Harvest and aquaculture of seaweeds are a multi-billion-dollar industry worldwide (FAO 2016). Sea vegetable aquaculture (SVA) will be increasingly dependent on a greater diversity of strains that are tolerant to coastal warming attributable to climate change. Market demand and interest in integrated aquaculture offer increased opportunities for development of sea vegetable crops in Maine. This study aimed to investigate the temperature tolerance of the edible kelp Alaria esculenta and to understand its potential as a sea vegetable crop in warming waters. Seedstocks from northern and southern populations were experimentally acclimated to higher temperatures expected in the future for the Gulf of Maine (GOM). Although some individuals were damaged by elevated water temperatures, a substantial proportion of seedstock remained healthy throughout the experiment. Seedstock source population did not influence health; however, gene expression patterns showed that southern strains exhibit different stress responses compared to northern strains. Alaria esculenta is not only able to acclimate to thermal stress, but this study suggests thermal adaptation in gametophyte seedstocks from a southern GOM population. Because the commercial life history stages obtained from seedstock might experience slower growth as part of stress tolerance, we are examining crop yield. Seedstocks that underwent experimental thermal acclimation had larger blades than seedstocks held at control temperatures. The protocols developed in this research will aid additional strain selection and will lead to further investigation of how to promote higher yields in Alaria esculenta aquaculture. This kelp can diversify sea vegetable landings in the State of Maine; sea farmers can incorporate sea vegetables into existing infrastructure in shellfish and finfish farms, increasing revenue on the same lease site. SVA can strengthen coastal communities by supplying opportunities to control landings and income to help working waterfronts flourish.
- FAO 2016. The State of the World Fisheries and Aquaculture 2016. FAO. Rome.
Seaweeds have been farmed and harvested world-wide for hundreds of years, yet sea vegetables are still considered novel in American mainstream culture and markets. Global aquatic algal landings in 2014 totaled 27.3 million tons, worth $US5.6 billion; over 95% of these landings were farmed (FAO 2016). Several kelps are valuable whole food crops (Wells et al. 2017). Capitalizing on this novel market can support coastal Maine communities and aid in restructuring the working waterfronts for the future. In order to have a successful sea vegetable aquaculture (SVA) industry in Maine, it will be important to diversify landings and ensure crops can withstand warming waters. The kelp Alaria esculenta has potential in US markets due to its nutritious and palatable characteristics, low iodine content, and its ability to be used in a variety of cooking applications (Wells et al. 2017). This work assessed the thermal acclimation of A. esculenta as a candidate for SVA in Maine, now and in the future.
In the northwestern Atlantic, Alaria esculenta is the dominant subtidal kelp, inhabiting water temperatures from -2 to ~24°C (Adey and Hayek 2011). There is strong, but weakly documented, evidence of range retraction since the late 1950s. While originally found in Long Island Sound and abundant in Massachusetts (Taylor 1957), extensive surveys and inquiries support a range retraction to Cape Ann, northern MA, (pers. obs., Adey and Hayak 2011), highlighting the importance of determining this kelp’s thermal tolerance.
Kelps have a complex life history, alternating between microscopic gametophytes and macroscopic sporophytes (Graham et al. 2016). Mature sporophytes produce basal bladelets called sporophylls that, when ripe, produce zoospores that are released into the environment. Zoospores settle and become either male or female haploid gametophytes. Under the correct conditions, gametophytes produce gametes that fuse to become diploid sporophytes. Investigation of the temperature tolerance of both life stages is necessary because the gametophytes act as seedstock for the commercial sporophyte crop.
Over 71% of coastal sea surface temperatures worldwide are warming significantly, and New England is a hotspot (Lima and Wethey, 2012). Thus, it is necessary to test whether Alaria esculenta can tolerate temperatures predicted for the Gulf of Maine, in the context of the temperature changes that already may have caused a range retraction up the East Coast in the past 50 years. Collection sites were selected to investigate any acclimations or adaptations to changing water temperature that might be useful for strain diversification and demonstration of thermal tolerance. Lubec, ME (30-year SST summer average of 10.9°C; 44.812819, -66.952376) was selected as a representative northern GOM site (northeastern GOM); Two Lights, Cape Elizabeth, ME (30-year SST summer average of 15.4°C; 43.564189, -70.197462), was selected as a representative southern GOM site, matching the range boundary temperatures (same SST as northern MA in the south-western GOM, despite being located in the central-western GOM). All temperature records are from the Satellite Oceanography Data Lab (School of Marine Sciences, University of Maine).
To assess temperature tolerance of the candidate crop, we examined the physiological response of the gametophyte seedstocks to higher temperatures with a gradual thermal acclimation. This response was measured at a cellular level by monitoring filament health, and at a genetic level by measuring variation in gene expression in response to temperature. By including gametophytes produced from Alaria growing at northern and southern sites in Maine, we can assess how health differs between seedstock sources, determine what genes are responsive to temperature, and consider whether populations in Maine have adapted to respond to thermal stress in different ways based on historical temperature exposures. To determine if thermal stress to the microscopic haploid seedstock, often considered the more sensitive of the life stages in kelps (Mueller et al. 2008), affected the future crop yield, these thermally acclimated seedstocks and their corresponding controls were crossed to produce diploid sporophyte blades. We have measured surface areas of juvenile blades from both source populations that were either thermally acclimated or maintained at a control temperature to determine how temperature stress to seedstocks may affect crop yields. Now outplanted into a coastal aquaculture farm, we further hope to determine by early 2018 how temperature history and seed source site may change crop yields.
- FAO 2016. The State of the World Fisheries and Aquaculture 2016. FAO. Rome.
- Wells, M., Potin, P., Craigie, J., Raven, J., Merchant, S., Helliwell, K., Smith, A., Camire, M. & S. H. Brawley. 2017. Algae as nutritional and functional food sources: Revisiting our understanding. Appl.Phycol. 29: 949-982.
- Adey, W. H. and L. A. C. Hayek. 2011. Elucidating marine biogeography with macrophytes: quantitative analysis of the North Atlantic supports the thermogeographic model and demonstrates a distinct subarctic region in the Northwestern Atlantic. Northeastern Naturalist 18:1-128.
- Taylor, W. R. 1957. Marine algae of the northeastern coast of North America. The University of Michigan Press, Ann Arbor.
- Graham, L., Graham, J., Wilcox, L. & M. Cook. 2016. Algae (3rd ).
- Lima, P. and D. S. Wethey. 2012. Three decades of high-resolution coastal sea surface temperatures reveal more than warming. Nature Communications 3:13.
- Müller, R., C. Wiencke, and K. Bischo 2008. Interactive effects of UV radiation and temperature on microstages of Laminariales (Phaeophyceae) from the Arctic and North Sea. Climate Research 37:203-213.
Objective 1: Identify temperature-tolerant seedstock strains of Alaria esculenta gametophytes for Maine sea vegetable aquaculture (SVA). During strain identification, regional differences can be determined.
Objective 2: Determine which temperature-responsive genes are associated with temperature tolerance of seedstocks (i.e. examine differences in gene expression).
Objective 3: Identify temperature-tolerant crop strains of Alaria esculenta sporophytes that demonstrate rapid growth for Maine SVA, examining regional differences, and determine whether temperature tolerance is heritable.
Objective 4: Promote SVA in Maine by attending conferences and forums and working directly with harvesters.
Four reproductively mature sporophytes were collected haphazardly from Two Lights (TL), Cape Elizabeth, ME in the southern Gulf of Maine (GOM), and an additional four sporophytes in Lubec (Lu), ME, in the northern GOM. Sporophylls were treated with a 0.01% betadine solution to eliminate protists and other contaminants and placed into circulating 12°C sterile seawater (4 µmol photons m2/s, 12:12 L:D photoperiod) to obtain zoospores. Zoospore cultures (i.e. TL1, TL2, TL3, TL4, Lu5, Lu6, Lu7, Lu8) were plated into replicate petri dishes and placed in constant light, to encourage zoospore settlement and gametogenesis. Gametophyte cultures from each seedstock were maintained in constant light (4 µmol photons m2/s) with biweekly sterile seawater changes and ¼ strength West-McBride (WES; Anderson, 2005) nutrient supplement until gametophytes reached 10+ cells in length. Replicate dishes from each seedstock were either maintained at 12 C as a control, or underwent a gradual thermal acclimation from 12 to 22 C, with a 1 C increase every 12 hours. Material was then maintained at the final temperature of 22 C for 3 days to understand the lasting effects of temperature. Replicate cultures were assessed for gametophyte health. The same gametophyte filaments were tracked throughout the experiment: filaments needed to maintain constant color and normal cell size to be categorized as healthy. Loss of color and plasmolysis (contraction of cell membrane from the cell wall) indicated stress, and categorized a filament as unhealthy. A complete loss of cell content was categorized as dead. Upwards of 100 gametophytes per replicate petri dish were monitored and categorized daily at an inverted light microscope. We performed a repeated-measures multivariate analysis of variance (MANOVA) in R Statistical Software, version 3.4.1, applying a Greenhouse-Geisser correction for departure from sphericity.
A subsample of material in each treatment from each seedstock was harvested at 12 C, 18 C, and 22 C steps throughout the experiment. Samples were placed in aluminum foil, flash-frozen in liquid nitrogen, and stored in a -80 C freezer for future RNA isolation. RNA was extracted from a subset of cultures (TL2, TL3, Lu6, Lu7, Lu8; two technical replicates each) using the Qiagen RNeasy Plant Mini Kit. RNA quality and quantity were assessed using a NanoDrop One/One Microvolume UV-Vis Spectrophotometer. Samples were sent to the Clinical Genomics Center at the Oklahoma Medical Research Foundation for cDNA library building and high-throughput sequencing. Samples were sequenced on the Illumina HiSeq 3000 platform for 150 bp paired-end reads, with an estimated 60M reads per sample. Sequences were mapped to Alaria esculenta contigs (Jackson et al. unpub.) available on the NCBI database, assembled using Trinity (Grabherr et al. 2011), and annotated using relevant genomes with TopHat (Trapnell et al. 2009). Gene expression levels were categorized as up- or down-regulated, or constantly maintained, with respect to the experimental treatment, and clustered by locus similarity, allowing for the centroid mobility to accommodate loci relations, using custom python scripts. Two Lights samples (TL2, TL3) were established as the baseline for comparison, and Lubec samples’ (Lu6, Lu7, Lu8) expression patterns that differed from the baseline were identified. All transcripts, or putative gene loci, were clustered based on the similarity of these expression patterns. We identified 51,469 unique transcripts, which most certainly include isoforms and alternative splicing transcripts, but annotated transcripts can be investigated as putative loci with functions specific to Alaria esculenta’s thermal response when biological function(s) of assigned annotations is/are considered. Co-regulated loci are now being analyzed, with a focus on heat shock factors and proteins.
After thermal acclimation experiments, cultures were gradually returned to the control temperature of 12 C (1 C decrease every 12 hours). Cultures were maintained in a vegetative state (constant light), for a recovery period of 3 months, with monthly sterile seawater changes and ¼ strength WES nutrient supplement. Control cultures from the same seedstocks were also maintained. To avoid effects of inbreeding, cultures from TL1 and TL2 were combined, blended, and sprayed onto kuralon-wound spools, as was TL3 and TL4; Lu cultures were treated similarly (Lu5 and Lu6, Lu7 and Lu8). Spools were placed under conditions to promote sporogenesis (10 C, 40 µmol photons m2/s, 14:10 L:D photoperiod). Control cultures (TL1/2-Ctrl, TL3/4-Ctrl, Lu5/6-Ctrl, Lu7/8-Ctrl) were also blended, seeded, and placed under the same conditions, so as to compare next-generation sporophyte grow-out based on previous thermal acclimation. Seeded line cultures were grown out for 3 months, with bi-weekly seawater changes and full-strength WES. After three months, sporophyte blades were selected at random to measure blade surface area using ImageJ (www.nih.gov) imaging software. We performed a two-factor analysis of variance to examine the effects of seedstock source location (north versus south) and treatment (previous thermal acclimation versus control).
Materials and Methods References:
- Andersen, R. A.. 2005. Algal Culturing Techniques. Elsevier Academic Press.
- Jackson, C.J., Salomaki, E.D., Lane, C.E., Saunders, G.W. Kelp transcriptomes provide robust support for interfamilial relationships and revision of the little known Arthrothamnaceae (Laminariales): Unpublished NCBI.
- Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I, Adiconis X, Fan L, Raychowdhury R, Zeng Q, Chen Z, Mauceli E, Hacohen N, Gnirke A, Rhind N, di Palma F, Birren BW, Nusbaum C, Lindblad-Toh K, Friedman N, Regev A. Full-length transcriptome assembly from RNA-seq data without a reference genome. Biotechnol. 2011 May 15; 29(7):644-52.
- Trapnell, C.; Pachter, L.; Salzberg, S. L. (16 March 2009). “TopHat: discovering splice junctions with RNA-Seq”. Bioinformatics. 25(9): 1105–1111.
We explored how the factors of treatment (thermal acclimation versus control) and location (Lu versus TL) affected the health of gametophytes over time using a repeated measures MANOVA, where the repeated measure was each day that the health of the gametophytes was assessed. The only significant effect was the treatment factor (p = 0.0032). Interestingly, whether seedstocks were sourced from Lubec or Two Lights did not contribute to gametophyte health (location factor p = 0.364). Sea surface temperature at Two Lights (southern GOM) can reach close to 22 C during the summer months; however, Lubec (northern GOM) barely reaches 18 C (Satellite Oceanography Data Lab, School of Marine Sciences, University of Maine). Regardless of treatment or location effects, a substantial proportion of gametophytes remained healthy through the experiment, suggesting that this critical stage of the A. esculenta life history can acclimate, at least over a few weeks, to temperatures expected to become more common in the Gulf of Maine (Figure 1).
As expected, many patterns of gene expression remained constant across strains and across exposure to elevated temperatures exposures, meaning these genes may be “housekeeping genes” that must be transcribed at all times to support cellular function. In many instances, we identified facultative heat responsive signatures where upregulation corresponded with increases in temperature, across all samples, regardless of source location, suggesting that each strain requires those types of gene expression to tolerate increasing temperature. Interestingly, we also found many instances where Lubec strains (Lu6, Lu7, and Lu8) had upregulated gene expression patterns with increased water temperature, while the Two Lights strains (TL2 and TL3) had upregulation those same pathways up to 18 C, yet maintain that level of expression at 22 C. Further study and analysis will be needed to investigate the functions of these pathways and to understand the differential expression pattern between strains from the two Maine sites.
An important group of genes to investigate are heat shock factors (HSF) and the heat shock proteins (HSP) that they regulate. HSPs are molecular chaperones involved in folding proteins for normal cell function, or refolding denatured proteins that have been damaged in response to stressful conditions, including heat (Buchanan et al. 2015). Overall, southern strains behave the same, as do northern strains. However, certain heat shock signatures appear to be site-specific, meaning that southern strains may already be adapted to respond differently in the warming southern waters (Figure 2). There is a clade of putative genes that respond to thermal stress (upregulation with increase in experimental temperature) only in the strains from the Two Lights site. This clade includes HSP70s, DnaJ (=Hsp40), HSFs 1 and 2, and Hsp33. HSP70s are involved in folding, unfolding, assembly, and disassembly of proteins that often work with cochaperones such as DnaJ (Buchanan et al. 2015). These can be constitutive, but in this case, are responding to thermal stress. HSF1 and 2 are involved in regulation of phosphorylation and protein phosphatase activity to regulate stress response (Buchanan et al. 2015). Hsp33 is a molecular chaperone that reacts to the accumulation of reactive oxygen species (ROS), that can accumulate in response to stress (Merchant et al. 2007). Southern strains employ different molecular strategies to deal with thermal stress, yet no statistically significant differences in health between populations was detected during thermal acclimation experiments up to 22 C. Thus, the gametophytic phase of the life history of Alaria esculenta has the ability to adapt to thermal stress associated with the increasing sea surface temperatures predicted for the GOM, and promises to be suitable for current and future SVA in Maine.
Great value to the industry is associated with understanding the stress response of seedstocks supplied from sites with different annual thermal stress, but it is equally important to assess how thermal stress applied to the haploid gametophytes affects crop yield in the next generation (sporophyte life cycle), and whether it is dependent on seedstock source location. A two-factor ANOVA shows that both location (Lu versus TL) and treatment (thermal acclimation versus control) have a significant effect on juvenile sporophyte blade surface area (location: p =0.00043, treatment: p =1 x 10-8). There is an interaction between factors, meaning that while there is a difference in crop yield between blades that were heat stressed and the controls, those differences are influenced by the source population location. Interestingly, sporophytes grown from northern strains that were exposed to thermal acclimation had the largest blade surface areas (Figure 3). Overall, sporophytes that were thermally acclimated had higher blade surface areas, and those from Lubec had blades up to twice the size of their counterparts from the warmer Two Lights site.
Results and Discussion References:
- Bob B. Buchanan, Wilhelm Gruissem, and Russell L. Jones. 2015. Biochemistry & Molecular Biology of Plants (2nd ed.). John Wiley & Sons, Ltd.
- Sabeeha S. Merchant, Simon E. Prochnik, Oliver Vallon, Elizabeth H. Harris, … Arthur R. Grossman. The Chlamydomonas Genome Reveals the Evolution of Key Animal and Plant Functions. Science 2007 12 October: 245-250
In the State of Maine, seedstocks for Alaria esculenta aquaculture can be collected and cultured from a variety of locations on the Maine coast. Applying a gradual thermal acclimation protocol may select for gametophytes that can withstand moderate heat stress, enabling broader use and handling within the industry.
Gene expression patterns show that Alaria esculenta seedstocks are capable of adapting to warming water; this is evident in the differences in gene expression between northern and southern strains that aren’t seen at a cellular physiology level in thermal acclimation experiments. These differences may be useful in future strain selection, and possibly in algal breeding programs.
Applying our thermal acclimation protocol in sea vegetable nurseries may be able to increase crop yield for farmers if grow-out to mature adults follows results already determined for juvenile sporophytes. This will be determined at harvest in early 2018. Previously acclimated seedstocks produce larger juvenile blades than seedstocks that were maintained at control temperatures. More research is needed to determine how seedstock location may affect sporophyte growth when this protocol is applied and it will be useful to include more Maine sites in following studies.
From this research, we have a better understanding of the temperature tolerance of Alaria esculenta in the warming Gulf of Maine: A. esculenta is an excellent, temperature resilient candidate for sea vegetable aquaculture now and in the future.
Education & Outreach Activities and Participation Summary
I work closely with Laurie Bragg, the Maine EPSCoR Program and Outreach Manager. She is designing education programs for all ages throughout the State that use seaweeds and sea vegetable aquaculture (SVA) as a teaching tool in the classroom. I act as a consultant for some of her projects, and as a seaweeds supplier for classroom activities and classroom kits designed by graduate students in the University of Maine’s Department of Science Education. I also act as a consultant to teachers throughout Maine who wish to use seaweed as a teaching tool. I have been asked to come into the classrooms and teach about SVA using hands-on activities.
One of the major outreach programs that I participate in is EPSCoR’s Seaweed Bootcamp, designed by Laurie Bragg. This program takes place over three days in the summers. Middle school and high school educators come to the University of Maine to learn how to use seaweed as a teaching tool in the classroom. We teach basic biology, ecology, sustainability, basic chemistry, and cooking, all through seaweeds. It is important to show hands-on activities that they can use in the classrooms, and provide write-ups on how to implement them. I demonstrate a basic chemistry activity using sodium alginate that shows how macroalgae are able to stay flexible yet firm in seawater. Sodium alginate is used in cooking, is used to make biodegradable water bottle, and is found in specialty bandages to promote flexible but durable would covering, so it can show how algal products are used in everyday life. The experiment also produces ooey-gooey gel structures that can entertain children of all ages (and adults too!). I create seaweeds snacks so that folks can learn new ways to incorporate sea vegetables into their diets (dulse and garlic popcorn, kelp chocolate cake, miso soup with seaweeds and tofu, etc.). Through this workshop, I produced one educational tool, and reached over 15 educators.
The University of Maine’s Communications and Reporting office published two newsletters highlighting this research. This research was on both the University of Maine’s home webpage, as well as the Maine EPSCoR webpage. They also produced a video of this research, and I was selected for a graduate Student Spotlight on the National EPSCoR website.
I have given tours of both my lab in Orono, Maine showing all of the Alaria esculenta kelp cultures, as well as the Center for Cooperative Aquaculture Research (CCAR, Franklin, Maine). Tour recipients have included visiting State Representatives, high school educators, and members of the National SeaGrant Office (NOAA) and the National Aquarium (Washington, D.C.).
I have been excited about this research and about how it can help different communities. I have participated in and presented at the following conferences, symposia, and summits, so as to reach as many folks as possible:
2014 Northeast Algal Symposium, Newport, RI
2014 UMaine School of Marine Science Graduate Student Symposium, Walpole, ME
2015 Northeast Algal Symposium, Syracuse, NY
2015 41st Annual Maine Fishermen’s Forum, Rockland, ME
2015 Seaweed Interdisciplinary Meeting, University of New England, Biddeford, ME
2015 UMaine School of Marine Science Graduate Student Symposium, Walpole, ME
2015 Northeast Aquaculture Conference & Exposition (NACE) and Milford Aquaculture
Seminar, Portland, ME
2016 2nd Annual Maine Aquaculture R&D Education Summit, Belfast, ME
2016 42nd Annual Maine Fishermen’s Forum, Rockland, ME
2016 Northeast Algal Symposium, Westfield, MA
2016 Sustainable and Ecological Network (SEANET) Stakeholder Advisory Meeting, Belfast, ME
2016 UMaine School of Marine Science Graduate Student Symposium, Walpole, ME
2017 3rd Annual Aquaculture R&D & Education Summit, Belfast, ME
2017 “Big Splash” Aquaculture Exhibit Public Opening, Bangor Discovery Museum, Bangor, ME
2017 Northeast Aquaculture Conference & Exposition, Providence, RI
2017 NSF National EPSCoR Meeting, Missoula, MT
2017 Sustainable and Ecological Network (SEANET) Stakeholder Advisory Meeting, Belfast, ME
In order to have a successful sea vegetable aquaculture (SVA) industry in Maine, it will be important to diversify landings. By introducing Alaria esculenta as a viable candidate crop, this research is enabling the expansion of aquaculture in Maine. This work also demonstrated that this subarctic kelp shows promise in the warming waters of the Gulf of Maine, and that we have gene expression analyses that can act as baseline information for future strain selection.
These efforts can expand the industry, while advocating for local, nutritious, food sources that require no fertilizer, irrigation, or pest control. SVA can offer off-season employment to Maine lobstermen, and can be incorporated onto existing sea farms that grow finfish, mussels, and oysters, providing additional income off the same lease site. Sea vegetable farms can also provide environmental services: Seaweeds are able to absorb excess nutrients (e.g. farm run-off, pollutants) that otherwise may cause plankton blooms, creating anoxic conditions that lead to fish kills on our coasts. Seaweeds also can sequester carbon, possibly aiding in ocean acidification remediation. SVA supports working waterfronts, and can enliven areas that are negatively affected by decreases or closures of certain fisheries, providing opportunities to control landings and harvests, and thus control income and allow coastal communities to thrive.
It is important to recognize how nascent sea vegetable aquaculture (SVA) is in mainstream business endeavors in the US, and how, currently, it can act as a complement to companies practicing wild harvest. Maine has wild harvest regulations that aim to protect resources and protect the marine habitats from which these seaweeds are gathered. I support both wild harvest and aquaculture, because both are currently sustainably practiced in Maine. However, as an advocate for incorporating seaweeds into everyday diets, I recognize the importance of working with and learning from wild harvesters to ensure SVA practices remain sustainable and that resources from both parties are valued. In order to increase production in a sustainable manner, the majority of the expansion must be within aquaculture, but, as a working waterfront community, it is important not to abandon the wild harvesters from whom our knowledge stems. I have learned how to use community outreach endeavors to talk to community members about SVA as a sustainable and ecologically-friendly way to produce more jobs, support coastal communities, diversify landings, and act as an educational tool in STEM programs throughout the State.
I would like to take this knowledge with me to my next endeavors. I aim to continue doing aquaculture research in Maine, but hope to learn more about how social and community aspects can help in these efforts. I am interested in comparing monoculture and polyculture systems that are currently developing along our coastline, to determine whether integrated polyculture is beneficial to increase landings. I’d also like to explore what social, economic, or educational roadblocks may inhibit further expansion of aquaculture in Maine.
To that aim, I secured an NSF EPSCoR fellowship to support these thermal acclimation projects, by supplying my stipend and tuition. I have also secured a modest University of Maine Graduate School Grant to help with transportation costs for my collection trips throughout Maine. With this support, I was able to continue this research and also start collections of microbial samples associated with sea vegetable farms and nearby wild sea vegetable beds, to determine if key microbes are important to aquaculture (e.g. How would growing shallow subtidal organisms in deeper offshore farms affect the microbiome and crop grow-out?). This is the future direction of my aquaculture research.