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
Summary
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。
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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。…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
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
Materials
| Algal culture tubes (55 mL sterile disposable glass culture tubes) | Any | NA | For algal cultures |
| Autoclave | Any | NA | For sterilizing equipment and seawater for algal cultures |
| Beakers (2 L glass) | Any | NA | For sorting diluted plankton net tow contents |
| Buckets (5 gallon, ~20L) | Any | NA | For diluting contents of planton net tow – should be seawater conditioned before first use |
| Carboys (20 L) | Any | NA | For storing seawater |
| Doliolid glass culturing jar (1.9 L narrow mouth glass jar with cap) | Qorpak | GLC-01882 | Container for culture |
| Doliolid glass culturing jar (3.8 L narrow mouth glass jar with cap) | Qorpak | GLC-01858 | Container for culture |
| Environmental Chamber (Temperature controlled enviromental chamber) | Any | NA | To accommodate plankton wheel and culture maintenance |
| Filtration apparatus for 47 mm filters | Any | NA | For filtering seawater for cultures |
| Glass microfiber filters, 47 mm | Whatman | 1825-047 | For filtering seawater for cultures |
| Glass pipette (borosillicate glass pipette (glass tubing), OD 10mm, ID 8 mm, wall thickness 1mm) | Science Company | NC-10894 | Custom cut and edges polished |
| Hose clamps, stainless steel, #104 (178 mm) | Any | NA | For holding culturing jars to the plankton wheel |
| Isochrysis galbana strain CCMP1323 | National Center for Marine Algae and Microbiota (NCMA) | strain CCMP1323 | For feeding doliolid cultures |
| L1 Media Kit, 50 L | National Center for Marine Algae and Microbiota (NCMA) | MKL150L | For culturing algae |
| Lamp (Fluorescent table lamp with an adjustable arm) | Any | NA | For illuminating doliolids in the jars and beakers |
| Lighted temperature controlled incubator | Any | NA | For algal cultures |
| Micropipettes and sterile tips (0-20 µl, 20-200 µl, 200-1000 µl) | Any | NA | For algal cultures |
| Plankton Net (202 µm 0.5 m, 5:1 length) with cod end ring and 4 L aquarium cod-end | Sea-Gear Corporation | 90-50×5-200-4A/BB | For collecting living doliolids (see Figure 4) |
| Plankton Wheel | NA | NA | Custom built (see Figure 2) |
| Plastic wrap | Any | NA | To cover inside of lid of doliolid culture jars |
| Potassium Permanganate | Fisher Scientific | P279-500 | Reagent for cleaning jars and glassware |
| Rhodomonas sp. strain CCMP740 | National Center for Marine Algae and Microbiota (NCMA) | strain CCMP740 | For feeding doliolid cultures |
| Rubber Tubing | NA | NA | For holding culturing jars to the plankton wheel (can be made from tygon tubing) |
| Sodium Bisulfite | Fisher Scientific | S654-500 | Reagent for cleaning jars and glassware |
| Sodium Hydroxide | Fisher Scientific | BP359-212 | Reagent for cleaning jars and glassware |
| Sterile serological pipettes (1 mL, 5 mL, 10 mL, 25 mL) | Any | NA | For algal cultures |
| Thalassiosira weissflogii strain CCMP1051 | National Center for Marine Algae and Microbiota (NCMA) | strain CCMP1051 | For feeding doliolid cultures |
| Tissue culture flasks (250 mL) | Any | NA | For algal cultures |
Referencias
- Banse, K. Zooplankton – Pivotal role in the control of ocean production. Ices Journal of Marine Science. 52 (3-4), 265-277 (1995).
- Wilson, S. E., Ruhl, H. A., Smith, K. L. Zooplankton fecal pellet flux in the abyssal northeast Pacific: A 15 year time-series study. Limnology and Oceanography. 58 (3), 881-892 (2013).
- Bode, A., Álvarez-Ossorio, M. T., Miranda, A., Ruiz-Villarreal, M. Shifts between gelatinous and crustacean plankton in a coastal upwelling region. Ices Journal of Marine Science. 70 (5), 934-942 (2013).
- Kiorboe, T. Zooplankton body composition. Limnology and Oceanography. 58 (5), 1843-1850 (2013).
- Holland, L. Z. Tunicates. Current Biology. 26 (4), R146-R152 (2016).
- Walters, T. L., et al. Diet and trophic interactions of a circumglobally significant gelatinous marine zooplankter, Dolioletta gegenbauri (Uljanin, 1884). Molecular Ecology. , (2018).
- Piette, J., Lemaire, P. Thaliaceans, the neglected pelagic relatives of ascidians: a developmental and evolutionay enigma. Quarterly Review of Biology. 90 (2), 117-145 (2015).
- Acuña, J. L. Pelagic tunicates: Why gelatinous?. American Naturalist. 158 (1), 100-107 (2001).
- Alldredge, A. L., Madin, L. P. Pelagic tunicates – unique herivores in the marine plankton. Bioscience. 32 (8), 655-663 (1982).
- Wada, H. Evolutionary history of free-swimming and sessile lifestyles in urochordates as deduced from 18S rDNA molecular phylogeny. Molecular Biology and Evolution. 15 (9), 1189-1194 (1998).
- Zeng, L. Y., Jacobs, M. W., Swalla, B. J. Coloniality has evolved once in stolidobranch ascidians. Integrative and Comparative Biology. 46 (3), 255-268 (2006).
- Delsuc, F., Brinkmann, H., Chourrout, D., Philippe, H. Tunicates and not cephalochordates are the closest living relatives of vertebrates. Nature. 439 (7079), 965-968 (2006).
- Garstang, W. The morphology of the Tunicata, and its bearing on the phylogeny of the chordata. Quarterly Journal of Microscopical Science. 72 (285), 51-187 (1929).
- Govindarajan, A. F., Bucklin, A., Madin, L. P. A molecular phylogeny of the Thaliacea. Journal of Plankton Research. 33 (6), 843-853 (2011).
- Godeaux, D., Bone, Q., Braconnot, J. C., Bone, Q. . The Biology of Pelagic Tunicates. , 1-24 (1998).
- Deibel, D., Lowen, B. A review of the life cycles and life-history adaptations of pelagic tunicates to environmental conditions). Ices Journal of Marine Science. 69 (3), 358369 (2012).
- Paffenhöfer, G., Köster, M. From one to many: on the life cycle of Dolioletta gegenbauri Uljanin (Tunicata, Thaliacea). Journal of Plankton Research. 33 (7), 1139-1145 (2011).
- Paffenhöfer, G. A., Gibson, D. M. Determination of generation time and asexual fecundity of doliolids (Tunicata, Thaliacea). Journal of Plankton Research. 21 (6), 1183-1189 (1999).
- Conley, K. R., Lombard, F., Sutherland, K. R. Mammoth grazers on the ocean’s minuteness: a review of selective feeding using mucous meshes. Proceedings of the Royal Society B-Biological Sciences. 285 (1878), (2018).
- Bouquet, J. M., et al. Culture optimization for the emergent zooplanktonic model organism Oikopleura dioica. Journal of Plankton Research. 31 (4), 359370 (2009).
- Denoeud, F., et al. Plasticity of Animal Genome Architecture Unmasked by Rapid Evolution of a Pelagic Tunicate. Science. 330 (6009), 1381-1385 (2010).
- Harrison, P. J., Waters, R. E., Taylor, F. J. R. A broad-spectrum artificial seawater medium for coastal and open ocean phytoplankton. Journal of Phycology. 16 (1), 2835 (1980).
- Ohman, M. D., et al. Zooglider: An autonomous vehicle for optical and acoustic sensing of zooplankton. Limnology and Oceanography-Methods. 17 (1), 69-86 (2019).
- Takahashi, K., et al. 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. Limnology and Oceanography. 60 (5), 1763-1780 (2015).
- Deibel, D., Bone, Q. . The biology of pelagic tunicates. , 171-186 (1998).
- Gibson, D. M., Paffenhöfer, G. A. Feeding and growth rates of the doliolid, Dolioletta gegenbauri Uljanin (Tunicata, Thaliacea). Journal of Plankton Research. 22 (8), 1485-1500 (2000).
- Haskell, A., Hofmann, E., Paffenhöfer, G., Verity, P. Modeling the effects of doliolids on the plankton community structure of the southeastern US continental shelf. Journal of Plankton Research. 21 (9), 1725-1752 (1999).
- Gibson, D. M., Paffenhöffer, G. A. Asexual reproduction of the doliolid, Dolioletta gegenbauri Uljanin (Tunicata, Thaliacea). Journal of Plankton Research. 24 (7), 703-712 (2002).
- Frischer, M. E., et al. Reliability of qPCR for quantitative gut content estimation in the circumglobally abundant pelagic tunicate Dolioletta gegenbauri (Tunicata, Thaliacea). Food Webs. 1, 7 (2014).
- Köster, M., Paffenhöfer, G., Baker, C., Williams, J. Oxygen consumption of doliolids (Tunicata, Thaliacea). Journal of Plankton Research. 32 (2), 171-180 (2010).
- Paffenhöfer, G. A hypothesis on the fate of blooms of doliolids (Tunicata, Thaliacea). Journal of Plankton Research. 35 (4), 919-924 (2013).
- Takahashi, K., et al. Sapphirinid copepods as predators of doliolids: Their role in doliolid mortality and sinking flux. Limnology and Oceanography. 58 (6), 1972-1984 (2013).
- Martí-Solans, J., et al. Oikopleura dioica Culturing Made Easy: A Low-Cost Facility for an Emerging Animal Model in EvoDevo. Genesis. 53 (1), 183-193 (2015).
- Nishida, H. Development of the appendicularian Oikopleura dioica: Culture, genome, and cell lineages. Development Growth & Differentiation. 50, S239-S256 (2008).
