Progress report for LNE21-431R
Seaweed farming is the second largest aquaculture industry worldwide, with global production used in cuisine, manufactured foods, fertilizer, and animal feed. Sea vegetables (the edible subset) are a $6 billion market worldwide and a growing industry, particularly in the Northeast US where commercial species are native. In the Northeast there are >200 established seaweed farms currently in operation as well as ~6 seaweed nurseries that supply these farms. Techniques for growing sea vegetables (both brown and red algae) are well established despite a complex two-part life cycle, but the US industry has been slow to adopt the more technically advanced germ line cultivation techniques used in Asia and Northern Europe. Instead, US farmers rely upon provision of tenuous wild-sourced seed to cultivate juvenile sporophytes for product, an outdated approach subject to natural vagaries in environmental conditions and stochastic changes in reproductive timing, fecundity, and seed quality. To increase production for novel emerging US seaweed markets, an investment into the technological methods for mechanization of seed stock development and preservation are needed. Annually, sea vegetable nurseries require access to sterilized seawater and sufficient lighting, space for care and maintenance of seed stocks, and expertise, making seedling cultivation a barrier to entry for most new aquaculturists. With the development of seed or broodstock cryopreservation, this provides ‘insurance’ for common and specialized strains, minimizing manual labor associated with long-term cultivation and shortened turnaround times for the production of healthy, viable, juvenile sporophytes on ‘seeded-lines’ that can be sold to farmers.
The proposed study seeks to develop long-term maintenance technologies for current (Sugar kelp - Saccharina latissima) and emerging seaweeds (Winged kelp – Alaria esculenta, Irish moss – Chondrus crispus, and Dulse – Palmaria palmata) in the Northeast by testing the efficacy of various sterile cryopreservation techniques for storage of vegetative cultures and spores.
Laboratory experiments will be used to develop cryopreservation approaches and will be initiated by harvesting wild broodstock (spores) of commercially emergent seaweeds and subjecting these spores to different freezing and thawing rates and cryoprotectants. Initial results for sugar kelp (Saccharina latissima) spores indicate that controlled-rate-freezing (CRF) is more effective than flash freezing. Using live-cultured spores, the traditional culturing technique will serve as a control and will be compared to revival culturing using preserved/thawed seed stocks. Accurate indication of viability is critical to developing quality control measures for nurseries seeking to offer cryopreservation services. We plan to engage both a hatchery/nursery (Atlantic Sea Farms) and a seaweed farm (Maine Sea Farms) to test the feasibility and ease of implementation of cryogenic protocols as well as provide seeded lines for grow-out tests of both brown and red algae for on-farm trials.
Edible seaweeds are a $6 billion USD market worldwide. Globally, the seaweed aquaculture sector is growing 7.7% annually, and a rate twice that in the Northeast US where many commercial species are native. To increase production for novel emerging US seaweed markets, an investment into the technological methods for mechanization of seed stock development and seed preservation are required. We propose to develop and optimize a well-established method for cryopreserving (freezing), thawing and growing juvenile seaweed vegetable species in biosecure nurseries. The method maintains frozen cultures of the microscopic life stage (spores) of current farmed seaweed vegetables and emerging species.
Research goal: To develop long-term maintenance technologies for current (sugar kelp - Saccharina latissima) and emerging seaweeds (Winged Kelp – Alaria esculenta and Reds – Dulse) in the Northeast by testing the efficacy of various sterile cryopreservation techniques for storage of vegetative cultures and spores.
Our research formally began in the Fall of 2021 for this project and as part of this project we established a cryogenic center with an auto-fill liquid nitrogen dewar for the storage of cryogenically preserved seaweed seed/spores.
During the fall of 2021 we targeted two phenotypes of sugar kelp (Saccharina latissima), which included skinny and wide blade phenotypes. Sporulation and cryogenic freezing activities were conducted in October and November 2021 for each phenotype, respectively. For each sporulation of seed the following methodology was applied:
Natural sources of sugar kelp (Saccharina latissima) reproductive tissue were obtained by the co-PIs from of mature sporophytes collected from two locations along the midcoast, Maine and key morphological features were recorded (stipe length, blade length and width). For each phenotype the surface of the reproductive tissue was soaked in sterile seawater for 5–10 min, cleaned with a damp cloth, scraped with a clean razor blade then stored on paper towels in the dark for 24 hours at 10°C. The following day the meiospores were harvested following the procedures of Flavin et al 2013 and Redmond et al 2014. Attempts were made in the fall of 2022 to obtain more natural sources of reproductive tissue for additional cryogenic tests but natural skinny kelp beds had been overharvested and were not available. We will be working with two farmers in 2023 (see Cooperators) to obtain all reproductive sorous tissue to complete this project and to provide cryogenic seed stock training.
Cyrogenic Freezing Procedure:
For the “skinny kelp” phenotype two cryoprotectants mixed 1:1 in 2 mL cryovials were used and the meiospores were frozen using a ThermoFisher CryoMed controlled rate freezer (CRF) at a rate of 1°C per min to a final temperature of -40°C followed by storage in liquid nitrogen vapor. For the “wide blade” phenotype three cryoprotectants were used and mixed 1:1 in 2 mL cryovials and frozen via CRF. For each phenotype the following number of vials were collected and stored:
Skinny blade Kelp: cryoprotectant 1 = 97 vials, cryoprotectant 2 = 96 vials
Wide blade Kelp: cryoprotectant 1 = 75, cryoprotectant 2 = 76, & cryoprotectant 3 = 55.
The number of vials depended on the meisopore density for each of the sporulation event. To minimize the genetic variation between different kelp blades, the meiospores from several fronds were combined for each sporulation event and downstream cryogenic freezing. There are advantages and disadvantages to doing this, as each kelp frond with have different meiospore vitality (as per previous research), but in order to test a broad range of cryoprotectants we chose to combine meiospores from different blades in order to generate many vials for downstream testing and differentiation into sporophytes.
For cyrogenic revival of meiospores both the freezing process and thawing process are important aspects of meiospore survival and differentiation. It is vitally important to remove / dilute the cryoprotectants as soon as possible. Two thawing techniques were examined for each cryoprotectant used for our two Saccharina latissima phenotypes (skinny and wide blade), 1) rapid thaw in water bath and 2) CryoThaw: a simple tube adapter to expedite and automate thawing.
- Rapid thaw in water bath: Each frozen meiospore vial is held in a 20 C water bath until a pea sized pellet appears and then the vial content is placed in 9 mL of chilled (4 C) Prov50/2 media, and inverted 5 times the evaluated for vitality using Fluorescein Diacetate (FDA) and observed for motility.
- CryoThaw (Beddall et. al, 2016 DOI: 1016/j.jim.2016.08.009): Using a small tube adaptor (commercially available) the frozen uncapped meiospore cryovial is placed at the entrance of a 15 mL centrifuge tube containing 9 mL Prov50/2 media (chilled to 4 C), the centrifuge is programmed to 22 C, 1500 RPM, for 5 min. Once the vial thaws the meiospore pellet is in the media within minutes and are diluted rapidly and within a controlled environment. The 15 mL vial is removed and inverted 5 times prior to analysis for vitality (as in step 1). The goal of this method is to provide a more controlled environment for thawing and diluting meiospores with the goal to remove the cryoprotectant as soon as possible.
After thawing, 1 mL of each thawed solution (from different cryoprotectants) is stained with 10 uL of FDA working stock and incubated at 10 C for 20 mins prior to flow cytometric analysis on a BioRad ZE5 Cell Analyzer. All vital meiospores will fluorescence green with blue laser excitation. The total number of meiospores is determined using red fluorescence (as an indicator of chlorophyll) as well as the number of vital cells. The percent vital cells is determined and recorded for each vial and cryoprotectant.
After thawing, 50 uL of each thawed solution is observed under the microscope using a hemocytometer to determine if any of the thawed cells are motile – an indication of vitality.
Meiospore Differentiation examination:
After thawing, X mL of each thawed solution is combined with 4 mL of Prov50/2 media and placed into a well of a 6-well tissue culture plate and incubated in 12/12 light/dark cycle to monitor for meiospore differentiation and eventual sporophyte formation.
Other Kelp species examined to date: In fall of 2022 a protocol for releasing Winged Kelp (Alaria esculenta) spores from reproductive fronds was developed in order to test spore cryopreservation techniques (TBD in 2023). The sporophylls were collected from natural sources (Pemaquid Point, ME), and returned to the lab on ice for processing. The sporophylls were removed from the fronds and scraped with a razor blade to remove mucus and contaminates from the surface. Afterwards the sporophylls were divided into two treatments to induce spore release. 1) Betadine dip: sporophylls were dipped in a Betadine solution (500 uL of Betadine to 500 mL of DIW) for 2 mins then removed into a tray of filtered seawater (FSW) and scraped with a razor blade again, sandwiched between paper towels, and incubated overnight in the dark at 12 C. 2) FSW treatment: sporophylls were dipped in FSW and scraped with a razorblade to remove mucus and surface contaminates, afterwards all scraped sporophylls were dipped in FSW for 30 seconds then removed and scraped again with a razor blade. All sporophylls were sandwiches between paper towels and incubated overnight at 12 C. Of the two treatments the one with the most success was the Betadine dip. Unfortunately, the season and all the reproductive tissue had been harvested the next time we attempted a sporulation for cryopreservation. We will plan to begin this process in Spring/Summer 2023 – using reproductive tissue provided by seaweed nurseries (see cooperators).
Preliminary results are available from our first cryopreservation tests of sugar kelp (Saccharina latissima) using three cryoprotectants and two thawing methods. Of the three cryoprotectants tested, cryoprotectant 3 (cryo 3) had the best % vitality results with the overall highest FDA fluorescence after thawing (Figure 1 - Percent vitality of Slatissima_wide blade). These results were significant using a Tukey T-Test. For both phenotypes of sugar kelp, cryoprotectant 2 was better than cryoprotectant 1 (Figure 1 - Percent vitality of Slatissima_wide blade & Figure 2 - Percent vitality of Slatissima_skinny blade). However, differences between the two thawing methods were inconclusive and require more replication. Additional cryopreservation experiments will be conducted on both sugar and winged kelp in 2023, to determine if cryoprotectant 3 provides more vital meiospores after thawing. In all cases no motility was observed after thawing.
No formal conclusions at this time.
Education & Outreach Activities and Participation Summary
Over the course of the past year, tours at Bigelow Laboratory have taken place for both the general public and for educational purposes (high school students and science educators) - during these tours the aspects of this seaweed project are highlighted and demonstrations of kelp reproduction have been presented.