Name: cultivation of the marine pelagic tunicate dolioletta gegenbauri (uljanin 1884) for experimental studies
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We are grateful to the many persons who have contributed accumulated knowledge to this project over the years including G.-A. Paffenh&#246;fer and D. Deibel who originally developed these protocols. M. K&#246;ster, and L. Lamboley have also contributed significantly to the development of these procedures.&#160; N.B. L&#243;pez-Figueroa and &#193;.E. Rodr&#237;guez-Santiago generated the estimates of doliolid abundance provided in Table 1. This study was supported in part by the US National Science Foundation awards OCE 082599, 1031263 to MEF, collaborative projects OCE 1459293 and OCE 14595010 to MEF and DMG and, the National Oceanic and Atmospheric Administration award NA16SEC4810007 to DMG. We are grateful to the hardworking and professional crew of the R/V Savannah. Lee Ann DeLeo prepared the figures, Charles Y. Robertson proofread the manuscript and, James (Jimmy) Williams manufactured the plankton wheel

1. Preparing culturing facilities for rearing D. gegenbauri
NOTE: All materials and equipment required are listed in the Table of Materials.
<ol>
	<li>Prepare 1 M Sodium Hydroxide (NaOH), 0.06 M Potassium Permanganate (KMnO4) solution. To prepare this solution, dissolve 400 g of NaOH into 10 L deionized water. Add 100 g of KMnO4 to the NaOH solution and mix well.</li>
	<li>Prepare a 0.1 M Sodium bisulfite (NaHSO3) solution by dissolv...

<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>

The capacity to culture doliolids has been established over the past several decades and has been used to support research in several areas. Experimental studies in our laboratories have supported the publication of at least 15 scientific studies focused on the feeding and growth18,26, reproduction18,28, diet6,29, physiology30</s...

Zooplankton account for the largest animal biomass in the ocean, are key components in marine food webs, and play important roles in ocean biogeochemical cycles1,2. Zooplankton, although comprised of a huge diversity of organisms, can be grossly distinguished into two categories: gelatinous and non-gelatinous with few intermediate taxa3,4. Compared to the non-gelatinous zooplankton, gelatinous zooplankton are especially difficult to study because of their complex life histories5, and their delicate tissues are easily damaged during capture and handling. Gelatinous zooplankton species are, therefore, notoriously difficult to culture in the laboratory and generally less studied compared to non-gelatinous species6.
Among gelatinous zooplankton groups, one abundant and of ecological importance in the world&#8217;s ocean are the Thaliaceans. Thaliaceans are a class of pelagic tunicates that include the orders Salpida, Pyrosomida, and Doliolida7. Doliolida, collectively referred to as doliolids, are small barrel-shaped free-swimming pelagic organisms that can reach high abundances in productive neritic regions of subtropical oceans. Doliolids are among the most abundant of all the zooplankton groups4,8. As suspension feeders, doliolids collect food particles from the water column by creating filter currents and capturing them on mucus nets9. Taxonomically, doliolids are classified in the phylum Urochordata10. Ancestral to the chordates, and in addition to their ecological significance as key components of marine pelagic systems, Thaliaceans are of significance to understanding the origins of colonial life history10,11 and the evolution of the chordates5,7,10,12,13,14.
The life history of doliolids is complex and contributes to the difficulty in culturing and sustaining them through their life cycle. A review of the doliolid life cycle and anatomy can be found in Godeaux et al.15. The doliolid life cycle, which involves an obligatory alternation between sexual and asexual life-history stages, is presented in Figure 1. Eggs and sperm are produced by the hermaphroditic gonozooids, the only solitary stage of the life cycle. Gonozooids release sperm to the water column and eggs are internally fertilized and released to develop into larvae. Larvae hatch and metamorphose into oozooids that can reach 1-2 mm. Presuming conducive environmental conditions and nutrition, oozooids become early nurses within 1-2 days at 20 &#176;C and initiate the colonial stages of the life cycle. Oozooids asexually produce buds on their ventral stolon. These buds leave the stolon and migrate to the dorsal cadophore where they line up in three paired rows. The central double rows become phorozooids and the outer two double rows become trophozooids. The latter provide food to both the nurse and the phorozooids16,17. The trophozooids supply the nurse with nutrition as she loses all internal organs. As the abundance of trophozooids increases, the size of the nurse can reach 15 mm in the laboratory. As the phorozooids grow, they increasingly ingest planktonic prey and reach ~ 1.5 mm in size prior to being released as individuals17. A single nurse may release &#62; 100 phorozooids during its lifespan18. After the phorozooids are released from the cadophore, they continue to grow and are the second colonial stage of the life cycle. Once they reach ~ 5 mm in size, each phorozooid develops a cluster of gonozooids on their ventral peduncle. These gonozooids can ingest particles when they reach ~1 mm in length. After the gonozooids have reached ~ 2 to 3 mm in size they are released from the phorozooid and become the only solitary stage of the life cycle. Once they reach ~ 6 mm in size, gonozooids become sexually mature17. Gonozooids can reach 9 mm or greater in length. Gonozooids are hermaphroditic, sperm is released intermittently while the fertilization of the eggs occurs internally16,17. When the gonozooid is &#8805; 6 mm in size, it releases up to 6 fertilized eggs. Successful culturing requires supporting the specific needs of each of these unique life history stages.
Due to the ecological and evolutionary significance of Thaliaceans, including doliolids, there is a need for the cultivation methodologies to advance the understanding of this organism&#8217;s unique biology, physiology, ecology, and evolutionary history19. Doliolids have considerable promise as experimental model organisms in developmental biology and functional genomics because they are transparent and likely have streamlined genomes20,21. The lack of reliable cultivation methods, however, impedes their usefulness as laboratory models. Although a handful of laboratories have published results based on cultured doliolids, to our knowledge cultivation approaches and detailed protocols have not been previously published. Based on years of experience, and trial and error cultivation attempts, the purpose of this study was to review experiences and to share protocols for the collection and cultivation of doliolids, specifically the species Dolioletta gegenbauri.

<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

Gelatinous zooplanktons play a crucial role in ocean ecosystems. However, it is generally difficult to investigate their physiology, growth, fecundity, and trophic interactions primarily due to methodological challenges, including the ability to culture them. This is particularly true for the doliolid, Dolioletta gegenbauri. D. gegenbauri commonly occurs in productive subtropical continental shelf systems worldwide, often at bloom concentrations capable of consuming a large fraction of daily primary production. In this study, we describe cultivation approaches for collecting, rearing, and maintaining D. gegenbauri for the purpose of conducting laboratory-based studies. D. gegenbauri and other doliolid species can be captured live using obliquely towed conical 202 &micro;m mesh plankton nets from a drifting ship. Cultures are most reliably established when water temperatures are below 21 &deg;C and are started from immature gonozooids, maturing phorozooids, and large nurses. Cultures can be maintained in rounded culture vessels on a slowly rotating plankton wheel and sustained on a diet of cultured algae in natural seawater for many generations. In addition to the ability to establish laboratory cultures of D. gegenbauri, we demonstrate that the collection condition, algae concentration, temperature, and exposure to naturally conditioned seawater are all critical to the culture establishment, growth, survival, and reproduction of D. gegenbauri.

Following the described procedures for collecting and culturing the doliolid, D. gegenbauri outlined in Figure 3, it is possible to maintain a culture of D. gegenbauri throughout its complex life history (Figure 1) and sustain it for many generations. Although cultivation of D. gegenbauri is described here, these procedures should also be relevant for the cultivation of other doliolid species.
Capturing heal...

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

Doliolids, including the species Dolioletta gegenbauri, are small gelatinous marine zooplankton of ecological significance found on productive subcontinental shelf systems worldwide. The difficulty of culturing these delicate organisms limits their investigation. In this study, we describe cultivation approaches for collecting, rearing, and maintaining the doliolid Dolioletta gegenbauri.

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

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

Availability of culturable model organisms has been fundamental to the advancement in many fields of study. This protocol, to our knowledge, is the only approach available for culturing Doliolids. Reliable Doliolid culture technology provides a unique experimental model to study organisms that are important to some of the most productive regions of the ocean and that represent the earliest evolutionary ancestors of vertebrates.This technique is unique as it keeps the algal food particles and the organisms suspended in the water column without contact with the walls of the container as they experience in nature. This culturing method can be applied to the culturing of other small gelatinous and crustaceans or plankton species. Because Doliolids have a complex life cycle, understanding and recognizing the needs of each life stage is critical.Learning the life cycle and how to recognize and handle them takes a lot of practice. In our experience, the only way to learn this culturing technique is to see it and do it. Several other project participants including Erin Arneson, Nicholas Castalaine, Natalia Lopez, Lauren Lamboye, Briana Pierce, Arya Rodriguez-Santiago, and Terrell Scarborough also appeared briefly in this video.Before establishing and rearing Doliolid cultures in the laboratory, clean and sterilize 1.9 and 3.8 liter glass culture jars by immersing them in the sodium hydroxide potassium permanganate solution. Allow the jars to soak overnight. Then transfer the jars into the sodium bisulfite solution.Allow the jars to soak overnight. Next rinse the jars thoroughly with deionized water. Allow the jars to dry.To maintain stock cultures, use rigorous axenic culture techniques. Every two weeks transfer 0.5 milliliters of old senescent culture to 25 milliliters of fresh growth media in sterile 55 milliliter glass culture tubes. To prepare larger volumes of phytoplankton for feeding Doliolids, transfer four milliliters of the phytoplankton from axenic stocks into 500 milliliter plastic tissue culture flasks containing 200 milliliters of growth media to inoculate at one to 50 dilution.Incubate the culture in an environmental incubator or walk-in environmental chamber at 20 degrees Celsius with a 12 to 12 hour light-dark cycle under cool white light illumination of 65 to 85 microEinsteins per square meter. Lay culture flasks flat to maximize illumination. Gently swirl the culture daily.Head out to sea. To locate Doliolids on the continental shelf, deploy oceanographic instrument CTD for identifying water conditions conducive to the presence of Doliolids by profiling water temperature, salinity, and chlorophyll A fluorescence. Confirm the water conditions by conducting exploratory net tows with specialized plankton nets.Alternatively, use an in situ profiling imaging system to detect Doliolids. Now operate the winch to retrieve the CTD. The Niskin bottles mounted on the CTD rosette collected seawater from the site where Doliolids are located and at the depth containing the highest estimates of chlorophyll.Rinse three 22-liter buckets with seawater and fill with seawater to a little more than half full. Fill the net cod end completely with seawater and attach the cod end to the net. Deploy the net.Lower and raise the net through the water column, maintaining an oblique towing angle of 15 to 25 degrees and vertical deployment and retrieval speed not greater than 15 meters per minute. Once the net is on board gently transfer and divide the contents of the cod end into the plastic buckets each containing surface seawater collected from the site. Using a wide-bore glass pipette gently mix the contents of the beaker with the pipette using vertical and horizontal motions.Using the index finger, create capillary suction to capture and transfer Doliolids. Capture the Doliolids from mixed phytoplankton in the beaker. After the addition of Doliolids, add Rhodomonas culture into the jar to a final concentration of about five times 10 to the third to 10 to the fourth cells per milliliter.Check the color of the digestive tract of Doliolids. The red color determines the Doliolids are actively feeding. To prevent Doliolids from being trapped at the air-water interface, completely fill the jars with unfiltered, particle-rich seawater to avoid the head space.Place a piece of plastic wrap over the jar opening. Avoid creating air bubbles that can also damage the animals. Carefully screw the cap onto the jar and gently invert the jar to determine if bubbles are present.If bubbles are present, fill more seawater to remove them. After the jars are filled reseal with the lid and wipe the excess water from the outside of the jar. Mount each jar onto the plankton wheel by placing the jar on the vertical metal bars covered with rubber tubing and between a stainless steel hose clamp.Ensure that the back of the jar is cushioned against the rubber tubing. Adjust the screw to tighten the hose clamp around the jar. Allow the jars to rotate at 0.3 rpm to keep the Doliolids in suspension.After three days acclimation to the laboratory conditions transfer 100 to 150 milliliters of culture water from the jar to a sample container and measure the algae concentration using a Coulter counter. Based on the estimation of algal concentration, add sufficient algae to bring the concentration in the culture jar to 40 to 95 micrograms of carbon per liter. Use the particle counter to monitor algal concentrations after the Doliolids have been feeding to guide the decision of how frequently and how much algae to add to the cultures.Maintain phytoplankton concentrations in the culture jars between 40 to 95 micrograms of carbon per liter. Culturing Doliolids requires supporting them through their complicated life cycle. Developing larvae and oozooids and early nurses.Once a minimum of eight trophozooids are visible on the nurse's cataphor, transfer at least four nurses into a new culturing jar. Once the phorozooids reach three millimeters in size, reduce the number of animals in the jar. When the phorozooids become larger than five millimeters and have developed gonozooid clusters remove all but four phorozooids.This protocol provides an overview of a Dolioletta gegenbauri collection and cultivation approach. Algal clearance rates were similar at concentrations from 20 to 60 micrograms of carbon per liter and decreased as the food concentrations increased. Clearance rates increased proportionately over temperature ranges supportive of D.gegenbauri growth.Growth rates ranging from 0.1 to 0.7 millimeters per day as a function of temperature and food availability were observed. The cultivation of Doliolids depends on successfully replicating oceanic conditions and accommodating the needs of the animals throughout their complicated life cycle. Consequently, gentle handling of the animals during their capture, isolation, and husbandry is required.Food concentrations must be monitored frequently and adjusted. This is particularly true during the developing larvae and oozooid life stages. This method is specifically designed for the rearing of small zooplankton species at the base of the marine food web.This method will be valuable for example to investigate zooplankton responses to climate change. Since its development, this technique has paved the way for zooplankton ecologists to determine the laboratory-based feeding, growth, and reproduction rates of Doliolids. Additionally this method paved the way for revolutionary molecular-based investigations of the Doliolid diet in nature.I would like to remind you that the glass cleaning protocol involves reagents that are respiratory irritants and should be used in well-ventilated areas. Working at sea can also be hazardous so follow the ship safety rules.

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