Field Collection and Laboratory Maintenance of Canopy-Forming Giant Kelp to Facilitate Restoration

Kelp forests are facing unprecedented losses due to climate-driven environmental stressors and shifts in trophic dynamics. This video illustrates a protocol and tools for culturing giant kelp, Macrocystis pyrifera, using the green gravel technique, and provides a valuable resource for further trials to address the successes and limitations of this method in different settings. Kelp culturing facilities must be able to control for temperatures ranging between 10 and 15 degrees Celsius, provide full spectrum light ranging between zero and 180 micromole photons per meter squared second, and filtered aeration.

Incubator systems with a built-in outlet, or an access port for wires and tubing can be adapted with lights and an air source. If an incubator system is not within the project's scope, budget, or intended scale, water baths tempered by either cool natural seawater, or a chiller can be used. Rearing temperatures are site and season specific.

Place a thermometer in growth media, or use a temperature gun to ensure that the temperature is between 10 and 18 degrees Celsius. Set full spectrum lights to a 12 hour light, 12 hour dark photo period using the timing settings on the light source, or by plugging the light source into a timer with a programmable cycle. Measure light intensity with a PAR quantum meter below the surface near the gravel and adjust using a dimmable light source, or by layering mesh over the light source.

Aeration is added to cultures with the use of air pumps. Use filters with a pore size of 02 micrometers to reduce airborne bacterial contamination. Materials and stations should be sterilized and prepared ahead of time.

See text for details. Gravel used for seeding should have a textured, or slightly pitted surface, since gametophytes are more likely to be retained on substrates with a high rugosity. Scrub and rinse gravel until the water runs clear to remove any dust, or debris.

Soak gravel in a 10%diluted bleach solution for at least 24 hours, and rinse with filter-sterilized seawater. Calculate the volume of filter-sterilized seawater needed to refresh culture containers each week, and schedule this filtration and sterilization task accordingly. Large batches of filter-sterilized seawater can be stored in dark containers for up to six months at eight to 10 degrees Celsius.

If refrigeration is not available, store in a dark, cool area. Filter water using a vacuum filtration system with a pore size of 55 to one micrometer. Turn the vacuum source off before all the water is pulled through to avoid damaging the filter, and pour the filtered water into a dedicated sterile container.

For larger volumes, a flow-through filtration system can be used. For example, run seawater through a series of three pleated filters, 10 micrometers, five micrometers, and one micrometer, arranged from largest to smallest pore size. This flow-through system is connected to an aquarium UV light.

Sterilize filtered seawater using UV and/or autoclaving methods. Enrichment of filter-sterilized seawater with nutrients and vitamins are critical to kelp growth. Common enrichment options include Provasoli-enriched seawater media and F/2 media.

See text for details. Obtain the necessary permits for kelp tissue collection that meet local laws and regulations. By scuba, select three to five sporophyll blades from 10 to 15 fertile Macrocystis pyrifera individuals with visible sori spaced two to five meters apart.

Select clean and intact sporophylls if possible with little to no fouling, or degradation. Store sporophyll blades separately according to the parent individual from this point forth. Sporophylls grow in a dense skirt at the base above the holdfast of the adult kelp, and can be identified by their lack of gas-filled pneumatocysts.

Transport sporophyll blades in dark collection bags to avoid overexposure to sunlight filled minimally with seawater from the site to keep blades wet and stored in coolers at approximately 12 degrees Celsius until arrival at the culturing space. Ensure that samples are not in direct contact with ice. Sporophylls can be shipped to, or from other locations.

Rinse sporophylls with filtered sterilized seawater. Wrap blades collected from a single Macrocystis pyrifera individual in moist paper towels soaked in filtered sterilized seawater, and again in aluminum foil to avoid light penetration and additional desiccation. This method of storage is commonly known as the burrito method.

Place these packages in a cooler with ice with a protective barrier, such as recycled bubble wrap, or cardboard. Prepare the cooler for overnight shipment and ensure that someone is available to receive the shipment and place the packages in refrigerated conditions. Optionally, store sporophylls for 12 to 48 hours in refrigerated conditions, which encourages spore release from sorus tissue.

To store, use the burrito method. If optionally stored, find evidence of partial sporulation on the paper towels indicating presence of fertile sorus tissue. Sorus tissue is often slightly raised and darker in color than surrounding tissue.

Select ripe sorus tissue, and cut into 25-centimeter squared sections using sterile scissors. Select one to two clean sori sections from 10 to 15 individual kelp parents to promote genetic diversity. To clean, gently scrub both sides of the sorus tissue in one direction only with a sterile gauze damped with filter-sterilized seawater.

If needed, scrape the sorus tissue gently with a sterile razor blade. Submerge the sori section in a freshwater bath for 30 seconds to one minute, and rinse with filter-sterilized seawater. Submerge each sori section in filter-sterilized seawater tempered to 10 to 15 degrees Celsius within a sterile 50 milliliter Falcon tube.

Alternatively, sori sections can sporulate in a single sterile container. Place tubes at four to 12 degrees Celsius in the dark to sporulate for a maximum of four hours. If no refrigerator is available, store in a low light, cool area.

Using a compound microscope and hemocytometer, observe the spore density of three to four samples every 30 minutes up to four hours. Change pipette tips between samples. If densities are at least 10, 000 spores per milliliter, move on to the next step.

If a sori section produces no spores after four hours, discard the sample. Spores can settle within hours after release, but may be observed swimming in a circular motion. Remove each of the sori sections from their Falcon tubes.

Combine the resulting spore solutions into a single sterilized container and quantify the final combined density. Calculate the final volume of spore solution needed for inoculation for a final concentration of 500 to 1, 000 spores per milliliter in culture containers. See text for details.

Place sterile glass slides within culture containers to monitor kelp development. Include at least 30 slides distributed randomly across culture containers for sufficient monitoring. Inoculate the calculated volume of spore solution into the culture container using a sterile pipette tip that contains substrates submerged in growth media.

Close the container, and gently stir to distribute spores. Place the container into your culture system. Set your temperature between 10 to 15 degrees Celsius based on the temperature at your deployment site.

Full spectrum lights for aquatic plants are set to a 12 hour light, 12 hour dark cycle with light intensities ranging between zero to 180 micromole photon per meter squared second. See text for a timeline of specific light conditions. After one day, provide light aeration with a filtered air source.

Monitor at least two random glass slides daily, or every other day for the first two weeks to assess development. After two weeks, monitor at least two random glass slides one to two times weekly for healthy growth and contamination until sporophytes reach one to two centimeters in size. To monitor, handle the slide with sterilized tweezers and place in a clean Petri dish containing enough sterilized seawater to submerge the glass slide.

Do not return glass slides to cultures after being removed to avoid cross-contamination. Use a compound, or inverted microscope at a magnification of 40 to 400x to observe early stage kelps. Development is expected to track the following timeline.

Settled spores are observed at zero to one day. Spores can germinate within a few hours demonstrated by the formation of a germ tube. Germination is typically observed at one to two days.

Early gametophytes are typically observed at one to four days. Gametogenesis, the process by which cells undergo division and differentiation to form male and female gametes, is typically observed within the first two weeks. Female cells are five to seven times larger than males.

Male gametophytes grow thin filamentous branches, whereas females are more round, or ovoid in shape. Females typically produce eggs within two to three weeks. Sperm released from the males fertilizes the eggs, which develop into embryonic sporophytes.

Sporophytes are typically observed within two to four weeks. The zygote undergoes rapid cell division, resulting in the growth of one to two centimeter blades within approximately six to eight weeks. Every week, change growth media to replenish necessary nutrients and minerals for Macrocystis pyrifera growth.

Chill fresh growth media to the appropriate temperature. Ensure growth media does not exceed 15 degrees Celsius during this process. Siphon media out of your culturing containers to avoid the disturbance of seeded substrates.

Let the media drain until the container is nearly empty. Refresh media immediately to minimize desiccation. Tilt growth containers slightly when refilling, so that media runs down the side of the culturing container to minimally disturb substrates.

Randomly rearrange container, or tub positions during weekly media changes to account for differences in light irradiance. See the text for a calendar to track activities and expectations for Macrocystis cultures. It indicates the timing of adjustments to light and aeration, as well as weekly media changes.

After six to eight weeks of laboratory culturing, juvenile sporophytes are one to two centimeters in length and ready for deployment. Refresh growth media in culture containers 24 hours before deployment. Obtain the necessary permits for gravel deployment that meet local laws and regulations.

Transport green gravel for up to six hours in a shaded cooler. Deployment should be timed to avoid the most direct sunlight. Utilize a shaded structure on the boat to avoid direct sun during the deployment process.

Carefully scatter green gravel from the surface onto the reef below, or via scuba when trialing at new sites and at small scales. The green gravel restoration technique is still in the academic phase with limited outplant survival data for other species and no data yet for Macrocystis pyrifera. Nonetheless, success in the field has been observed in a recent pilot study, indicated by haptera attachment to surrounding substrate and juvenile kelps growing up to 1.2 meters after two months in the field.

Using the field collection and laboratory maintenance outlined in this protocol, we cultured two distinct donor kelp populations at 12 degrees Celsius and 20 degrees Celsius to elucidate temperature-dependent effects on the development of micro-stages of Macrocystis pyrifera through time. After 24 days, gametophytes were counted from microscope images. Temperature had an effect for the cooler K1 population, but not for the K4 population, suggesting a possible adaptive divergence in thermal tolerance traits.

This highlights the importance of site-specific rearing temperatures. After 32 days, visible sporophytes were counted on each glass slide. Temperature had a similar effect for both K1 and K4 populations.

Samples reared at 20 degrees Celsius grew few visible sporophytes compared to those reared at 12 degrees Celsius. This result suggests that sporophyte production is more sensitive to temperature than the gametophyte stage, and that culturing temperatures must not exceed 15 degrees Celsius to achieve sporophyte development, as outlined in the protocol. With this visualized protocol, we hope to encourage further trials with the green gravel method to upscale restoration of valuable underwater kelp forests by researchers and managers globally.

Best of luck on your green gravel journey with Macrocystis pyrifera.