Total Mycosporine-like Amino Acids Concentrations Research Articles

UV-absorbing mycosporine-like amino acids in the eyes of temperate marine and freshwater fish species

Ultraviolet radiation (UVR) is the photochemically most reactive waveband of incident solar irradiation. Despite high absorption in aquatic environments, UVR causes numerous biochemical, genetic, and cytotoxic effects in aquatic organisms. To counteract UVR stress, many of those species are able to synthesize, accumulate, or acquire UV-sunscreen compounds for photoprotection from their diet. The most abundant UV sunscreens in marine and freshwater organisms are mycosporine-like amino acids (MAAs), which exhibit high molar extinction coefficients in the UVR range along with a strong photo- and heat stability. In this study, we investigated the qualitative and quantitative MAA distribution patterns in the eyes of 39 fish species, mainly from the temperate northern hemisphere (Baltic Sea, Northern Atlantic), using state-of-the-art analytical methods. The fish eyes of the most investigated species (33 taxa) contained MAAs, between one and seven different compounds. The MAAs palythine, asterina-330, palythene, and usujirene were present (as previously reported), and three new compounds, aplysiapalythine A, porphyra-334, and shinorine, were identified. Total MAA concentrations covered a wide range from trace amounts to > 4.2 mg g−1 dry weight, thereby providing the first quantitative data on MAAs in fish eyes. The highest MAA contents were measured in Sprattus sprattus, which are comparable to those of intertidal red seaweeds. The trophic transfer of MAAs from primary producers via zooplankton to the fish is discussed, along with the localization in the fish eye as well as possible additional functions.

Assessment of the photoprotective potential and toxicity of Antarctic red macroalgae extracts from Curdiea racovitzae and Iridaea cordata for cosmetic use

Botanical and marine extracts have been used in cosmetic products as natural antioxidants or UV-absorbers to reduce the use of organic UV filters. However, despite the high UV exposure of Antarctic macroalgae that induces the production of photoprotective compounds, the use of marine macroalgae extracts for photoprotection has been little explored. Thus our objective was to assess the photoprotective potential and the toxicity of extracts from two red macroalgae Curdiea racovitzae and Iridaea cordata. Chemical characterization was based on the qualitative and quantitative analyses of mycosporine-like amino acids (MAAs) by high performance liquid chromatography coupled to mass spectrometry. Photoprotective properties were assessed by UV spectrophotometry and photostability assay. Antioxidant capacity was studied by DPPH and superoxide radical scavenging assays. UVB and UVA photoprotection was assessed in keratinocytes by cell viability analysis after UVB irradiation and quantification of intracellular reactive oxygen species (ROS) induced by UVA, respectively. Toxicity was estimated by cytotoxicity, phototoxicity and HET-CAM (Hen's egg test-chorioallantoic membrane) assays. Red algae extracts had high concentrations of MAAs, mainly consisted of palythine. The extracts absorbed in the UVB and UVA-II and were photostable. The C. racovitzae extract showed antioxidant activity against superoxide radicals. In addition, the C. racovitzae (1.0 and 0.5 mg/mL) and I. cordata (1.0 mg/mL) extracts had an UVB photoprotection similar to benzophenone-3, which was dependent on the total MAAs concentration. Besides, the C. racovitzae extract decreased ROS generation by 31 and 24% at 100 and 50 μg/mL, respectively. Furthermore, it was more effective in reducing ROS generation than quercetin and Helioguard 365®. Finally, both extracts were considered to be non-cytotoxic, non-phototoxic and non-irritant. Therefore, the C. racovitzae extract is a good candidate to be added to anti-aging and sunscreen formulations in order to protect against oxidative stress and to improve the absorption spectra of UV filters.

Quantitative and Qualitative HPLC Analysis of Mycosporine-Like Amino Acids Extracted in Distilled Water for Cosmetical Uses in Four Rhodophyta.

Mycosporine-like amino acids (MAAs) have gained considerable attention as highly active photoprotective candidates for human sunscreens. However, more studies are necessary to evaluate the extraction efficiencies of these metabolites in cosmetic compatible solvents, as well as, their subsequent HPLC analysis. In the present study, MAA extraction using distilled water and 20% aqueous methanol in four Rhodophyta was investigated. Different re-dissolution solvents and a C8 and C18 columns were tested for the HPLC analysis. Porphyra-334, shinorine, palythine, palythine-serine, asterina-330, and palythinol were identified by HPLC/ESI-MS. The separation of these MAAs were improved employing the C8-column, and using methanol as re-dissolution solvent. Regarding total MAAs concentrations, no differences between the two solvents were found. The highest MAA amounts were observed injecting them directly in the HPLC. According to these results, distilled water could be an excellent extraction solvent for MAAs. Nevertheless, the re-dissolution in pure methanol after dryness would be the best option for the qualitative analysis of the most common MAAs in these red algae. Our results entail important implications regarding the use of red macroalgae as promising candidates as environment-friendly sources of natural sunscreens.

Stress metabolite pattern in the eulittoral red alga Pyropia plicata (Bangiales) in New Zealand – mycosporine-like amino acids and heterosides

Intertidal rocky shore ecosystems are affected by steep environmental gradients such as fluctuating solar irradiation and salinity along the marine-terrestrial interface. The eulittoral red alga Pyropia plicata (Bangiales) is endemic and abundant to coastal regions of New Zealand and almost unstudied in terms of ecophysiological performance under radiation and salinity stress. Therefore, the acclimation potential of this species against enhanced ultraviolet radiation (UVR) and osmotic stress was evaluated in a combination of field and laboratory experiments with an emphasis on stress metabolite concentrations (UV-sunscreens, organic osmolytes). Samples of P. plicata were collected at the same site in the intertidal zone of Wellington, New Zealand over three seasons (April–November 2016) and used in independent UV and salt stress experiments under controlled conditions. The mycosporine-like amino acids (MAA) shinorine and porphyra-334 were the quantitatively dominant UV-sunscreen compounds, and the total concentrations varied over the year between 5 and 14 mg g−1 dry weight (DW), but neither UVR nor PAR had a significant impact on the total or individual MAA concentrations. A UV radiation stress experiment was conducted, but the total MAA concentrations of 6–8 mg g−1 DW did not change, neither did the contents of shinorine (~ 3 mg g−1 DW) nor that of porphyra-334 (4–5 mg g−1 DW). This suggests, that P. plicata has sufficiently high UV-sunscreen amounts and hence does not respond to changes in UV radiation. Pyropia plicata contained three heterosides (floridoside, D- and L-isofloridoside), which act as organic osmolytes. Seasonally the total concentrations of these compounds varied between 203 and 1226 mmol kg−1 DW, with L-isofloridoside quantitatively dominating all samples. A salt stress experiment showed an increase in the total heteroside concentrations in P. plicata with increasing salinities. However, floridoside was the most up-regulated heteroside under hypersaline conditions indicating its key role in osmotic acclimation. Our data indicate that P. plicata always contains various stress metabolites in consistently high concentrations which mitigate against environmental changes typical of the intertidal zone of New Zealand.

Effects of Light and Salinity Stresses in Production of Mycosporine-Like Amino Acids by Gymnodinium catenatum (Dinophyceae).

Mycosporine-like amino acids (MAAs) were analyzed in a Portuguese Gymnodinium catenatum strain when transferred to high salinity and high light conditions. Total MAA concentrations increased progressively between 30 and 36 psu, attaining at 36 psu 2.9-fold the 30 psu treatment. When abruptly transferred to solar light in an outdoor shadowed location, MAA concentration increased steadily along the day for most compounds. After 8h, mycosporine-glycine, palythene and M-319 attained or surpassed 25-fold their initial concentration, while M-370 only attained 4-fold concentration. When transferred from halogen to fluorescent light, polar MAAs such as shinorine and porphyra-334, increased until day two and then declined, while M-370 increase slowly, becoming the dominant compound from the profile after 1week. These experiments put into evidence the relation of palythene with M-319, which was further identified as its acid degradation product, palythine. Acid degradation of M-370 originated M-324, while M-311 seems to be the precursor of M-370. Under high salinity and high light conditions chain formation was altered toward shorter chains or solitary cells. This alteration can represent a morphological stress sign, which in the natural environment could affect average population speed during daily vertical migrations.

The relationship between UV-irradiance, photoprotective compounds and DNA damage in two intertidal invertebrates with contrasting mobility characteristics

The photoprotective role of mycosporine-like amino acids (MAA) against the generation of DNA cyclobutane pyrimidine dimers (CPD) was studied in the sessile intertidal anemone Actinia tenebrosa and the mobile intertidal gastropod Diloma aethiops through 27months at a mid-latitude New Zealand location. MAA were sequestered by A. tenebrosa and D. aethiops from their diet, although maximum total MAA levels in both species were not correlated with seasonal variation in maximum ambient UV-B levels recorded at the collection site. Temporal changes in total MAA in A. tenebrosa showed a six months lag-time in their concentration regarding to the environmental UV-B levels. This lag period corresponded to an observed increase in CPD production from spring to summer; suggesting that MAA do not completely protect the anemone from UV-B during summer. For D. aethiops, total MAA concentrations did not change significantly during the study, although qualitative changes in MAA were apparent. A month lag-time in MAA concentration in D. aethiops and possibly the physical barrier that the shell confers to the animal, can explain reduced CPD levels in comparative terms with A. tenebrosa. Although MAA are used by invertebrates for photoprotection, contrasting mobility characteristics and the presence of physical adaptations can confer them important protection levels during temporal changes of UV-B at mid-latitude places of the Southern Hemisphere.

The effects of NO3(-) supply on Mazzaella laminarioides (Rhodophyta, Gigartinales) from southern Chile.

The effects of nitrate supply on growth, pigments, mycosporine-like amino acids (MAAs), C:N ratios and carrageenan yield were investigated in Mazzaella laminarioides cultivated under solar radiation. This species is economically important in southern Chile where an increase of nitrogen in coastal waters is expected as a consequence of salmon aquaculture activity. Apical segments were cultivated in enriched seawater with five different NO3(-) concentrations (0, 0.09, 0.18, 0.38 and 0.75 mm) during 18 days. Although phycoerythrin and phycocyanin content, as well as C:N ratios, were reduced in the control treatment (without NO3(-) supply), when compared to NO3(-) treatments, total MAA concentration, carrageenan yield and growth rates were similar in all tested conditions. Nevertheless, during the experiment, an important synthesis of mycosporine-glycine took place in a nitrate concentration-dependent manner, with accumulation being saturated around 0.18 mm of nitrate. These results indicate that exposure to high NO3(-) concentration of more than 100 times the values observed in the nature did not impair the photoprotection system, as determined by MAAs, nor did it have a deleterious effect on growth or carrageenan yield of M. laminarioides, a late successional species from Chile.

The relative contributions of diet and associated microbiota to the accumulation of UV‐absorbing mycosporine‐like amino acids in the freshwater copepod Boeckella antiqua

Summary1. Mycosporine‐like amino acids (MAAs) are ubiquitous compounds in aquatic organisms that are usually considered sunscreens that protect them from harmful ultraviolet radiation. Given that virtually all animals lack the metabolic pathways to synthesise MAAs de novo, they must acquire them either from their diet or from microorganisms living in close association. In freshwater copepods, accumulation of MAAs is stimulated by exposure to ultraviolet and/or visible radiation.2. A 2 × 2 factorial experiment was performed to assess the contributions of dietary and microbial sources of MAAs in the freshwater copepod Boeckella antiqua. The treatments consisted of two different diets: an MAA‐free diet, including only Chlamydomonas reinhardtii, and an MAA‐rich diet, including both C. reinhardtii and Peridinium inconspicuum, crossed with two antibiotic treatments, with and without chloramphenicol. Treatment with chloramphenicol was intended to inhibit the development of bacteria associated with the copepods.3. MAA concentration in B. antiqua was affected by the experimental conditions: (i) exposure to artificial PAR + UVR stimulated the accumulation of several MAAs (up to 62% increase in total MAA concentration with respect to the initial concentration); (ii) the presence of chloramphenicol in the culture media reduced the MAA concentration in copepods fed an MAA‐free diet; (iii) in the absence of chloramphenicol, copepods fed the MAA‐rich diet had significantly higher total MAA concentration than those fed the MAA‐deficient diet; but (iv) dietary supplementation with an MAA‐rich algae in the presence of chloramphenicol failed to significantly increase total MAA concentration.4. Analysis of profiles from denaturing gradient gel electrophoresis (DGGE) showed that the prokaryotic community associated with the copepods was affected by chloramphenicol. Dendograms constructed from digitalised DGGE images consistently grouped the antibiotics treatments separately from the initial samples and the treatments without antibiotics. Two band positions were exclusive to treatments without antibiotics.5. We conclude that when offered an MAA‐rich diet, B. antiqua may accumulate a proportion of MAAs from diet. However, we suspect that in the absence of an MAA‐rich dietary source (as in its natural habitat), virtually all MAAs present in B. antiqua are produced by copepod‐associated prokaryotes.

UV-Induced Mortality in Encapsulated Intertidal Embryos: Are Mycosporine-Like Amino Acids an Effective Sunscreen?

Mycosporine-like amino acids (MAAs) are believed to protect a variety of marine organisms against the negative effects of ultraviolet radiation (UVR). However, their role in protecting developing intertidal encapsulated embryos remains untested. In the present study, we focused on the UV protective role of natural concentrations of MAAs for two intertidal gastropod species, Bembicium nanum and Siphonaria denticulata, which lay egg masses in habitats exposed to direct sunlight. We predicted that in both species, a higher concentration of MAAs within the egg mass would increase the likelihood of embryonic survivorship in the presence of UVR. Egg masses from both species were collected along the rocky shores of southeastern New South Wales, and a portion from each was subjected to one of three separate spectral treatments: full spectrum, UV-B block, and UV block. Proportions of surviving embryos were recorded following 72 hr exposure to spectral treatment. In addition, MAAs in each egg mass were quantified. Levels of variation in MAA concentration were striking, with S. denticulata egg masses showing more intraspecific variation than those of B. nanum. Surprisingly, survivorship under all spectral treatments was extremely high for both species, irrespective of MAA concentration. Under full spectrum treatments, B. nanum survivorship and total MAA concentration were significantly and positively correlated; however, MAA accounted for just 23.6% (R = 0.486) of the variation in survivorship. In contrast, survivorship in S. denticulata was not correlated with MAA concentration under full spectrum light. We conclude that the dependence on MAAs as photoprotection may be species-specific; however, it is likely that both species possess alternative mechanisms that minimize the negative effects of UVR.

Mycosporines from freshwater yeasts: a trophic cul-de-sac?

Mycosporine-like amino-acids (MAAs) are found in aquatic bacteria, algae, and animals. A related compound, the mycosporine-glutaminol-glucoside (myc-glu-glu), has recently been reported in freshwater yeasts. Although animals depend on other organisms as their source of MAAs, they can efficiently accumulate them in their tissues. In this work we assessed the potential transfer of the yeast mycosporine myc-glu-glu from the diet into the copepod Boeckella antiqua and the ciliate Paramecium bursaria. For this purpose, we performed experiments to study the feeding of B. antiqua and P. bursaria on the yeast Rhodotorula minuta and their ability to bioaccumulate myc-glu-glu. Bioaccumulation of myc-glu-glu in B. antiqua was assessed through long-term factorial experiments manipulating the diet (Chlamydomonas reinhardii and C. reinhardii + yeasts) and radiation exposure (PAR and PAR + UVR). Shorter term experiments were designed in the case of P. bursaria. The composition and concentration of MAAs in the diet and in the consumers were determined by HPLC analyses. Our results showed that even though both consumers ingested yeast cells, they were unable to accumulate myc-glu-glu. Moreover, when exposed to conditions that stimulated the accumulation of photoprotective compounds (i.e. UVR exposure), an increase in MAAs concentration occurred in copepods fed C. reinhardii plus yeasts as well as in those fed only C. reinhardii. This suggests that the copepods were able to modify their tissue concentrations of MAAs in response to environmental clues but also that the contribution of yeast mycosporines to total MAAs concentration was negligible.

Ontogenetic change in the abundance of mycosporine-like amino acids in non-zooxanthellate coral larvae

Although mycosporine-like amino acids (MAAs) have been extensively investigated in reef-building corals, the sources of these MAAs and the process of their interconversion remain a topic of interest. Here we examined ontogenetic change in the abundance of MAAs in planula larvae of the spawning scleractinian coral Goniastrea retiformis in the absence of zooxanthellae and other dietary input. In order to examine the potential contribution of prokaryotes in the synthesis of MAAs in animal tissue, one group of larvae were treated with the antibiotic rifampicin. High concentrations of MAAs (mycosporine-glycine, shinorine, palythinol, asterina-330), were present in the asymbiotic eggs and adults; however, no MAAs were present in the endosymbiotic zooxanthellae. We documented a steady decline in the total MAA concentrations through time in larvae treated with rifampicin; however, in the absence of antibiotic there was a significant increase in the concentration of MAAs, driven by a sharp increase in the abundance of shinorine and palythinol between day 3 and 7. Our results suggest that MAA synthesis and conversion in G. retiformis larvae occurred in the absence of symbiotic zooxanthellae, and indicate a possible contribution of prokaryotes associated with the animal tissue to these processes.

Chemical sunscreens in intertidal gastropod egg masses

Summary Many marine organisms contain mycosporine-like amino acids (MAAs), a suite of chemical sunscreens that absorb potentially damaging ultraviolet radiation. No previous study has quantified MAAs in invertebrate larvae from a broad range of species. In this study, intertidal egg masses of varying developmental stages from 44 gastropods were collected from the southeastern Australian coast to determine potential patterns of MAA occurrence. Total MAA concentration did not change as egg masses matured. MAA concentration differed significantly according to gastropod order. Herbivores generally deposited egg masses with more MAAs than carnivores, but spawning habitat and egg mass structure had no effect on total MAA concentration. Furthermore, fewer MAAs occurred in inviable egg masses than viable ones. In capsular egg masses, MAAs existed only in embryos or intracapsular fluid.

The effect of different methanol concentrations and temperatures on the extraction of mycosporine-like amino acids (MAAs) in algae and Zooplankton

Mycosporine-like amino acids (MAAs) are a family of intracellular UV-absorbing compounds involved in the protection of aquatic organisms against solar UV radiation. These compounds have been identified and quantified in many marine and freshwater organisms, however, no standard method to analyze these compounds is available. Consequently, protocols to extract MAAs largely differ, for example, in the type of solvent, concentrations, temperatures, and times used. In this study, we tested whether the concentrations of MAAs are affected by extraction at different temperatures and methanol (MeOH) concentrations. Natural assemblages of freshwater phytoplankton, the cyclopoid copepod Cyclops abyssorum tatricus, and the marine alga Porphyra sp. were used as test organisms. The MAAs shinorine, palythine, MAA 331, asterina-330, and porphyra-334 present in the organisms examined were generally best extracted at 45 °C in 25 % aqueous MeOH. In Porphyra sp. and natural freshwater phytoplankton, the mean total MAA concentrations obtained with this protocol were respectively -13 and ∼3 times higher than when extracted in 100% MeOH at 4 °C. In Cyclops, concentrations of MAAs were also highest when extracted in 25 % MeOH at 45 °C, but there was no statistically significant difference among the different protocols (P = 0.079). Depending on the organism examined, both MeOH concentration and temperature affected extraction efficiency and final MAA concentration. Our results stress the need for a priori testing the influence of these variables to assure that the highest concentration is obtained without altering the qualitative MAA composition. Based on these results, direct comparison of MAA concentrations reported in the scientific literature should be done with caution.

Effects of ultraviolet radiation and water motion on the reef coral, Porites compressa Dana: a transplantation experiment

Scleractinian corals have adapted to live in habitats were the level of ultraviolet radiation (UVR, 280–400 nm) is extremely high. The putative photoprotective molecules called mycosporine-like amino acids (MAAs) contained in the corals' tissues absorb UVR and release it harmlessly as heat. MAA concentration in corals is quite plastic and correlates well with UVR dose, but other ecological factors such as water motion may influence MAA production as well. In this study, the effects of ambient UVR and water motion on MAA concentration and several physiological parameters of the reef coral Porites compressa Dana were investigated in a two by two factorial transplantation experiment. Replicate branches from nine morphologically distinct colonies were transplanted from the windward side of Coconut Island (Kaneohe Bay, HI) to a control area on the windward side (ambient water motion) and to an area on the leeward side (low water motion). The transplanted corals were placed under UV-opaque (UVO) or UV-transparent (UVT) filters fixed to the reef. Initially and at 3 and 6 weeks, coral branches were weighed to determine calcification rate and tissues were extracted in methanol for photosynthetic pigment and MAA analysis via high performance liquid chromatography (HPLC). UVR was a significant factor determining MAA concentration. When UVR was screened from the corals' environment, total MAA concentration decreased by 33% over 6 weeks. However, UVR-exposed corals moved to low water motion also decreased MAA levels, while UVR-exposed corals moved to the control area retained initial levels. Photosynthetic pigments and calcification rate were also significantly reduced in corals moved to low water motion. There was no UVR effect on photosynthetic pigments or calcification rate. This study provides evidence that water motion is important for the maintenance of MAAs. However, there were interesting colony-specific patterns in MAA composition and response to the UVR treatment; some colonies had high total concentrations of MAAs in all treatments, while others displayed a pronounced UVR effect. Also, each genotype seemed to have its own signature MAA composition. These findings indicate a genetic (host, zooxanthellae or both) component to UVR resistance in this population of P. compressa.

Large variability in the concentration of mycosporine‐like amino acids among zooplankton from lakes located across an altitude gradient

The qualitative and quantitative composition of mycosporine‐like amino acids (MAAs), a family of intracellular UV‐absorbing compounds, were investigated in zooplankton from 15 lakes located in the Central Alps between 913 and 2,485 m above sea level. The lakes differed in their UV water transparency (1% attenuation depth, Z1%, at 320 nm: 1.1–25.6 m) and maximum depth (Zmax: 3–133 m), thus offering the possibility to test the influence of different UV exposure conditions of zooplankton on the concentration of MAAs. Seven distinct MAAs were detected, but shinorine (maximum absorption: 334 nm) was the predominant compound. In the copepods Cyclops abyssorum, C. abyssorum tatricus, and Acanthodiaptomus denticornis, the total MAA concentration ranged from 0.01 to 3.1% of the dry weight. In the rotifers Keratella cochlearis and Polyarthra dolichoptera, MAAs were also found; however, these compounds were undetectable in Asplanchna priodonta as well as in the cladocerans Daphnia hyalina, D. longispina, Bosmina longispina, and Chydorus sphaericus. The total concentration of MAAs in populations of Cyclops spp. and phytoplankton collected simultaneously was not associated (r2 = 0.09, P > 0.05), suggesting a different dynamic in the accumulation of these compounds. The variability in the concentration of MAAs, however, was related with the diffuse attenuation coefficient at 320 nm (r2 = 0.74, P < 0.001) and the fraction of the water column to which 1% of the surface irradiance at 320 nm (Z1% : Zmax) penetrated (r2 = 0.86, P < 0.001). These relationships suggest that the prevailing UV exposure condition in the lakes is a major determinant of the concentration of MAAs found in zooplankton. Our data support the hypothesis that MAAs, together with other photoprotective compounds, play a major role in minimizing the damaging effects of solar UV radiation in zooplankton from transparent lakes.