Alexandra Campbell

Global habitat degradation has prompted an urgent need for efficient restoration strategies at appropriate spatial scales. “Applied nucleation” is a restoration technique used in terrestrial forests, which starts with planting small vegetation patches that set the trajectory for natural propagation and recovery, resulting in large-scale restoration outcomes that require fewer resources. This concept could provide a framework for the restoration of marine seaweed forests, which are declining at unprecedented scales. We used the loss and 13-year restoration efforts of crayweed (Phyllospora comosa) forests along 70 km of Sydney’s coastline, Australia, to determine the feasibility and challenges of applied nucleation in marine forests. After transplanting events at 14 sites, 43% of transplanted sites led to the establishment and expansion of crayweed, covering ~19,000 m2 along Sydney’s coastline. Recruitment 9 months post-transplantation was negatively associated with grazing and positively related to survival of transplanted adults—a source of propagules and canopy cover in the short-term. In the longer term, crayweed expansion was positively associated with other canopy-forming seaweed species, suggesting canopy provision as an important factor influencing recovery. Small-scale efforts, such as applied nucleation, that consider factors influencing seaweed establishment and expansion can help re-establish marine forests at relevant scales.

Bromoform is the key antimethanogenic bioactive in Asparagopsis species, yet its variation in culture and relationship with other halogenated compounds remain poorly understood. Here, the profile of 9 halogenated compounds in A. taxiformis sporophytes cultured at 0.5 g/L over five weeks were quantified, comparing control cultures to those exposed to gentamicin (10 and 20 mg/L) which temporarily reduced surface bacteria by 81–95 %. Chemical analyses (n = 75) identified three classes of compounds (haloacids, halomethanes and haloacetones) comprising totals of 1–3.3 % dry weight (dw). Haloacids represented half the total; dibromoacetic acid (DBAA, 31.4 %), tribromoacetic acid (TBAA, 10 %) and bromochloroacetic acid (1.3 %). Halomethanes accounted for 39.5 % with bromoform (36.4 %) the single most abundant compound at 0.44–1.38 % dw. DBAA and TBAA concentrations reached 0.47–0.82 % and 0.13–0.38 % dw, respectively. Haloacetones contributed 10.7 % of the total, positively correlating with five other halogenated compounds. However, DBAA and TBAA concentrations correlated more closely to growth, cell morphology and bacterial densities than to other halogenated compounds. Overall, there was a temporal shift in growth rates for both antibiotic treatments – initially suppressing growth by 10–25 % before stabilising with all treatments converging to a specific growth rate of ~8.5 % day−1 by week 5. Extending the cultures for an additional 10 weeks confirmed no long-term impacts of antibiotic treatments on growth, halogenated compounds or the composition of surface bacterial community. This study is the first comprehensive evaluation of halogenated compound loads in cultured Asparagopsis, identifying TBAA as a major component that may contribute to antimethanogenic activity in ruminant feed.

The farming of the red seaweed Asparagopsis taxiformis is expanding due to its ability to mitigate methane emissions from livestock when supplemented into cattle feed, with increasing focus on developing land-based, recirculating aquaculture systems to enhance production. The exudates of Asparagopsis are allelopathic towards a range of organisms; however, there remains a lack of data on the potential autotoxicity of exudate-derived metabolites from the A. taxiformis holobiont, which may have implications on the outcomes of land-based A. taxiformis aquaculture. To address this research gap, we conducted an omics-wide examination of conspecific allelopathy in the holobiont of A. taxiformis tetrasporophytes. Following a 5-day cultivation in seawater pre-conditioned with its own exudates, the growth rate of tetrasporophytes was dramatically reduced at some stocking densities compared to those in control seawater without pre-existing A. taxiformis exudates. An untargeted exometabolomic analysis identified that various peptide and amino acid-based compounds, alkaloids, terpenoids, and fatty acids were at least three to four times more abundant in cultures that experienced reduced growth, suggesting these compounds may be associated with autotoxicity-like effects. Furthermore, in cultures experiencing reduced growth, we detected increased expression of stress-related genes involved in the production and scavenging of reactive oxygen species (e.g. vanadium-dependent haloperoxidases, animal heme peroxidases and manganese superoxide dismutase). Altogether, our study provides the first characterisation of A. taxiformis secreted molecules, some of which may be considered autotoxicity candidates and further emphasises that regular water exchange or ultrafiltration is vital for successful cultivation of A. taxiformis in closed or low-exchange systems.

In plants and animals, the microRNA (miRNA) class of small regulatory RNA plays an essential role in controlling gene expression in all aspects of development, to respond to environmental stress, or to defend against pathogen attack. This well-established master regulatory role for miRNAs has led to each protein-mediated step of both the plant and animal miRNA pathways being thoroughly characterized. Furthermore, this degree of characterization has led to the development of a suite of miRNA-based technologies for gene expression manipulation for fundamental research or for use in industrial or medical applications. In direct contrast, molecular research on the miRNA pathway of macroalgae, specifically seaweeds (marine macroalgae), remains in its infancy. However, the molecular research conducted to date on the seaweed miRNA pathway has shown that it shares functional features specific to either the plant or animal miRNA pathway. In addition, of the small number of seaweed species where miRNA data is available, little sequence conservation of individual miRNAs exists. These preliminary findings show the pressing need for substantive research into the seaweed miRNA pathway to advance our current understanding of this essential gene expression regulatory process. Such research will also generate the knowledge required to develop novel miRNA-based technologies for use in seaweeds. In this review, we compare and contrast the seaweed miRNA pathway to those well-characterized pathways of plants and animals and outline the low degree of miRNA sequence conservation across the polyphyletic group known as the seaweeds.

The red seaweed Asparagopsis taxiformis (Bonnemaisoniaceae, Rhodophyta) produces a bioactive natural product, bromoform, which, when fed to ruminant livestock, can eradicate methane emissions. However, to cultivate enough A. taxiformis to produce a yield that would have a meaningful impact on global greenhouse gas emissions, we need to advance our current understanding of the biology of this seaweed species. Here, we used both a domesticated diploid tetrasporophyte (>1.5 years in culture) and wild samples to establish a high-quality draft nuclear genome for A. taxiformis (lineage 6 based upon phylogenetic analyses using the cox2-3 spacer). The constructed nuclear genome is 142 Mb in size (including 70.67% repeat regions) and was determined to encode for approximately 10,474 protein-coding genes, including those associated with secondary metabolism, photosynthesis, and defence. To obtain information regarding molecular differences between cultured and wild tetrasporophytes, we further explored differential gene expression relating to their different growth environments. Cultured tetrasporophytes, which contained a relatively higher level of bromoform compared to wild tetrasporophytes, demonstrated an enrichment of regulatory factors, such as protein kinases and transcription factors, whereas wild tetrasporophytes were enriched for the expression of defence and stress-related genes. Wild tetrasporophytes also expressed a relatively high level of novel secretory genes encoding proteins with von Willebrand factor A protein domains (named rhodophyte VWAs). Gene expression was further confirmed by proteomic investigation of cultured tetrasporophytes, resulting in the identification of over 400 proteins, including rhodophyte VWAs, and numerous enzymes and phycobiliproteins, which will facilitate future functional characterisation of this species. In summary, as the most comprehensive genomic resource for any Asparagopsis species, this resource for lineage 6 provides a novel avenue for seaweed researchers to interrogate genomic information, which will greatly assist in expediating production of Asparagopsis to meet demand by both aquaculture and agriculture, and to do so with economic and environmental sustainability.

Epiphytic bacteria can play a crucial role in regulating seaweed traits, yet their specific contributions to both host growth and natural products remain unclear for most seaweeds. Here, we explored the effects of manipulating the surface-associated bacteria on the growth and halogenated compounds of two domesticated strains of Asparagopsis taxiformis sporophytes under intensive culture. Under standard conditions, sporophytes had an average surface bacterial density of 1 per 32 µm2. Following five different antibiotic treatments at 10 mg L−1 for 24 h (i.e., penicillin, streptomycin, kanamycin, gentamicin, and vancomycin), bacterial densities were significantly reduced depending on the antibiotic, averaging 1 per 90 µm2. Antibiotics treatment led to 22–89% reductions in bacterial density compared to controls after 2 weeks, with gentamicin and streptomycin most effective (86–89% reductions to ~ 1 per 260 µm2), followed by vancomycin (77%), kanamycin (23%) and penicillin (22%). We examined the impacts on growth and the concentration of two major halogenated compounds, bromoform and dibromoacetic acid, with a combined concentration of 1–2% dry weight. Our results highlight that the antibiotic treatments with the lowest bacterial densities (gentamicin and streptomycin) had higher growth rates than the control (> 50% increase). We observed a negative impact on the bromoform and dibromoacetic acid concentrations, with 42% and 35% reductions, respectively, dependent on strain and antibiotic. Overall, our study demonstrates that the epiphytic bacteria in A. taxiformis cultures can be manipulated to enhance seaweed biomass production, but there may be a trade off in natural product levels in some culture lines.

Aquaculture of the red seaweed genus Asparagopsis is rapidly expanding due to its potential to mitigate enteric methane emissions from cattle rumen. These developments include sea-based culture of gametophytes and land-based culture of tetrasporophytes. Successful establishment of land-based aquaculture relies on providing species-specific optimal conditions, including stocking density of the culture. However, an understanding of the gene expression across the Asparagopsis holobiont at different stocking densities is lacking. Here, we applied a meta-transcriptomic approach to investigate the gene expression of Asparagopsis taxiformis tetrasporophytes and their associated microbiome at different stocking densities ranging from 0.18 g FW L−1 to 5 g FW L−1. Overall, the specific growth rate of A. taxiformis decreased as stocking density increased (from 10.7 % day−1 to −0.2 % day−1). The highest density was characterised by downregulation of photosynthesis-related genes and upregulation of some putative defense-related genes in the seaweed. The microbial transcript abundance was positively correlated to stocking density, with prominent activity of Actinomycetes as seaweed growth rate slowed. Additionally, several microbial virulence factors such as OmpA were upregulated in the highest seaweed density. Altogether, our transcriptomic data suggested the onset of senescence in A. taxiformis cultured at the highest stocking density, although the appearance of the filaments was visibly unaffected. This “hidden” senescence was accompanied by a shift to virulence in the associated microbiome. The meta-transcriptome could therefore be used as a tool to monitor system health for commercial Asparagopsis farms. This work also demonstrates the potential of employing omics to assess optimal culture conditions of any seaweed species.

The range-expansion of tropical herbivores due to ocean warming can profoundly alter temperate reef communities by overgrazing the seaweed forests that underpin them. Such ecological interactions may be mediated by changes to seaweed-associated microbiota in response to warming, but empirical evidence demonstrating this is rare. We experimentally simulated ocean warming and marine heatwaves (MHWs) to quantify effects on two dominant temperate seaweed species and their microbiota, as well as grazing by a tropical herbivore. The kelp Ecklonia radiata's microbiota in sustained warming and MHW treatments was enriched with microorganisms associated with seaweed disease and tissue degradation. In contrast, the fucoid Sargassum linearifolium's microbiota was unaffected by temperature. Consumption by the tropical sea-urchin Tripneustes gratilla was greater on Ecklonia where the microbiota had been altered by higher temperatures, while Sargassum's consumption was unaffected. Elemental traits (carbon, nitrogen), chemical defences (phenolics) and tissue bleaching of both seaweeds were generally unaffected by temperature. Effects of warming and MHWs on seaweed holobionts (host plus its microbiota) are likely species-specific. The effect of increased temperature on Ecklonia's microbiota and subsequent increased consumption suggest that changes to kelp microbiota may underpin kelp-herbivore interactions, providing novel insights into potential mechanisms driving change in species' interactions in warming oceans.

Seaweed farming is the single largest aquaculture commodity with >30 million tonnes produced each year. Furthermore, the restoration of lost seaweed forests is gaining significant momentum, particularly for kelps in warming temperate areas. Whether in aquaculture settings, following restoration practices, or in the wild, all seaweeds undergo biotic interactions with a diverse range of co-occurring or cocultured organisms. To date, most research assessing such biotic interactions has focused on the response of the organism interacting with seaweeds, rather than on the seaweeds themselves. However, understanding how seaweeds respond to other organisms, particularly on a molecular scale, is crucial for optimizing outcomes of seaweed farming or restoration efforts and, potentially, also for the conservation of natural populations. In this systematic review, we assessed the molecular processes that seaweeds undergo during biotic interactions and propose priority areas for future research. Despite some insights into the response of seaweeds to biotic interactions, this review specifically highlights a lack of characterization of biomolecules involved in the response to chemical cues derived from interacting organisms (four studies in the last 20 years) and a predominant use of laboratory-based experiments conducted under controlled conditions. Additionally, this review reveals that studies targeting metabolites (70%) are more common than those examining the role of genes (22%) and proteins (8%). To effectively inform seaweed aquaculture efforts, it will be crucial to conduct larger scale experiments simulating natural environments. Also, employing a holistic approach targeting genes and proteins would be beneficial to complement the relatively well-established role of metabolites.Seaweed farming is the single largest aquaculture commodity with >30 million tonnes produced each year. Furthermore, the restoration of lost seaweed forests is gaining significant momentum, particularly for kelps in warming temperate areas. Whether in aquaculture settings, following restoration practices, or in the wild, all seaweeds undergo biotic interactions with a diverse range of co-occurring or cocultured organisms. To date, most research assessing such biotic interactions has focused on the response of the organism interacting with seaweeds, rather than on the seaweeds themselves. However, understanding how seaweeds respond to other organisms, particularly on a molecular scale, is crucial for optimizing outcomes of seaweed farming or restoration efforts and, potentially, also for the conservation of natural populations. In this systematic review, we assessed the molecular processes that seaweeds undergo during biotic interactions and propose priority areas for future research. Despite some insights into the response of seaweeds to biotic interactions, this review specifically highlights a lack of characterization of biomolecules involved in the response to chemical cues derived from interacting organisms (four studies in the last 20 years) and a predominant use of laboratory-based experiments conducted under controlled conditions. Additionally, this review reveals that studies targeting metabolites (70%) are more common than those examining the role of genes (22%) and proteins (8%). To effectively inform seaweed aquaculture efforts, it will be crucial to conduct larger scale experiments simulating natural environments. Also, employing a holistic approach targeting genes and proteins would be beneficial to complement the relatively well-established role of metabolites.

Interactions between hosts and their microbiota are vital to the functioning and resilience of macro-organisms. Critically, for hosts that play foundational roles in communities, understanding what drives host-microbiota interactions is essential for informing ecosystem restoration and conservation. We investigated the relative influence of host traits and the surrounding environment on microbial communities associated with the foundational seaweed Phyllospora comosa. We quantified 16 morphological and functional phenotypic traits, including host genetics (using 354 Single Nucleotide Polymorphisms) and surface-associated microbial communities (using 16S rRNA-gene amplicon sequencing) from 160 individuals sampled from eight sites spanning Phyllospora's entire latitudinal distribution (1300 km). Combined, these factors explained 54% of the overall variation in Phyllospora's associated microbial community structure, much of which was related to the local environment (~32%). We found that putative "core" microbial taxa (i.e. present on all Phyllospora individuals sampled) exhibited slightly higher association with host traits when compared to "variable" taxa (not present on all individuals). We identified several key genetic loci and phenotypic traits in Phyllospora that were strongly related to multiple microbial amplicon sequence variants, including taxa with known associations to seaweed defense, disease and tissue degradation. This information on how host-associated microbial communities vary with host traits and the environment enhances our current understanding of how "holobionts" (hosts plus their microbiota) are structured. Such understanding can be used to inform management strategies of these important and vulnerable habitats.