopus japonicus plays important ecological and economic roles in the coastal areas of Japan. Here, we aim to reveal the relation between habitat characteristics and density of juvenile and adult sea cucumbers in Maizuru Bay. Density of adult sea cucumbers was estimated by underwater visual surveys and bottom trawls and that of newly settled individuals by seedling collectors distributed in the bay. The physical environment of the sea bottom was evaluated from median diameter, total organic carbon, total nitrogen, carbon-to-nitrogen ratio, carbon isotope ratio, nitrogen isotope ratio, depth, slope, and distance from the coastline. Adult sea cucumber density was higher in the east side than the west side of Maizuru Bay, the former having a no-take zone for sea cucumbers. Juvenile sea cucumbers were most abundantly collected in the central part of the bay. High organic matter from the sea, short distance from the coastline, and high seabed gradient (steepness) had a positive effect on the density of adult sea cucumbers, and depth had positive effects on their body size. We suggest that sustainable management of sea cucumbers can be achieved by a combination of the establishment of no-take zones and increased protection of nursery habitats.     

Combined influence of water temperature, salinity and body size on energy budget in the sea cucumber Apostichopus japonicus Selenka

The worldwide demand for sea cucumber is outpacing the sustainable harvest capabilities of fisheries. Sea cucumber ranches and farms are striving to supplement wild harvest, but variable temperature and salinity conditions in pond culture systems make sea cucumber production challenging. In this study, we evaluated how water temperature, salinity and body size affected the energy budget of pond-cultured sea cucumber Apostichopus japonicus Selenka. An orthogonal design was used to identify the most suitable conditions for energy consumption and scope for growth (SFG). After the 42-day experimental period, we found that water temperature, salinity and body size significantly influenced energy consumption, while salinity and body size were the main influencing factors on SFG. Based on these results, we suggest that a water temperature of 16 °C and a salinity of 30 g L^?1 are optimal conditions for stocking sea cucumber A. japonicus with a body size of 37.34 ± 4.63 g. As such, the optimum stocking seasons for sea cucumber A. japonicus may be April in the spring and October in the autumn.     

Spatial variation of quantitative color traits in green and black types of sea cucumber <Emphasis Type="Italic">Apostichopus japonicus</Emphasis> (Stichopodidae) using image processing

It has been suggested that the Japanese sea cucumber, Apostichopus japonicus, has three color types (red, green, and black), although the qualitative difference between the color types, particularly between the green and black types, is unclear because of continuous color variation among color types. This study elucidated the color variation between green and black types using image processing (RGB, red–green–blue system) and multivariate analysis to demonstrate whether or not the black and green types can be quantitatively classified. Moreover, spatial variation of the RGB value among various local sites was clarified to estimate potential environmental factors that may affect the color variation. The series of analyses revealed that a quantitative boundary between green and black types could be provisionally established, and also that spatial variability in the intermediate (continuous) color trait between green and black types was significant. Potential environmental factors (depth and industrial activity index) were correlated with the color traits in both color types. These results suggest that the green and black types cannot be regarded as independent color traits and that the color variation between green and black types may be influenced by local environmental factors.     

基于微卫星标记的地理群体仿刺参遗传结构判别分析模型的建立

通过主成因判别分析法(discriminant analysis of principal component,DAPC)建立仿刺参个体水平地理亚群体来源的判别模型。实验运用了20个微卫星引物,对俄罗斯海参崴(RS)和中国大连两个亚群(CN1、CN2)3个仿刺参地理群体进行了产物扩增和标记分型,获得3个地理群体分子标记表型数据。开创性地运用主成因分析和判别分析法建立判别模型,以研究不同群体仿刺参个体水平上遗传背景及其群体来源。结果显示,运用20对微卫星引物在仿刺参不同地理群体中稳定扩增的136个等位基因位点数据建立的判别模型可以准确判别刺参个体的群体来源。用75%的原有数据集建立模型,剩余的25%数据对模型的准确度进行验证,3个群体时模型准确率大于80%,2个群体时模型准确性超过90%。传统的遗传多样性和遗传结构分析显示,刺参的3个地理群体间存在中度的遗传分化,且遗传距离较近(D1=0.18,D2=0.159)。传统分析方法和新方法结果良好的一致性表明,利用DAPC和微卫星等位基因信息建立的刺参地理群体判别模型,可以很好地用于未知遗传背景刺参种质的来源鉴别。     

Release size and stocking density for grow-out of <Emphasis Type="Italic">Apostichopus japonicus</Emphasis> in the sea with raft-cultured macroalgae

Sea cucumber, Apostichopus japonicus, is the main cultured species in China. The main culture style for this species is the sea ranching model. Field trials were conducted in bottom-cages to preliminary reveal optimal releasing size, as well as maximum density of A. japonicus in an integrated multi-trophic aquaculture sea ranching area. Different sizes (4–30 g ind^?1) and densities (336–1342 g m^?2) of sea cucumbers were cultured for 13 months in Rongcheng Bay, Shandong Province, China. The size experiment showed that sea cucumber of all sizes grew throughout the experimental period. Sea cucumbers <15 g had high mortality in summer and low SGR in winter, while larger individual (>20 g ind^?1) had no advantage of growth. Sea cucumber sizes of 15–20 g may be suitable for release, considering their higher survival rate and SGR. The density experiment showed that the high biomass group had a low SGR and that the maximum release biomass was 793 g m^?2 based on a regression analysis. The optimal practical release season for sea cucumber was spring based on the results of two field experiments.     

The influence of genetics factor on key growth traits and quantitative genetic analysis of sea cucumber <Emphasis Type="Italic">Apostichopus Japonicus</Emphasis> (Selenka) heat-resistant and fast-growing strain

Using incomplete diallels cross and North Carolina II design, this study established sea cucumber families with heat-resistant and fast-growing Apostichopus japonicus strains from different ecological areas. Then, this study analyzed the influence of paternal and maternal genetic effects on the growth of A. japonicus, evaluated the growth effect of different A. japonicus families in the early growth stage and estimated the combining ability and heritability. The study showed that, in the early growth stage of A. japonicus, the influence of paternal genetic effects on the growth was higher than that of maternal effects. The best five parents and eight offspring families in terms of genetic potential breeding and growth were selected. The 3-month weight, 9-month weight, 12-month weight, 5-month to 9-month specific growth rate, 9-month coefficient of variation (CV) and 12-month CV of A. japonicus are at moderate heritability level, in order, 28.18, 40.82, 43.56, 27.87, 44.39 and 38.59 %, respectively; these traits played a leading role in parental additive gene effect on the formation of hybrids offspring traits.     

A preliminary identification of genomic loci for body colour variation in the sea cucumber Apostichopus japonicus

The sea cucumber Apostichopus japonicus is one of the most economically important aquaculture species in China, which shows a distinct characteristic of body colour variation. A number of purple variants were obtained in the coast of China, which shows great potential to develop a niche market. In the present study, to understand the genetic basis of green‐purple colour variation, a genome‐wide association study based on the 2b‐RAD sequencing was conducted. As a result, 8,795 genome‐wide single nucleotide polymorphism (SNP) markers were obtained and then applied to the association analysis. Ten most associated SNP loci with p‐value <1.0 × 10^–8 were obtained, and they were assigned to 4 chromosomes (chr17, chr21, chr15 and chr05). In genomic regions neighbouring to those SNPs, 33 candidate genes with annotation were obtained and the endothelin‐converting enzyme‐1, which was reported to be involved in pigmentation variation, was identified. To our knowledge, this is the first report of the genetic locus for body colour variation in this species. These findings will be useful for the fine mapping of the determining gene for body colour variation of A. japonicus.     

刺参养殖池塘中新敌害生物澳洲异尾涡虫的鉴定及其危害

为了对导致辽宁大连、山东东营的2家养殖场池塘养殖刺参大量化皮死亡的新的敌害生物进行鉴定并确定其对养殖刺参的危害。本实验通过形态学观察、分子鉴定及系统发育分析确定了涡虫的分类地位,通过生态学方法确定了其生态适应条件,通过切割后培养的方法观测了其再生能力,通过与刺参苗种的共培养实验测试了该物种对刺参的危害及其危害方式。形态学观察结果显示,该涡虫体长0.96～3.26 mm,体宽0.49～1.93 mm,外观黄色或黄褐色,头部钝圆,具一对暗红色棒状眼点,尾部具两条并列的尾垂;显微镜镜检发现其表皮下分布密集的虫黄藻,体表周生纤毛,雌雄同体,口后具有两个生殖孔;对该物种COⅠ及18S r DNA基因片段扩增测序结果进行分析,并构建基于18S rDNA基因的系统发育树,结果显示该生物与澳洲异尾涡虫序列同源性达99.64%,根据其形态学特征,并结合18S rDNA分子鉴定结果,将该生物鉴定为澳洲异尾涡虫;进一步对其生活习性进行了研究,结果显示,该生物具有避光性,其适宜温度为18～24°C,适宜pH为5.5～8.0,适宜盐度为20～40;再生实验表明,该物种具有很强的前后轴极性再生能力;该生物与刺参的共培养实验表明,澳洲异尾涡虫对刺参体表表现出很强的趋向性,可以吸附在刺参体表导致刺参苗种溃疡、化皮甚至死亡,但刺参的体腔、肠道、呼吸树内均未发现虫体寄生。研究表明,澳洲异尾涡虫是营自由生活的池塘养殖刺参的一种新的敌害生物,在养殖过程中需要密切关注并防范该敌害生物。     

Effects of mud substrate and water current on the behavioral characteristics and growth of the sea cucumber Apostichopus japonicus in the Yuehu lagoon of northern China

To determine whether marine mud substrate is suitable for sea cucumber aquaculture, we studied the effects of sea mud on the behavioral characteristics, growth and survival of Apostichopus japonicus, in both the field and laboratory. Our results showed that sea mud is beneficial for the growth of A. japonicus, but was unfavorable for its locomotion and attachment when a water current was present. In the field experiment in Yuehu lagoon, juvenile A. japonicus preferred to inhabit the base of seaweeds or dead leaves, which provide a favorable substrate for both their locomotion and ingestion. The mud substrate was not suitable for the small juveniles to inhabit; therefore, >3.25 g ind^?1 is the preferred size for bottom-sowing culture of sea cucumbers on the seabed in a field environment such as the Yuehu lagoon. Water current is a key factor influencing sea cucumber distribution on the bare mud substrate, with 90 % of juvenile sea cucumbers (<40 g ind^?1) being unable to keep still in a current speed of 0.115 m s^?1 for 10 min. In conclusion, a mud substrate is suitable for A. japonicus aquaculture, although hard substrates or shelters are a prerequisite for successful rearing. The water current is a key factor that influences substrate selection by A. japonicus and, thus, is an important factor to be considered in the bottom-sowing culture of these organisms.     

Toxicity of chlorothalonil to marine organisms

We evaluated the acute toxicity of the antifouling biocide 2,4,5,6-tetrachloro-1,3-benzenedicarbonitrile (chlorothalonil, TPN) to an alga (Skeletonema costatum), crustacea (Tigriopus japonicus, kuruma prawn Marsupenaeus japonicus), and a teleost fish (red sea bream Pagrus major) commonly found in the coastal waters of Japan. We also performed acute toxicity and early-life-stage tests for TPN using mummichog Fundulus heteroclitus and used the results to predict the chronic toxicity of TPN to red sea bream. The acute toxicity values of TPN (based on measured concentrations) for S.?costatum, T.?japonicus, M.?japonicus, red sea bream, and mummichog were 0.95, 16, 290, 35, and 61???g/l, respectively. The early-life-stage test with mummichog embryos revealed that hatchability and growth were the most sensitive indicators, and the lowest- and no-observed-effect concentrations, based on the measured concentrations, were 32 and 11???g/l, respectively. We estimated the chronic toxicity value for red sea bream to be 11???g/l based on these toxicity values. Considering the current measured concentrations of TPN in coastal waters of Japan, its environmental impact would appear to be limited.     

An annual faecal 16S amplicon sequencing of individual sea cucumber (Apostichopus japonicus) demonstrates the feeding behaviours against eukaryotes in natural environments

To fill in the gaps in knowledge as to how individual sea cucumber Apostichopus japonicus behave consuming eukaryotic food sources in natural environments, eukaryotic communities in the faeces of sea cucumbers and sediments were analysed through one whole year based on 16S rRNA gene sequencing of the organelle genomes. A total of 390 eukaryotic features were obtained, and 99.7% of the features were assigned to chloroplasts. The eukaryotic communities in faeces and sediments showed seasonal fluctuations through one whole year based on Bray‐Curtis distance and community composition. Comparison of eukaryotic communities between faeces and sediments showed that 12 families including Chaetocerotaceae and Laminariaceae were more abundant in faeces than in sediments, suggesting that sea cucumbers may choose sediment containing these algal taxa more often compared with others in natural environments. All features of Laminariaceae were assigned to Saccharina japonica, which is consistent with the fact that this alga is one of the most suitable diets in the aquaculture of A. japonicus. Assessments of individual 16S amplicon sequences of both faecal and sediment samples could be an alternative tool to help us understand dynamic feeding behaviours of sea cucumber populations in contributing to bioresource conservation and development of a superior approach to aquaculture.     

Genetic connectivity supports recovery of gorgonian populations affected by climate change

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Larval growth rates differ in response to seasonal temperature variations among clupeoid species inhabiting the Pacific coastal waters of Japan

Growth rates of round herring Etrumeus teres, Japanese anchovy Engraulis japonicus, and Japanese sardine Sardinops melanostictus larvae respond differently to seasonal temperature variations in Sagami Bay on the Pacific coast of central Japan. Larvae of these three clupeoid species were collected from small seine net catches between February 2008 and April 2009. Correlations between otolith growth increment widths and ambient temperatures were examined at five body sizes and indexed to otolith radii of 50–175 μm, which corresponded to notochord lengths of 15–24 mm. Round herring larvae showed positive correlations between growth rate and habitat temperature at all five body sizes. In contrast, Japanese sardine larvae did not show significant correlations between growth rate and ambient temperature at any of the five body sizes. Japanese anchovy larvae showed significant but weaker correlations between growth rate and temperature than round herring. Although these three species share habitats with some differences in seasonal occurrence in the bay, they showed different species-specific responses to seasonal habitat temperature variations. These differences in response to temperature variations may constitute an ecological basis for different migration patterns among the three species.     

Development of Gene‐derived SNP Markers and Their Application for the Assessment of Genetic Diversity in Wild and Cultured Populations in Sea Cucumber,Apostichopus japonicus

The Apostichopus japonicus is a valuable aquaculture species in China. In this study, 51 single nucleotide polymorphisms (SNPs) were identified from expressed sequence tags of sea cucumber using high‐resolution melting. The average observed heterozygosity (Ho) and expected heterozygosity (He) were 0.2462 and 0.2897, respectively. Thirty‐two of these loci were used for estimating the genetic similarity and variation between the five hatchery stocks from China and two wild stocks from Japan. No significant differences in Ho or He were observed between the wild and hatchery populations. The pairwise Fst (which ranged from 0.0119 to 0.0236) and the genetic identity (which varied from 0.9802 to 0.9915) showed no significant differentiation between the wild and cultured stocks. The analysis of molecular variance indicated the source of variation was at the level of “within the populations.” The information on the genetic variation and differentiation in cultured and wild populations of A. japonicus obtained in this study is useful for setting up suitable guidelines for founding and maintaining of cultured stocks and for future genetic improvement by selective breeding.     

Incorporating accessibility limitation into the surplus production model

Limited access to aquatic populations hinders estimation of their status and establishment of effective management measures. We propose a modified surplus production model to cope with this problem. The model provides population parameters and biological reference points from a time series of annual accessible abundance data. Simulation tests showed that the model provided biological reference point estimates with little bias when sufficiently long time series were available. Even for short time series, we could obtain nearly unbiased estimates by providing information on the exploitation rate at the maximum sustainable yield (F _MSY). As an application, we fit the modified surplus production model to 7-year accessible biomass estimates of a local population of Japanese spiky sea cucumber Apostichopus japonicus using a Bayesian approach. The results indicated that the stock in the area studied was likely to have experienced recent overfishing and had a high probability of being overfished in the future.