Diverse inorganic carbon uptake strategies in Antarctic seaweeds: Revealing species-specific responses and implications for Ocean Acidification.

Seaweeds are important components of coastal benthic ecosystems along the Western Antarctic Peninsula (WAP), providing refuge, food, and habitat for numerous associated species. Despite their crucial role, the WAP is among the regions most affected by global climate change, potentially impacting the ecology and physiology of seaweeds. Elevated atmospheric CO2 concentrations have led to increased dissolved inorganic carbon (Ci) with consequent declines in oceanic pH and alterations in seawater carbonate chemistry, known as Ocean Acidification (OA). Seaweeds possess diverse strategies for Ci uptake, including CO2 concentrating mechanisms (CCMs), which may distinctly respond to changes in Ci concentrations. Conversely, some seaweeds do not operate CCMs (non-CCM species) and rely solely on CO2. Nevertheless, our understanding of the status and functionality of Ci uptake strategies in Antarctic seaweeds remains limited. Here, we investigated the Ci uptake strategies of seaweeds along a depth gradient in the WAP. Carbon isotope signatures (δ13C) and pH drift assays were used as indicators of the presence or absence of CCMs. Our results reveal variability in CCM occurrence among algal phyla and depths ranging from 0 to 20 m. However, this response was species specific. Among red seaweeds, the majority relied solely on CO2 as an exogenous Ci source, with a high percentage of non-CCM species. Green seaweeds exhibited depth-dependent variations in CCM status, with the proportion of non-CCM species increasing at greater depths. Conversely, brown seaweeds exhibited a higher prevalence of CCM species, even in deep waters, indicating the use of CO2 and HCO3-. Our results are similar to those observed in temperate and tropical regions, indicating that the potential impacts of OA on Antarctic seaweeds will be species specific. Additionally, OA may potentially increase the abundance of non-CCM species relative to those with CCMs.

Tetrasporophytic bias coupled with heterozygote deficiency in Antarctic Plocamium sp. (Florideophyceae, Rhodophyta).

Meiosis and syngamy generate an alternation between two ploidy stages, but the timing of these two processes varies widely across taxa, thereby generating life cycle diversity. One hypothesis suggests that life cycles with long-lived haploid stages are correlated with selfing, asexual reproduction, or both. Though mostly studied in angiosperms, selfing and asexual reproduction are often associated with marginal habitats. Yet, in haploid-diploid macroalgae, these two reproductive modes have subtle but unique consequences whereby predictions from angiosperms may not apply. Along the western Antarctic Peninsula, there is a thriving macroalgal community, providing an opportunity to explore reproductive system variation in haploid-diploid macroalgae at high latitudes where endemism is common. Plocamium sp. is a widespread and abundant red macroalga observed within this ecosystem. We sampled 12 sites during the 2017 and 2018 field seasons and used 10 microsatellite loci to describe the reproductive system. Overall genotypic richness and evenness were high, suggesting sexual reproduction. Eight sites were dominated by tetrasporophytes, but there was strong heterozygote deficiency, suggesting intergametophytic selfing. We observed slight differences in the prevailing reproductive mode among sites, possibly due to local conditions (e.g., disturbance) that may contribute to site-specific variation. It remains to be determined whether high levels of selfing are characteristic of macroalgae more generally at high latitudes, due to the haploid-diploid life cycle, or both. Further investigations of algal life cycles will likely reveal the processes underlying the maintenance of sexual reproduction more broadly across eukaryotes, but more studies of natural populations are required.

Neosuberitenone, a New Sesterterpenoid Carbon Skeleton; New Suberitenones; and Bioactivity against Respiratory Syncytial Virus, from the Antarctic Sponge Suberites sp.

Respiratory syncytial virus (RSV) is a highly contagious human pathogen that poses a significant threat to children under the age of two, and there is a current need for new small molecule treatments. The Antarctic sponge Suberites sp. is a known source of sesterterpenes, and following an NMR-guided fractionation procedure, it was found to produce several previously unreported metabolites. Neosuberitenone (1), with a new carbon scaffold herein termed the 'neosuberitane' backbone, six suberitenone derivatives (2-7), an ansellane-type terpenoid (8), and a highly degraded sesterterpene (9), as well as previously reported suberitenones A (10) and B (11), were characterized. The structures of all of the isolated metabolites including absolute configurations are proposed on the basis of NMR, HRESIMS, optical rotation, and XRD data. The biological activities of the metabolites were evaluated in a range of infectious disease assays. Suberitenones A, B, and F (3) were found to be active against RSV, though, along with other Suberites sp. metabolites, they were inactive in bacterial and fungal screens. None of the metabolites were cytotoxic for J774 macrophages or A549 adenocarcinoma cells. The selectivity of suberitenones A, B, and F for RSV among other infectious agents is noteworthy.

Tongalides, Halogenated Butenolides from an Antarctic Delisea sp. Rhodophyte.

Six new halogenated butenolides, tongalides A-C (1-3) and their acetylated congeners (4-6), were isolated from an extract of the Antarctic rhodophyte Delisea sp. that displayed significant antibiotic activity. The structures of the compounds were determined by analysis of data acquired by spectroscopic and spectrometric techniques including NMR, HRESIMS, optical rotation, and X-ray diffraction studies. The newly isolated compounds were assayed for antibacterial activity, but exhibited no growth inhibition of ESKAPE pathogens. The extract bioactivity was attributed to the previously reported Z-acetoxyfimbrolide A also isolated from the extract, providing further evidence that the exocyclic double bond is essential to the antibacterial activity of the structurally related fimbrolide class of metabolite.

Who Cares More about Chemical Defenses - the Macroalgal Producer or Its Main Grazer?

The consequences of defensive secondary metabolite concentrations and interspecific metabolite diversity on grazers have been extensively investigated. Grazers which prefer certain food sources are often found in high abundance on their host and as a result, understanding the interaction between the two is important to understand community structure. The effects of intraspecific diversity, however, on the grazer are not well understood. Within a single, localized geographic area, the Antarctic red seaweed Plocamium sp. produces 15 quantitatively and qualitatively distinct mixtures of halogenated monoterpenes ("chemogroups"). Plocamium sp. is strongly chemically defended which makes it unpalatable to most grazers, except for the amphipod Paradexamine fissicauda. We investigated differences in the feeding and growth rates of both Plocamium sp. and P. fissicauda, in addition to grazer reproductive output, in relation to different chemogroups. Some chemogroups significantly reduced the grazer's feeding rate compared to other chemogroups and a non-chemically defended control. The growth rate of Plocamium sp. did not differ between chemogroups and the growth rates of P. fissicauda also did not show clear patterns between the feeding treatments. Reproductive output, however, was significantly reduced for amphipods on a diet of algae possessing one of the chemogroups when compared to a non-chemically defended control. Hence, intraspecific chemodiversity benefits the producer since certain chemogroups are consumed at a slower rate and the grazer's reproductive output is reduced. Nevertheless, the benefits outweigh the costs to the grazer as it can still feed on its host and closely associates with the alga for protection from predation.

Hidden Diversity in an Antarctic Algal Forest: Metabolomic Profiling Linked to Patterns of Genetic Diversification in the Antarctic Red Alga Plocamium sp.

The common Antarctic red alga Plocamium sp. is rich in halogenated monoterpenes with known anticancer and antimicrobial properties and extracts of Plocamium sp. have strong ecological activity in deterring feeding by sympatric herbivores. Plocamium sp. collected near Anvers Island, Antarctica showed a high degree of secondary metabolite diversity between separate individuals. GC/MS results revealed 15 different combinations of metabolites (chemogroups) across individuals, which were apparent at 50% or greater Bray-Curtis similarity and also clearly distinguishable by eye when comparing chromatographic profiles of the secondary metabolomes. Sequencing of the mitochondrial cox1 gene revealed six distinct haplotypes, of which the most common two had been previously reported (now referred to as Haplotypes 1 and 2). With the exception of one individual, three of the chemogroups were only produced by individuals in Haplotype 1. All the other 12 chemogroups were produced by individuals in Haplotype 2, with five of these chemogroups also present in one of the four new, less common haplotypes that only differed from Haplotype 2 by one base pair. The functional relevance of this metabolomic and genetic diversity is unknown, but they could have important ecological and evolutionary ramifications, thus potentially providing a foundation for differential selection.

Every Rule Has an Exception: a Cheater in the Community-Wide Mutualism in Antarctic Seaweed Forests.

Dense macroalgal forests on the Western Antarctic Peninsula serve important ecological roles both in terms of considerable biomass for primary production as well as in being ecosystem engineers. Their function within the Antarctic ecosystem has been described as a crucial member of a community-wide mutualism which benefits macroalgal species and dense assemblages of associated amphipod grazers. However, there is a cheater within the system that can feed on one of the most highly chemically defended macroalgal hosts. The amphipod Paradexamine fissicauda has been found to readily consume the finely branched red macroalga Plocamium cartilagineum. This amphipod grazer not only feeds on its host, but also appears to sequester its host's chemical defenses for its own utilization. This review summarizes what we know about both of these exceptions to the community-wide mutualism.

Bioactivity of Spongian Diterpenoid Scaffolds from the Antarctic Sponge Dendrilla antarctica.

The Antarctic sponge Dendrilla antarctica is rich in defensive terpenoids with promising antimicrobial potential. Investigation of this demosponge has resulted in the generation of a small chemical library containing diterpenoid secondary metabolites with bioactivity in an infectious disease screening campaign focused on Leishmania donovani, Plasmodium falciparum, and methicillin-resistant Staphylococcus aureus (MRSA) biofilm. In total, eleven natural products were isolated, including three new compounds designated dendrillins B-D (10-12). Chemical modification of abundant natural products led to three semisynthetic derivatives (13-15), which were also screened. Several compounds showed potency against the leishmaniasis parasite, with the natural products tetrahydroaplysulphurin-1 (4) and dendrillin B (10), as well as the semisynthetic triol 15, displaying single-digit micromolar activity and low mammalian cytotoxicity. Triol 15 displayed the best profile against the liver-stage malaria parasites, while membranolide (5) and dendrillin C (11) were strong hits against MRSA biofilm cultures.

Spongian Diterpenoids Derived from the Antarctic Sponge Dendrilla antarctica Are Potent Inhibitors of the Leishmania Parasite.

From the CH2Cl2 extract of the Antarctic sponge Dendrilla antarctica we found spongian diterpenes, including previously reported aplysulphurin (1), tetrahydroaplysulphurin-1 (2), membranolide (3), and darwinolide (4), utilizing a CH2Cl2/MeOH extraction scheme. However, the extracts also yielded diterpenes bearing one or more methyl acetal functionalities (5-9), two of which are previously unreported, while others are revised here. Further investigation of diterpene reactivity led to additional new metabolites (10-12), which identified them as well as the methyl acetals as artifacts from methanolysis of aplysulphurin. The bioactivity of the methanolysis products, membranoids A-H (5-12), as well as natural products 1-4, were assessed for activity against Leishmania donovani-infected J774A.1 macrophages, revealing insights into their structure/activity relationships. Four diterpenes, tetrahydroaplysulphurin-1 (2) as well as membranoids B (6), D (8), and G (11), displayed low micromolar activity against L. donovani with no discernible cytotoxicity against uninfected J774A.1 cells. Leishmaniasis is a neglected tropical disease that affects one million people every year and can be fatal if left untreated.

Anverenes B⁻E, New Polyhalogenated Monoterpenes from the Antarctic Red Alga Plocamium cartilagineum.

The subtidal red alga Plocamium cartilagineum was collected from the Western Antarctic Peninsula during the 2011 and 2017 austral summers. Bulk collections from specific sites corresponded to chemogroups identified by Young et al. in 2013. One of the chemogroups yielded several known acyclic halogenated monoterpenes (2-5) as well as undescribed compounds of the same class, anverenes B-D (6-8). Examination of another chemogroup yielded an undescribed cyclic halogenated monoterpene anverene E (9) as its major secondary metabolite. Elucidation of structures was achieved through one-dimensional (1D) and 2D nuclear magnetic resonance (NMR) spectroscopy and negative chemical ionization mass spectrometry. Compounds 1-9 show moderate cytotoxicity against cervical cancer (HeLa) cells.

Effects of ocean acidification on the shells of four Mediterranean gastropod species near a CO2 seep.

Marine CO2 seeps allow the study of the long-term effects of elevated pCO2 (ocean acidification) on marine invertebrate biomineralization. We investigated the effects of ocean acidification on shell composition and structure in four ecologically important species of Mediterranean gastropods (two limpets, a top-shell snail, and a whelk). Individuals were sampled from three sites near a volcanic CO2 seep off Vulcano Island, Italy. The three sites represented ambient (8.15pH), moderate (8.03pH) and low (7.73pH) seawater mean pH. Shell mineralogy, microstructure, and mechanical strength were examined in all four species. We found that the calcite/aragonite ratio could vary and increased significantly with reduced pH in shells of one of the two limpet species. Moreover, each of the four gastropods displayed reductions in either inner shell toughness or elasticity at the Low pH site. These results suggest that near-future ocean acidification could alter shell biomineralization and structure in these common gastropods.

Declines in plant palatability from polar to tropical latitudes depend on herbivore and plant identity.

Long-standing theory predicts that the intensity of consumer-prey interactions declines with increasing latitude, yet for plant-herbivore interactions, latitudinal changes in herbivory rates and plant palatability have received variable support. The topic is of growing interest given that lower-latitude species are moving poleward at an accelerating rate due to climate change, and predicting local interactions will depend partly on whether latitudinal gradients occur in these critical biotic interactions. Here, we assayed the palatability of 50 seaweeds collected from polar (Antarctica), temperate (northeastern Pacific; California), and tropical (central Pacific; Fiji) locations to two herbivores native to the tropical and subtropical Atlantic, the generalist crab Mithraculus sculptus and sea urchin Echinometra lucunter. Red seaweeds (Rhodophyta) of polar and temperate origin were more readily consumed by urchins than were tropical reds. The decline in palatability with decreasing latitude is explained by shifts in tissue organic content along with the quantity and quality of secondary metabolites, degree of calcification or both. We detected no latitudinal shift in palatability of red seaweeds to crabs, nor any latitudinal shifts in palatability of brown seaweeds (Phaeophyta) to either crabs or urchins. Our results suggest that evolutionary pressure from tropical herbivores favored red seaweeds with lower palatability, either through the production of greater levels of chemical defenses, calcification, or both. Moreover, our results tentatively suggest that the "tropicalization" of temperate habitats is facilitated by the migration of tropical herbivores into temperate areas dominated by weakly defended and more nutritious foods, and that the removal of these competing seaweeds may facilitate the invasion of better-defended tropical seaweeds.

Darwinolide, a New Diterpene Scaffold That Inhibits Methicillin-Resistant Staphylococcus aureus Biofilm from the Antarctic Sponge Dendrilla membranosa.

A new rearranged spongian diterpene, darwinolide, has been isolated from the Antarctic Dendroceratid sponge Dendrilla membranosa. Characterized on the basis of spectroscopic and crystallographic analysis, the central seven-membered ring is hypothesized to originate from a ring-expansion of a spongian precursor. Darwinolide displays 4-fold selectivity against the biofilm phase of methicillin-resistant Staphylococcus aureus compared to the planktonic phase and may provide a scaffold for the development of therapeutics for this difficult to treat infection.

Structure and Function of Macroalgal Natural Products.

Since the initial discovery of marine phyco-derived secondary metabolites in the 1950s there has been a rapid increase in the description of new algal natural products. These metabolites have multiple ecological roles as well as commercial value as potential drugs or lead compounds. With the emergence of resistance to our current arsenal of drugs as well as the development of new chemotherapies for currently untreatable diseases, new compounds must be sourced. As outlined in this chapter algae produce a diverse range of chemicals many of which have potential for the treatment of human afflictions.In this chapter we outline the classes of metabolites produced by this chemically rich group of organisms as well as their respective ecological roles in the environment. Algae are found in nearly every environment on earth, with many of these organisms possessing the ability to shape the ecosystem they inhabit. With current challenges to climate stability, understanding how these important organisms interact with their environment as well as one another might afford better insight into how they respond to a changing climate.

The immediate wound-induced oxidative burst of Saccharina latissima depends on light via photosynthetic electron transport.

Reactive oxygen species (ROS) produced by an oxidative burst are an important component of the wound response in algae, vascular plants, and animals. In all taxa, ROS production is usually attributed solely to a defense-related enzyme like NADPH-oxidase (Nox). However, here we show that the initial, wound-induced oxidative burst of the kelp Saccharina latissima depends on light and photosynthetic electron transport. We measured oxygen evolution and ROS production at different light levels and in the presence of a photosynthetic inhibitor, and we used spin trapping and electron paramagnetic resonance as an orthogonal method. Using an in vivo chemical probe, we provide data suggesting that wound-induced ROS production in two distantly related and geographically isolated species of Antarctic macroalgae may be light dependent as well. We propose that electron transport chains are an important and as yet unaddressed component of the wound response, not just for photosynthetic organisms, but for animals via mitochondria as well. This component may have been obscured by the historic use of diphenylene iodonium, which inhibits not only Noxes but also photosynthetic and respiratory electron transport as well. Finally, we anticipate physiological and/or ecological consequences of the light dependence of macroalgal wound-induced ROS since pathogens and grazers do not disappear in the dark.

Chemical mediation of mutualistic interactions between macroalgae and mesograzers structure unique coastal communities along the western Antarctic Peninsula.

Hard bottom communities along the western Antarctic Peninsula region are dominated by thick macroalgal forests, which support high densities of mesograzers, particularly amphipods, and also numerous gastropods. The macroalgae are chemically defended from consumption by the mesograzers and other herbivores and they provide the mesograzers a chemically defended refuge from predation by omnivorous fish. The macroalgae benefit in return because the mesograzers remove epiphytic algae from them. Since these two assemblages are major components of the community, this can be viewed as a community-wide mutualism. Most subcomponents of these interactions have also been documented in lower latitude communities and the similarities and differences between the communities in Antarctica and in other regions are discussed.

Reactive oxygen species and the Antarctic macroalgal wound response.

Reactive oxygen species (ROS) are commonly produced by algal, vascular plant, and animal cells involved in the innate immune response as cellular signals promoting defense and healing and/or as a direct defense against invading pathogens. The production of reactive species in macroalgae upon injury, however, is largely uncharacterized. In this study, we surveyed 13 species of macroalgae from the Western Antarctic Peninsula and show that the release of strong oxidants is common after macroalgal wounding. Most species released strong oxidants within 1 min of wounding and/or showed cellular accumulation of strong oxidants over an hour post-wounding. Exogenous catalase was used to show that hydrogen peroxide was a component of immediate oxidant release in one of five species, but was not responsible for the entire oxidative wound response as is common in vascular plants. The other component(s) of the oxidant cocktail released upon wounding are unknown. We were unable to detect protein nitration in extracts of four oxidant-producing species flash frozen 30 s after wounding, but a role for reactive nitrogen species such as peroxynitrite cannot be completely ruled out. Two species showed evidence for the production of a catalase-activated oxidant, a mechanism previously known only from the laboratory and from the synthetic drug isoniazid used to kill the human pathogen Mycobacterium tuberculosis. The rhodophyte Palmaria decipiens, which released strong oxidants after wounding, also produced strong oxidants upon grazing by a sympatric amphipod, suggesting that oxidants are involved in the response to grazing.

Site-specific variability in the chemical diversity of the Antarctic red alga Plocamium cartilagineum.

Plocamium cartilagineum is a common red alga on the benthos of Antarctica and can be a dominant understory species along the western Antarctic Peninsula. Algae from this region have been studied chemically, and like "P. cartilagineum" from other worldwide locations where it is common, it is rich in halogenated monoterpenes, some of which have been implicated as feeding deterrents toward sympatric algal predators. Secondary metabolites are highly variable in this alga, both qualitatively and quantitatively, leading us to probe individual plants to track the possible link of variability to genetic or other factors. Using cox1 and rbcL gene sequencing, we find that the Antarctic alga divides into two closely related phylogroups, but not species, each of which is further divided into one of five chemogroups. The chemogroups themselves, defined on the basis of Bray-Curtis similarity profiling of GC/QqQ chromatographic analyses, are largely site specific within a 10 km² area. Thus, on the limited geographical range of this analysis, P. cartilagineum displays only modest genetic radiation, but its secondary metabolome was found to have experienced more extensive radiation. Such metabogenomic divergence demonstrated on the larger geographical scale of the Antarctic Peninsula, or perhaps even continent-wide, may contribute to the discovery of cryptic speciation.