Name: streamlined sampling and cultivation of the pelagic cosmopolitan larvacean, oikopleura dioica
Uploaded: 2020-06-16T15:00:00
Duration: 11 min 55 s
Description: Watch this Scientific Journal Video about Streamlined Sampling and Cultivation of the Pelagic Cosmopolitan Larvacean, Oikopleura dioica at JoVE.com

We are grateful to Garth Ilsley for his support in establishing the culture system. We acknowledge Ritsuko Suyama&#8217;s and Sylvain Guillot&#8217;s contributions to early sampling and species identification efforts. Special thanks are due to Hiroki Nishida, Takeshi Onuma, and Tatsuya Omotezako for their generous support and guidance throughout, including initial establishment of the local culturing system and sharing animals and microalgal culture. We also thank Daniel Chourrout, Jean-Marie Bouquet, Anne Aasjord, Cristian Ca&#241;estro, and Alfonso Ferr&#225;ndez-Rold&#225;n for sharing their expertise on sampling and culturing. Jai Denton, Charles Plessy, and Jeffrey Jolly provided invaluable feedback on the manuscript. Charlotte West formulated a generalized equation for algal calculation. Finally, we thank OIST for funding, Mary Collins and the OIST Fieldwork Safety Committee for advice on safe sampling procedures, the staff of OIST machine shop for the construction of culturing and sampling equipment, and Koichi Toda for delivering seawater.

The author has nothing to declare.

To facilitate flexibility in the establishment of O. dioica culture, it is important to understand the animals&#8217; natural habitat. Seasonal data provides information about the ranges of physical parameters, which can be used to guide laboratory culturing conditions. It also helps in understanding seasonal fluctuations in the abundance of animals. In Okinawa, O. dioica is most reliably found from June to October. However, in Tokyo bay, populations peak in February and October41. Although culturing of O. dioica is often reported at 20 &#176;C or lower27,28,29, Okinawan O. dioica shows better survival at temperatures above 20 &#176;C; this might be explained by the fact the minimum surface seawater temperature in Okinawa is ~20 &#176;C (Figure 6). The abundance of O. dioica might also be influenced by phytoplankton blooms42 and predator abundance43,44. Regardless of where O. dioica are collected, understanding the seasonality of local populations maximizes the chance of sampling and culturing success.
Given the appropriate season and location, net sampling is an effective way of collecting large numbers of Oikopleura with minimal effort. Plankton nets with smaller mesh size (60-70 &#181;m) may also be used to collect all stages of the animals. Fully mature animals are rarely found in the net, perhaps due to their fragility at the end of the lifecycle. Therefore, species identification followed by sampling is achieved by microscopic observation of subchordal cells. Mature individuals usually appear one or two days post-sampling as animals continue to grow in the laboratory. Although net sampling is efficient, alternative sampling methods might be necessary in different circumstances. For example, net sampling near urban areas can collect large numbers of phytoplankton, making it difficult to isolate Oikopleura. In such cases, simple bucket sampling to collect surface seawater or boat sampling from areas beyond the harbor are recommended. The results showed that the gradual change in salinity due to consecutive days of rain did not affect the abundance of O. dioica; however, shore sampling immediately after extreme weather events such as tropical cyclones should be avoided. These events cause sudden and drastic biogeochemical changes in a sheltered body of water45,46. The stormwater runoff may carry pollutants, sediments, and excess nutrients, which increase turbidity and lower water quality47. Filter-feeding plankton, such as Oikopleura, may be especially susceptible to these changes due to their mode of feeding and limited mobility. In such a circumstance, we recommend postponing sampling for a few days until the local conditions return to normal.
The introduction of a multi-step filter system is essential to maintain small, filter-feeding organisms such as O. dioica. Using poorly filtered seawater (for example, a 25 &#181;m mesh in the previous culture system), the culture was often unstable especially during summer, potentially due to the higher abundance of phytoplankton. Although some phytoplankton are beneficial to O. dioica growth, others produce biotoxins that can cause abnormal development of O. dioica embryos48. In addition, a high concentration of diatoms such as Chaetoceros spp. are potentially harmful to O. dioica growth as they can possess long setae which can clog the house and prevent efficient feeding49. We frequently observed houses of small animals being clogged by C. calcitrans setae; therefore, we now feed C. calcitrans only to animals at Day 2 and older (Table 3).
Although it was not a problem here, small scale long-term culturing of O. dioica can experience sudden drops in population size due to a genetic bottleneck; in such cases, Mart&#237;-Solans et al.29 recommend adding new wild individuals to the culture every 20 generations.
The Oikopleura culture system is flexible. A stable culture can be established within a week. Long-term culturing of O. dioica is possible on a modest budget with non-specialist equipment. The daily effort required for the maintenance of 5-10 beakers of Oikopleura is generally less than 2 hours with 2 people. O. dioica can also be maintained in artificial seawater, which is beneficial to those without access to natural seawater28. Long-term storage of algal food is possible using solid-culture and cryopreservation29. Moreover, O. dioica sperm can be cryopreserved, and remain viable for more than a year50. All of these factors mean that cultures can be easily re-established. Finally, past experience with accidental culturing of Pleurobrachia sp. may suggest that the culturing system developed for Oikopleura could potentially be extended to a broader community of fragile pelagic organisms.
O. dioica continues to provide powerful insights into various biological fields. An understanding of local seasonality, a meticulous culture system, and a few dedicated individuals allow effective culture to be established with little effort. Oikopleura culture system provides the baseline resources to investigate a wide range of biological fields relating to ecology, development, genomics, and evolution of this unique marine chordate.

Model organisms have been instrumental in addressing many biological questions including those relating to development, genetics, and physiology. Moreover, additional model organisms facilitate new discoveries and therefore are crucial to achieve a greater understanding of nature1,2. Marine zooplankton are diverse groups of organisms that play an important role in ocean ecosystems3,4,5,6. Despite their abundance and ecological importance, gelatinous organisms such as planktonic tunicates are often under-represented in plankton biodiversity studies because their transparency and fragility make field collection and identification challenging7,8. Adapted sampling techniques and laboratory culturing allow closer observation of the animals in vitro, which has furthered the knowledge in the biology of planktonic tunicates9,10,11,12.
Larvaceans (Appendicularians) are a class of free-swimming marine tunicates comprising around 70 described species worldwide8,13. As they are one of the most abundant groups within zooplankton communities14,15,16,17, larvaceans represent a primary food source for larger planktonic organisms such as fish larvae18,19. Unlike ascidians-the sessile tunicates-larvaceans retain a tadpole-like morphology and remain planktonic throughout their lives20. Each animal lives inside a self-built, intricate filter-feeding structure known as a house. They accumulate particulates in their houses by creating water currents through the undulating motion of their tails21. Clogged houses are discarded throughout the day, some of which form carbon aggregates and eventually sink to the seabed22; thus, larvaceans play a major role in global carbon flux23. Most species are reported to live in the pelagic zone within the upper 100 m of the water column13; however, the giant larvacean Bathochordaeus is known to inhabit the depths of 300 m24. A study on Bathochordaeus in Monterey Bay, California revealed that the animals also serve as a biological vector of microplastics, suggesting a potential importance in understanding the role of appendicularians in the vertical transport and distribution of microplastics in the oceans25.
Oikopleura dioica, a species of larvacean, has attracted attention in recent years as a model organism due to several remarkable characteristics. It is commonly reported throughout the world&#8217;s oceans. It is especially abundant in coastal waters26, which allows easy sampling from the shore. Long-term, stable culturing is possible with both natural and artificial seawater27,28,29. Temperature dependent generation times are as short as 4-9 days in laboratory conditions. It has high fecundity with each female capable of producing &#62;300 eggs throughout the year. As a tunicate, it occupies an important phylogenetic position for understanding chordate evolution30,31. At 70 Mb, O. dioica has the smallest identified genome amongst all chordates32. Among larvaceans, O. dioica is the only described non-hermaphroditic species thus far33.
The first successful O. dioica culture with laboratory grown microalgae was reported by Paffenho&#776;fer34. The original culture protocol using synchronous motors and paddles was developed by Fenaux and Gorsky35 and later adopted by multiple laboratories. More recently, Fujii et al.36 reported O. dioica culturing in artificial seawater, a robust culture system and field collection were described by Bouquet et al.27 and an optimized protocol for a simplified, affordable system was reported by Marti-Solans et al.29. Aside from the traditional Oikopleura culture system, a newly reported design with a double tube rearing tank also has the potential to culture Oikopleura sp.37.
We present a detailed protocol for initiating an O. dioica monoculture based on a combination of protocols developed by major Oikopleura research groups at the Sars International Centre for Marine Molecular Biology27, the University of Barcelona29, Osaka University28, and our own observations. In previously published culture protocols, detailed information regarding the composition of algal media, shore sampling techniques, and Oikopleura identification were only roughly described, leaving a lot of ambiguity. Here, with the aid of visual information in the video protocol, we have assembled all the critical information needed to set up an O. dioica culture from the ground up in a straightforward, step-by-step manner. We describe how to distinguish O. dioica from another commonly reported species, O. longicauda, which is one of the most challenging steps. Although the existing culture systems are applicable for the cultivation of O. dioica worldwide, we highlight the importance of protocol adjustment based on local environmental conditions. The presented information combines widely published data as well as knowledge gained through experience. The current protocol is ideally suited for researchers interested in establishing a culture from scratch.

<table>
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			<td>Activated charcoal</td>
			<td>Sigma</td>
			<td>C2764-2.5KG</td>
			<td></td>
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			<td>Alluminum pulley</td>
			<td>Rainbow Products</td>
			<td>10604-10607</td>
			<td></td>
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			<td>Biotin</td>
			<td>Sigma</td>
			<td>B4501-100MG</td>
			<td></td>
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			<td>Boric acid</td>
			<td>Wako</td>
			<td>021-02195</td>
			<td></td>
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		<tr>
			<td>Cobalamin (B12)</td>
			<td>Sigma</td>
			<td>V2876-100MG</td>
			<td></td>
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		<tr>
			<td>Cobalt(II) chloride hexahydrate</td>
			<td>Wako</td>
			<td>036-03682</td>
			<td></td>
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			<td>Copper(II) sulfate pentahydrate</td>
			<td>Wako</td>
			<td>039-04412</td>
			<td></td>
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			<td>Disodium edetate hydrate</td>
			<td>Wako</td>
			<td>044-29525</td>
			<td></td>
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		<tr>
			<td>Hexaammonium heptamolybdate tetrahydrate</td>
			<td>Wako</td>
			<td>019-03212</td>
			<td></td>
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		<tr>
			<td>Hexagon wrench</td>
			<td>Anex</td>
			<td>No.6600</td>
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			<td>Hydrochloric acid</td>
			<td>Wako</td>
			<td>080-01066</td>
			<td></td>
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			<td>Iron(III) chloride hexahydrate</td>
			<td>Wako</td>
			<td>091-00872</td>
			<td></td>
		</tr>
		<tr>
			<td>Jebao programmable auto dosing pump</td>
			<td>Jebao</td>
			<td>DP-4</td>
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			<td>Magnet pump</td>
			<td>REI-SEA</td>
			<td>RMD-201</td>
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			<td>Manganese(II) chloride tetrahydrate</td>
			<td>Wako</td>
			<td>134-15302</td>
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			<td>Polypropylene wound cartridge filter</td>
			<td>Advantec</td>
			<td>TCW-10N-PPS</td>
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			<td></td>
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			<td>TCW-5N-PPS</td>
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			<td></td>
			<td></td>
			<td>TCW-1N-PPS</td>
			<td></td>
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			<td>Screwless terminal block</td>
			<td>SATO PARTS</td>
			<td>SL4500</td>
			<td></td>
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			<td>Simple plankton net</td>
			<td>RIGO, Japan</td>
			<td>5512-C</td>
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			<td>Sodium metasilicate</td>
			<td>Sigma</td>
			<td>307815-1KG</td>
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			<td>Sodium nitrate</td>
			<td>Wako</td>
			<td>195-02545</td>
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			<td>Sodium phosphate monobasic anhydrous</td>
			<td>MP Biomedicals</td>
			<td>194740</td>
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			<td>Streptomycin sulfate salt</td>
			<td>Sigma</td>
			<td>S6501-25G</td>
			<td></td>
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		<tr>
			<td>Synchronous electric motor</td>
			<td>Servo</td>
			<td>D5N6Z15M</td>
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			<td>Thiamin hydrochloride</td>
			<td>Wako</td>
			<td>201-00852</td>
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			<td>UV sterilizer</td>
			<td>Iwaki</td>
			<td>UVF-1000</td>
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			<td>Zinc chloride</td>
			<td>MP Biomedicals</td>
			<td>194858</td>
			<td></td>
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streamlined sampling and cultivation of the pelagic cosmopolitan larvacean, oikopleura dioica

Oikopleura dioica is a planktonic chordate with exceptional filter-feeding ability, rapid generation time, conserved early development, and a compact genome. For these reasons, it is considered a useful model organism for marine ecological studies, evolutionary developmental biology, and genomics. As research often requires a steady supply of animal resources, it is useful to establish a reliable, low-maintenance culture system. Here we describe a step-by-step method for establishing an O. dioica culture. We describe how to select potential sampling sites, collection methods, target animal identification, and the set-up of the culturing system. We provide troubleshooting advice based on our own experiences. We also highlight critical factors that help sustain a robust culture system. Although the culture protocol provided here is optimized for O. dioica, we hope our sampling technique and culture setup will inspire new ideas for maintaining other fragile pelagic invertebrates.

Watch this Scientific Journal Video about Streamlined Sampling and Cultivation of the Pelagic Cosmopolitan Larvacean, Oikopleura dioica at JoVE.com

Streamlined Sampling and Cultivation of the Pelagic Cosmopolitan Larvacean, Oikopleura dioica

Oikopleura dioica is a tunicate model organism in various fields of biology. We describe sampling methods, species identification, culturing setup, and culturing protocols for the animals and algal feed. We highlight key factors that helped strengthen the culture system and discuss the possible problems and resolutions.

Streamlined Sampling and Cultivation of the Pelagic Cosmopolitan Larvacean, Oikopleura dioica

Oikopleura dioica is a planktonic chordate with exceptional filter-feeding ability, rapid generation time, conserved early development, and a compact ...

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