Evidence of local and regional declines in the canopy-forming alga Ericaria amentacea, a foundation species of diverse marine forest communities on exposed Mediterranean coasts, have spurred restoration efforts focused on sustainable ex-situ techniques. The need to balance the costs of culture maintenance and the susceptibility of early life stages to stressors in the native habitat, including rapid, often extreme shifts in temperature, hydrodynamics and nutrient availability, have driven current efforts to create a culture environment that primes seedlings for outplant, increasing their resilience rather than maximizing growth. We tested the effects of 1) higher culture temperature (25 degrees C) combined with wave simulation and 2) reduced nutrient loads (10% of standard protocol) with wave simulation on post-culture and post-outplant outcomes relative to optimal growth conditions in established protocols (20 degrees C, no waves, high-nutrient culture medium). While increased temperature and water motion negatively affected seedling growth in culture, and higher nutrients caused oxidative stress likely associated with enhanced epiphyte overgrowth, these effects were not clearly translated into patterns of long-term growth in the field. Instead, survival in the initial days post-outplant appeared to be the bottleneck for restoration potential, where substrates with persisting seedlings at one month were generally found with flourishing juveniles at four months. Larger clumps of seedlings, in turn, were strongly associated with both initial survival and future growth. These results underscore the importance of the zygote settlement phase to establish high seedling densities, which may be optimized by phenological monitoring of the donor population. They also suggest that less-controlled, more environmentally-realistic culture conditions involving the introduction of mild stress may enhance the survival of early life stages of E. amentacea during the transition to the native environment, providing a means to simultaneously reduce human resource costs in culture and move toward scaling up.

1. Restoration of foundation species in historical habitat may be difficult if adult facilitation is obligatory for survival of early life stages. 2. On intertidal Mediterranean coasts, large-scale loss of the dominant forest-forming macroalga Ericaria amentacea have prompted restoration efforts using recruits. Yet, early life stages may be more susceptible to the abiotic stress that characterizes their habitat. 3. We tested strategies to enhance resilience of lab-cultured juveniles of E. amentacea to environmental stress in historical habitat lacking conspecifics. Juveniles were exposed in culture to fluctuations of the dominant physical stressors, irradiance and temperature, and then outplanted in upper and lower zones of their native intertidal range. 4. Without adult canopy, juvenile outplant survival was limited to the lower tidal range, with nearly complete mortality in the upper zone. Survival was also strongly determined by spatial clumping of recruits within the outplant substrate. Longer-term growth in the lower zone was enhanced by fluctuating mild stress in culture, with variable irradiance and concurrent heat pulses increasing post-outplant cover by 40%-60% after 4 months. Clumping also promoted growth across experimental treatments. 5. Synthesis and applications. Reliance on self-facilitation feedbacks is a common barrier to foundation species restoration in high-stress habitats. Our results suggest that without adult habitat amelioration, environmental stress limits recruit survival in intertidal algal forest. Yet, exposure to transient, low environmental stress in culture and outplanting of clumped individuals may confer resilience and allow successful establishment of early life stages in zones of reduced abiotic stress, providing a source for gradual colonization of more high-stress areas.

Ciguatoxins (CTXs), potent neurotoxins produced by dinoflagellates of the genera Gambierdiscus and Fukuyoa, accumulate in commonly consumed fish species, causing human ciguatera poisoning. Field collections of Pacific reef fish reveal that consumed CTXs undergo oxidative biotransformations, resulting in numerous, often toxified analogs. Following our study showing rapid CTX accumulation in flesh of an herbivorous fish, we used the same laboratory model to examine the tissue distribution and metabolization of Pacific CTXs following long-term dietary exposure. Naso brevirostris consumed cells of Gambierdiscus polynesiensis in a gel food matrix over 16 weeks at a constant dose rate of 0.36 ng CTX3C equiv g−1 fish d−1. CTX toxicity determination of fish tissues showed CTX activity in all tissues of exposed fish (eight tissues plus the carcass), with the highest concentrations in the spleen. Muscle tissue retained the largest proportion of CTXs, with 44% of the total tissue burden. Moreover, relative to our previous study, we found that larger fish with slower growth rates assimilated a higher proportion of ingested toxin in their flesh (13% vs. 2%). Analysis of muscle extracts revealed the presence of CTX3C and CTX3B as well as a biotransformed product showing the m/z transitions of 2,3-dihydroxyCTX3C. This is the first experimental evidence of oxidative transformation of an algal CTX in a model consumer and known vector of CTX into the fish food web. These findings that the flesh intended for human consumption carries the majority of the toxin load, and that growth rates can influence the relationship between exposure and accumulation, have significant implications in risk assessment and the development of regulatory measures aimed at ensuring seafood safety.

Coral reefs are increasingly shifting to algal dominance after disturbances. While herbivory that controls algal biomass may influence the stability of coral versus algal states, most reefs are overfished. Despite this, little is known about the contribution of smaller fishes to herbivory, especially in algal versus coral post-disturbance habitats, where differences in structural complexity may also influence herbivory. After coral loss driven by a predator outbreak in Mo'orea, French Polynesia, the benthic community comprised a mosaic patchwork of remnant coral, the brown macroalga Turbinaria ornata on the reef flat, and turf algae (< 1 cm filaments) along the reef slope. We partitioned fish herbivory by size by deploying palatable macroalgae within cages with variable opening sizes in coral versus algal habitats on a reef flat and a reef slope. Herbivory was similar to 2x higher in Turbinaria habitats than coral habitats on the reef flat, while herbivory in turf and coral habitats on the slope were similar. The only measurable herbivory in coral habitats on the reef flat occurred in open treatments that allowed access by the biggest fishes, while smaller fishes were more important in Turbinaria habitats, evidenced by consumption of algae in cages with smaller openings. Equal or higher herbivory in algal habitats compared to coral may indicate an important feedback post-disturbance, and contribute to the rapid recovery of Mo'orean reefs.

Two predominant anthropogenic impacts on ecosystems, nutrient enrichment and the removal of consumers, are predicted to interact in their effects on producer diversity. Yet, measures of diversity alone may not capture changes occurring in the underlying mechanisms structuring communities. Furthermore, evidence for these interactions in rocky intertidal systems is mixed and may be confounded by variable baseline productivity or short experimental durations that do not capture seasonality, environmental heterogeneity or successional processes. We conducted a 2-year experiment examining the main and interactive effects of nutrients and herbivores in a low-productivity environment on the northern shore of Cook Strait near Wellington, New Zealand. We explicitly accounted for the small-scale spatial environmental heterogeneity characteristic of rocky shores. Rapid and dramatic shifts in diversity metrics and cover of algal communities in the first year were driven by removal of herbivores. Within 2 months of herbivore exclusion, open space decreased from >90% to <15%, regardless of nutrient addition, and both algal diversity and evenness plummeted while species richness rose. In contrast to theoretical predictions, strong interactions between top-down and bottom-up forces on diversity were rare; rather, over time, the addition of nutrients negatively impacted diversity. Further, where herbivores were removed, nutrients inhibited succession beyond ephemeral forms, contrasting with the tolerance model of succession observed under ambient nutrients. The effects of nutrients were only elucidated by accounting for variation in habitat complexity and seasonality, demonstrating that environmental heterogeneity can mask bottom-up processes. Moreover, shifts in relative species abundances revealed that after 10 months, nutrient addition caused increased community turnover regardless of herbivore presence or absence, indicating reduced stability not apparent in simple measures of diversity. Synthesis. Our results suggest that nutrient addition and herbivore reduction exert strong but mostly independent control on community biodiversity, where context dependency in habitat complexity, seasonality and successional stage played key roles in determining community outcomes. These findings demonstrate the importance of longer-term field studies incorporating environmental context and multiple metrics of diversity to reveal the mechanisms underlying effects of nutrient enrichment and herbivore loss on producer community structure.

Foundation species facilitate associated communities and provide key ecosystem functions, making anthropogenically driven phase-shifts involving these species critically important. One well-documented such phase-shift has been from coral to algal domination on tropical reefs. On South Pacific coral reefs, the macroalga Turbinaria ornata has expanded its range and habitat but, unlike algae that often dominate after phase-shifts, T. ornata is structurally complex and generally unpalatable to herbivores. Therefore, it may serve a foundational role on coral reefs, such as providing habitat structure to more palatable primary producers and corresponding trophic support to fishes. We predicted increasing T. ornata density would facilitate growth of associated algae, resulting in a positive trophic cascade to herbivorous fish. An experiment manipulating T. ornata densities showed a unimodal relationship between T. ornata and growth of understory algae, with optimal growth occurring at the most frequent natural density. Epiphyte cover also increased with density until the same optimum, but remained high with greater T. ornata densities. Foraging by herbivorous fishes increased linearly with T. ornata density. An herbivore exclusion experiment confirmed T. ornata facilitated epiphytes, but resource use of epiphytes by herbivores, though significant, was not affected by T. ornata density. Therefore, T. ornata performs foundational roles because it provides novel habitat to understory and epiphytic macroalgae and trophic support to consumers, though likely this function is at the expense of the original foundational corals.

Ciguatera poisoning is caused by the consumption of reef fish or shellfish that have accumulated ciguatoxins, neurotoxins produced by benthic dinoflagellates of the genera Gambierdiscus or Fukuyoa. Although ciguatera constitutes the primary cause of seafood intoxication in Cuba, very little information is available on the occurrence of ciguatoxins in the marine food web and the causative benthic dinoflagellate species. This study conducted on the south-central coast of Cuba reports the occurrence of Gambierdiscus and Fukuyoa genera and the associated benthic genera Ostreopsis and Prorocentrum. Gambierdiscus/Fukuyoa cells were present at low to moderate abundances depending on the site and month of sampling. This genus was notably higher on Dictyotaceae than on other macrophytes. PCR analysis of field-collected samples revealed the presence of six different Gambierdiscus and one Fukuyoa species, including G. caribaeus, G. carolinianus, G. carpenteri, G. belizeanus, F. ruetzleri, G. silvae, and Gambierdiscus sp. ribotype 2. Only Gambierdiscus excentricus was absent from the eight Gambierdiscus/Fukuyoa species known in the wider Caribbean region. Eleven clonal cultures were established and confirmed by PCR and SEM as being either G. carolinianus or G. caribaeus. Toxin production in each isolate was assessed by a radioligand receptor binding assay and found to be below the assay quantification limit. These novel findings augment the knowledge of the ciguatoxin-source dinoflagellates that are present in Cuba, however further studies are needed to better understand the correlation between their abundance, species-specific toxin production in the environment, and the risk for fish contamination, in order to develop better informed ciguatera risk management strategies.

In Cuba, ciguatera poisoning associated with fish consumption is the most commonly occurring non-bacterial seafood-borne illness. Risk management through fish market regulation has existed in Cuba for decades and consists of bans on selected species above a certain weight; however, the actual occurrence of ciguatoxins (CTXs) in seafood has never been verified. From this food safety risk management perspective, a study site locally known to be at risk for ciguatera was selected. Analysis of the epiphytic dinoflagellate community identified the microalga Gambierdiscus. Gambierdiscus species included six of the seven species known to be present in Cuba (G. caribaeus, G. belizeanus, G. carpenteri, G. carolinianus, G. silvae, and F. ruetzleri). CTX-like activity in invertebrates, herbivorous and carnivorous fishes were analyzed with a radioligand receptor-binding assay and, for selected samples, with the N2A cell cytotoxicity assay. CTX activity was found in 80% of the organisms sampled, with toxin values ranging from 2 to 8 ng CTX3C equivalents g−1 tissue. Data analysis further confirmed CTXs trophic magnification. This study constitutes the first finding of CTX-like activity in marine organisms in Cuba and in herbivorous fish in the Caribbean. Elucidating the structure–activity relationship and toxicology of CTX from the Caribbean is needed before conclusions may be drawn about risk exposure in Cuba and the wider Caribbean.

Essential nutrients are critical for physiological processes of organisms. In fish, they are obtained primarily from the diet, and their transfer and accumulation are known to be impacted by environmental variables such as water temperature, pH and salinity, as well as by diet composition and matrices. Yet, prey items consumed by fish may also contain toxic compounds such as marine toxins associated with harmful algae. These biotoxins have the potential to affect essential trace element assimilation in fish through chemical interactions such as the formation of trace element-toxin complexes or by affecting general fish physiology as in the modification of ion specific transport pathways. We assessed the influence of dietary exposure to brevetoxins (PbTxs), ichthyotoxic neurotoxins produced by the dinoflagellate Karenia brevis, on trophic transfer of two essential trace elements, Mn and Zn, in a fish model. Using ecologically relevant concentrations of PbTxs and trace elements in controlled laboratory conditions, juvenile turbots Scophthalmus max-Onus were given food containing PbTxs before or at the same time as a feeding with radiotracers of the chosen essential elements (Mn-54 and Zn-65). Treatments included simultaneous exposure (PbTxs + Mn-54 + Zn-65) in a single-feeding, 3-week daily pre-exposure to dietary PbTx followed by a single feeding with Mn-54 and Zn-65, and a control (Mn-54 and Zn-65 only). After a 21-day depuration period, turbot tissue brevetoxin levels were quantified and assimilation efficiencies of Mn-54 and Zn-65 were assessed. PbTxs were found in turbot tissues in each exposure treatment, demonstrating dietary trophic transfer of these toxins; yet, no differences in assimilation efficiencies of Mn or Zn were found between treatments or the control (p > 0.05). These results indicate that, in our experimental conditions, PbTx exposure does not significantly affect the trophic transfer of Mn and Zn in fish.

Marine organisms are exposed to and affected by a multitude of chemicals present in seawater and can accumulate in their tissues a wide range of contaminants as well as natural biotoxins associated with harmful algal blooms (HABs). Trace elements and biotoxins may modify physiological functions in exposed organisms, and studies have been conducted to better understand their respective kinetics and effects in marine species. Despite the increasing concern of concurrent toxic HABs and pollution events due to anthropogenic pressures and global change, very little information is available on their combined effects. Chemical interactions between biotoxins and trace elements have been reported, and exposure to certain biotoxins is known to modify ion transport pathways, suggesting that biotoxins have the potential to alter trace element uptake. Using specific and sensitive radiotracer techniques (radioligand receptor binding assay and γ-spectrometry), this laboratory study examined the influence of pre-exposure to the brevetoxins (PbTxs)-producing microalgae Karenia brevis on the bioaccumulation of selected non-essential (Cd) and essential (Co, Mn and Zn) trace elements in the blue mussel Mytilus edulis. PbTxs are a group of neurotoxins known to accumulate in bivalves but also to have lethal effects on a number of marine organisms including fish and mammals. We found that, over 23 days exposure to the radiotracers, the bioaccumulation of the dissolved essential trace elements Co, Mn and Zn in M. edulis was not significantly affected by pre-exposure to toxic K. brevis. In contrast, the uptake rate constant ku of Cd was significantly higher in the pre-exposed group (p < 0.05), likely caused by a decrease in mussel clearance rates after K. brevis exposure. These results suggest that the effects of algal toxin exposure on bioaccumulation of trace elements in mussels may be trace element-dependent.

Ciguatoxins (CTXs) are potent algal toxins that cause widespread ciguatera poisoning and are found ubiquitously in coral reef food webs. Here we developed an environmentally-relevant, experimental model of CTX trophic transfer involving dietary exposure of herbivorous fish to the CTX-producing microalgae Gambierdiscus polynesiensis. Juvenile Naso brevirostris were fed a gel-food embedded with microalgae for 16 weeks (89 cells g-1 fish daily, 0.4 μg CTX3C equiv kg-1 fish). CTXs in muscle tissue were detectable after 2 weeks at levels above the threshold for human intoxication (1.2 ± 0.2 μg CTX3C equiv kg-1). Although tissue CTX concentrations stabilized after 8 weeks (∼3 ± 0.5 μg CTX3C equiv kg-1), muscle toxin burden (total μg CTX in muscle tissue) continued to increase linearly through the end of the experiment (16 weeks). Toxin accumulation was therefore continuous, yet masked by somatic growth dilution. The observed CTX concentrations, accumulation rates, and general absence of behavioural signs of intoxication are consistent with field observations and indicate that this method of dietary exposure may be used to develop predictive models of tissue-specific CTX uptake, metabolism and depuration. Results also imply that slow-growing fish may accumulate higher CTX flesh concentrations than fast-growing fish, which has important implications for global seafood safety.

Ciguatoxins are algal toxins responsible for tens of thousands of human intoxications yearly, both in tropical and subtropical endemic regions as well as worldwide through fish exportation. Previously developed methods for biotoxin surveillance in the environment and seafood include analytical methods and in vivo and in vitro bioassays. The radioligand receptor binding assay (r-RBA) is among the in vitro methodologies currently used for the detection and quantification of marine biotoxins. For the ciguatoxin group, the r-RBA has been widely used as a means to characterize the mode of action and as detection method in various biological matrices. Yet, screening methods have not been standardized, and the details of the ciguatoxin-specific r-RBA are not well-documented, which limit interlaboratory comparison and progress toward method validation. This work presents the development of an optimized r-RBA for ciguatoxins and provides guidance on its use and quality control checks for analysis of environmental samples. We focus on the analysis of critical parameters involved in determining assay acceptability. Calculation of toxin concentrations in fish samples is illustrated with four examples. Thus, this paper provides the detailed information required for a full validation of the r-RBA, a necessary step toward the development and implementation of a regulatory monitoring programme for ciguatoxins in seafood products using the r-RBA.

As blooms of harmful algae continue to increase worldwide, the consequential risk for contamination of seafood with algal toxins is also growing. Mounting efforts to develop methods for efficient, reliable detection and quantification of these biotoxins have led to advancement of various chemical and analytical techniques, as well as different functional bioassays. One such bioassay, the radioligand receptor-binding assay (RBA), is based on the principle of competition for common receptor-binding sites between a toxin ligand labelled with radioactive hydrogen atoms and the toxin present in an environmental sample. This method has been optimized into a high-throughput microplate format for three important groups of neurotoxins (saxitoxins (STXs), brevetoxins and ciguatoxins (CTXs)) and was recently adopted as an AOAC Official Method of Analysis for the saxitoxins. As a functional assay that measures composite toxic potency, the radioligand RBA is a particularly useful tool for analysis of seafood samples containing a melange of known and/or unknown toxin analogues, as commonly occurs with chemically diverse toxin families such as STXs and CTXs. Moreover, the radioligand RBA can be used not only for sample screening and toxin quantification but also for research purposes including determination of receptor structure/function and target molecules as well as analysis of structure–activity relationships and toxic-equivalency factors among individual toxin analogues. Finally, the radioligand RBA has the potential to support biotoxin risk assessment and regulatory action.

Herbivores balance resource requirements with predation risk, which can differ among landscapes; hence, landscape can shape these trade-offs, influencing herbivore distribution and behavior. While this paradigm has been well established on coral-dominated reefs, tropical reefs worldwide are shifting to algal dominance. If herbivores avoid algae due to higher risk and forage in coral, these algal states may be stabilized. However, if herbivores forage more in resource-rich algal states, this may promote coral recovery. We assessed the distribution and behavior of herbivorous fishes in Moorea, French Polynesia in coral and algal turf-dominated fringing reef sites. Acanthuridae were more abundant in coral states and Labridae, tribe Scarinae, in algal turf states, though total fish abundances were equivalent in the two states. Fish in both families spent more time feeding in algal states and hiding/swimming in coral states. Thus, behavior reflects the trade-off between resource acquisition and refuge in these two landscapes and may promote recovery to coral.

Despite increasing concern that sediment loads from disturbed watersheds facilitate algal dominance on tropical reefs, little is known of how sediments interact with two primary drivers of algal communities, nutrients and herbivory. We examined the effects of sediment loads on the thalli of two increasingly abundant genera of macroalgae, Galaxaura and Padina, in a bay subject to terrestrial sediment influx in Mo’orea, French Polynesia. Field experiments examining (1) overall effects of ambient sediments and (2) interacting effects of sediments (ambient/removal) and herbivores (caged/uncaged) demonstrated that sediments had strong but opposite effects on both species’ biomass accumulation. Sediment removal increased accumulation of Padina boryana Thivy 50% in the initial field experiment but had no effect in the second; rather, in a novel interaction, herbivores overcompensated for increases in tissue nutrient stores that occurred with sediments loads, likely by preferential consumption of nutrient-rich meristematic tissues. Despite negative effects of sediments on biomass, Padina maintained rapid growth across treatments in both experiments. In contrast, positive growth in Galaxaura divaricata Kjellman only occurred with ambient sediment loads. In mesocosm experiments testing interactions of added nutrients and sediments on growth, Galaxaura grew at equivalent rates with sediments (collected from thalli on the reef) as with additions of nitrate and phosphate, suggesting sediments provide a nutrient subsidy. For Padina, however, the only effect was a 50% reduction in growth with sediment. Overall, retention of thallus sediments creates a positive feedback that Galaxaura appears to require to sustain net growth, while Padina merely tolerates sediments. These results indicate that sediments can modify nutrient and herbivore control of algae in ways that differ among species, with the potential for strong and unexpected effects on the abundance and composition of tropical reef macroalgae.

Summary Humans have a long history of changing species' ranges and habitat distributions, making studies of the ecological processes that may facilitate these changes of key importance, particularly in cases where a primary foundation species is replaced by another, less desirable species. We investigated the impact of nutrients and herbivory on Turbinaria ornata, a secondary foundational macroalga that depends on and likely competes with coral, the primary foundational community. T. ornata is also rapidly expanding in range and habitat across the South Pacific. We conducted (i) a mesocosm experiment assessing relative nutrient limitation, (ii) a field experiment comparing importance of nutrients (+/−) and herbivory (+/−) to biomass accumulation, and (iii) an herbivory assay and toughness test comparing enriched and ambient thalli to assess changes to anti‐herbivory defences. We found no evidence of growth being nutrient limited in T. ornata; rather than stimulating growth, nutrient addition deterred herbivores. However, when physical toughness was removed, enriched algae were preferred, with consumption rates 25‐fold those of unenriched algae. Additionally, enriched thalli were tougher than ambient thalli, suggesting physical defences were bolstered by nutrient enrichment. Synthesis. We found a unique interaction where nutrients inhibit herbivory and facilitate Turbinaria ornata biomass accumulation. While concern is often placed on degradation of foundation species via anthropogenic change, instead here we show that anthropogenic change can facilitate secondary foundation species. This facilitation may allow a secondary foundation species to better compete with primary foundation species.

Nitrogen (N) or phosphorus (P) limits primary productivity in nearly every ecosystem worldwide, yet how limitation changes over time, particularly in connection to variation in environmental drivers, remains understudied. We evaluated temporal and species-specific variability in the relative importance of N and P limitation among tropical macroalgae in two-factor experiments conducted twice after rains and twice after dry conditions to explore potential linkages to environmental drivers. We studied three common macroalgal species with varying ecological strategies: a fast-growing opportunist, Dictyota bartayresiana; and two calcifying species likely to be slower growing, Galaxaura fasciculata and Padina boryana. On the scale of days to weeks, nutrient responses ranged among and within species from no limitation to increases in growth by 20 and 40 % over controls in 3 d with N and P addition, respectively. After light rain or dry conditions, Dictyota grew rapidly (up to ~60 % in 3 d) with little indication of nutrient limitation, while Padina and Galaxaura shifted between N, P, or no limitation. All species grew slowly or lost mass after a large storm, presumably due to unfavorable conditions on the reef prior to the experiment that limited nutrient uptake. Padina and Galaxaura both became nutrient limited 3 d post-storm, while Dictyota did not. These results suggest that differing capabilities for nutrient uptake and storage dictate the influence of nutrient history and thus drive nutrient responses and, in doing so, may allow species with differing ecological strategies to coexist in a fluctuating environment. Moreover, the great variability in species’ responses indicates that patterns of nutrient limitation are more complex than previously recognized, and generalizations about N versus P limitation of a given system may not convey the inherent complexity in governing conditions and processes.