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Sea lettuce (Ulva) is a genus of green macroalgae present along all the coasts of the world's oceans. It represents about 100 species with diverse habitats. Inter- and intra-species natural variation is very large, both in terms of growth characteristics and biomass biochemical composition. As a result, Ulva biomass has a wide range of applications and strain selection can achieve significant increases in yield(s). Establishing solid, long term and cost-effective methodologies for the conservation of Ulva genetic diversity is then required to safeguard and reuse selected strains. Here, we report a cryopreservation-based protocol for the long-term preservation of foliose Ulva strains. Strains from seven different Ulva species were cryopreserved for 15 and/or 120 days in liquid nitrogen, and of the 3 replicates cryopreserved, at least one survived, allowing us to successfully recover all strains. On average, among all specimen cryo-preserved, 82% of them survived and grew post cryo-preservation. [ABSTRACT FROM AUTHOR]

Ulvan lyase is a useful tool which can degrade ulvan into oligosaccharides with a range of biological activities. In this study, the ulvan lyase ULA-3 from Alteromonas sp. 76–1, which was a 1467-bp encoded 489-amino acid residue protein, has been cloned, heterologously expressed and biochemically characterized. ULA-3 showed the maximum activity at 45 ˚C and pH 9.0. The Km is 1.879 mg mL−1 and Vmax is 2.597 µmol min−1 mL−1. Its enzyme activity was outstanding at 11.754 U mg−1. Moreover, its metal ion stability is exceptionally good and most metal ions at a concentration of 1 mM have no inhibitory effect on its activity. It can endolytically degrade ulvan into oligosaccharides with a degree of polymerization (Dp) of 2–4 and with unsaturated double bond through the β-elimination reaction, which mainly cleaves the glycosidic bond between rhamnose and uronic acids. All in all, our study has increased the ulvan lyase database and offers the opportunity to fully utilize ulvan, a green biomass resource from marine algae. [ABSTRACT FROM AUTHOR]

The genus Ulva is globally distributed and has been thoroughly studied because of its functional biochemical composition, rapid growth rates and opportunistic features, and interest in Ulva cultivation is growing worldwide. In Europe, mostly near- and on-shore flow-through cultivation systems are used and land-based recirculating aquaculture systems (RAS) using fresh water or artificial seawater have not been developed for Ulva. While RAS provides quality control and can be located inland, maintenance costs are high. Using selected strains adapted to low-salinity could reduce seawater production costs and improve the economic feasibility. Therefore, our study assessed how salinity can be used as a tool for strain selection and optimization of functional traits. Growth rates and antioxidant activity of three species (four strains) of tubular and foliose Ulva from the NE-Atlantic and Mediterranean (foliose: Ulva lacinulata – two geographical strains, tubular: Ulva linza and Ulva flexuosa) were followed for three weeks at salinities ranging from 10 to 30 PSU. The tubular strains achieved optimal growth at a lower salinity than U. lacinulata. However, growth rates of both foliose strains were higher than of tubular strains, even at sub-optimal salinity. Therefore, U. lacinulata is a good candidate for RAS with artificial seawater, and the cost of salt can be reduced by up to 33.3% (20 PSU) without significantly reducing the growth rate of U. lacinulata. Higher antioxidant activity was achieved by reducing the salinity to 10 PSU for 10 days, suggesting that the functional traits of cultivated Ulva lacinulata can be optimized prior to harvest. [ABSTRACT FROM AUTHOR]

The ongoing climate changes have resulted from high levels of anthropogenic CO2 being released into the atmosphere in the past decades. Carbon-cutting measures have been proposed to reduce global warming by 2 °C, bringing it back to preindustrial levels. There are several options on hand to meet the need for carbon reduction on a global basis, and one such option refers to CO2 sequestration via the photosynthesis of macroalgae. Species of the green seaweed Ulva are ubiquitous throughout the world oceans; they can maintain fast growth during most seasons thanks to their unique photosynthetic capacity combined with high nitrogen and phosphorous uptake rates. High commodity productions of seaweeds, such as green species of Ulva, can also deliver important additional benefits and ecosystem services, including human food, animal feed, proteins, minerals, or active natural molecules, therefore alleviating human needs shortly. Still, whether seaweeds, in general, can trap CO2 for long periods (100 years) is controversial. Technical and biological gaps need to be resolved before an effective and sustainable seaweed-based mass production intended for the sequestration of CO2 is established. This study elaborates on the potential global use of seaweeds in sequestering carbon and addresses the efficient inorganic carbon uptake and fixation traits of Ulva species. This study also overviews the photosynthetic traits and growth of seaweeds in offshore, semi-controlled, and land-based cultivation settings, focusing on total carbon budgets and footprints for Ulva sp. from the Israeli Mediterranean Sea. Altogether this work addresses the potential use of Ulva sp. as a valuable tool to moderate climate changes. [ABSTRACT FROM AUTHOR]

The ubiquitous and species rich genus Ulva comprises entities of green macroalgae with variable morphologies. Ulva species are important from ecological and economic perspectives, but their identification is often problematic. Current knowledge on Ulva diversity has focused mainly on foliose individuals of temperate regions, but genetic and morphological data on tubular species are often insufficient and the species richness is ambiguous due to the lack of molecularly identified type vouchers. Together with a previous study, our study demonstrates that due to the crypticity of tubular entities of the genus Ulva present in the Atlantic-Baltic Sea transect, certain species remained undetected until recently whereas molecular evidence of other historically identified species is missing. An entity which appears to be a relatively frequent species in the Atlantic-Baltic Sea transect and which was probably mis-identified with other species in the past is here described as Ulva capillata sp. nov.. The description is based on molecular identification using tufA and rbcL sequences, and by comparing the species´ phylogenetic relationships, distribution and range margins in the Atlantic-Baltic Sea transect, as well as on morpho-anatomical characters, and early ontogenetic development. By comparisons with closely related and potentially morphologically overlapping species concepts we were able to identify the uniqueness of U. capillata. Therefore, the description of U. capillata as a new species within the genus Ulva is supported by a combination of molecular, morphological, and ontogentic evidence which confirm their uniqueness in comparison to other species concepts. Furthermore, our results strongly emphasize the importance and necessity to molecularly investigate especially tubular historic type vouchers within the genus Ulva to facilitate a clear species identification to omit continuing with taxonomic confusion and ongoing misapplication of names of e.g. cryptic species concepts within this important green algal genus. [ABSTRACT FROM AUTHOR]

Recent evidence suggests that seaweed extracts can protect plants against microbial disease by upregulating the host's immune system. However, if this upregulation comes at a cost to host vegetative and reproductive growth it will make the use of seaweed extracts in crop disease protection less attractive. In a series of experiments, using Arabidopsis thaliana infected by white rust (Albugo sp.) and treated with crude extracts of Ulva lactuca and Saccharina latissima, we test the hypothesis that a growth-immunity trade-off exists. We include temporal samples of the two seaweed species because the composition of bioactive molecules in them is known to vary over the year. Our results confirm that extracts of both species have the potential to protect against disease, but that both the timing of seaweed sampling and extraction procedures can introduce considerable variation to this effect. Importantly, these seaweed treatments were also associated with reduced host vegetative mass, though treatment with S. latissima could compensate for this as it also stimulated increased seed mass. We conclude that the effects of crude seaweed extracts from U. lactuca and S. latissima on plant fitness are not immediately predictable, and benefits in terms of disease protection may come with costs. [ABSTRACT FROM AUTHOR]

Sulfated polysaccharides derived from green marine macroalgae are known for their wide range of therapeutic properties, including the mitigation of inflammatory disorders. A sulfated polysaccharide, URP-2, composed of (1 → 4) linked α-rhamnopyranose, β-xylopyranose, and β-glucuropyranose, was isolated from Ulva reticulata (Ulvaceae). URP-2 exhibited significant anti-inflammatory effects by increasing interferon (IFN)-α expression (four to ten-fold) in lipopolysaccharide (LPS)-induced CALU-1 cells in a dose-dependent manner (31.25 to 125 μg mL−1), compared to LPS-induced cells. Additionally, the elevated IFN-γ levels observed in LPS-induced cells were substantially reduced (by five-fold) following treatment with URP-2 at 125 μg mL−1. URP-2, at a concentration of 125 μg mL−1, effectively reduced the elevated levels of interleukin (IL)-1β in LPS-induced cells, decreasing it from approximately 95% to 1.3%. Tumor necrosis factor (TNF)-α level was also reduced from 30% to approximately 1% in LPS induced CALU-1 cells. Structure–activity relationship analyses of URP-2 suggest that its potent anti-inflammatory properties are likely attributed to the presence of sulfate groups and (1 → 4) linkages. Therefore, URP-2 demonstrated promising potential as a natural alternative for the treatment of inflammatory-related disorders.Graphical abstract: Sulfated polysaccharides derived from green marine macroalgae are known for their wide range of therapeutic properties, including the mitigation of inflammatory disorders. A sulfated polysaccharide, URP-2, composed of (1 → 4) linked α-rhamnopyranose, β-xylopyranose, and β-glucuropyranose, was isolated from Ulva reticulata (Ulvaceae). URP-2 exhibited significant anti-inflammatory effects by increasing interferon (IFN)-α expression (four to ten-fold) in lipopolysaccharide (LPS)-induced CALU-1 cells in a dose-dependent manner (31.25 to 125 μg mL−1), compared to LPS-induced cells. Additionally, the elevated IFN-γ levels observed in LPS-induced cells were substantially reduced (by five-fold) following treatment with URP-2 at 125 μg mL−1. URP-2, at a concentration of 125 μg mL−1, effectively reduced the elevated levels of interleukin (IL)-1β in LPS-induced cells, decreasing it from approximately 95% to 1.3%. Tumor necrosis factor (TNF)-α level was also reduced from 30% to approximately 1% in LPS induced CALU-1 cells. Structure–activity relationship analyses of URP-2 suggest that its potent anti-inflammatory properties are likely attributed to the presence of sulfate groups and (1 → 4) linkages. Therefore, URP-2 demonstrated promising potential as a natural alternative for the treatment of inflammatory-related disorders. [ABSTRACT FROM AUTHOR]

Mass aquaculture production of the green seaweed Ulva is advancing under the stimulus of many interesting applications. Increasing the efficiency of carbon use in Ulva mass cultures is important to improve its production and diversify biomass applications. Given the high capacity of Ulva species to use bicarbonate as a C source, mass cultures of Ulva ohnoi carried out under natural conditions and controlled pH were stoichiometrically supplied with sodium bicarbonate on demand. The specific growth rate (average 0.108 ± 0.008 day−1) and the biochemical composition of U. ohnoi remained stable over three consecutive production cycles, in which C conversion from added bicarbonate into net produced biomass ranged between 88.5 ± 4.2 and 95.9 ± 0.9%. C conversion was significantly higher (p < 0.05) than conversion of nitrogen (73.4 to 87.4%) and phosphorus (68.7 to 80.2%). By enriching the total bicarbonate at 4% with H13CO3−, the algal isotopic signature δ13C increased from − 7.5 ± 0.38 to 1505 ± 56‰ after 8 days of growth. Uptake of 13C was steady throughout the culturing period and resulted in biomass production estimates very similar to those calculated from the growth rate. The use of bicarbonate in Ulva cultures represents an important improvement in C conversion efficiency. It is also an easy alternative to produce biomass sufficiently enriched in 13C, which is demanded in studies on the nutritional value of algae. Reliable production estimates in mass cultures can be expected from the 13C uptake rate in algae with high bicarbonate utilization capacity. [ABSTRACT FROM AUTHOR]

Ulva sp. is a cosmopolitan green macroalga with fast growth and a well-balanced amino acid composition, making it an interesting potential protein resource. Generally, the protein digestibility in unprocessed raw seaweed is low, which is why extraction and concentration of the protein is needed. During protein extraction from fresh Ulva sp. using double screw pressing, sulfite was added as antioxidant in order to increase protein quality by inhibiting oxidative reactions. By pressing, 13–14% of the total biomass protein was extracted. Isoelectric precipitation at pH 2 resulted in a yield of 36% of the extracted protein corresponding to 5% of the total protein, independent on sulfite treatment. However, size exclusion chromatography showed that sulfite addition resulted in a higher amount of native state Rubisco protein. This was explained by inhibited redox enzyme activity and improved polyphenol levels, suggesting prevention of polyphenol-protein crosslinking, resulting in higher amount of soluble protein and improved protein quality. [ABSTRACT FROM AUTHOR]

The green algal genus Ulva is widely distributed around all continents. Plants with both distromatic or monostromatic thalli now form a single taxon based on ITS rDNA and rbcL gene sequencing. Ulva is known to occupy several ecological niches including freshwater and marine (intertidal and subtidal) habitats, attributed to its tolerance to key determinants such as light, temperature, and salinity. The genus is perceived as model system to study life cycle, morphogenesis and development from simple to complex multicellularity. The life cycle is isomorphic and biphasic type, knowledge of which is important in developing viable cultivation techniques. The culture of Ulva is by photo-bioreactor, land-based, and open-sea farming producing about 1500 t dry annum−1 biomass. The understanding of scientific basis for eutrophication-driven green tide events is of paramount importance for coastal ecosystem management. Studies related to cross-kingdom cross-talk between Ulva and surrounding microbes have been recently undertaken through high-throughput techniques to understand their role in growth, development, and morphogenesis. Several regional species are rich in vital nutrients and thus qualify in the functional food sector, and recent research is poised to develop a bio-refinery model for complete utilization of feedstock. Ulva spp. are also used as a feed source in aquaculture and for environmental bioremediation. [ABSTRACT FROM AUTHOR]

Blue economy, the sustainable and effective use of ocean resources for economic growth, is a major challenge to coastal communities. Marine macroalgae are potentially sustainable feedstock for future food, materials, chemicals, and fuels. For seaweed biorefinery, the fractionation of the biomass to co-produce multiple products is crucial in the efficient valorization of the marine biomass. In this work, we developed a protocol for co-production of six different products from the green macroalga Ulva ohnoi using green extraction methods. A total of 90.31 ± 1.94% of the initial biomass was recovered in separated products. The fraction of the recovered products from initial dry weight biomass was 45.42 ± 1.91% salts, 3.67 ± 1.38% starch, 3.81 ± 1.26% lipids, 13.88 ± 0.40% ulvan, 14.83 ± 1.06% proteins, and 8.70 ± 1.87% cellulose. A potential revenue analyses, based on these experimental data and current market prices, suggests that total the revenue fluctuates between US$1.56 and US$3.93 kg−1 of dry biomass and depends on recovered products fraction in the seaweed biomass and products market value. [ABSTRACT FROM AUTHOR]

The green algal genus Ulva (Ulvophyceae) has been recently perceived as a model organism, due to its tractability for genetic analyses. This biomass is drawing considerable attention as a potential feedstock for energy security, including emerging applications in functional foods, bio-fuels and energy, pharmaceutical, biotechnological industries, etc. Nevertheless, unlike other commercially farmed seaweeds where clonal propagation is practiced, this genus solely relies on motile reproductive cells (zoospores or gametes) for seeding or seedling production. The culture practices encompass photobioreactor, land-based and open sea farming. The knowledge of reproduction is pivotal for efficient seeding technologies, selecting and domesticating fast growing varieties as well as genetic improvement. The successful induction of reproduction in Ulva spp. is being achieved through external environmental stimuli as well as by manipulating sporulation and swarming inhibitor concentration. However, comprehensive biochemical and molecular evidences are underdeveloped and further knowledge is imperative to improve aquaculture practice. This mini-review provides comprehensive insights into life cycle, spore formation, mechanisms for the induction of reproduction, biochemical and molecular aspects of sporulation besides advances in the farming of Ulva spp. with future prospective. The existing commercial trade of Japan about 1500 t dry annum−1 is poised to become the leading industry with focus on niche, high-value products in the coming decade. [ABSTRACT FROM AUTHOR]

The long-term inclusion of 10% seaweed meal (Ulva rigida and Undaria pinnatifida) was evaluated in diets for Senegalese sole (Solea senegalensis). Triplicate groups of fish with an initial body weight of 23 g were fed the experimental diets (ULVA and UNDARIA) and compared to fish fed a control diet with no seaweed included (CTRL), both in juvenile (after 5 months of feeding, up to 60 g) and on-growing stages (after 9 months of feeding, up to 160 g). In the early juvenile phase, the inclusion of seaweeds did not affect fish growth, but final body weight was significantly lower in on-growing fish fed the UNDARIA diet. Overall, growth performance was also reduced in fish fed the UNDARIA diet, with a significantly lower daily growth index. Whole-body composition and nutrient retention (% intake) remained unaffected by the dietary treatment, but by the end of the growth trial fish fed the UNDARIA diet had a significantly lower protein gain associated with significantly lower intestinal villi width. At the end of the experiment, fish fed the UNDARIA diet had a significantly higher iodine flesh content (375.7 μg kg−1) than those fed the CTRL (187.5 μg kg−1) or ULVA (199.3 μg kg−1) diets, whereas selenium content was similar in all groups of fish. In conclusion, U. rigida seems a valid ingredient for Senegalese sole diets without affecting fish growth or nutrient utilization. Undaria pinnatifida was an effective way of naturally fortifying the nutritional value of sole fillets for human consumption, but resulted in growth impairment, so a lower inclusion level should be further evaluated. [ABSTRACT FROM AUTHOR]

Key factors affecting the economic sustainability of any aquaculture industry and in particular the seaweed industry are its ecological interactions and impacts. Understanding these issues requires an extended production analysis and simulation, given the natural variability and dynamics of external factors that affect those interdependencies. As such, making sense of production data is required for suitable planning and resource optimization in a seaweed farm. The present work calculates the required water renewal rates for seaweed flow-through production units, using a novel simple user-friendly nitrogen budget model. The user interface is straightforward and the model parameter inputs and outputs are minimal, whereby the target users are commercial seaweed farmers. The model was parameterized for production kinetics of Ulva spp. based on an extensive literature survey, and was evaluated with published data on seaweed-growing experiments. Results for the estimated number of volume renewals per day are in agreement with the experimental data. The outputs indicate that this application can be used to estimate the envelope, i.e. average lower and upper ranges for water renewal rates for a given production in a given site. This model and corresponding parameterization for Ulva spp. are available to be used by farmers, managers and researchers in the form of a spreadsheet file (available as Supplementary Material). The conceptual model and application presented herein represent the basis for future developments to incrementally increase complexity, regarding additional seaweed species and production settings (in recirculating and integrated multitrophic aquaculture systems) by farmers and planners. [ABSTRACT FROM AUTHOR]

This study evaluated five experimental diets (40 % protein, 11 % lipids): the first diet (FM) was formulated with 10 % fish meal content and no fish oil; the second diet (FO) contained 0.5 % fish oil, while fish meal was totally replaced with poultry by-product meal; the third (DHA) and the fourth (LO) diets were formulated to replace fish oil with 0.5 % microalga oil and 0.5 % linseed oil, respectively; the fifth diet (SLO) used soybean meal and corn gluten as main protein sources, with 0.5 % linseed oil. A conventional, commercial-like diet (Ctrl) included 36 % fish meal and 1.4 % fish oil (36 % protein, 7.5 % lipids). Brown shrimp Farfantepenaeus californiensis juveniles (0.12 g) were stocked into 50-L tanks at ten shrimp tank, and eight feeding treatments were applied: shrimp fed Ctrl diet ad libitum (100 % pelleted feed ration: CtrlA); shrimp fed a 50 % ration of Ctrl diet (CtrlB), FM, FO, DHA, LO, or SLO in presence of fresh Ulva offered ad libitum; and, finally, shrimp fed only fresh Ulva (UC). After the 8-week feeding trial, survival was over 87 %; CtrlA, CtrlB, FM, and SLO treatments presented significantly better growth ( p < 0.05); CtrlB, FM, and SLO improved significantly ( p < 0.05) the feed conversion ratio and protein efficiency ratio by approximately 100 % compared to CtrlA. Results confirm that with a plant-based diet in the presence of live Ulva clathtrata available ad libitum, it is possible to obtain a sustainable shrimp product. [ABSTRACT FROM AUTHOR]

Low-technology practices are generally the rule when cultivating marine macroalgae, and they do not necessarily comply with sustainability requirements. When integrated with other marine organisms in land-based setups, seaweed culture can be sustainable also providing environmental benefits. Major challenges of such integrated aquaculture systems, however, are in sea-based setups. The current study examined the growth rates of Ulva rigida and Gracilaria bursa-pastoris, as well as their protein and carbohydrate contents, when exposed to different distances from an offshore, fed-fish cage system. Nutrient levels in seawater were consistently high downstream from the fish cages, significantly enhancing the specific growth rates and cellular contents of starch and soluble protein in these two seaweeds. Specifically, daily maximal growth rates were 17 % day for U. rigida and 10 % day for G. bursa-pastoris, maximal starch contents were 22 and 21 %, respectively, and maximal protein contents were on a dry weight basis 7 and 13 %, respectively. When repositioned at low ambient nutrient levels for 48 h, the starch and the carbohydrate levels increased by 129 and 131 % for U. rigida and by 198 and 150 % for G. bursa-pastoris, respectively. Altogether, this study supports implementing future viable mean of sustainable macroalgae cultivation by taking advantage of excessive nutrients released from an offshore fish farm. [ABSTRACT FROM AUTHOR]

Integrated multi-trophic aquaculture (IMTA) is being explored on both economic and environmental grounds in many traditional aquaculture regions. To test a variety of suitable macroalgae species and management scenarios, a numerical model is developed to quantify the remediation of dissolved nutrients and production of macroalgae near a nutrient source. Differences in the morphological, physiological, and economic characteristics of different macroalgae species can provide flexibility when considering the cost and benefit of farming macroalgae. Results show that of the three species studied, Macrocystis pyrifera removed 75 % of dissolved inorganic nitrogen (DIN) input from a point source, while Porphyra umbilicalis and Ulva lactuca removed 5 % . Both M. pyrifera and P. umbilicalis have reduced bioremediation capacity at increasing flow rates. U. lactuca showed increased bioremediation potential as flow rate increased from low to moderate flows. Increasing the optical depth increased the bioremediation potential of M. pyrifera for moderate values of the light attenuation coefficient, whereas bioremediation was unaffected by optical depth for both U. lactuca and P. umbilicalis. Harvesting increased bioremediation capacity of all species by up to 25-fold dependent on the establishment phase and harvesting frequency. We conclude that the choice of macroalgae species greatly affects the success of IMTA and that both harvesting and farm arrangements can be used to greatly optimize bioremediation. [ABSTRACT FROM AUTHOR]

Increasing levels of a mixture of Ulva spp. produced in an integrated multi-trophic aquaculture (IMTA) system were evaluated in Nile tilapia juveniles for partial replacement of dietary fish meal. A control diet (CTRL) was compared with three experimental diets containing 10 % (U10), 15 % (U15), and 20 % (U20) of Ulva spp. meal. Triplicate groups of fish (13 g initial body weight) were fed each diet for 63 days at 26 °C. Nutrient apparent digestibility coefficients and nitrogen retention efficiency did not vary significantly among diets. By the end of the trial, all groups of fish more than tripled their initial body weight. Specific growth rate and final body weight of U10 diet were similar to CTRL and significantly higher than U15 and U20 diets. Increasing Ulva dietary incorporation levels significantly increased feed conversion ratio (FCR), from 1.0 (CTRL) to 1.4 (U20). Fish fed with U10 diet had the highest protein efficiency ratio and nitrogen retention efficiency allowing this fish to growth and reach a final body weight similar to the CTRL group. Protein content was highest in fish fed with the CTRL diet, whereas the highest lipid content was observed in fish fed with U20 diet. The results show that the incorporation of IMTA-produced Ulva meal in Nile tilapia diets is possible up to 10 % without compromising growth performance, protein utilization, and protein retention of juveniles. The high capacity of Nile tilapia to digest all experimental diets suggests that Ulva meal is a practical partial replacement for fish meal in Nile tilapia diets. [ABSTRACT FROM AUTHOR]

The aim of this study was to evaluate the suitability of two marine macroalgae, Ulva lactuca (Chlorophyta) and Pterocladia capillacea (Rhodophyta), meals as a supplement to enhance the nutritional value of formulated feeds for European seabass Dicentrarchus labrax fry. Seven isoproteic (50 % crude protein), isocaloric (500 Kcal/100 g gross energy) diets containing four levels (0 or control, 5, 10, and 15 %) of either Ulva meal (UM) or Pterocladia meal (PM) were tested. Each diet was fed to triplicate groups of D. labrax fry (initial body weight, 0.23 ± 0.02 g for Ulva- and 0.14 ± 0.01 g for Pterocladia-fed fish), to apparent visual satiety for 8 weeks. The results indicated that feeding seabass at 5 % UM or PM level (U and P diets) produced the best growth, feed utilization, nutrient retention, and survival rates among all the dietary groups. Feeding fish with a 5 % PM-added diet has also improved stress response after a 5-min air exposure test, prior to the termination of the feeding trial. These findings suggest that both Pterocladia and Ulva meals could be potentially used as an additional feed component (at 5 %) for enhancement of seabass D. labrax fry performance, nutrients composition, and stress resistance especially when subjected to transportation from hatchery to weaning ponds/tanks. [ABSTRACT FROM AUTHOR]

Since 2007, green tides have occurred along the coast of the Yellow Sea, China. The green tide extended to 50,000 km (floating area) within 2-3 months and the calculated covering area was about 400 km in 2010. These facts implied that the growth and reproduction of the dominant species Ulva prolifera were stimulated. We observed that 1 cm blades (single layer) released 2.84-6.62 × 10 spores or 1.14-2.65 × 10 gametes and that 91.6-96.4 % of them germinated into younger seedlings. This means that, in theory, 1 g (fresh weight) of blades was able to produce about 2.8 × 10-2.7 × 10 new younger seedlings. From 2009 to 2011, the growth rate of green tide algae was measured in situ in enclosure experiments in Rudong coast, Jiangsu Province and the growth curve of the algae was divided into four phases: lag phase, accelerated phase, stationary phase, and decline phase. Usually, the average daily specific relative growth rate was about 23.2-23.6 % d for a whole growth period, and it reached up to 56.2 % d in the accelerated phase. Correspondingly, the morphology of green tide algae in enclosures also showed periodic variation as follows: blades presented new filamentous branches from old thallus in the lag phase, longer filamentous branches in the accelerated phase, tubular and cystic blades in the stationary phase, and folded blades in the decline stage. Those studies may be useful for understanding the green tide blooming mechanism. [ABSTRACT FROM AUTHOR]

The South African abalone aquaculture industry is expanding and there is a lack of information on the nutritional profiles of the seaweeds that are used as feed. The current study quantified the protein contents of Ulva rigida and Ulva capensis from natural populations and Ulva lactuca from a commercial, integrated seaweed/abalone aquaculture system. Three methods of protein quantification were used: the Bradford method, crude protein, and specific nitrogen to protein (N-Prot) conversion factors, and results were compared to each other. The results showed that values obtained with the 'traditional' conversion factor of 6.25 were on average higher than Bradford values by factors of 64.1 % in U. capensis, 77.1 % in U. rigida and 58.9 % in U. lactuca. This pattern is in line with other published work on seaweed and microalgae. Analyses of amino acid composition showed that aspartic acid was the most abundant amino acid and that these species were also rich in glycine and alanine but poor in histidine, methionine and cysteine. N-Prot factors were as follows: U. capensis, 5.58; U. rigida, 5.12 and U. lactuca, 5.65. An average N-Prot factor of 5.45 provides a more accurate estimate of the protein content of the Ulva species studied than the 'traditional' factor of N × 6.25. [ABSTRACT FROM AUTHOR]

During the summer of 2008 and 2009, massive algal blooms repeatedly broke out in the Yellow Sea of China. These were undoubtedly caused by the accumulations of one or more species in the macroalgal genus Ulva. In previous reports, morphological observation indicated that the species involved in this phenomenon is Ulva prolifera but molecular analyses indicated that the species belongs to an Ulva linza-procera-prolifera (LPP) clade. Correct identification of the bloom species is required to understand and manage the blooms, but the taxonomic status of the bloom species remains unclear. In the current study, the taxonomic status of 22 selected specimens from the Yellow Sea was assessed by using both morphological and molecular (ITS and rbcL sequences) data. In addition, 5S rDNA analyses were performed for those samples clustering in the LPP clade, and phylogenetic tree and ribotype analyses were constructed for determining the possible origin of the bloom. Three free-floating and two attached Ulva species were distinguished and described: Ulva compressa Linnaeus and Ulva pertusa Kjellman were found in free-floating samples; U. linza Linnaeus was found on rocks; and U. prolifera O.F. Müller was found in both habitats. Diversity in free-floating Ulva of the Yellow Sea appears to be greater than previously thought. The dominant free-floating Ulva species, U. prolifera, was not closely related to local populations attached to rocks but was closely related to populations from Japan. [ABSTRACT FROM AUTHOR]

In this study, homologous cloning coupled with the rapid amplification of cDNA ends was used to clone a full-length cytosolic heat shock protein 70 of Ulva pertusa (designated as UPHsp70). Bioinformatics was used to analyze structural features, homologous relationship, and phylogenetic position of UPHsp70. The full length of UPHsp70 cDNA was 2,283 bp, with a 5′ untranslated region of 65 bp, a 3′ untranslated region of 247 bp, and an open reading frame of 1,971 bp encoding a polypeptide of 656 amino acids with an estimated molecular weight of 71.13 kDa and an estimated isoelectric point of 5.04. The UPHsp70 had four degenerate repeats of tetrapeptide GGMP and three typical Hsp70 signature motifs. The specific C-terminus amino acid sequence of cytosolic UPHsp70 was EEVD, and the conservation of Hsp70s of N-terminus was higher than that of C-terminus. The homology between UPHsp70 and Hsp70s of other known algae and land plants was more than 70%. Under different stress conditions, mRNA expression levels of UPHsp70 were quantified by quantitative reverse transcriptase-polymerase chain reaction. When U. pertusa samples were kept in different temperatures (5-40°C) for 1 h, the expression level of UPHsp70 at 5°C, 35°C, or 40°C was over onefold higher than that at 25°C. When U. pertusa samples were kept at 30°C for different times (0-12 h), the mRNA expression level of UPHsp70 had a trend of rise first then fall. The expression level of UPHsp70 reached maximum level after 5 h. When U. pertusa samples were kept in different salt concentrations (0-45‰) for 2 h, the expression level of UPHsp70 at 0‰ or 5‰ salt concentration was twofold higher than that at 30‰ for 2 h. The expression levels of UPHsp70 at 30‰, 35‰, and 40‰ were low and had no difference ( P < 0.05). When U. pertusa samples were kept at ultraviolet irradiation or desiccated for different times (0-4 h), the mRNA expression level of UPHsp70 reached maximum level after 3.0 h; after that, it was maintained at high level. [ABSTRACT FROM AUTHOR]

Controlled cultivation of marine macroalgal biomass such as Ulva species, notably Ulva lactuca, is currently studied for production of biofuels or functional food ingredients. In a eutrophic environment, this macrophyte is exposed to varying types of nutrient supply, including different and fluctuating levels of nitrogen sources. Our understanding of the influences of this varying condition on the uptake and growth responses of U. lactuca is limited. In this present work, we examined the growth response of U. lactuca exposed to different sources of nitrogen (NH; NO; and the combination NHNO) by using photo-scanning technology for monitoring the growth kinetics of U. lactuca. The images revealed differential increases of the surface area of U. lactuca disks with time in response to different N-nutrient enrichments. The results showed a favorable growth response to ammonium as the nitrogen source. The NHCl and NaNO rich media (50 μM of N) accelerated U. lactuca growth to a maximum specific growth rate of 16.4 ± 0.18% day and 9.4 ± 0.72% day, respectively. The highest biomass production rate obtained was 22.5 ± 0.24 mg DW m·day. The presence of ammonium apparently discriminated the nitrate uptake by U. lactuca when exposed to NHNO. Apart from showing the significant differential growth response of U. lactuca to different nitrogen sources, the work exhibits the applicability of a photo-scanning approach for acquiring precise quantitative growth data for U. lactuca as exemplified by assessment of the growth response to two different N-sources. [ABSTRACT FROM AUTHOR]

Seaweeds growing in the intertidal zone are exposed to fluctuating nitrate and ultraviolet radiation (UVR) levels. While it has been shown that elevated UVR levels and the decrease of nitrate concentration can reduce photosynthetic levels in seaweeds, less is known about the combined effect of nitrate levels and UVR on metabolism and photoprotection mechanisms of intertidal species. Consequently, the objective of this study was to evaluate the effect of nitrate concentration and UVR treatments on photosynthesis, respiration, nitrate reductase activity and phenolic compound levels of Ulva rigida (Chlorophyta). There was a two- to threefold increase in maximal gross photosynthesis (GP) and respiration rates, as nitrate increased from 0 to 50 μM NO. Similarly, nitrate reductase activity increased linearly from low values in algae incubated at 0 μM NO to high values in tissue incubated at 50 μM NO. Phenolic compounds in the tissue of U. rigida increased approximately 60% under 50 μM NO relative to those incubated at 0 μM NO. Algae exposed to UVR (8 h) showed a significant decrease in the effective quantum yield and respiration, however, no effect was observed in the phenolic compounds levels. Full recovery of effective quantum yield was observed after U. rigida was transferred for 48 h to low PAR. Nitrate reductase also decreased after an 8-h UVR exposure, but no differences were observed among the nitrate treatments. This study shows that high nitrate levels reduced the negative effect of UVR on the effective quantum yield and increased the recovery of key metabolic enzymes. It is possible that the increase of phenolic compounds in the thallus of U. rigida under high nitrate levels provide a photoprotective mechanism when exposed to high UV levels during low tides. [ABSTRACT FROM AUTHOR]

Samples of the massive drifting green alga, Ulva linza, were collected from the coastal waters of the Yellow Sea, southwest of Korea, in early July 2009, and cultured under laboratory conditions. The effects of various temperature (10-30°C) and irradiance levels (0-1,000 μmol photons m s) on photosynthesis, growth, and tissue nutrient content of U. linza were investigated in laboratory for both individuals of the late-stage vegetation (LSV) and the early-stage vegetation (ESV). After 1 h acclimation to various irradiance and temperature conditions, maximum gross photosynthetic rate of ESV was significantly higher than those of LSV. In the long-term (7-d) acclimation experiments to various irradiance and temperature levels, gross photosynthetic rates of ESV individuals were also significantly higher than those of LSV individuals. High photosynthetic rate of ESV individuals induced increase in mass of about 60% over the growth saturation irradiance (136 μmol photons m s) and about 20% under low temperature conditions (10-15°C) during 7-d. The gross photosynthesis of LSV individuals was low when examined under temperature and irradiance conditions that were optimum for ESV growth. Consequently, free-floating U. linza exhibits cellular senescence beginning in early July in the Yellow Sea, and green tides formed by this species cannot be maintained beyond this time in the open sea. However, we expect that U. linza can proliferate quickly after settlement on new coastal habitats of the Yellow Sea because of the high tissue nitrogen utilization for photosynthesis in ESV, which is formed by germination of reproductive cells. [ABSTRACT FROM AUTHOR]

Disorders in blood coagulation can lead to an increased risk of bleeding (hemorrhage) or clotting (thrombosis). These illnesses have increased over the last decades and no useful new substances have been discovered to remediate them. In search of new compounds from marine natural resources, macroalgae from the Northwest Mexican Pacific coast were investigated in order to detect anticoagulant activity. Egregia menziesii, Ulva neumatoidea, Porphyra perforata, Silvetia compressa, and Codium fragile were collected from Ensenada coasts. Collected materials were cleaned, dried, milled, and stored until use. Proximate chemical composition and sulfate content were determined in dried powder. Hot and cold aqueous extracts were obtained from the dried algae in order to isolate polysaccharides and similar compounds. Methanol-soluble compounds were separated by means of Soxhlet extraction. Organic and aqueous extracts were screened for anticoagulant activity in both intrinsic and extrinsic pathways of clot formation. Clotting activity was studied by standardized plasma coagulation tests (activated partial thromboplastin time (aPTT) and prothrombin time (PT)). Heparin, a sulfated glycosaminoglycan widely used in anticoagulant therapy, was used as reference. Effects were defined either as aPTT index (Sample aPTT/Control aPTT ratio) or PT index (Sample PT/Control PT ratio). Some of the fractions showed anticoagulant activity over intrinsic pathways, whereas they were found to be coagulants on the extrinsic pathway. The highest aPTT index was 1.8 for U. nematoidea (1 μg mL). Hot aqueous extracts from E. menziesii (1 μg mL) showed the highest potency, with an aPTT index of 1.4. Sulfate content and anticoagulant activity were not correlated. [ABSTRACT FROM AUTHOR]

In order to obtain information on the content and composition of the water-soluble polysaccharides from Ulva clathrata, an extraction at 60°C, in different media, was performed: water, EDTA and HCl (F-I), each followed by a sequential extraction in NaOH 0.1 M (F-II). The extracts were recovered and analyzed for total carbohydrates, proteins, rhamnose, uronic acids and sulfate content. Differences were obtained in the yield and composition in both fractions of the different media (F-I and F-II). Higher yields resulted in the first fraction on all media. HCl extraction was the best in both fractions (14.83 ± 1.5% and 5.96 ± 1.1%, F-I and F-II, respectively). In all cases, F-I was more sulfated ranging from 27.87% to 35.8% and higher in rhamnose content, whereas F-II had higher protein and slightly higher uronic acid content. FTIR spectra showed that soluble polysaccharides from the green seaweed U. clathrata are sulfated polysaccharides, similar to ulvan obtained from other Ulva species and confirmed by the H-NMR spectrum, where the characteristic signal for the deoxy sugar (rhamnose) is present. [ABSTRACT FROM AUTHOR]

Seaweed cultivation is imperative to augment increasing industrial demand. Ulva fasciata Delile is a potential seaweed for cultivation with applications in food industries. There is a renewed interest in large-scale aquaculture of this species in India due to its envisaged demand in snack food products. In the present study, we have successfully demonstrated the possibility of inducing zoospores in vegetative tissue, effective regeneration and improved growth in this seaweed by manipulating salinity (from 15 to 30 psu) and temperature (from 15 to 35°C). The optimum salinity and temperature requirement for zoospores induction were found to be 15 psu and 25°C, respectively. The quadriflagellate zoospores showed negative phototaxis and the settlement and germination pattern similar to several other green seaweeds. The optimum regeneration (78.53 ± 10.05%) was recorded at 25°C and 30 psu salinity. The maximum daily growth rate (16.1 ± 0.28%) was at 25°C and 30 psu salinity which corresponded to the field conditions. This method could be further refined at nursery culture to achieve artificial seeding essential for the success of commercial cultivation of this seaweed. [ABSTRACT FROM AUTHOR]

In the present study, the use of seaweeds for phosphate absorption was examined as a tertiary treatment in sewage treatment plants, to improve the water quality and reduce eutrophication risks. The data came from both laboratory and field experiments that took place on Ios Island sewage treatment plant. Three different macroalgae were tested and Ulva lactuca was finally chosen thanks to its high survivability in low salinity waters. Since the main restrictive factor was low salinity, we initially established the ratio of seawater:effluent that combined satisfactory viability with maximum phosphate absorption. The biomass growth under these conditions was also examined. Based on the above results, we designed a continuous-flow system with a 1/4 volume per hour water turnover, in a mixture of 60% sewage effluent: 40% sea water and 30 g L-1 initial biomass of U. lactuca that must be renewed every 10 days. Under these conditions and time frame, the phosphate content of the effluent was reduced by about 50%. [ABSTRACT FROM AUTHOR]

Abstract  A new “germling cluster” method is proposed for tank cultivation of seaweed in a free-floating form. This method was applied to the tank cultivation of Ulva prolifera using deep seawater (DSW) pumped up from over 300 m depth off the cape of Muroto in southwest Japan. Numerous zoids of U. prolifera were induced by cutting thalli into 1–2 mm long pieces. Three days after fragment production, the zoids were released. The zoid suspension was concentrated to a density of more than 104 zoids per mL medium, and placed in a Petri dish for culture. The dense, germinating zoids began to adhere to each other and form aggregations. The germling aggregations were then removed from the bottom of the dish and torn into a large number of small “germling clusters” using an electric mixer. Each cluster contained 10–100 germlings. Once the germling clusters had attained more than 5 mm diameter in culture, they were transplanted as free-floating forms to a 500 L outdoor tank with continuous aeration, to which DSW was supplied at an exchange rate of 3 volumes per day. As a result, the average daily growth rate (DGR) in the tank throughout the year was 37%, though the DGR fluctuated with seasonal temperature changes. [ABSTRACT FROM AUTHOR]

Trends in wrack composition and biomass, and its relationship with the anthropogenic impact were studied along a coastal area in Nuevo Gulf (south Patagonia) in front of Puerto Madryn city. Beach-cast macroalgae composition was sampled from 1992 to 1999 in the Puerto Madryn beaches and in several other nearby beaches in 1993, 1996 and 1998. Historical information was based on local knowledge and observations reported by marine biologists who worked in the area. The botanical composition of the beach-cast macroalgae in Puerto Madryn indicates a succession in the dominance from Codium spp. to Ulva spp. during the 1990s and from Ulva to Undaria pinnatifida since 1998, accompanied by a significant decrease in biomass of Gracilaria gracilis and Macrocystis pyrifera. The increase of the opportunist species such as Ulva may be supported by the continuous delivery of waste waters into the Nuevo Gulf while the dominance of U. pinnatifida may be associated with port activities. During the sampling period the highest wrack biomass values were recorded in spring and summer. The beach-cast seaweed biomass harvested by the municipality of Puerto Madryn during beach cleaning operation, ranged between 2500 and 12000 t year−1 ( 200 and 960 t dry weight). Wrack harvesting produces an environmental impact by removing sand from the beach and affecting coastal communities. Composting of wrack is proposed as one of the environmental alternatives to land disposal. [ABSTRACT FROM AUTHOR]

The potential of three estuarine macroalgae ( Ulvarotundata, Enteromorpa intestinalis and Gracilaria gracilis) as biofilters for phosphate ineffluents of a sea bass ( Dicentrarchus labrax) cultivationtank was studied. These seaweeds thrive in Cádiz Bay and were alsoselected because of their economic potential, so that environmental andeconomicadvantages may be achieved by future integrated aquaculture practices in thelocal fish farms. The study was designed to investigate the functioning of Pnutrition of the selected species. Maximum velocity of phosphate uptake (2.86μmol PO4 g−1 dry wth−1) was found in U. rotundata.This species also showed the highest affinity for this nutrient. At low flowrates (< 2 volumes d−1), the three species efficientlyfiltered the phosphate dissolved in the waste water, with a minimum efficiencyof 60.7% in U. rotundata. Net phosphate uptake rate wassignificantly affected by the water flow, being greatest at the highest rateassayed (2 volumes d−1). The marked decrease in tissue P shownby the three species during a flow-through experiment suggested that growth wasP limited. However, due to the increase in biomass, total P biomass increasedinthe cultures. A significant correlation was found between growth rates and thenet P biomass gained in the cultures. A three-stage design under low water flow(0.5 volumes d−1) showed that the highest growth rates (up to0.14 d−1) and integrated phosphate uptake rates(up to 5.8 μmol PO4 3− g−1dry wt d−1) were found in E.intestinalis in the first stage, with decreasing rates in thefollowing ones. As a result, phosphate become limiting and low increments oreven losses of total P biomass in these stages were found suggesting thatphosphate was excreted from the algae. The results show the potential abilityofthe three species to reduce substantially, at low water flow, the phosphateconcentration in waste waters from a D. labrax cultivationtank, and thus the quality of effluents from intensive aquaculture practices. [ABSTRACT FROM AUTHOR]

Three estuarine macroalgae ( Ulva rotundata, Enteromorpha intestinalis, Gracilariagracilis) of economic potential were cultivated in the laboratory toassess their biofiltering capacities for ammonium in waste effluents from a seabass ( Dicentrarchus labrax) cultivation tank. The studywasdeveloped to investigate the functioning of N nutrition of the three species.Atlow water flow (< 2 volumes d−1) the three species strippedefficiently the ammonium dissolved in the waste water from the fish tank, withaminimum biofiltering efficiency estimate of 61% in unstarved cultures of G. gracilis at a water flow of 2 volumesd−1. Maximum velocity for ammonium uptake (89.0 μmolNH4 + g−1 dry wth−1) was found in U. rotundata,whereas G. gracilis showed the highest affinity for thisnutrient. The net ammonium uptake rate was significantly affected by the waterflow, being greatest at the highest flow assayed (2 volumesd−1). Variations of tissue N and C:N ratios during aflow-through experiment suggested that N was not limiting macroalgal growth.However, when ammonium was supplied at a flow rate of 0.5 volumesd−1, specially in a three-stage design, the marked reductionintissue N and the biomass C:N:P ratios suggested a more general nutrientdeficiency. A significant correlation was found between growth rates and the Nbiomass gained in the cultures. The three-stage design under low water flow(0.5volumes d−1) showed that the highest ammonium uptake rates (upto 80.9 μmol NH4 + g−1 dry wtd−1 in U. rotundata) were found inthe first stage, with decreasing rates in the following ones. As a result, lowincrements or even losses of total N biomass in these stages were found,suggesting that ammonium was excreted from the algae. We conclude that thesespecies present a potential ability to biofilter the ammonium dissolved inwastewater from a D. labrax cultivation tank, suggesting thatscaling up the biofiltration designs, future practises using these macroalgaemay be implemented in the local fish farms, resulting in both environmental andeconomical advantages. [ABSTRACT FROM AUTHOR]

A simple enzyme mixture containing 2% Cellulase Onozuka R–10 and1% Macerozyme R–10 prepared in deionised water supplemented with 3% NaCland 1 mM CaCl2 was developed for isolating rapidlyprotoplasts from different species of Monostroma, Enteromorpha and Ulva. The yield fordifferent species of Monostroma ranged from 9.6 ×106 to 10.2 × 106 cells g−1f. wt thallus, and for Enteromorpha from 3.48 × 106 to 11.7× 106 cells g−1 f. wt and for Ulva from 4.58 × 106 to 26.8 ×106 cells g−1 f. wt. The overallregeneration rate of the protoplasts isolated was usually > 90% and showednormal morphogenesis. The method yields rapid mass production of viableprotoplasts with high regeneration rates. [ABSTRACT FROM AUTHOR]

The responses of Gracilaria lemaneiformis, an easily epiphytized host,and the relatively resistant G. cornea mutant, to the green alga Ulva lactuca were studied using biculture experiments with and withoutantibiotics. Both Gracilaria species grown with and without U.lactuca showed different levels of growth rate, release of hydrogenperoxide and of halogenated hydrocarbons. These quantitative differencesled to a successful response against Ulva lactuca in the case of G.cornea mutant and to a failure in response in the case of G.lemaneiformis. The response of each Gracilaria species to U.lactuca was qualitatively similar to its response to bacteria. This suggeststhe involvement of oligosaccharide elicitors produced in the presence ofepiphytes and bacteria. A clear Gracilaria inhibition was demonstratedwith extracts of the culture medium. It appears that hydrogen peroxide,halogenated hydrocarbons and oligosaccharides may be components of theinhibitory activity of the extracts. The responses of Gracilaria speciesto the presence of U. lactuca suggest the characterization of adefence response. [ABSTRACT FROM AUTHOR]

‘Green tides’ Ulva is often harvested for environmental reasons, and put in a dump. Observations on degradation of Ulva in such dumps led us to consider recovery of hydrolysis juice in order to methanize this rather than the entire alga. The hydrolysis step was then studied in the laboratory and under real conditions. The decomposition of Ulva was rapid (7.1% C d−1), but its degradation incomplete (38% C remaining after 52 days). After 9 months in a dump, VFA contents in the flows were insignificant and N and C contents in the remaining material were due to the non-degradable fraction. Modifications of the physical or chemical conditions of hydrolysis didn't improve suffisently significantly the results to be used on a large scale. On the other hand, the techniques which could allow a rapid recovery of the juice improved together the recovery of the COD. The hydrolysis juice is a very good substrate for methanisation: the methane yield reached 330 L kg−1 VS, and the epuration rate 93%. The process combining the two steps, hydrolysis and juice methanisation, is one which offers a reasonable compromise between methane output, productivity of the system and treatment cost. However, there are still two problems, dependence on climatic conditions, and too low a recovery rate of the initial organic material. [ABSTRACT FROM AUTHOR]

The chemical composition and structures of several ulvan extracts isolated from various Ulva species were studied. They were all composed mainly of rhamnose, glucuronic acid, xylose, glucose and sulphate with smaller amounts of iduronic acid and traces of galactose. Proteins were also present, most likely as contaminants. Precise quantification of the uronic acid content by chemical-enzymatic hydrolysis coupled to HPAEC-PAD analysis and by colorimetry was not achieved, most likely due to the incomplete hydrolysis of glucuronan segments, inadequate HPAEC-pulsed-amperometric response factor for iduronic acid and to a possible differential colorimetric response of the two uronic acids. 13C NMR spectroscopic investigation of different ulvans demonstrated that they were all based on ulvanobiuronic acid 3-sulphate A and B repeating units [β-D-Glc pA-(1->4)-α-L-Rhap3S and α-L-IdopA-(1->4)-α-L-Rha p3S, respectively] as well as contiguous β 1->4 linked D-glucuronic acids possibly occurring either in ulvan or as a separate glucuronan. Marked variations in the content of the repeating structures were seen among the different samples. However, due to the limited number of samples studied, no conclusion was reached concerning the effects of species and ecophysiological conditions on the chemistry of ulvan. [ABSTRACT FROM AUTHOR]