An examination of the epiphytic nature of Gambierdiscus toxicus, a dinoflagellate involved in ciguatera fish poisoning

Twenty-four specimen of macroalgae were collected in nearshore waters of the island of Hawaii, identified, and maintained to examine how the epiphytic relationship between Gambierdiscus toxicus (isolate BIG12) varied among the macroalgal species. Gambierdiscus cells were introduced to Petri dishes containing 100 g samples of each macroalgal host, which were examined at two, 16, 24, and every 24–72 h thereafter, over a 29-day period. Gambierdiscus proliferated in the presence of some host species (e.g., Galaxaura marginata and Jania sp.), but grew little in the presence of other species (e.g., Portieria hornemannii). Gambierdiscus exhibited high survival rates (>99%) in the presence of Chaetomorpha sp., but died before the end of the experiment (after 21 days) with other host species (e.g., Dictyota and Microdictyon spp.). Gambierdiscus avoided contact with P. hornemannii, but averaged up to 30% attachment with other host species. The numbers of Gambierdiscus cells belonging to one of three classes (alive and attached; alive and unattached; and dead) were determined for each time point. The 24 algal hosts were grouped according to their commonalities relative to these three classes using a Bray-Curtis similarity index, similarity profile (SIMPROF) permutation tests, and Multi-Dimensional Scaling (MDS) analysis (PRIMER 6). The resultant six groupings were used to construct different Gambierdiscus growth profiles for the different algal hosts. Group A is characterized by a preponderance of unattached cells and high mortality rates. Groups B, C, E, and F also displayed high proportions of unattached cells, but mortality either occurred later (Groups B and C) or rates were lower (Groups E and F). Group D had the highest proportion of attached cells. Group E contained three out of the four chlorophyte species, while Group F contained the majority of the rhodophytes. Over 50% of the species in Group F are considered to be palatable, whereas Groups A, B, and C are composed of species that exhibit chemical defenses against herbivory. The results of this study coupled with previous findings indicate that Gambierdiscus is not an obligate epiphyte; it can be free-swimming and found in the plankton. The conditions that lead to changes between epiphytic and planktonic stages need to be better studied in order to determine how they affect Gambierdiscus growth and physiology, connectivity and dispersion mechanisms, and toxin movement up into the foodweb.     

Coral settlement onto Halimeda opuntia: a fatal attraction to an ephemeral substrate?

Degraded reefs with a high abundance of macroalgae usually also have low densities of coral recruits. Few studies, however, have examined whether these algae affect coral larval settlement. This study demonstrates, experimentally, that larvae of the Caribbean coral Favia fragrum can settle on the green alga Halimeda opuntia even when another substrate more suitable for settlement is present. Larval settlement onto experimental substrates was quantified under three treatments: rubble only, rubble plus plastic algal mimic, and rubble plus live H. opuntia. Similar total larval settlement was observed in all treatments. No larvae settled on the algal mimic, but total settlement was similar on the rubble in the first two treatments, showing that the rubble alone offered sufficient substrate for high settlement success. About half the larvae in the live algal treatment settled on H. opuntia instead of on the rubble, showing that larvae did not reject this substrate as they did the algal mimic. This result raises the possibility that corals will settle on some macroalgae when their abundance is high. Most macroalgae, including H. opuntia, are ephemeral substrates unsuitable for post-settlement survival. Such unexpected settlement may therefore have significant consequences for coral recruitment success on algal-dominated reefs.     

Melanothamnus maniticola sp. nov. (Ceramiales,Rhodophyta): an epizoic species evolved for living on the West Indian Manatee

Over 35 macroalgae have been documented growing epizoically on sea turtles, and macroalgae are also known to grow on the West Indian Manatee, but the number and identity of these latter species have not been determined. Analysis of DNA sequences of 12 samples collected from different manatees captured in three areas of Florida indicated that they represented a single undescribed species within the Rhodomelaceae genus Melanothamnus. Morphological analysis revealed Melanothamnus characteristics but also a previously undescribed combination of character states. These include eight to nine, but as many as 11, pericentral cells; heavy cortication restricted to the base of thalli, and a sharp transition between the corticated and ecorticate sections of the thallus; cells surrounding the ostiole being similar in size to the outer pericarp cells immediately below, and robust rhizoids that have no terminal lobes and develop from central axial cell filaments instead of pericentral cells. The unique characteristics of the rhizoids may be evolutionary adaptations for anchoring the thalli to manatee epidermis. This species is described as M. maniticola sp. nov.     

Macroalgal palatability and the flux of ciguatera toxins through marine food webs

The benthic dinoflagellate Gambierdiscus toxicus produces polyether toxins that cause ciguatera fish poisoning in humans. The toxins initially enter food webs when fish forage on macroalgae, or other substrates, hosting this epiphytic dinoflagellate. Population studies of G. toxicus and risk assessments in ciguatera-prone regions often rely on quantifying dinoflagellates on macroalgae. Underlying these studies is the assumption that the algae sampled represent a readily consumable resource equally available for benthic grazers. However, many algal hosts of G. toxicus possess a variety of defenses against grazing, and host–dinoflagellate associations may act as toxin sources or sinks depending on their palatability. Marine macroalgae may tolerate or avoid herbivory by exhibiting fast growth, by having poor nutritional quality, by utilizing spatial or temporal escapes or by using chemical or structural defenses. Thus, rapidly consumed algae that cope with herbivores by growing fast, such as many filamentous turfs, could be responsible for a high toxin flux even at low dinoflagellate densities. In contrast, ubiquitous unpalatable algae with much higher dinoflagellate densities might contribute little to toxin flux, and effectively act as refuges for G. toxicus. To date, G. toxicus has been reported from 56 algal genera, two cyanobacteria, one diatom, and one seagrass; 63% of these contain species that are defended from fish grazing and other grazers via chemical, morphological or structural defenses, by low nutritional quality, or by a combination of defensive strategies. High dinoflagellate densities on unpalatable macroalgae could indicate passive accumulation of cells on undisturbed hosts, rather than population explosions or active toxin sources for food webs. Understanding the flow of ciguatoxins in nature requires consideration of the ecology of both G. toxicus and its algal hosts. The complexity of marine algal–herbivore interactions also has consequences for other benthic dinoflagellates that produce toxins, which accumulate in consumers.     

Unique distribution of epiphytic Neosiphonia harveyi (Rhodomelaceae,Rhodophyta) along sargassacean hosts

Although epiphytism is generally heaviest on older portions of the host thallus and both variation and abundance of epiphytes decrease with proximity to meristematic apices, Neosiphonia harveyi (Bailey) Kim, Choi, Guiry and Saunders was frequently found on upper parts of sargassacean hosts. This study compared density, thallus length and fertility of epiphytic N. harveyi among different regions of Sargassum patens C. Agardh thalli to reveal a unique distribution pattern of this epiphyte. The majority of epiphytic N. harveyi was observed on the upper part of host thalli and was seldom seen near the basal part. Over a 3‐month period, mean epiphyte density was greater on host apices while thallus length and fertility were greater on subapical portions of host thalli, suggesting the colonization by this epiphyte near the apical part of the host. Distribution patterns of this epiphyte were similar among S. patens growing at different depths. Apical portions of S. patens appear to be more suitable substrata for N. harveyi settlement and colonization compared with other portions of the host thallus, regardless of depth.     

Algal contact as a trigger for coral disease

Diseases are causing alarming declines in reef‐building coral species, the foundation blocks of coral reefs. The emergence of these diseases has occurred simultaneously with large increases in the abundance of benthic macroalgae. Here, we show that physical contact with the macroalga Halimeda opuntia can trigger a virulent disease known as white plague type II that has caused widespread mortality in most Caribbean coral species. Colonies of the dominant coral Montastraea faveolata exposed to algal transplants developed the disease whereas unexposed colonies did not. The bacterium Aurantimonas coralicida, causative agent of the disease, was present on H. opuntia sampled close to, and away from diseased corals, indicating that the alga serves as a reservoir for this pathogen. Our results suggest that the spread of macroalgae on coral reefs could account for the elevated incidence of coral diseases over past decades and that reduction of macroalgal abundance could help control coral epizootics.     

DIATOMS AS HOSTS FOR OTHER DIATOMS: OBSERVATIONS FROM SOUTHERN CALIFORNIA

Large epiphytic and epilithic diatom species hosting other diatoms were observed in several fresh‐ and brackish water sites in Southern California. The most commonly encountered hosts were species forming long filaments attached to rocks or macroalgae, Hydrosera whampoensis (Schwarz) Hendey, Melosira varians Agardh, Pleurosira laevis (Ehrenberg) Compere and Terpsinoe musica Ehrenberg. These large diatoms often had smaller diatoms attached, usually to the girdle bands and occasionally to the mucilage pads connecting the cells. For example, cells of T. musica were observed supporting growth of a diverse diatom assemblage composed of species of the genera Achnanthes, Achnanthidium, Amphora, Cocconeis and Tabularia; Synedra sp. was attached to M. varians and B. paxillifer; and Cocconeis placentula was seen on H. whampoensis. Thus, large epiphytic and epilithic diatoms seem to provide suitable sites for attachment of small epiphytic diatom species, and it appears that this phenomenon is more common than previously thought.     

Epiphytic diatom communities on intertidal seaweeds from Iceland

Epiphytic diatom communities on macroalgae from Iceland coastal waters were investigated during July 2005. Ten species of seaweeds have been collected belonging to brown, red and green algae. The analysis of epiphytic diatom community was carried out under scanning electron microscopy. The epiphytic diatom abundances varied from 7 ± 5 to 7524 ± 3491 cells mm⁻². Erect growth forms were the most abundant, representing on average 50% of the total diatoms (Achnanthes cf. brevipes var. parvula, Tabularia investiens, T. fasciculata, Hyalosira cf. delicatula, Gomphoseptatum aestuarii, Pseudogomphonema plinskii), followed by adnate (29%) (Cocconeis stauroneiformis, C. scutellum) and motile forms (21%) (Nitzschia cf. amphibia and Navicula perminuta). Highly branched seaweeds with articulated thallus surface, offering a number of microenvironments to be occupied by the epiphytes, showed a high level of colonization, mainly due to erect and motile diatoms. Flat thalli with smooth surface allowed for the growth of mainly erect diatoms.     

Depth-related variation in epiphytic communities growing on the brown alga Lobophora variegata in a Caribbean coral reef

Lobophora variegata is a dominant macroalga on coral reefs across the Caribbean. Over the last two decades, it has expanded its vertical distribution to both shallow and deep reefs along the leeward coast of the island of Cura?ao, Southern Caribbean. However, the ecological implications of this expansion and the role of L. variegata as a living substratum are poorly known. This study compared epiphytic algal communities on L. variegata blades along two depth transects (6?C40?m). The epiphytic community was diverse with a total of 70 species of which 49 were found directly attached to L. variegata. The epiphytic community varied significantly between blade surface, depth and site. The greatest number of genera per blade was found growing on the underside of the blades regardless of site and depth. Filamentous red algae (e.g. Neosiphonia howei) were commonly found on the upperside of the blades over the whole depth gradient, whereas the underside was mainly colonized by calcifying (e.g. Hydrolithon spp., Jania spp., Amphiroa fragillissima), fleshy red algae (e.g. Champia spp., Gelidiopsis spp., Hypnea spinella) and foliose brown alga (e.g. Dictyota spp.). Anotrichum tenue, a red alga capable of overgrowing corals, was a common epiphyte of both blade surfaces. L. variegata plays an important role as a newly available substratum. Thus, its spread may influence other algal species and studies of benthic macroalgae such as L. variegata should also take into consideration their associated epiphytic algal communities.     

Ontogenetic Variation in Blade Toughness May Contribute to the Spread of Turbinaria ornata Across the South Pacific

Coral reefs are shifting from coral to algal-dominated ecosystems worldwide. Recently, Turbinaria ornata, a marine alga native to coral reefs of the South Pacific, has spread in both range and habitat usage. Given dense stands of T. ornata can function as an alternative stable state on coral reefs, it is imperative to understand the factors that underlie its success. We tested the hypothesis that T. ornata demonstrates ontogenetic variation in allocation to anti-herbivore defense, specifically that blade toughness varied nonlinearly with thallus size. We quantified the relationship between T. ornata blade toughness and thallus size for individual thalli within algal stands (N = 345) on seven fringing reefs along the north shore of Moorea, French Polynesia. We found that blade toughness was greatest at intermediate sizes that typically form canopies, with overall reduced toughness in both smaller individuals that refuge within the understory and older reproductive individuals that ultimately detach and form floating rafts. We posit this variation in blade toughness reduces herbivory on the thalli that are most exposed to herbivores and may facilitate reproduction in dispersing stages, both of which may aid the proliferation of T. ornata.     

Screening of epiphytic dinoflagellates for radical scavenging and cytotoxic activities

In the search for antioxidant substances/radical scavengers and cytotoxic substances, extracts from four species (10 strains) of epiphytic dinoflagellates in laboratory cultures were subjected to screening for production of bioactive metabolites. Assays for antioxi-dants were performed using microsomal lipids prepared from rat livers, which were oxidized with Fe³⁺-nitrilotriacetic acid complex (Fe³⁺-NTA). Generated lipid peroxides were determined using the thiobarbituric acid (TBA) colorimetric method. Screening tests for cytotoxic activity were carried out using P388 leukemic cells of which the survival ratio was assessed using the tetrazolium salt (MTT) method. Extracts from two strains (Gymnodinium sp. and Gambierdiscus toxicus) and those from five strains (two of G. toxicus, two of Coolia monotis, and one of Prorocentrum sp.) were found to contain active constituents for antioxidant activity and for cytotoxic activity, respectively.     

Extracts of North Sea macroalgae reveal specific activity patterns against attachment and proliferation of benthic diatoms: a laboratory study

A variety of macroalgae (Ceramium rubrum, Corallina officinalis, Palmaria palmata, Mastocarpus stellatus, Fucus vesiculosus, Cladophora rupestris, Ulva sp.) were investigated by scanning electron microscopy to visualize epiphytic colonizers. The macroalgae differed in terms of their epiphytic coverage of bacteria, fungi and diatoms. Macroalgae, largely devoid of epiphytic diatoms, were hypothesized to employ effective antifouling means to reduce epiphytic coverage, whilst heavily fouled macroalgae were proposed to lack antifouling strategies. To test these hypotheses from an allelochemical perspective with regard to fouling diatoms, dichloromethane-methanol (1:1) crude extracts of macroalgae were concentrated in dimethylsulfoxide and investigated in diatom attachment and proliferation assays using four benthic diatoms (Nitzschia sp., Navicula phyllepta, Navicula arenaria and Amphora sp.). Algal extracts exhibited a distinct pattern of activity against the test diatoms, suggesting a targeted and selective effect of macroalgal metabolites on individual fouling diatoms. The main outcome of this study was that visual inspection and quantitative categorization of epiphytic colonizers on macroalgal thalli could not be used to predict reliably whether macroalgae employed a chemical defense mechanism.     

Phenology and thallus size in a non-native population of Gracilaria vermiculophylla

Phenology, or seasonal variation in life cycle events, is poorly described for many macroalgal species. We describe the phenology of a non-native population of Gracilaria vermiculophylla whose thalli are free-living or anchored by decorating polychaetes to tube caps. At a site in South Carolina, USA, we sampled 100 thalli approximately every month from January 2014 to January 2015. We assessed the reproductive state and measured thallus size based on wet weight, thallus length, and thallus surface area from herbarium mounts. Because life cycle stage cannot be assigned using morphology, we implemented a PCR assay to determine the life cycle stage—tetrasporophyte, female gametophyte, or male gametophyte—of each thallus. Tetrasporophytes dominated throughout the year, making up 81%–100% of thalli sampled per month. Reproductive tetrasporophytes varied between 0% and 65% of monthly samples and were most common in warm summer months (July through September) when thalli also tended to be larger. The vast majority of the reproductive thalli were worm-anchored and not fixed to hard substratum via a holdfast. Thus, free-living thalli can be reproductive and potentially seed new non-native populations. Given G. vermiculophylla reproduction seems tied closely to temperature, our work suggests phenology may change with climate-related changes in seawater temperatures. We also highlight the importance of understanding the natural history of macroalgae to better understand the consequence of range expansions on population dynamics.     

Development of adventive embryos in cauline leaves of Sargassum macrocarpum (Fucales, Phaeophyta)

Spontaneous formation and development of adventive embryos were observed in cauline leaves of Sargassum macrocarpum in laboratory culture. Semi-spherical swellings, which were 200–250 μm in diameter, arose from the surface of cauline leaves of thalli cultured for 4 months from zygotic embryos. Swellings became cylindrical protuberances and grew into ‘daughter’ thalli with one or two small cauline leaves. These thalli detached from ‘mother’ thalli and attached to the surface of culture vessels by rhizoids produced within 1 week after detachment. Each daughter thallus developed into an individual thallus exhibiting the same morphological processes as zygotic embryos.     

Cell wall polysaccharides are informative in Porphyra species taxonomy

As part of systematic studies of the genus Porphyrain New Zealand, constituent sugar analyses of cell wall polysaccharidesin situ in dry thalli were found to yield data that weretaxonomically informative. Variation in constituent sugar levels betweenspecieswas sufficient in some cases to be useful in species differentiation. Thereproductive state of thallus regions had a significant impact on the levels ofconstituent sugars, whereas storage of dried thalli for eight months had noeffect. Three epiphytic taxa currently classified as species ofPorphyra appear to be incorrectly placed within the genus,as their constituent sugars and the levels of these sugars differed markedlyfrom those of all other species examined.     

Effect of grazing by isopods and amphipods on growth of Ulva spp. (Chlorophyta)

Eutrophication of shallow coastal waters often leads to blooms of macroalgae. Grazing by crustaceans, such as amphipods and isopods, can reduce macroalgal biomass accumulation. At the same time, growth of the macroalgae can be stimulated by epiphyte removal. The role of grazing by isopods and amphipods on Ulva spp. biomass development was investigated in the Veerse Meer, a brackish lagoon situated in the southwest Netherlands. Exclusion of grazing in the field did not stimulate Ulva spp. growth. In fact, growth rates were higher in exclosures that allowed grazers to enter. Edibility tests identified the amphipod Gammarus locusta, and the isopods Idotea chelipes and Sphaeroma hookeri as potential grazers on Ulva spp. However, when epiphytic diatoms were present on the Ulva spp. thalli, Gammarus and Sphaeroma grazed on ephiphytes and not on Ulva tissue. Only Idotea continued to graze on Ulva spp. A laboratory growth experiment revealed a positive effect of Gammarus presence on Ulva spp. growth, probably caused by preferential removal of epiphytic diatoms from the Ulva spp. thalli. The growth stimulation by epiphyte removing grazers such as Gammarus may explain the higher growth rates in the presence of grazers observed in the field. When determining the potential role of invertebrate grazers in controlling macroalgal biomass accumulation, it is important to include an assessment of the epiphyte abundance on the macroalgae, as preferential removal of epiphytes may stimulate growth and thus have the opposite effect.     

Macroalgal Extracts Induce Bacterial Assemblage Shifts and Sublethal Tissue Stress in Caribbean Corals

Benthic macroalgae can be abundant on present-day coral reefs, especially where rates of herbivory are low and/or dissolved nutrients are high. This study investigated the impact of macroalgal extracts on both coral-associated bacterial assemblages and sublethal stress response of corals. Crude extracts and live algal thalli from common Caribbean macroalgae were applied onto the surface of Montastraea faveolata and Porites astreoides corals on reefs in both Florida and Belize. Denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene amplicons was used to examine changes in the surface mucus layer (SML) bacteria in both coral species. Some of the extracts and live algae induced detectable shifts in coral-associated bacterial assemblages. However, one aqueous extract caused the bacterial assemblages to shift to an entirely new state (Lobophora variegata), whereas other organic extracts had little to no impact (e.g. Dictyota sp.). Macroalgal extracts more frequently induced sublethal stress responses in M. faveolata than in P. astreoides corals, suggesting that cellular integrity can be negatively impacted in selected corals when comparing co-occurring species. As modern reefs experience phase-shifts to a higher abundance of macroalgae with potent chemical defenses, these macroalgae are likely impacting the composition of microbial assemblages associated with corals and affecting overall reef health in unpredicted and unprecedented ways.     

Immunological detection of the fungal parasite,Pythium sp.; the causative organism of red rot disease in Porphyra yezoensis

The red rot disease of Porphyra yezoensis Ueda (Rhodophyta) is caused by a parasitic fungus, Pythium sp. To facilitate the detection of this pathogen in infected thalli of P. yezoensis, polyclonal and monoclonal antibodies were prepared. Antibodies were raised against antigen prepared from an isolate of fungal hyphae obtained from red-rot infected thallus of P. yezoensis from Aichi Prefecture. Polyclonal antibody was obtained from the antisera of immunized rabbits. Monoclonal antibody was obtained from the culture supernatant of a hybridoma which had been established by cell fusion between a myeloma cell line and spleen cells of immunized mice. Hyphae were detected by means of indirect fluorescent antibody technique. Titers of polyclonal antibodies obtained were too low to recognize fungal hyphae that had penetrated the thalli of P. yezoensis; however, monoclonal antibody was useful for the detection of fungi that had penetrated algal thalli. The monoclonal antibody was specific for the Pythium sp. from red-rot infected thalli of P. yezoensis from Saga (western Japan) and from Aichi Prefectures (central Japan), but was ineffective for infections from Miyagi Prefecture (northern Japan). It is evident, therefore, that Pythium sp. can give rise to immunologically distinct groups of red rot disease. Based on chemical and enzymatic treatments, the antigenic determinant appeared to localize on the sugar chains of glycoconjugates or the polysaccharides of the hyphal cell wall.     

Phenotypic plasticity and morphological adaptation of Halimeda opuntia (Bryopsidales,Chlorophyta) to light intensity

Plasticity gives plants the ability to adapt their morphology and growth to various environments. Environmental factors can lead to changes in the growth forms of algae. Halimeda opuntia was used as a model organism to determine the effects of highly irradiated and shade conditions in the field and its responses to low light conditions in the laboratory, which are known to be some of the main factors driving its morphological plasticity. To investigate the morphological and anatomical characteristics of the species, a total of 61 characteristics and 54 samples from Thailand, Japan and Indonesia were examined. In addition, we also tested the response under different light conditions in the laboratory. A phylogenetic tree was constructed using the tufA marker to confirm the species identification. We found that the thallus forms, branching patterns and segment shapes and sizes responded to the different light conditions. A brittle thallus network was found under shade conditions with low light intensity, while a compact thallus form was found under highly irradiated conditions. The H. opuntia segment shape was reniform under highly irradiated conditions, but its segments were adapted to be deeply trilobed in the lower thallus and tripartite shape in the upper thallus under shade conditions. The segment size also adapted; the segments in shaded areas were longer than those in sunny areas, which might have helped them increase their thallus height to ensure exposure to the high light habitat. This morphological variation and plasticity expanded our understanding of the range of H. opuntia plasticity present in the field and could help explain why this species is common and successfully colonizes various habitats worldwide.     

PARTICULATE ORGANIC MATTER AS AN ALTERNATIVE NUTRIENT SOURCE FOR TROPICAL SARGASSUM SPECIES (FUCALES, PHAEOPHYCEAE)

Large Fucales are abundant on coastal coral reefs of the Great Barrier Reef, but are often limited by the availability of inorganic nutrients. Particle loads in these reef waters are high, which is generally perceived as detrimental for aquatic plants due to a reduction of light. Here, I provide evidence that several abundant Sargassum species supplement their nutrient supply with nutrients derived from the layer of particulate matter (PM) deposited on their thalli. In experiments involving removal or addition of PM, growth rates of Sargassum spp. were up to 180% higher when particles were present on the thalli. Tissue nitrogen and phosphorus levels of thalli with a surface PM layer were 10%–30% higher than those of thalli without PM. The amount of PM deposited in situ on thalli of five species of brown algae ranged from 0.6 to 0.9 g C_org·g⁻¹ dry weight alga, depending on the species' morphology. I suggest that a nutrient-rich diffusive boundary layer is created on the thallus surface by an epiphytic microbial community that remineralizes the particulate nutrients. When algal growth is nutrient limited, the use of particle-derived nutrients as a source alternative to nutrients in the water column may outweigh any potential adverse effects of the thallus particle layer.