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

This protocol visually demonstrates how to establish a long-term, reliable culture system for the marine pelagic tunicate Oikopleura dioica. Oikopleura culture system provides the baseline resources to investigate a wide range of biological fields relating to ecology, development, genomics, and evolution. This species has a short lifecycle, high fecundity, and it can be easily collected from the shore, making it an excellent model organism.

Though this protocol is optimized for O.dioica, we hope it can be applied to other planktonic organisms. To begin, initiate algal food to sustain Oikopleura culture, starting by autoclaving filtered seawater. To initiate a stock culture of Chaetoceros calcitrans, 60 mils of autoclaved seawater was supplemented with the following:30 microliters of vitamins, 30 microliters of solution A, 15 microliters of sodium silicate, and 60 microliters of streptomycin.

Finally, 30 microliters of C.calcitrans from a previous stock culture was added as an inoculant. Refer to table two in the manuscript regarding supplement and inoculation volumes for the other algal species and culture types. After inoculation, the stock and subcultures are maintained in an incubator at 17 degrees Celsius with continuous lighting.

After approximately 10 days, algal growth is confirmed by the flasks becoming colored. When this occurs, move the stock cultures to long-term storage at four degrees Celsius for up to one month while maintaining the subculture in the same incubator. To create a working culture, add necessary supplements to 400 mils of autoclaved seawater and inoculate with 100 mils of the previous subculture.

Seal the flask with a rubber cap, insert a one-mil, disposable pipette, and move them to an algae station and maintain them at room temperature with an eight-hour photoperiod and constant aeration. Manually swirl algal cultures twice a day to prevent aggregation at the bottom of the flask. Once the algal cultures have been established, collect field samples at a local site.

Cast a plankton net, and allow the cod end to sink one to two meters below the surface. Tow the net at a speed of approximately 5 to one meters per second. Continue towing back and forth for between two and five minutes.

Towing time should be adjusted according to plankton abundance on site. After the tow, carefully lift out the net and slowly transfer the contents of the cod end to a 500-mil, glass bottle. Fill the bottle to the rim to avoid air bubbles that could cause stress to the animals during transport.

At this stage, the presence of Oikopleura can be confirmed by viewing the sample bottles against a dark background. However, microscopic observation is required for species-level identification. Repeat this process until three 500-mil bottles have been collected.

Collect surface seawater to record salinity, temperature, and chlorophyll concentration with a CTD profiler. Additionally, collect 10 to 15 liters of on-site seawater to help acclimatize the animals to laboratory conditions. To acclimatize the animals, prepare a one-to-one ratio of surface seawater from the sample site and filtered seawater maintained in the lab.

The final volume should be adjusted to between five to 10 liters, depending on the concentration of the plankton sample. Carefully add the 500-mil sample to the prepared beaker. Using a paddle attached to a synchronous motor rotating at 15 RPM, keep the plankton in suspension overnight.

The following morning, check for the presence of Oikopleura by eye. Transfer the animals to a Petri dish, and observe under a dark-field microscope set to between magnification 20 to 40 times for a positive identification. Oikopleura are characterized as tadpole-shaped animals undulating their tails inside a translucent house.

To confirm O.dioica, look for fully mature males with yellow gonads or females with eggs. If the animals are immature, look for the presence of two subchordal cells at the distal half of the tail. These are present in both mature and immature animals.

Once the species is confirmed, transfer them to a new Petri dish. Repeat this process until 10 to 20 individuals have been confirmed at species level. If no O.dioica are found, keep the beaker stirring for an extra day or two.

Small O.dioica that weren't initially observable will continue to grow and become easier to see. If none appear after one week, discard the sample and try sampling again. In order to establish a monoculture, isolate 120 O.dioica from a field sample in five liters of filtered seawater.

The following morning, look for fully mature males identified by yellow gonads and for females with eggs that appear as shimmering, golden spheres. Prepare a spawning beaker by gently transferring 15 males and 30 females with a five-mil, blunt-ended pipette to a beaker containing 2.5 liters of filtered seawater. To minimize physical stress during the manual transfer, slowly release the animals under the surface of the new seawater.

Allow isolated adults to spawn naturally. After spawning, successful fertilization can be confirmed by extracting five to 10 mils of seawater from the bottom of the spawning beaker and identifying eggs with cleavages under a microscope. On the first morning after spawning, day one, a new generation of young animals with inflated houses should appear in the beaker.

Use a 500-mil, handheld beaker to gently transfer animals to a new beaker containing 7.5 liters of fresh, filtered seawater. Slowly pour the content at the surface to minimize turbulence and shear forces. On the second morning after spawning, day two, manually transfer 150 animals to a new beaker containing five liters of fresh, filtered seawater.

On the third morning post-spawning, day three, manually transfer 120 animals to a new beaker with five liters of fresh, filtered seawater. By the fourth day, sexually mature, adult animals should be present. Collect 15 males and 30 females for a new spawning beaker with 2.5 liters of filtered seawater to initiate the next generation.

This process is repeated and maintained for the life of the culture. In order to maintain a monoculture of O.dioica, algal food is prepared daily from working cultures. The animals are fed three times a day.

Prepare daily food by measuring absorbance of the working culture using a spectrophotometer set at 660 nanometers. Absorbance values, target O.dioica culture volumes, and animal maturity are placed into a prepared spreadsheet based on a previously determined algal growth curve to calculate the exact volume of algae required for the day's feeding. Pipette the calculated volumes into 50-mil tubes, and centrifuge at 5, 000g for five minutes at room temperature.

Slowly pour off supernatant, and carefully pipette up and down to resuspend the pelleted algae. Refill the tubes to their original volume with fresh, filtered seawater. Based on the daily algal feeding chart from the spreadsheet, feed the specified amount to each beaker at 9 a.m.

12 p.m. and 5 p.m. An automated dosing pump can be set to feed the animals at 5 p.m.

on weekends without the presence of culturing staff. After each feeding, maintain the prepared algal food at four degrees Celsius until ready to be used for the next feed. O.dioica can be collected from a harbor by the gentle towing of a modified plankton net fitted with 100-micron mesh and a non-filtering cod end.

Sampling between 2015 and 19 showed seasonal variation in the presence of O.dioica in the harbors of Okinawa. Surface seawater temperature seemed to influence the presence of the animals. O.dioica dominated when surface seawater temperature was 28 degrees or higher.

O.dioica coexisted with Oikopleura longicauda at temperatures between 24 to 27 degrees. However, O.longicauda dominated at temperatures below 23 degrees. Changes in salinity did not correlate with the abundance of O.dioica.

The three most important factors for establishing stable O.dioica culture systems are maintaining high water quality with a multi-step filtration system, identifying an optimal feeding regime, preferably by creating algal standard curves, and preparation of spawning beakers with sufficient numbers of males and females. Following the provided protocol, the lifecycle of O.dioica is four days at 23 degrees. We've reliably established six independent populations of O.dioica, all of which lasted more than 20 generations.

In conclusion, establishing a stable culture of O.dioica can be done quickly and cost-effectively. With a proper setup, a stable culture can be achieved within one week. In conjunction with its small genome and quick lifecycle, this makes O.dioica a powerful chordate model species.

O.dioica can be maintained in both artificial and natural seawater. Long-term storage of algal food is possible using solid culture and cryopreservation. In addition, O.dioica sperm can also be cryopreserved and remain viable for more than a year.

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.