A ~40-kb flavi-like virus does not encode a known error-correcting mechanism.

It is commonly held that there is a fundamental relationship between genome size and error rate, manifest as a notional "error threshold" that sets an upper limit on genome sizes. The genome sizes of RNA viruses, which have intrinsically high mutation rates due to a lack of mechanisms for error correction, must therefore be small to avoid accumulating an excessive number of deleterious mutations that will ultimately lead to population extinction. The proposed exceptions to this evolutionary rule are RNA viruses from the order Nidovirales (such as coronaviruses) that encode error-correcting exonucleases, enabling them to reach genome lengths greater than 40 kb. The recent discovery of large-genome flavi-like viruses (Flaviviridae), which comprise genomes up to 27 kb in length yet seemingly do not encode exonuclease domains, has led to the proposal that a proofreading mechanism is required to facilitate the expansion of nonsegmented RNA virus genomes above 30 kb. Herein, we describe a ~40 kb flavi-like virus identified in a Haliclona sponge metatranscriptome that does not encode a known exonuclease. Structural analysis revealed that this virus may have instead captured cellular domains associated with nucleic acid metabolism that have not been previously found in RNA viruses. Phylogenetic inference placed this virus as a divergent pesti-like lineage, such that we have provisionally termed it "Maximus pesti-like virus." This virus represents an instance of a flavi-like virus achieving a genome size comparable to that of the Nidovirales and demonstrates that RNA viruses have evolved multiple solutions to overcome the error threshold.

Artificial light at night and warming impact grazing rates and gonad index of the sea urchin Centrostephanus rodgersii.

Artificial light at night (ALAN) is a growing threat to coastal habitats, and is likely to exacerbate the impacts of other stressors. Kelp forests are dominant habitats on temperate reefs but are declining due to ocean warming and overgrazing. We tested the independent and interactive effects of ALAN (dark versus ALAN) and warming (ambient versus warm) on grazing rates and gonad index of the sea urchin Centrostephanus rodgersii. Within these treatments, urchins were fed either 'fresh' kelp or 'treated' kelp. Treated kelp (Ecklonia radiata) was exposed to the same light and temperature combinations as urchins. We assessed photosynthetic yield, carbon and nitrogen content and C : N ratio of treated kelp to help identify potential drivers behind any effects on urchins. Grazing increased with warming and ALAN for urchins fed fresh kelp, and increased with warming for urchins fed treated kelp. Gonad index was higher in ALAN/ambient and dark/warm treatments compared to dark/ambient treatments for urchins fed fresh kelp. Kelp carbon content was higher in ALAN/ambient treatments than ALAN/warm treatments at one time point. This indicates ocean warming and ALAN may increase urchin grazing pressure on rocky reefs, an important finding for management strategies.

Effect of marine heatwaves and warming on kelp microbiota influence trophic interactions.

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.

Recruitment of a threatened foundation oyster species varies with large and small spatial scales.

Understanding how habitat attributes (e.g., patch area and sizes, connectivity) control recruitment and how this is modified by processes operating at larger spatial scales is fundamental to understanding population sustainability and developing successful long-term restoration strategies for marine foundation species-including for globally threatened reef-forming oysters. In two experiments, we assessed the recruitment and energy reserves of oyster recruits onto remnant reefs of the oyster Saccostrea glomerata in estuaries spanning 550 km of coastline in southeastern Australia. In the first experiment, we determined whether recruitment of oysters to settlement plates in three estuaries was correlated with reef attributes within patches (distances to patch edges and surface elevation), whole-patch attributes (shape and size of patches), and landscape attributes (connectivity). We also determined whether environmental factors (e.g., sedimentation and water temperature) explained the differences among recruitment plates. We also tested whether differences in energy reserves of recruits could explain the differences between two of the estuaries (one high- and one low-sedimentation estuary). In the second experiment, across six estuaries (three with nominally high and three with nominally low sedimentation rates), we tested the hypothesis that, at the estuary scale, recruitment and survival were negatively correlated to sedimentation. Overall, total oyster recruitment varied mostly at the scale of estuaries rather than with reef attributes and was negatively correlated with sedimentation. Percentage recruit survival was, however, similar among estuaries, although energy reserves and condition of recruits were lower at a high- compared to a low-sediment estuary. Within each estuary, total oyster recruitment increased with patch area and decreased with increasing tidal height. Our results showed that differences among estuaries have the largest influence on oyster recruitment and recruit health and this may be explained by environmental processes operating at the same scale. While survival was high across all estuaries, growth and reproduction of oysters on remnant reefs may be affected by sublethal effects on the health of recruits in high-sediment estuaries. Thus, restoration programs should consider lethal and sublethal effects of whole-estuary environmental processes when selecting sites and include environmental mitigation actions to maximize recruitment success.

Seaweeds as holobionts: Current state, challenges, and potential applications.

Seaweeds play a strong ecological and economical role along the world's coastlines, where they support industries (e.g., aquaculture, bioproducts) and essential ecosystem services (e.g., biodiversity, fisheries, carbon capture). Evidence from wild and cultured seaweeds suggests that microorganisms play crucial roles in their health and functioning, prompting the need for considering seaweeds and their microbiome as a coherent entity or "holobiont." Here we show that the number of studies investigating seaweed hosts and their microbiome have increased in the last two decades. This likely reflects the increase in the appreciation of the importance of microbiomes for eukaryotic hosts, improved molecular approaches used to characterize their interactions, and increasing interest in commercial use of seaweeds. However, although increasing, most studies of seaweed holobionts have focused on (i) a few seaweed species of ecological or commercial significance, (ii) interactions involving only bacteria, and (iii) descriptive rather than experimental approaches. The relatively few experimental studies have mostly focused on manipulating abiotic factors to examine responses of seaweeds and their microbiome. Of the few studies that directly manipulated microorganisms to investigate their effects on seaweeds, most were done in laboratory or aquaria. We emphasize the need to move beyond the descriptions of patterns to experimental approaches for understanding causation and mechanisms. We argue that such experimental approaches are necessary for a better understanding of seaweed holobionts, for management actions for wild and cultivated seaweeds, and to better integrate studies of seaweed holobionts with the broader fields of seaweed ecology and biology, which are strongly experimental.

Functional guilds and drivers of diversity in seaweed-associated bacteria.

Comparisons of functional and taxonomic profiles from bacterial communities in different habitats have suggested the existence of functional guilds composed of taxonomically or phylogenetically distinct members. Such guild membership is, however, rarely defined and the factors that drive functional diversity in bacteria remain poorly understood. We used seaweed-associated bacteria as a model to shed light on these important aspects of community ecology. Using a large dataset of over 1300 metagenome-assembled genomes from 13 seaweed species we found substantial overlap in the functionality of bacteria coming from distinct taxa, thus supporting the existence of functional guilds. This functional equivalence between different taxa was particularly pronounced when only functions involved in carbohydrate degradation were considered. We further found that bacterial taxonomy is the dominant driver of functional differences between bacteria and that seaweed species or seaweed type (i.e. brown, red and green) had relatively stronger impacts on genome functionality for carbohydrate-degradation functions when compared to all other cellular functions. This study provides new insight into the factors underpinning the functional diversity of bacteria and contributes to our understanding how community function is generated from individual members.

Disentangling direct vs indirect effects of microbiome manipulations in a habitat-forming marine holobiont.

Host-associated microbiota are critical for eukaryotic host functioning, to the extent that hosts and their associated microbial communities are often considered "holobionts". Most studies of holobionts have focused on descriptive approaches or have used model systems, usually in the laboratory, to understand host-microbiome interactions. To advance our understanding of host-microbiota interactions and their wider ecological impacts, we need experimental frameworks that can explore causation in non-model hosts, which often have highly diverse microbiota, and in their natural ecological setting (i.e. in the field). We used a dominant habitat-forming seaweed, Hormosira banksii, to explore these issues and to experimentally test host-microbiota interactions in a non-model holobiont. The experimental protocols were aimed at trying to disentangle microbially mediated effects on hosts from direct effects on hosts associated with the methods employed to manipulate host-microbiota. This was done by disrupting the microbiome, either through removal/disruption using a combination of antimicrobial treatments, or additions of specific taxa via inoculations, or a combination of thew two. The experiments were done in mesocosms and in the field. Three different antibiotic treatments were used to disrupt seaweed-associated microbiota to test whether disturbances of microbiota, particularly bacteria, would negatively affect host performance. Responses of bacteria to these disturbances were complex and differed substantially among treatments, with some antibacterial treatments having little discernible effect. However, the temporal sequence of responses antibiotic treatments, changes in bacterial diversity and subsequent decreases in host performance, strongly suggested an effect of the microbiota on host performance in some treatments, as opposed to direct effects of the antibiotics. To further test these effects, we used 16S-rRNA-gene sequencing to identify bacterial taxa that were either correlated, or uncorrelated, with poor host performance following antibiotic treatment. These were then isolated and used in inoculation experiments, independently or in combination with the previously used antibiotic treatments. Negative effects on host performance were strongest where specific microbial antimicrobials treatments were combined with inoculations of strains that were correlated with poor host performance. For these treatments, negative host effects persisted the entire experimental period (12 days), even though treatments were only applied at the beginning of the experiment. Host performance recovered in all other treatments. These experiments provide a framework for exploring causation and disentangling microbially mediated vs. direct effects on hosts for ecologically important, non-model holobionts in the field. This should allow for better predictions of how these systems will respond to, and potentially mitigate, environmental disturbances in their natural context.