Complete mitochondrial genome of the sea cucumber Apostichopus japonicus (Echinodermata: Holothuroidea): The first representative from the subclass Aspidochirotacea with the echinoderm ground pattern

Complete mitochondrial genome plays an important role in the accurate revelation of phylogenetic relationships among metazoans. Here we present the complete mitochondrial genome sequence from a sea cucumber Apostichopus japonicus (Echinodermata: Holothuroidea), which is the first representative from the subclass Aspidochirotacea. The mitochondrial genome of A. japonicus is 16,096 bp in length. The heavy strand consists of 31.8% A, 20.2% C, 17.9% G, and 30.1% T bases (AT skew = 0.027: GC skew = 0.062). It contains thirteen protein-coding genes (PCGs), twenty-two transfer RNA genes, and two ribosomal RNA genes. There are a total of 3793 codons in all thirteen mitochondrial PCGs, excluding incomplete termination codons. The most frequently used amino acid is Leu (15.77%), followed by Set (9.73%), Met (8.62%), Phe (7.94%), and Ala (7.28%). Intergenetic regions in the mitochondrial genome of A. japonicus are 839 bp in total, with three relatively large regions of Unassigned Sequences (UAS) greater than 100 bp. The gene order of A. japonicus is identical to that observed in the five studied sea urchins, which confirms that the gene order shared by the two classes (Holothuroidea and Echinoidea) is a ground pattern of echinoderm mitochondrial genomes. Bayesian tree based on the cob gene supports the following relationship: (outgroup, (Crinoids, (Asteroids, Ophiuroids, (Echinoids, Holothuroids)))). (C) 2009 Elsevier B.V. All rights reserved.

Role of animal pole protuberance and microtubules during meiosis in sea cucumber Apostichopus japonicus oocytes

Fully grown oocytes of Apostichopus japonicus have a cytoplasmic protuberance where the oocyte attaches to the follicle. The protuberance and the oolamina located on the opposite side of the oocyte indicate the animal-vegetal axis. Two pre-meiotic centrosomes are anchored to the protuberance by microtubules between centrosomes and protuberance. After meiosis reinitiation induced by DTT solution, the germinal vesicle (GV) migrates towards the protuberance. The GV breaks down after it migrates to the oocyte membrane on the protuberance side. The protuberance then contracts back into the oocyte and the first polar body extrudes from the site of the former protuberance. The second polar body forms beneath the first. Thus the oocyte protuberance indicates the presumptive animal pole well before maturation of the oocyte.

Effects of body size and water temperature on food consumption and growth in the sea cucumber Apostichopus japonicus (Selenka) with special reference to aestivation

To investigate the effects of body size and water temperature on feeding and growth in the sea cucumber Apostichopus japonicus (Selenka), the maximum rate of food consumption in terms of energy (C-maxe; J day(-1)) and the specific growth rate in terms of energy (SGRe; % day(-1)) in animals of three body sizes (mean +/- SE) - large (134.0 +/- 3.5 g), medium (73.6 +/- 2.2 g) and small (36.5 +/- 1.2 g) - were determined at water temperatures of 10, 15, 20, 25 and 30 degrees C. Maximum rate of food consumption in terms of energy increased and SGRe decreased with increasing body weight at 10, 15 and 20 degrees C. This trend, however, was not apparent at 25 and 30 degrees C, which could be influenced by aestivation. High water temperatures (above 20 degrees C) were disadvantageous to feeding and growth of this animal; SGRe of A. japonicus during aestivation was negative. The optimum temperatures for food consumption and for growth were similar and were between 14 and 15 degrees C, and body size seemed to have a slight effect on the optimal temperature for food consumption or growth. Because aestivation of A. japonicus was temperature dependent, the present paper also documented the threshold temperatures to aestivation as indicated by feeding cessation. Deduced from daily food consumption of individuals, the threshold temperature to aestivation for large and medium animals (73.3-139.3 g) was 24.5-25.5 degrees C, while that for small animals (28.9-40.7 g) was between 25.5 and 30.5 degrees C. These values are higher than previous reports; differences in sign of aestivation, experimental condition and dwelling district of test animals could be the reasons.

Effect of water temperature on digestive enzyme activity and gut mass in sea cucumber Apostichopus japonicus (Selenka), with special reference to aestivation

The effect of water temperature on gut mass and digestive enzyme activity in sea cucumber Apostichopus japonicus, including relative gut mass (RGM), amylase, lipase, pepsin and trypsin activities were studied at temperatures of 7, 14, 21, and 28A degrees C over a period of 40 days. Results show that RGM significantly decreased after 40 days at 21A degrees C and markedly decreased over the whole experiment period at 28A degrees C; however, no significant effect of duration was observed at 7 or 14A degrees C. At 14A degrees C, trypsin activity significantly decreased over 10 and 20 days, then increased; amylase and trypsin activity significantly decreased after 40 days at 28A degrees C. However, no significant effect of duration was found on amylase, pepsin or trypsin activities in the other temperature treatment groups. At 28A degrees C, lipase activity peaked in 20 days and then markedly decreased to a minimum at the end of the experiment. On the other hand, pepsin activity at 28A degrees C continuously increased over the whole experimental period. Principle component analysis showed that sea cucumbers on day 40 in the 21A degrees C group and in the previous 20 days in the 28A degrees C group were in the prophase of aestivation. At 28A degrees C, sea cucumbers aestivated at 30-40 days after the start of the experiment. It is concluded that the effect of temperature on the digestion of A. japonicus is comparatively weak within a specific range of water temperatures and aestivation behavior is accompanied by significant changes in RGM and digestive enzyme activities.

Bioenergetic responses of sub-adult sea cucumber Apostichopus japonicus (Selenka) (Echinodermata: Holothuroidea) to temperature with special discussion regarding its southernmost distribution limit in China

1. This paper investigated the bioenergetic responses of the sea cucumber Apostichopus japonicus (wet weights of 36.5 +/- 1.2 g) to different water temperatures (5, 10, 15, 20, 25 and 30 degrees C) in the laboratory. 2. Results showed that theoretically the optimal temperatures for energy intake and scope for growth (SFG) of sub-adult A. japonicus was at 15.6 and 16.0 degrees C, respectively. The aestivation threshold temperature for this life-stage sea cucumber could be 29.0 degrees C by taking feeding cessation as the indication of aestivation. 3. Our data suggests that A. japonicus is thermo-sensitive to higher temperature, which prevents it from colonising sub-tropical coastal zones. Therefore, water temperature plays an important role in its southernmost distribution limit in China. 4. The potential impact of global ocean warming on A. japonicus might be a northward shift in the geographical distribution. Crown Copyright (C) 2009 Published by Elsevier Ltd, All rights reserved.

Effects of acute temperature or salinity stress on the immune response in sea cucumber, Apostichopus japonicus

Invertebrates are increasingly raised in mariculture, where it is important to monitor immune function and to minimize stresses that could suppress immunity. The activities of phagocytosis, superoxide dismutase (SOD), catalase (CAT), myeloperoxiclase (MPO), and lysozyme (LSZ) were measured to evaluate the immune capacities of the sea cucumber, Apostichopus japonicus, to acute temperature changes (from 12 degrees C to 0 degrees C, 8 degrees C, 16 degrees C, 24 degrees C, and 32 degrees C for 72 h) and salinity changes (from 30 parts per thousand to 20 parts per thousand, 25 parts per thousand, and 35 parts per thousand for 72 h) in the laboratory. Phagocytosis was significantly affected by temperature increases in 3 h, and by salinity (25 parts per thousand and 35 parts per thousand) changes in 1 h. SOD activities decreased significantly in 0.5 h to 6 h samples at 24 degrees C. At 32 degrees C, SOD activities decreased significantly in 0.5 h and 1 h exposures, and obviously increased for 12 h exposure. CAT activities decreased significantly at 24 degrees C for 0.5 h exposure, and increased significantly at 32 degrees C in 3 h to 12 h exposures. Activities of MPO increased significantly at 0 degrees C in 0.5 h to 6 In exposures and at 8 degrees C for 1 h. By contrast, activities of MPO decreased significantly in 24 degrees C and 32 degrees C treatments. In elevated-temperature treatments, activities of LSZ increased significantly except at 32 degrees C for 6 h to 12 h exposures. SOD activity was significantly affected by salinity change. CAT activity decreased significantly after only 1 h exposure to salinity of 20 parts per thousand.. Activities of MPO and LSZ showed that A. japonicus tolerates limited salinity stress. High-temperature stress had a much greater effect on the immune capacities of A. japonicus than did low-temperature and salinity stresses. Crown Copyright (C) 2008 Published by Elsevier Inc. All rights reserved.

Effects of salinity on energy budget in pond-cultured sea cucumber Apostichopus japonicus (Selenka) (Echinodermata: Holothuroidea)

Pond farming for sea cucumber has developed rapidly along the northern coast of China in the recent years. Holothurians inhabiting ponds undergo seasonal fluctuations of salinity. This study deals with the bioenergetic responses of pond-cultured sea cucumbers Apostichopus japonicus (wet weight of 37.5 +/- 1.8 g) to different water salinities [22, 27, 31.5, and 36 practical salinity units (psu)] at 15 degrees C in the laboratory to determine the influence of water salinity on growth and energy allocation in this species. Results show that ingested energy and scope for growth (SFG) were highest at 31.5 psu and then decreased when water salinity was below or above this point. Although energy ingested was lowest at 36 psu, the lowest SFG occurred at 22 psu (only 102.68 +/- 14.26 J g(-1) d(-1)) because animals reared at 22 psu spent much more consumed energy on feces (72.19%), respiration (21.70%), and excretion (2.59%), leaving less energy for growth (3.52%). Results suggest that pond-cultured sea cucumbers could tolerate chronic salinity fluctuations at a range of 22 to 36 psu and grew better between 27 and 31.5 psu, but decreased at salinities above and below the mentioned salinity range. Crown Copyright (C) 2010 Published by Elsevier B.V. All rights reserved.

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

Metabolic characteristics of the sea cucumber Aposticholpus 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 degrees 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 degrees 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 T, while that of the immature animals peaked at 25 degrees 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 degrees 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. Oil the other hand, apart from small individuals maintained at 10 degrees 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. (c) 2005 Elsevier B.V. All rights reserved.

The influence of diets containing dried bivalve feces and/or powdered algae on growth and energy distribution in sea cucumber Apostichopus japonicus (Selenka) (Echinodermata : Holothuroidea)

In recent years, bivalve feces and powdered algae have been used as the food sources of holothurians in China. In this study, growth and energy budget for sea cucumber Apostichopus japonicus (Selenka) with initial wet body,veights of 32.5 1.0 g (mean +/- SE, n=45) when fed with five different granule diets containing dried bivalve feces and/or powdered algae in water temperature 13.2-19.8 degrees C and salinity 30-32ppt were quantified in order to investigate how diets influence growth and energy distribution and to find out the proper diet for land-based intensive culture of this species. Results showed that diets affected the food ingestion, feces production, food conversion efficiency and apparent digestive ratios, hence the growth and energy budget. Sea cucumbers fed with dried feces of bivalve showed poorer energy absorption, assimilation and growth than individuals fed with other four diets; this could be because feces-drying process removed much of the benefits. Dried bivalve feces alone, therefore, were not a suitable diet for sea cucumbers in intensive cultivation. The mixed diets of feces and powered algae showed promising results for cultivation of sub-adult Apostichopus japonicus, while animals fed with powdered algae alone, could not obtain the best growth. According to SGR of tested animals, a formula of 75% feces and 25% powdered algae is the best diet for culture of this species. Extruded diets were used in the present experiment to overcome shortcomings of the traditional powdered feeds, however, it seems a conflict exists between drying bivalve feces to form extruded diets and feeding sea cucumbers with fresh feces which contain beneficial bacteria. Compared with other echinoderms, in holothurians the energy deposited in growth is lower and the energy loss in feces accounts for the majority of the ingested energy. Such detailed information could be helpful in further development of more appropriate diets for culture of holothurians. (c) 2006 Elsevier B.V. All rights reserved.

Impact of CO2-driven acidification on the development of the sea cucumber Apostichopus japonicus (Selenka) (Echinodermata: Holothuroidea)

We evaluated the impact of ocean acidification on the early development of sea cucumber Apostichopus japonicus. The effect of pH-levels (pH 8.04, 7.85, 7.70 and 7.42) were tested on post-fertilization success, developmental (stage duration) and growth rates. Post-fertilization success decreased linearly with pH leading to a 6% decrease at pH 7.42 as compared to pH 8.1. The impact of pH on developmental time was stage-dependent: (1) stage duration increased linearly with decreasing pH in early-auricularia stage; (2) decreased linearly with decreasing pH in the mid-auricularia stage; but (3) pH decline had no effect on the late-auricularia stage. At the end of the experiment, the size of doliolaria larvae linearly increased with decreasing pH. In conclusion, a 0.62 unit decrease in pH had relatively small effects on A. japonicus early life-history compared to other echinoderms, leading to a maximum of 6% decrease in post-fertilization success and subtle effects on growth and development.

Bioenergetic trade-offs in the sea cucumber Apostichopus japonicus (Echinodermata: Holothuroidea) in response to CO2-driven ocean acidification

Ocean acidification (OA) caused by excessive CO2 is a potential ecological threat to marine organisms. The impacts of OA on echinoderms are well-documented, but there has been a strong bias towards sea urchins, and limited information is available on sea cucumbers. This work examined the effect of medium-term (60 days) exposure to three pH levels (pH 8.06, 7.72, and 7.41, covering present and future pH variability) on the bioenergetic responses of the sea cucumber, Apostichopus japonicus, an ecologically and economically important holothurian in Asian coasts. Results showed that the measured specific growth rate linearly decreased with decreased pH, leading to a 0.42 %/day decrease at pH 7.41 compared with that at pH 8.06. The impacts of pH on physiological energetics were variable: measured energy consumption and defecation rates linearly decreased with decreased pH, whereas maintenance energy in calculated respiration and excretion were not significantly affected. No shift in energy allocation pattern was observed in A. japonicus upon exposure to pH 7.72 compared with pH 8.06. However, a significant shift in energy budget occurred upon exposure to pH 7.41, leading to decreased energy intake and increased percentage of energy that was lost in feces, thereby resulting in a significantly lowered allocation into somatic growth. These findings indicate that adult A. japonicus is resilient to the OA scenario at the end of the twenty-first century, but further acidification may negatively influence the grazing capability and growth, thereby influencing its ecological functioning as an "ecosystem engineer" and potentially harming its culture output.

Acute peristome edema disease in juvenile and adult sea cucumbers Apostichopus japonicus (Selenka) reared in North China

Acute peristome edema disease (APED) is a new disease that broke out in cultured sea cucumber along the Shangdong and Liaoning province coasts in China, PR, and has caused a great deal of death in Apostichopus japonicus (Selenka) since 2004. Here we report virus-like particles found in intestine epithelium of sea cucumbers reared in North China. It is the first time that sea cucumbers are reported to be infected by virus. Histological examinations showed that the viral inclusion bodies existed in intestine epithelium cells. Electron microscopic examinations show that the virions were spherical, 80-100 nm in diameter, and composed of a helical nucleocapsid within an envelope with surface projections. Detailed studies on the morphogenesis of these viruses found many characteristics previously described for coronaviruses. Virus particles always congregated, and formed a virus vesicle with an encircling membrane. The most obvious cellular pathologic feature is large granular areas of cytoplasm, relatively devoid of organelles. Tubular structures within virus-containing vesicles, nucleocapsid inclusions, and double-membrane vesicles are also found in the cytopathic cells. No rickettsia, chlamydia, bacteria, or other parasitic organisms were found. (c) 2007 Elsevier Inc. All rights reserved.

刺参Apostichopus japonicus (Selenka)耐高温品系选育的基础研究

刺参是目前支撑我国海水增养殖业高效、稳定、持续发展的重要物种之一，但该产业面临着种质匮乏、病害严重、养殖环境局部恶化和产品品质难以保证等诸多问题。本文围绕刺参增养殖过程中的种质苗种问题，以培育耐高温品系为研究目标，在评价我国沿海不同地理群体刺参及其子一代在热刺激条件下HSP70基因mRNA表达差异的基础上，以低纬度地理群体为亲本，经温度刺激后比较了幼体及幼参的生长及HSP70 mRNA表达差异，获得了性状优良的耐高温群体。主要研究结果如下： 1、研究了我国沿海刺参不同地理群体在热激后HSP70基因mRNA的表达差异，比较了在不同温度下不同地理群体幼参的生长特性。青岛、日照和养殖等低纬度群体成参HSP70基因mRNA表达变化较大连、长岛等高纬度群体明显；热激后低纬度群体幼参HSP70基因mRNA表达量较高纬度群体高；高温条件下，高纬度群体子一代成活率低生长。温度升高对高纬度群体生长的影响较大。 2、利用荧光实时定量PCR检测了HSP70基因在不同组织、不同温度、不同时间mRNA水平的表达差异。刺参的各组织都有HSP70的mRNA表达，且对热刺激都有应答。自然水温12℃时，在＋12℃温度刺激时刺参的呼吸树中的HSP70 mRNA水平较高；刺参成参、幼参呼吸树中HSP70基因的mRNA水平在热激后6-12h达到最高；低温刺激后HSP70 mRNA下调表达。 3、分别从受精卵和初耳幼体开始在不同温度下进行幼体培育， 27℃以上温度培育的刺参幼体，其生长和成活率显著低于24℃以下温度培育的幼体；不同起始刺激阶段和不同温度都是显著影响因子。从初耳幼体进行高温培育，幼体的变态受到明显影响，26℃以上温度培育幼体的变态率显著低于24℃以下培育温度。 4、以日照刺参为亲本，在26℃培育温度下获得了耐高温群体幼参，并从生长、HSP70mRNA水平的表达差异与常温培育的群体（普通群体）进行了比较。耐高温群体幼参在较高温度下成活率和HSP70mRNA表达水平明显高于普通群体，而二者在特定生长率随温度变化上没有差异。 5、在浙江苍南进行了选择群体耐高温能力和生长验证实验。耐温群体的温度耐受能力提高了0.7℃，进入夏眠时间延后15d左右。耐温刺参群体的特定生长率和成活率在3月温度回升后远高于对照组。

Morphological and ultrastructural characterization of the coelomocytes in Apostichopus japonicus

The coelomocytes suspended in the coelomic fluid and occurring in the coelomic epithelial layer of the sea cucumber Apostichopus japonicus (Selenka) (Holothuroidea: Aspidochirota: Stichopodidae) function as mediators of the immune system, trephocytic cells and nutrient transport cells. Types of coelomocytes are characterized based on their morphological and ultrastructural features. Flow cytometry plus light and electron microscopic analyses were conducted in order to characterize the coelomocytes of A. japonicus. Six types of coelomocytes were identified: lymphocytes, morula cells, amoebocytes, crystal cells, fusiform cells and vibratile cells. Within these major categories, several distinctive cell types occurred that might represent developmental stages. The mean +/- SD coelomocyte concentration in the individuals (body length: 10 to 15 cm; weight: 100 to 150 g) was (3.79 +/- 0.65) X 10(6) cells ml(-1). The coelomic fluid contained mainly hyalinocytes (76.69%) and granulocytes (23.31 %).

Immune condition of Apostichopus japonicus during aestivation

Aestivation is an indispensable state in the life history of sea cucumbers, Apostichopus japonicus. The immune characteristics of the coelomic fluid of A. japonicus, were investigated during aestivation. Samples were collected between July and November 2006 from a coastal pond located off the Yellow Sea in Jiaonan, Shandong Province, China. The total coelomocytes counts (TCC), total superoxide dismutase (T-SOD), catalase (CAT), myeloperoxidase (MPO), and lysozyme (IZM) in the coelomic fluid were measured. The activities of catecholamines, [adrenaline (AD), noradrenaline (NA), and dopamine (DOP)] were estimated. TCC decreased from July to September, indicating weakness of the cellular immune activity at that time. Activities of SOD, CAT, MPO, and LZM changed significantly from July to October. Catecholamines AD and NA in coelomic fluid were significantly higher on August 21 and November 27. There was no significant variation in DOP during the sampling period. Thus, immune characters in coelomic fluid of A. japonicus changed significantly during aestivation. Water temperature was significantly and negatively correlated with TCC, and salinity was significantly and positively correlated with AD and NA. The mechanism of aestivation in A. japonicus is complex and might not be attributed only to environmental changes, such as temperature and salinity, as shown in previous studies. (C) 2008 Elsevier B.V. All rights reserved.