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Effects of Chaetoceros muelleri and Rhodotorula sp. on the development, growth and survival of the planktonic larvae of sea cucumber (Holothuria leucospilota)

  • Corresponding author: YU Zonghe, yuzh@scau.edu.cn
  • Received Date: 2020-05-27
    Accepted Date: 2020-12-27
    Available Online: 2021-04-04
  • The sea cucumber Holothuria leucospilota is an edible sea cucumber species, which is widely distributed in the shallow waters of tropical and subtropical Indo-Pacific Ocean. Nowdays, H. leucospilota has become an important fishery resource owing to the continuously increasing market demand, however, overfishing activities will put this species at risk of extinction in the future. Artificial breeding of H. leucospilota is considered as a useful strategy to solve this problem. The use of suitable substitute diets is an economical and efficient approach for breeding of planktonic larvae of sea cucumbers. In this study, the microalgae Chaetoceros muelleri and Rhodotorula sp. were fed to planktonic larvae of sea cucumber H. leucospilota, either singly or in combination, to evaluate their nutritional quality. C. muelleri and Rhodotorula sp. were mixed in 4∶0, 3∶1, 2∶2, 1∶3 and 0∶4, by volume, and they were labeled as groups A, B, C, D and E, respectively. Results showed that larvae in group A, which fed solely on C. muelleri, exhibited the fastest growth and development among the five groups, larvae could develop to the auricularia stage on the 10th day and the doliolaria stage on the 18th day; meanwhile, survival of the larvae in group A was the highest, and the survival rate in this group was 58.73%±2.75% at the end of the experiment. By contrast, the larvae in groups D and E always stayed in the early auricularia stage, and individuals in these two groups could not form the hyaline spheres during the whole study period. The larvae in group E exhibited negative growth during the whole study period, and survival rate of this group was only 22.22% ± 5.50% at the end of the experiment. In general, negative effects on the development, growth and survival of the planktonic larvae of H. leucospilota were found with the increase in the proportion of the Rhodotorula sp. in the diet, and therefore Rhodotorula sp. was not a suitable substitute for breeding of H. leucospilota.
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Effects of Chaetoceros muelleri and Rhodotorula sp. on the development, growth and survival of the planktonic larvae of sea cucumber (Holothuria leucospilota)

    Corresponding author: YU Zonghe, yuzh@scau.edu.cn
  • 1. College of Marine Sciences, South China Agricultural University, Guangzhou    510642, China
  • 2. South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou    510301, China
  • 3. Institute of Animal Science, Guangdong Academy of Agricultural Sciences, Guangzhou    510640, China
  • 4. College of Marine Science, Hainan University, Haikou    570228, China

Abstract: The sea cucumber Holothuria leucospilota is an edible sea cucumber species, which is widely distributed in the shallow waters of tropical and subtropical Indo-Pacific Ocean. Nowdays, H. leucospilota has become an important fishery resource owing to the continuously increasing market demand, however, overfishing activities will put this species at risk of extinction in the future. Artificial breeding of H. leucospilota is considered as a useful strategy to solve this problem. The use of suitable substitute diets is an economical and efficient approach for breeding of planktonic larvae of sea cucumbers. In this study, the microalgae Chaetoceros muelleri and Rhodotorula sp. were fed to planktonic larvae of sea cucumber H. leucospilota, either singly or in combination, to evaluate their nutritional quality. C. muelleri and Rhodotorula sp. were mixed in 4∶0, 3∶1, 2∶2, 1∶3 and 0∶4, by volume, and they were labeled as groups A, B, C, D and E, respectively. Results showed that larvae in group A, which fed solely on C. muelleri, exhibited the fastest growth and development among the five groups, larvae could develop to the auricularia stage on the 10th day and the doliolaria stage on the 18th day; meanwhile, survival of the larvae in group A was the highest, and the survival rate in this group was 58.73%±2.75% at the end of the experiment. By contrast, the larvae in groups D and E always stayed in the early auricularia stage, and individuals in these two groups could not form the hyaline spheres during the whole study period. The larvae in group E exhibited negative growth during the whole study period, and survival rate of this group was only 22.22% ± 5.50% at the end of the experiment. In general, negative effects on the development, growth and survival of the planktonic larvae of H. leucospilota were found with the increase in the proportion of the Rhodotorula sp. in the diet, and therefore Rhodotorula sp. was not a suitable substitute for breeding of H. leucospilota.

  • 玉足海参(Holothuria leucospilota)广泛分布于印度-西太平洋海域,在我国主要分布于台湾、福建、广东、广西、海南以及西沙群岛等地的近岸浅水区[1-3]。玉足海参具有较高的食用和药用价值,采捕玉足海参是许多太平洋岛屿传统渔业的一部分,近年来随着海参自然资源的枯竭和市场需求量的持续增加,其价格水涨船高[3-7]。研究发现,玉足海参不仅对自然海域环境有较强的修复作用,还可以与鱼虾等进行混养用于养殖环境调控,因此对其进行人工养殖可带来较高的环境与经济效益[3, 8-10]。随着我国南海主要经济海参种类资源的枯竭,玉足海参已被列为新的捕捞对象,无序捕捞导致玉足海参自然资源日益减少,因此,通过人工繁育与增殖放流对玉足海参资源进行补充迫在眉睫。目前,热带海参中糙海参(H. scabra)的人工繁育和养殖技术已经比较成熟[11-12],而玉足海参等热带海参的人工繁育尚处于起步阶段[13-14]

    饵料是关乎水产苗种培育成功与否的关键环节之一,海参育苗所用的饵料应遵循营养价值高、易摄食与消化吸收的原则。国内外培育海参浮游幼体所用的饵料多为单胞藻[11, 13]或是单胞藻与酵母的混合物[12, 14]。牟氏角毛藻(Chaetoceros muelleri)和海洋红酵母(Rhodotorula sp.)为海参、虾蟹以及贝类幼体的优质饵料[15-17]。其中,牟氏角毛藻具有营养丰富、耐高温、抗污染及生长速率快等优点[18]。玉足海参的繁殖期为每年6—10月[14],相较于其他常见的单胞藻,牟氏角毛藻更适合于在南方进行规模化培养,用于投喂玉足海参浮游幼体。海洋红酵母富含蛋白质、肝糖、不饱和脂肪酸、类胡萝卜素及动物幼体生长激素等多种营养物质[19],并且海洋红酵母已经实现商业化生产,市场供应充足,如能将其作为替代饵料用于热带海参浮游幼体培育,可以大幅度地降低培养单胞藻所需要的人力和物力成本,并能够避免投喂单胞藻所引入的病、敌害等风险。

    本研究利用牟氏角毛藻和海洋红酵母投喂玉足海参的浮游幼体,探讨两种饵料单独投喂和混合投喂对幼体发育、生长及成活率的影响。研究结果可以为热带海参的人工繁育提供一定的科学依据。

1.   材料与方法
  • 牟氏角毛藻藻种购自中国科学院海洋研究所藻种库,利用f/2培养基培育至指数生长期后用于海参浮游幼体投喂。用血球计数板测定其密度。取500 mL藻液用事先灼烧并称重的WhatmanGF/C玻璃纤维滤膜进行抽滤,按照Yu等[20]的方法测定藻液中悬浮颗粒物浓度(total particulate matter,TPM)及颗粒物中有机物含量(organic matter content,OMC)。结果表明牟氏角毛藻的平均密度为48×104个/mL,TPM为(24.20±0.85) mg/L,OMC为71.92%±1.35%。

  • 海洋红酵母购自山东省烟台市某生物科技有限公司,为瓶装粉红色液体,其原料成分为海洋红酵母、虾青素和有益菌代谢产物等的混合物,有效菌总数≥100×108 CFU/mL。摇匀后取一定体积的酵母液,按照“牟氏角毛藻”的方法测定其TPM及OMC。结果表明,海洋红酵母液TPM为(42.50±3.78) mg/mL,OMC为84.39%±3.43%。海洋红酵母在使用前摇匀并与清洁海水按照1∶1800的体积比进行稀释,稀释液现配现用,其TPM为24 mg/L。

  • 玉足海参亲参采捕自深圳大亚湾桔钓沙海域(22°33'38"N, 114°33'12"E),将其暂养、清空肠道内含物后,于2019年9月4日,挑选体质量200 g以上,体表无明显损伤的个体,采用阴干和流水刺激法获取精卵[14]。将海参精卵混合授精10 min后,洗掉多余的精子,转移至数个500 L的玻璃钢桶中进行孵化,孵化水体保持24 h充气,使受精卵均匀悬浮在水体中。孵化密度为1个/mL,孵化期间每天换水1/3,孵化2 d,待受精卵发育至小耳幼体后,利用牟氏角毛藻液与海洋红酵母稀释液(见“海洋红酵母”)按照体积比1∶1的比例混合进行投喂,日投喂量约为20 L/m3水体。

  • 2019年9月7日正式开始进行投喂实验,此时玉足海参浮游幼体处于小耳幼体阶段。向孵化桶中添加清洁海水,使幼体平均密度降至0.7个/mL,然后将水体混匀,采取虹吸法将幼体连同培养水体一起转移至实验桶中至50 L刻度线。实验过程中,每个实验桶水位始终保持在50 L,每天中午换水1次,每次换水量为总体积的1/2。每个实验桶底部均布设2个气头,保持适度充气,使幼体一直处于悬浮状态。定期利用虹吸法清理桶底残饵、粪便和死亡幼体。实验所用海水经孔径为5 μm的微孔过滤袋进行过滤后,添加消毒粉(20 mg/L)处理12 h,最后利用硫代硫酸钠(10 mg/L)中和余氯后使用。实验期间水温为27.8~30.7 °C,pH为7.9~8.1,盐度为31.3~33.4。

    牟氏角毛藻在使用前进行镜检,如藻液中存在原生动物,则利用电鱼杆通电予以杀灭;海洋红酵母则按照“海洋红酵母”中的方法稀释后使用。本实验设5个处理组,即将牟氏角毛藻液与海洋红酵母稀释液分别按照4∶0、3∶1、2∶2、1∶3和0∶4的体积比混合后投喂玉足海参浮游幼体,依次标记为A、B、C、D和E组,每组设3个重复。实验前期每桶早晚各投喂1 L饵料,中后期投喂量加倍,同一时期不同处理组投饵量(以TPM为基准)保持一致。

  • 实验开始后,每2天镜检观察并记录各组玉足海参浮游幼体的发育状态。每隔4天对幼体进行计数,计数前先将实验桶水体混匀,然后利用玻璃烧杯随机取10 mL水体,在灯光下观察其中幼体个数并记录。每个实验桶计数3次,取平均值,将计数结果与初始值进行比较以计算成活率。计数结束后,每个实验桶均随机取5~10个幼体存于离心管中,带回实验室镜检测量其体长。实验开始时,玉足海参浮游幼体的平均密度为0.7个/mL,平均体长为(460.67±21.78) μm。本实验一共持续20 d,至9月27日结束。

  • 利用SPSS 19.0软件对数据进行统计分析。其中,利用单因素方差分析(One-Way ANOVA)检验同一取样时间各组玉足海参浮游幼体体长及成活率差异的显著性,当数据之间差异显著时,则利用Duncan氏法对数据进行多重比较。成活率数据经过平方根反正弦变换后再进行统计分析。实验结果用平均值±标准差(mean±SD)表示。P<0.05表示差异显著。

2.   结果
  • 各处理组玉足海参浮游幼体发育状态随时间的变化如表1所示。其中,第4、10和20天各组幼体的图片见图1。实验开始后第0~8天内,A、B、C这3组玉足海参浮游幼体发育状态保持一致,均从第4天开始进入中耳幼体阶段,而D和E组幼体一直停滞在小耳幼体阶段(表1)。第10~14天,A和B组幼体进入大耳幼体阶段,而同时期C组一直处于中耳幼体阶段,D和E组继续停滞在小耳幼体阶段。第16天,A组幼体进入大耳幼体后期阶段,同期其他组幼体发育状态未发生变化。第18~20天(受精后第21~23天),A组幼体进入樽形幼体阶段,B组在第20天进入大耳幼体阶段后期,C组进入大耳幼体阶段,而D和E组继续停滞在小耳幼体阶段。总体来说,饵料中牟氏角毛藻的比例越高,玉足海参浮游幼体的发育速率越快,反之,海洋红酵母的比例越高,则幼体发育速率越慢。对于同一取样时期,A、B、C组幼体胃部的饱满度相对较高,而D和E组幼体胃部饱满度则很低;A和B组耳状幼体球状体较明显,而C、D和E组幼体球状体不明显或者缺失(图1)。

    时间/d
    time
    组别    groups
    ABCDE
    0小耳小耳小耳小耳小耳
    2小耳小耳小耳小耳小耳
    4中耳中耳中耳小耳小耳
    6中耳中耳中耳小耳小耳
    8中耳中耳中耳小耳小耳
    10大耳大耳中耳小耳小耳
    12大耳大耳中耳小耳小耳
    14大耳大耳中耳小耳小耳
    16大耳后期大耳中耳小耳小耳
    18樽形大耳大耳小耳小耳
    20樽形大耳后期大耳小耳小耳
    注:A~E组分别表示牟氏角毛藻液与海洋红酵母稀释液按照4∶0、3∶1、2∶2、1∶3和0∶4的体积比混合;发育状态取1/3以上个体达到的发育阶段进行描述。下同
    Notes: groups A-E indicate the C. muelleri and Rhodotorula sp. are mixed in 4∶0, 3∶1, 2∶2, 1∶3 and 0∶4, by volume; the larval stage is described by more than 1/3 of the individuals develop to this stage. The same below

    Table 1.  Temporal variations in development of the planktonic larvae of H. leucospilota at different diet treatments

    Figure 1.  Temporal variations in development of the planktonic larvae of H. leucospilota at different diet treatments

  • 各处理组玉足海参浮游幼体体长随时间的变化如图2所示。实验开始后,A、B、C组幼体均以较快的速率生长,其中A组幼体生长最快,至第8天时该组幼体体长已达到(698.67±119.82) μm;而D和E组幼体生长则非常缓慢,特别是E组幼体体长在整个实验过程中一直表现为负增长。除第4天外,同期各组幼体体长之间均存在显著差异(P<0.05),其中A和B组体长均显著高于D和E组。第16天以后A、B两组幼体体长减小是由于部分耳状幼体发育为樽形幼体,而其他各组体长减小则是由于发育不良造成的(图1)。总体来说,饵料中牟氏角毛藻的比例越高,玉足海参浮游幼体生长越快,反之,饵料中海洋红酵母的比例越高,则幼体生长越慢。因此,投喂海洋红酵母不利于玉足海参浮游幼体的生长。

    Figure 2.  Temporal variations in body length of the planktonic larvae of H. leucospilota at different diet treatments

  • 饵料对各时期玉足海参浮游幼体的成活率均有显著影响(P<0.05) (图3)。实验开始后,各组幼体成活率均随着时间的延长而下降,其中只投喂牟氏角毛藻的A组下降幅度最小,实验结束时该组幼体成活率为58.73%±2.75%。由于A组幼体发育较快,实验后期部分樽形幼体可能已经附着而未进行计数,因此A组幼体实际成活率应该会更高一些。相比之下,随着饵料中海洋红酵母比例增加,幼体成活率下降幅度增大,实验结束时D和E组幼体成活率均显著低于A、B、C组(P<0.05),其中E组幼体成活率仅为22.22%±5.50%,尚不及A、B、C组的1/2。对于同一取样时期,A组幼体成活率均显著高于E组(P<0.05)。实验后期E组幼体的成活率多低于同期其他组。总体来说,饵料中牟氏角毛藻的比例越高,玉足海参浮游幼体死亡率越低,反之,饵料中海洋红酵母的比例越高,则幼体死亡率越高。因此,投喂海洋红酵母不利于玉足海参浮游幼体的存活。

    Figure 3.  Temporal variations in survival rate of the planktonic larvae of H. leucospilota at different diet treatments

3.   讨论
  • 对海参进行人工繁育和养殖是维持海参资源可持续开发的重要途经[11-14]。我国仿刺参(Apostichopus japonicus)人工繁育起步较早,经过几十年的发展,仿刺参的人工育苗技术已经十分成熟。仿刺参浮游幼体阶段通常利用牟氏角毛藻、杜氏盐藻(Dunaliella salina)、小新月菱形藻(Nitzschia closterium f. minutissima)以及湛江等鞭金藻 (Isochrysis zhanjiangensis)等单胞藻作为饵料[16]。海洋红酵母富含蛋白质、不饱和脂肪酸和虾青素等,具有较高的营养价值,在单胞藻饵料中适量添加海洋红酵母作为替代饵料,可以增强仿刺参浮游幼体肠道消化酶活性,加速幼体的生长与发育,还可以抑制水体中有害细菌的滋生[17, 21-22]。近年来,一些不具备单胞藻培养条件的育苗场完全利用海洋红酵母和食用酵母等替代饵料投喂仿刺参浮游幼体,也取得了很好的效果[16, 23]。研究表明,将海洋红酵母与单胞藻混合投喂能提高仿刺参浮游幼体的生长率,而单独投喂海洋红酵母的幼体变态成活率要显著高于投喂单胞藻[17]。当然,海洋红酵母并不适合所有水产动物的幼体,如在饵料中添加海洋红酵母就不利于长肋日月贝(Amusium pleuronectes)浮游幼虫的生长与存活[15]。本研究表明,随着饵料中海洋红酵母比例的增加,玉足海参浮游幼体的生长发育及成活率均受到不利影响,这直接表明海洋红酵母并不适合作为替代饵料用于玉足海参浮游幼体培育。

    目前,玉足海参的人工繁育尚处于起步阶段,相关的技术还不成熟,培育效果也不稳定。如Dabbagh等[24]的研究表明,利用牟氏角毛藻、钙质角毛藻(C. calcitrans)以及扁藻(Tetraselmis sp.)作为饵料投喂玉足海参浮游幼体,在水温为29°C,盐度为40,培养密度为0.15个/mL条件下,幼体可以在受精后第22~27天发育为樽形幼体,最终有4.2%的幼体能够发育为五触手幼体。近年来,我国学者经过努力,进一步提高了玉足海参浮游幼体的发育速率和附着效率,如Huang等[14]的研究表明,利用牟氏角毛藻和面包酵母(Saccharomyces cerevisiae)混合投喂玉足海参浮游幼体效果更好,在水温为29~33 °C,盐度为27~30,培养密度为0.2~0.5个/mL条件下,幼体可以在受精后第17~20天发育为樽形幼体,该阶段幼体成活率为54.2%,最终有8.5%的幼体能够发育为五触手幼体。本研究发现,只投喂角毛藻的A组玉足海参浮游幼体发育最快,该组幼体可以在受精后第20天左右发育为樽形幼体,其发育速率优于混合投喂牟氏角毛藻、钙质角毛藻和扁藻的幼体[24],但是不如混合投喂牟氏角毛藻和面包酵母的幼体[14]。本研究中A组玉足海参浮游幼体发育至樽形幼体阶段其成活率为58.73%±2.75%,该值要优于Huang等[14]的研究结果,但是大耳幼体和大耳幼体后期阶段体长却相对较小,究其原因可能是幼体培养密度过大,投喂强度较低造成的;或者是由于测量间隔时间较长,错过了个体较大的发育阶段—玉足海参大耳幼体后期向樽形幼体转变阶段,体长会急剧减小[14]。另外,盐度和水温等环境因素也会对海参浮游幼体的生长和发育造成影响,如高盐度会降低玉足海参浮游幼体的生长速率[24]。本研究中玉足海参浮游幼体培育水温与盐度等条件与已有研究存在较大的差别,这可能是造成幼体生长发育存在差异的原因。

    球状体可以为海参浮游幼体提供浮力,并为其变态提供营养,海参浮游幼体能否形成球状体是育苗成功与否的关键[14, 25]。本研究中投喂角毛藻比例较高的A、B组玉足海参浮游幼体球状体较明显,据此可以推断这两组幼体更容易变态附着。投喂海洋红酵母比例较高的C、D、E组幼体发育速率缓慢,球状体不明显或者缺失,据此可以推断这3组幼体变态附着的希望十分渺茫。未来需要进一步加强玉足海参繁殖生物学等基本内容的研究,并探讨单胞藻的选择与搭配以及面包酵母与螺旋藻粉等替代饵料在玉足海参浮游幼体培育中的应用。

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