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

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.