Name: cultivation of the marine pelagic tunicate dolioletta gegenbauri (uljanin 1884) for experimental studies
Uploaded: 2019-08-09T14:30:00
Description: Watch this Scientific Journal Video about Cultivation of the Marine Pelagic Tunicate Dolioletta gegenbauri Uljanin 1884 for Experimental Studies at JoVE.com

我们感谢多年来为这个项目贡献积累的知识的许多人,包括G.A.帕芬霍夫和D.迪贝尔最初开发了这些协议。M. Köster和L.兰博利也为这些程序的发展作出了重大贡献。 N.B. 洛佩斯-菲格罗亚和罗德里格斯-圣地亚哥提出了表1中提供的多利奥利德丰度估计数。这项研究部分得到了美国国家科学基金会授予OCE 082599、1031263给MEF的合作项目、OCE 1459293和OCE 14595010对MEF和DMG的合作项目的支持,以及国家海洋和大气管理局授予DMGNA16SEC4810007的合作项目。我们感谢R/V萨凡纳号辛勤工作和专业船员。李·安·德利奥准备了数字,查尔斯·罗伯逊校对手稿,詹姆斯(吉米)威廉姆斯制造了浮游生物轮

1. 为饲养D.gegenbauri准备养殖设施
注:所有所需的材料和设备都列在材料表中。
<ol><li>制备1M氢氧化钠(NaOH),0.06M高锰酸钾(KMnO4)溶液。为了制备此溶液,将 400 克 NaOH 溶解到 10 L 去离子水中。将 100 g KMnO4添加到 NaOH 解决方案中,并混合均匀。</li>
	<li>通过将 100 g 的 NaHSO3溶解成 10 L 去离子水并混合良好,制备 0.1M 双硫酸钠 (NaHSO3) 溶液...

<ol>
	<li>Banse, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zooplankton+&#8211;+Pivotal+role+in+the+control+of+ocean+production.">Zooplankton &#8211; Pivotal role in the control of ocean production.</a> Ices Journal of Marine Science. 52 (3-4), 265-277 (1995).</li><li>Wilson, S. E., Ruhl, H. A., Smith, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zooplankton+fecal+pellet+flux+in+the+abyssal+northeast+Pacific:+A+15+year+time-series+study.">Zooplankton fecal pellet flux in the abyssal northeast Pacific: A 15 year time-series study.</a> Limnology and Oceanography. 58 (3), 881-892 (2013).</li><li>Bode, A., &#193;lvarez-Ossorio, M. T., Miranda, A., Ruiz-Villarreal, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shifts+between+gelatinous+and+crustacean+plankton+in+a+coastal+upwelling+region.">Shifts between gelatinous and crustacean plankton in a coastal upwelling region.</a> Ices Journal of Marine Science. 70 (5), 934-942 (2013).</li><li>Kiorboe, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zooplankton+body+composition.">Zooplankton body composition.</a> Limnology and Oceanography. 58 (5), 1843-1850 (2013).</li><li>Holland, L. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tunicates.">Tunicates.</a> Current Biology. 26 (4), R146-R152 (2016).</li><li>Walters, T. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diet+and+trophic+interactions+of+a+circumglobally+significant+gelatinous+marine+zooplankter,+Dolioletta+gegenbauri+(Uljanin,+1884).">Diet and trophic interactions of a circumglobally significant gelatinous marine zooplankter, Dolioletta gegenbauri (Uljanin, 1884).</a> Molecular Ecology. , (2018).</li><li>Piette, J., Lemaire, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thaliaceans,+the+neglected+pelagic+relatives+of+ascidians:+a+developmental+and+evolutionay+enigma.">Thaliaceans, the neglected pelagic relatives of ascidians: a developmental and evolutionay enigma.</a> Quarterly Review of Biology. 90 (2), 117-145 (2015).</li><li>Acu&#241;a, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pelagic+tunicates:+Why+gelatinous?.">Pelagic tunicates: Why gelatinous?.</a> American Naturalist. 158 (1), 100-107 (2001).</li><li>Alldredge, A. L., Madin, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pelagic+tunicates+&#8211;+unique+herivores+in+the+marine+plankton.">Pelagic tunicates &#8211; unique herivores in the marine plankton.</a> Bioscience. 32 (8), 655-663 (1982).</li><li>Wada, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+history+of+free-swimming+and+sessile+lifestyles+in+urochordates+as+deduced+from+18S+rDNA+molecular+phylogeny.">Evolutionary history of free-swimming and sessile lifestyles in urochordates as deduced from 18S rDNA molecular phylogeny.</a> Molecular Biology and Evolution. 15 (9), 1189-1194 (1998).</li><li>Zeng, L. Y., Jacobs, M. W., Swalla, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coloniality+has+evolved+once+in+stolidobranch+ascidians.">Coloniality has evolved once in stolidobranch ascidians.</a> Integrative and Comparative Biology. 46 (3), 255-268 (2006).</li><li>Delsuc, F., Brinkmann, H., Chourrout, D., Philippe, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tunicates+and+not+cephalochordates+are+the+closest+living+relatives+of+vertebrates.">Tunicates and not cephalochordates are the closest living relatives of vertebrates.</a> Nature. 439 (7079), 965-968 (2006).</li><li>Garstang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+morphology+of+the+Tunicata,+and+its+bearing+on+the+phylogeny+of+the+chordata.">The morphology of the Tunicata, and its bearing on the phylogeny of the chordata.</a> Quarterly Journal of Microscopical Science. 72 (285), 51-187 (1929).</li><li>Govindarajan, A. F., Bucklin, A., Madin, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+molecular+phylogeny+of+the+Thaliacea.">A molecular phylogeny of the Thaliacea.</a> Journal of Plankton Research. 33 (6), 843-853 (2011).</li><li>Godeaux, D., Bone, Q., Braconnot, J. C., Bone, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Biology of Pelagic Tunicates. , 1-24 (1998).</li><li>Deibel, D., Lowen, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+the+life+cycles+and+life-history+adaptations+of+pelagic+tunicates+to+environmental+conditions).">A review of the life cycles and life-history adaptations of pelagic tunicates to environmental conditions).</a> Ices Journal of Marine Science. 69 (3), 358369 (2012).</li><li>Paffenh&#246;fer, G., K&#246;ster, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+one+to+many:+on+the+life+cycle+of+Dolioletta+gegenbauri+Uljanin+(Tunicata,+Thaliacea).">From one to many: on the life cycle of Dolioletta gegenbauri Uljanin (Tunicata, Thaliacea).</a> Journal of Plankton Research. 33 (7), 1139-1145 (2011).</li><li>Paffenh&#246;fer, G. A., Gibson, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+generation+time+and+asexual+fecundity+of+doliolids+(Tunicata,+Thaliacea).">Determination of generation time and asexual fecundity of doliolids (Tunicata, Thaliacea).</a> Journal of Plankton Research. 21 (6), 1183-1189 (1999).</li><li>Conley, K. R., Lombard, F., Sutherland, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammoth+grazers+on+the+ocean's+minuteness:+a+review+of+selective+feeding+using+mucous+meshes.">Mammoth grazers on the ocean's minuteness: a review of selective feeding using mucous meshes.</a> Proceedings of the Royal Society B-Biological Sciences. 285 (1878), (2018).</li><li>Bouquet, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+optimization+for+the+emergent+zooplanktonic+model+organism+Oikopleura+dioica.">Culture optimization for the emergent zooplanktonic model organism Oikopleura dioica.</a> Journal of Plankton Research. 31 (4), 359370 (2009).</li><li>Denoeud, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasticity+of+Animal+Genome+Architecture+Unmasked+by+Rapid+Evolution+of+a+Pelagic+Tunicate.">Plasticity of Animal Genome Architecture Unmasked by Rapid Evolution of a Pelagic Tunicate.</a> Science. 330 (6009), 1381-1385 (2010).</li><li>Harrison, P. J., Waters, R. E., Taylor, F. J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+broad-spectrum+artificial+seawater+medium+for+coastal+and+open+ocean+phytoplankton.">A broad-spectrum artificial seawater medium for coastal and open ocean phytoplankton.</a> Journal of Phycology. 16 (1), 2835 (1980).</li><li>Ohman, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zooglider:+An+autonomous+vehicle+for+optical+and+acoustic+sensing+of+zooplankton.">Zooglider: An autonomous vehicle for optical and acoustic sensing of zooplankton.</a> Limnology and Oceanography-Methods. 17 (1), 69-86 (2019).</li><li>Takahashi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+observations+of+a+doliolid+bloom+in+a+warm+water+filament+using+a+video+plankton+recorder:+Bloom+development,+fate,+and+effect+on+biogeochemical+cycles+and+planktonic+food+webs.">In situ observations of a doliolid bloom in a warm water filament using a video plankton recorder: Bloom development, fate, and effect on biogeochemical cycles and planktonic food webs.</a> Limnology and Oceanography. 60 (5), 1763-1780 (2015).</li><li>Deibel, D., Bone, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The biology of pelagic tunicates. , 171-186 (1998).</li><li>Gibson, D. M., Paffenh&#246;fer, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feeding+and+growth+rates+of+the+doliolid,+Dolioletta+gegenbauri+Uljanin+(Tunicata,+Thaliacea).">Feeding and growth rates of the doliolid, Dolioletta gegenbauri Uljanin (Tunicata, Thaliacea).</a> Journal of Plankton Research. 22 (8), 1485-1500 (2000).</li><li>Haskell, A., Hofmann, E., Paffenh&#246;fer, G., Verity, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+the+effects+of+doliolids+on+the+plankton+community+structure+of+the+southeastern+US+continental+shelf.">Modeling the effects of doliolids on the plankton community structure of the southeastern US continental shelf.</a> Journal of Plankton Research. 21 (9), 1725-1752 (1999).</li><li>Gibson, D. M., Paffenh&#246;ffer, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asexual+reproduction+of+the+doliolid,+Dolioletta+gegenbauri+Uljanin+(Tunicata,+Thaliacea).">Asexual reproduction of the doliolid, Dolioletta gegenbauri Uljanin (Tunicata, Thaliacea).</a> Journal of Plankton Research. 24 (7), 703-712 (2002).</li><li>Frischer, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reliability+of+qPCR+for+quantitative+gut+content+estimation+in+the+circumglobally+abundant+pelagic+tunicate+Dolioletta+gegenbauri+(Tunicata,+Thaliacea).">Reliability of qPCR for quantitative gut content estimation in the circumglobally abundant pelagic tunicate Dolioletta gegenbauri (Tunicata, Thaliacea).</a> Food Webs. 1, 7 (2014).</li><li>K&#246;ster, M., Paffenh&#246;fer, G., Baker, C., Williams, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen+consumption+of+doliolids+(Tunicata,+Thaliacea).">Oxygen consumption of doliolids (Tunicata, Thaliacea).</a> Journal of Plankton Research. 32 (2), 171-180 (2010).</li><li>Paffenh&#246;fer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+hypothesis+on+the+fate+of+blooms+of+doliolids+(Tunicata,+Thaliacea).">A hypothesis on the fate of blooms of doliolids (Tunicata, Thaliacea).</a> Journal of Plankton Research. 35 (4), 919-924 (2013).</li><li>Takahashi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sapphirinid+copepods+as+predators+of+doliolids:+Their+role+in+doliolid+mortality+and+sinking+flux.">Sapphirinid copepods as predators of doliolids: Their role in doliolid mortality and sinking flux.</a> Limnology and Oceanography. 58 (6), 1972-1984 (2013).</li><li>Mart&#237;-Solans, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oikopleura+dioica+Culturing+Made+Easy:+A+Low-Cost+Facility+for+an+Emerging+Animal+Model+in+EvoDevo.">Oikopleura dioica Culturing Made Easy: A Low-Cost Facility for an Emerging Animal Model in EvoDevo.</a> Genesis. 53 (1), 183-193 (2015).</li><li>Nishida, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+appendicularian+Oikopleura+dioica:+Culture,+genome,+and+cell+lineages.">Development of the appendicularian Oikopleura dioica: Culture, genome, and cell lineages.</a> Development Growth &amp; Differentiation. 50, S239-S256 (2008).</li></ol>

在过去几十年中,已经建立了培养多利利德的能力,并被用于支持若干领域的研究。我们实验室的实验研究支持了至少15项科学研究的出版,这些研究侧重于喂养和生长18,26,繁殖18,28,饮食6, 29,生理学30,生态学31,生态模型27多利奥利德。...

浮游生物是海洋中最大的动物生物量,是海洋食物网中的关键成分,在海洋生物地球化学周期1、2中发挥重要作用。浮游动物,虽然由巨大的生物多样性组成,可以完全区分为两类:胶状和非胶状,很少中间分类3,4。与非胶状浮游动物相比,胶状浮游动物由于生命历史复杂,在捕获和处理过程中容易受损,因此特别难以研究。因此,以实验室中难以养殖为名,与非胶状物种6相比,研究较少。
在胶状浮游动物群中,一个在世界海洋中具有丰富和生态重要性的植物群是塔利亚人。塔利亚人是一类中上层的突子,包括萨尔皮达,火焰兵米达和多利奥利达7号订单。多利奥利达,统称为多利奥利德,是小型桶形自由游泳的中上层生物,可以在亚热带海洋的生产性神经区达到高丰度。多利奥利德是所有浮游动物群中最丰富的,有4、8个。作为悬浮喂食器,多利奥利德通过产生滤流并在粘液网9上捕获食物颗粒,从水柱中收集食物颗粒。分类学上,多利奥利德被分类在植物乌罗霍达10。与和弦的祖先,除了其生态意义作为海洋中上层系统的关键组成部分,塔利亚人是了解殖民生命历史的起源10,11和进化的意义和弦5,7,10,12,13,14。
多利奥利德的寿命是复杂的,并造成在它们生命周期中培养和维持它们的困难。多利奥利德生命周期和解剖学的回顾可以在哥德奥等人15。多利奥利德生命周期 , 涉及性和非性生命史阶段的强制替 , 如图 1 所示。卵子和精子是由她的血性淋病产生,这是生命周期中唯一的单独阶段。Gonozooid将精子释放到水柱中,卵子在内部受精并释放成幼虫。幼虫孵化和变质成可达到1-2毫米的卵虫。 假定有利的环境条件和营养,在20°C的1-2天内成为早期护士,并启动生命周期的殖民阶段。无性性地在腹腔上产生芽。这些芽离开龙,并迁移到背毛,在那里他们排队成三对成行。中央双行变为双排,外两排变为节流。后者为护士和16、17的食虫提供食物。营养动物为护士提供营养,因为她失去了所有的内部器官。随着营养动物的丰度增加,护士在实验室中的大小可以达到15毫米。随着磷化物的生长,它们越来越多地摄入浮游动物,在作为个体17被释放之前,其大小达到1.5毫米。一名护士在其寿命18期间可释放>100个磷。在从结虫中释放后,它们继续生长,是生命周期的第二殖民阶段。一旦它们达到5毫米的大小,每个磷化物在其腹腔的足刺上形成一簇淋杂虫。这些淋病在长度达到±1毫米时可以摄录颗粒。在淋病达到2至3毫米的大小后,它们从磷化物中释放出来,成为生命周期中唯一的单独阶段。一旦他们达到 + 6 毫米的大小, 淋病成为性成熟17.淋病长度可达9毫米或更大。果子病是她的造血剂,精子间歇性地释放,而受精的卵子发生在内部16,17。当淋病大小为6毫米时,它释放多达6个受精卵。成功的培养需要支持这些独特的生命历史阶段的具体需求。
由于塔利亚人(包括多利奥利德)的生态和进化意义,有必要采用栽培方法,以增进对这种生物体独特的生物学、生理学、生态学和进化史的理解19.多利奥利德在发育生物学和功能基因组学中作为实验模型生物体具有相当大的前景,因为它们是透明的,并且可能具有简化的基因组20,21。然而,缺乏可靠的栽培方法妨碍了它们作为实验室模型的作用。虽然少数实验室已经发布了基于培养多利利德的结果,但对我们的知识培养方法和详细协议,以前没有发表过。根据多年的经验和试验和错误栽培尝试,本研究的目的是审查经验,并分享协议,收集和栽培多利奥利德,特别是多利奥莱塔gegenbauri物种。

<table border="0" cellpadding="0" cellspacing="0" style="width:1860px;" width="1860">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:681px;">Algal culture tubes (55 mL sterile disposable glass culture tubes)</td>
			<td style="width:417px;">Any</td>
			<td style="width:167px;">NA</td>
			<td style="width:595px;">For algal cultures</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:681px;">Autoclave</td>
			<td style="width:417px;">Any</td>
			<td style="width:167px;">NA</td>
			<td>For sterilizing equipment and seawater for algal cultures</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:681px;">Beakers (2 L glass)</td>
			<td style="width:417px;">Any</td>
			<td style="width:167px;">NA</td>
			<td>For sorting diluted plankton net tow contents</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:681px;">Buckets (5 gallon, ~20L)</td>
			<td style="width:417px;">Any</td>
			<td style="width:167px;">NA</td>
			<td>For diluting contents of planton net tow - should be seawater conditioned before first use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:681px;">Carboys (20 L)&#160;</td>
			<td style="width:417px;">Any</td>
			<td style="width:167px;">NA</td>
			<td>For storing seawater</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Doliolid glass culturing jar (1.9 L narrow mouth glass jar with cap)</td>
			<td>Qorpak</td>
			<td>GLC-01882</td>
			<td>Container for culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Doliolid glass culturing jar (3.8 L narrow mouth glass jar with cap)</td>
			<td>Qorpak</td>
			<td>GLC-01858</td>
			<td>Container for culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:681px;">Environmental Chamber (Temperature controlled enviromental chamber)</td>
			<td style="width:417px;">Any</td>
			<td style="width:167px;">NA</td>
			<td>To accommodate plankton wheel and culture maintenance</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:681px;">Filtration apparatus for 47 mm filters</td>
			<td style="width:417px;">Any</td>
			<td style="width:167px;">NA</td>
			<td>For filtering seawater for cultures</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass microfiber filters, 47 mm</td>
			<td>Whatman</td>
			<td>1825-047</td>
			<td>For filtering seawater for cultures</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass pipette (borosillicate glass pipette (glass tubing), OD 10mm, ID 8 mm, wall thickness 1mm)</td>
			<td>Science Company</td>
			<td>NC-10894</td>
			<td>Custom cut and edges polished</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:681px;">Hose clamps, stainless steel, #104 (178 mm)</td>
			<td style="width:417px;">Any</td>
			<td style="width:167px;">NA</td>
			<td>For holding culturing jars to the plankton wheel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isochrysis galbana strain CCMP1323</td>
			<td>National Center for Marine Algae and Microbiota (NCMA)</td>
			<td>strain CCMP1323</td>
			<td>For feeding doliolid cultures</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L1 Media Kit, 50 L</td>
			<td>National Center for Marine Algae and Microbiota (NCMA)</td>
			<td>MKL150L</td>
			<td>For culturing algae</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:681px;">Lamp (Fluorescent table lamp with an adjustable arm)</td>
			<td style="width:417px;">Any</td>
			<td style="width:167px;">NA</td>
			<td>For illuminating doliolids in the jars and beakers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:681px;">Lighted temperature controlled incubator</td>
			<td style="width:417px;">Any</td>
			<td style="width:167px;">NA</td>
			<td>For algal cultures</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:681px;">Micropipettes and sterile tips (0-20 &#181;l, 20-200 &#181;l, 200-1000&#160;&#181;l)</td>
			<td style="width:417px;">Any</td>
			<td style="width:167px;">NA</td>
			<td>For algal cultures</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plankton Net (202 &#181;m 0.5 m, 5:1 length) with cod end ring and &#160;4 L aquarium cod-end</td>
			<td>Sea-Gear Corporation</td>
			<td>90-50x5-200-4A/BB</td>
			<td>For collecting living doliolids (see Figure 4)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:681px;">Plankton Wheel</td>
			<td style="width:417px;">NA</td>
			<td style="width:167px;">NA</td>
			<td>Custom built (see Figure 2)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plastic wrap</td>
			<td>Any</td>
			<td>NA</td>
			<td>To cover inside of lid of doliolid culture jars</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium Permanganate</td>
			<td>Fisher Scientific</td>
			<td>P279-500</td>
			<td>Reagent for cleaning jars and glassware</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rhodomonas&#160;sp. strain CCMP740</td>
			<td>National Center for Marine Algae and Microbiota (NCMA)</td>
			<td>strain CCMP740</td>
			<td>For feeding doliolid cultures</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:681px;">Rubber Tubing</td>
			<td style="width:417px;">NA</td>
			<td style="width:167px;">NA</td>
			<td>For holding culturing jars to the plankton wheel (can be made from tygon tubing)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Bisulfite</td>
			<td>Fisher Scientific</td>
			<td>S654-500</td>
			<td>Reagent for cleaning jars and glassware</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Hydroxide</td>
			<td>Fisher Scientific</td>
			<td>BP359-212</td>
			<td>Reagent for cleaning jars and glassware</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:681px;">Sterile serological pipettes (1 mL, 5 mL, 10 mL, 25 mL)</td>
			<td style="width:417px;">Any</td>
			<td style="width:167px;">NA</td>
			<td>For algal cultures</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thalassiosira weissflogii strain CCMP1051</td>
			<td>National Center for Marine Algae and Microbiota (NCMA)</td>
			<td>strain CCMP1051</td>
			<td>For feeding doliolid cultures</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:681px;">Tissue culture flasks (250 mL)</td>
			<td style="width:417px;">Any</td>
			<td style="width:167px;">NA</td>
			<td>For algal cultures</td>
		</tr>
	</tbody>
</table>

cultivation of the marine pelagic tunicate dolioletta gegenbauri (uljanin 1884) for experimental studies

胶状动物在海洋生态系统中起着至关重要的作用。然而,通常很难研究它们的生理、生长、肥力和营养性相互作用,这主要是由于方法上的挑战,包括培养它们的能力。多利奥利利德,多利奥莱塔·格根本古里尤其如此。D. Gegenbauri通常发生在全世界生产性的亚热带大陆架系统中,其浓度往往能消耗大量日常初级生产。在这项研究中,我们描述了收集、饲养和维护D.Gegenbauri的培养方法,以便进行基于实验室的研究。D. Gegenbauri和其他多利奥利物种可以通过从漂流船上斜拖锥形202 μm网状浮游生物网进行实时捕获。当水温低于21°C,并且从不成熟的淋病、成熟的红细胞和大型护士开始时,培养是最可靠的。培养物可以在缓慢旋转的浮游生物轮上保持在圆形培养容器中,并在天然海水中养殖藻类的饮食中维持多年。除了建立D.gegenbauri实验室培养物的能力外,我们还证明了采集条件、藻类浓度、温度和暴露于自然条件海水对培养物都至关重要。建立,成长,生存和繁殖的D.gegenbauri。

按照图3中概述的收集、培育多利奥利的所述程序,可以在其复杂的生命史中保持D.gegenbauri文化(图1)和维持它许多代。虽然这里描述了D.gegenbauri的栽培,但这些程序也应与其他多利利德物种的种植相关。
捕获健康和未受损的多利奥利德动物需要应用专用网和牵引程序(图4)。作为没有硬结构的?...

Watch this Scientific Journal Video about Cultivation of the Marine Pelagic Tunicate Dolioletta gegenbauri Uljanin 1884 for Experimental Studies at JoVE.com

Cultivation of the Marine Pelagic Tunicate Dolioletta gegenbauri Uljanin 1884 for Experimental Studies

用于实验研究的海洋可点多利奥莱塔·格根巴里(Uljanin 1884)的培养

多利奥利德,包括多利奥莱塔gegenbauri物种,是在世界生产次大陆架系统中发现的具有生态意义的小型胶状海洋浮游动物。培养这些微妙生物的难度限制了他们的调查。在这项研究中,我们描述了收集、饲养和维护多利奥利莱塔·格根巴里的栽培方法。

Gelatinous zooplanktons play a crucial role in ocean ecosystems. However, it is generally difficult to investigate their physiology, growth, fecundity, ...

可培养模型生物的可得性是许多研究领域发展的根本。据我们所知，该协议是培养多利利德的唯一方法。可靠的多利奥利德养殖技术提供了一个独特的实验模型，用于研究对海洋一些生产力最高的地区非常重要，并且代表了脊椎动物最早的进化祖先。这种技术是独一无二的，因为它保持藻类食物颗粒和悬浮在水柱中的有机体，而不会与容器的墙壁接触，因为它们在自然界中体验。这种栽培方法可应用于其他小明胶和甲壳类或万有生物的栽培。由于多利利德具有复杂的生命周期，因此了解和认识到每个生命阶段的需求至关重要。学习生命周期以及如何识别和处理它们需要大量的实践。根据我们的经验，学习这种栽培技术的唯一方法就是看到它并做到这一点。其他几个项目参与者，包括艾琳·阿内森、尼古拉斯·卡斯塔琳、娜塔莉亚·洛佩兹、劳伦·兰博耶、布瑞娜·皮尔斯、艾莉亚·罗德里格斯-圣地亚哥和特雷尔·斯卡伯勒也出现在这段视频中。在实验室中建立和培养多利利德培养物之前，通过浸入氢氧化钾高锰酸钠溶液中，清洁和消毒 1.9 升和 3.8 升玻璃培养罐。让罐子浸泡过夜。然后将罐子转移到双硫酸钠溶液中。让罐子浸泡过夜。接下来用去维水彻底冲洗罐子。让罐子晾干。为了保持库存文化，使用严格的斧头文化技术。每两周将0.5毫升的旧衰老培养剂转移到无菌55毫升玻璃培养管中的25毫升新鲜生长培养剂中。为了准备大量浮游植物来喂养多利利兹，请将四毫升浮游植物从轴类库存转移到含有 200 毫升生长介质的 500 毫升塑料组织培养瓶中，以 1 至 50 稀释进行接种。在20摄氏度的环境孵化器或步入式环境室内孵化养殖，在每平方米65至85微微的冷白光照明下，有12至12小时的光暗循环。平整培养瓶，以最大限度地提高照明效果。每天轻轻旋转文化。出海去。为了在大陆架上定位多利利叶，部署海洋学仪器CTD，通过分析水温、盐度和叶绿素甲荧光，确定有利于多利利的存在的水条件。使用专门的木板网进行探索性网托，确认水条件。或者，使用原位分析成像系统来检测多利利德。现在操作车机以检索 CTD。安装在CTD玫瑰花环上的尼斯金瓶从多利奥利德的地点和含有叶绿素最高估计值的深度收集海水。用海水冲洗三个 22 升桶，然后用海水填充半点多一点。用海水将鳕鱼网端完全填满，将鳕鱼端附在网中。部署网络。通过水柱降低并提升网，保持15至25度的倾斜牵引角度，垂直部署和取回速度不超过每分钟15米。一旦网在船上轻轻转移，并分裂鳕鱼端的内容到塑料桶，每个含有从现场收集的地表海水。使用宽孔玻璃移液器，使用垂直和水平运动将烧杯的内容与移液器轻轻地混合。使用食指，创建毛细管吸力来捕获和转移多利利德。从烧杯中的混合浮游植物中捕获多利利德。加入多利奥利兹后，将罗多蒙纳培养物加入罐中，最终浓度约为每毫升的约5倍10至第三至10至第四细胞。检查多利利德消化道的颜色。红色决定了多利利德正在积极喂食。为了防止多利利德被困在空气-水界面，完全填充罐子与未经过滤的，富含颗粒的海水，以避免头部空间。将一块塑料包裹放在罐子开口上。避免产生气泡，以免损害动物。小心地将盖子拧到罐子上，轻轻反转罐子，以确定是否存在气泡。如果存在气泡，请填充更多的海水以清除它们。装满罐子后，用盖子重新密封，从罐子外擦去多余的水。将罐子放在用橡胶管覆盖的垂直金属棒上，在不锈钢软管夹之间，将每个罐子安装到木板轮上。确保罐的背面靠橡胶管缓冲。调整螺钉以拧紧罐周围的软管夹。让罐以 0.3 rpm 转速旋转，以保持多利利德处于悬架状态。三天后，适应实验室条件后，将100至150毫升的培养水从罐子转移到样品容器中，并使用库尔特计数器测量藻类浓度。根据藻类浓度的估计，加入足够的藻类，使培养罐中的浓度为每升40至95微克碳。使用颗粒计数器监测多利利德喂养后藻类浓度，以指导向培养物添加藻类的频率和程度。保持培养罐中浮游植物浓度在每升40至95微克碳之间。培养多利利德需要支持他们度过复杂的生命周期。发展幼虫和卵巢和早期护士。一旦在护士的阴极上至少看到八个三磷酮，至少将四名护士转移到一个新的培养罐中。一旦磷类动物达到三毫米的大小，减少罐子里的动物数量。当磷类体大于五毫米并发展出戈诺佐类星团时，除四个磷类体而去掉所有类星体。该协议概述了多利奥莱塔·格根鲍里收集和栽培方法。藻类清除率与每升20微克碳的浓度相似，随着食物浓度的增加而降低。在支持 D.gegenbauri 生长的温度范围内，清除率按比例增加。观察到，由于温度和食物供应情况，每天的生长量从0.1毫米到0.7毫米不等。多利奥利德的栽培取决于能否成功复制海洋条件，并满足动物在其复杂的生命周期中的需求。因此，在动物捕获、隔离和饲养过程中，需要对动物进行温和的处理。必须经常监测和调整食物浓度。这在幼虫和卵母体生命阶段尤其如此。这种方法是专门为在海洋食物网底部饲养小浮游动物物种而设计的。这种方法对于研究浮游动物对气候变化的反应将很有价值。自开发以来，这项技术为浮游动物生态学家确定多利利德的实验室喂养、生长和繁殖率铺平了道路。此外，这种方法为革命性的分子研究多利利德饮食的性质铺平了道路。我想提醒你，玻璃清洗协议涉及试剂，呼吸刺激物，应该用于通风良好的区域。在海上工作也可能很危险，因此请遵守船舶安全规则。

Research

JoVE Journal

Environment

Cultivation of the Marine Pelagic Tunicate Dolioletta gegenbauri (Uljanin 1884) for Experimental Studies

A Noninvasive Method For In situ Determination of Mating Success in Female American Lobsters (Homarus americanus)

A Noninvasive Method For In situ Determination of Mating Success in Female American Lobsters Homarus americanus

一种非侵入性的方法<em&gt;原位</em&gt;交配成功的女美国龙虾​​（测定<em&gt;美洲螯龙虾</em&gt;）

Despite being one of the most productive fisheries in the Northwest Atlantic, much remains unknown about the natural reproductive dynamics of American ...

科研

环境科学

A Fish-feeding Laboratory Bioassay to Assess the Antipredatory Activity of Secondary Metabolites from the Tissues of Marine Organisms

了鱼喂食实验室生物测定，从海洋生物的组织评估次生代谢产物的Antipredatory活动

Marine chemical ecology is a young discipline, having emerged from the collaboration of natural products chemists and marine ecologists in the 1980s with ...

Clean Sampling and Analysis of River and Estuarine Waters for Trace Metal Studies

清洁取样和痕量金属研究河和河口水域分析

Most of the trace metal concentrations in ambient waters obtained a few decades ago have been considered unreliable owing to the lack of contamination ...

Experimental Protocol for Biodiesel Production with Isolation of Alkenones as Coproducts from Commercial Isochrysis Algal Biomass

Experimental Protocol for Biodiesel Production with Isolation of Alkenones as Coproducts from Commercial Isochrysis Algal Biomass

实验协议生产生物柴油与饱和酮的分离从商业余积<em&gt;等鞭金藻</em&gt;藻类生物量

The need to replace petroleum fuels with alternatives from renewable and more environmentally sustainable sources is of growing importance. ...

VacuSIP, an Improved InEx Method for In Situ Measurement of Particulate and Dissolved Compounds Processed by Active Suspension Feeders

VacuSIP, an Improved InEx Method for In Situ Measurement of Particulate and Dissolved Compounds Processed by Active Suspension Feeders

VacuSIP，对改进的方法INEX<em&gt;原位</em&gt;颗粒物与测量溶解的化合物加工后主动悬挂给料机

Benthic suspension feeders play essential roles in the functioning of marine ecosystems. By filtering large volumes of water, removing plankton and ...

Ecotoxicological Method with Marine Bacteria Vibrio anguillarum to Evaluate the Acute Toxicity of Environmental Contaminants

Ecotoxicological Method with Marine Bacteria Vibrio anguillarum to Evaluate the Acute Toxicity of Environmental Contaminants

生物毒理学方法与海洋细菌<em&gt;鳗弧菌</em&gt;评估环境污染物的急性毒性

Bacteria are an important component of the ecosystem, and microbial community alterations can have a significant effect on biogeochemical cycling and food ...

Vegetated Treatment Systems for Removing Contaminants Associated with Surface Water Toxicity in Agriculture and Urban Runoff

用于去除与农业和城市径流中地表水毒性有关的污染物的植被处理系统

Urban stormwater and agriculture irrigation runoff contain a complex mixture of contaminants that are often toxic to adjacent receiving waters. Runoff may ...

Extraction of Organochlorine Pesticides from Plastic Pellets and Plastic Type Analysis

从塑料颗粒中提取有机氯农药和塑料类型分析

Plastic resin pellets, categorized as microplastics (&#8804;5 mm in diameter), are small granules that can be unintentionally released to the environment ...

Development of New Methods for Quantifying Fish Density Using Underwater Stereo-video Tools

利用水下立体视频工具量化鱼类密度的新方法研究进展

The use of video camera systems in ecological studies of fish continues to gain traction as a viable, non-extractive method of measuring fish lengths and ...

Methods for Image-based Surveys of Benthic Macroinvertebrates and Their Habitat Exemplified by the Drop Camera Survey for the Atlantic Sea Scallop

基于图像的海底无脊椎动物及其栖息地调查方法大西洋扇贝落相机调查

Underwater imaging has long been used in the field of marine ecology but decreasing costs of high-resolution cameras and data storage have made the ...