Aminoácidos tipo micosporinas (MAAs) são compostos responsáveis pela fotoproteção no ultravioleta de diversos organismos aquáticos. São sintetizados pela via do ácido chiquímico por algas, bactérias e fungos, de maneira similar à síntese de flavonóides em plantas superiores. Neste trabalho foram conduzidos estudos relacionados a estes compostos em algas. Protocolos de extração de diferentes algas foram testados alterando-se parâmetros como solventes, temperatura e condições de incubação. Os resultados mostraram que dependendo da alga estudada, diferentes condições podem mudar a concentração de MAAs extraída, ressaltando a importância de se testar diversos parâmetros na extração, evitando assim valores sub- ou superestimados de concentrações. O desenvolvimento de um método por HPLC permitiu a separação de 6 MAAs com boa resolução. A caracterização estrutural foi realizada majoritariamente por espectrometria de massas utilizando diferentes tipos de analisadores. Estas análises permitiram a proposição de mecanismos de fragmentação descritos pela primeira vez para esta classe de compostos e possibilitaram a identificação de diferentes MAAs em algumas micro e macroalgas. Ensaios in vitro foram realizados com o extrato obtido da macroalga Gracilaria domingensis no intuito de avaliar seu potencial uso em formulações cosméticas direcionadas à fotoproteção. Os testes de estabilidade quanto ao pH, temperatura e exposição à radiação ultravioleta bem como os ensaios de citotoxicidade, fototoxicidade e avaliação do fator de proteção solar sugeriram que este extrato pode ser promissor quando incorporado em formulações direcionadas para a fotoproteção. Este extrato não apresentou atividade antioxidante significativa. Os estudos com o dinoflagelado Prorocentrum minimum isolado de duas regiões diferentes, quando exposto às radiações ultravioleta, mostraram que houve uma indução das MAAs em ambas linhagens em todos os tratamentos realizados no período de 72 h. A indução foi mais rápida e pronunciada na linhagem oriunda de Lisboa, em Portugal do que na linhagem proveniente de Kattegat, na Dinamarca. Estes dados estão de acordo com as características do local de origem das linhagens, uma vez que os dinoflagelados originários de Portugal em seu meio natural estavam sujeitos a maiores irradiações. Medidas do rendimento quântico do fotossistema II indicaram que a síntese e acúmulo de MAAs em P. minimum isolada de Lisboa ofereceu vantagens na proteção do sistema fotossintético e na supressão de espécies reativas de oxigênio desta microalga quando comparada aos indivíduos da mesma espécie de regiões com menores irradiações. / Mycosporine-like amino acids (MAAs) are chemically related water soluble compounds responsible for UV photoprotection in many aquatic organisms. They are biosynthesized via the shikimate pathway by algae, bacteria and fungi in a similar manner to the biosynthesis of UV-screening flavonoids in terrestrial plants. In this work, studies related to this class of compounds were performed with algae. Extraction protocols of some algae were tested using diverse solvents at different temperature. The results showed that depending on the organism examined, both solvent concentration and temperature affected extraction efficiency and final MAA concentration. The improvement of a HPLC method separated a mix of 6 MAAs with good resolution. The structural characterization was made by mass spectrometry using different analyzers. The analysis by mass spectrometry allowed the proposition of new mechanisms of fragmentation and identification of different MAAs in some micro and macroalgae. In vitro preliminary assays were performed to evaluate the potential use of Gracilaria domingensis extract in suncare products. The extract showed no antioxidant activity, however the pH, temperature and UV exposure stability, as well the citotoxicity, phototoxicity and sun protection factor showed a potential commercial utilization for the extracts. The UV exposure experiments with the dinoflagellate Prorocentrum minimum isolated from Lisbon, Portugal and Kattegat, Denmark, showed that MAAs were induced in all treatments during 72 h. The induction was faster in the species from Lisbon than the ones from Kattegat. These data are in agreement with local isolation place, since the dinoflagellates from Portugal are submitted to high irradiance regime in their natural enviroment. The quantum yield of photosystem II suggested that the over production of MAAs by P. Minimum from Lisbon protects the photosynthetic apparatus, indicating that MAAs act as spectrally specific UV sunscreens in phytoplankton.

Ultraviolet light is damaging but fish have evolved protective mechanisms, which allows them to live in shallow water reefs, high in UV radiation. This thesis details my investigation into the physiological ecology of solar ultraviolet (UV) absorbing compounds, known as mycosporine-like amino acids found in the external epithelial mucus, and examines the supporting role potentially played by a UV-induced DNA repair mechanism in coral reef fish of the Indo-Pacific. Using reverse phase chromatography and UV spectrophotometry, I examined whether the distribution of MAA compounds across different areas of the body is correlated with differential UV exposure. Comparisons were made between the MAA content and the absorbance spectra of mucus from the dorsal, ventral, caudal and head body surface areas in five species of Scaridae (Chlorurus sordidus, Scarus schlegeli, S. niger, S. psittacus and S. globiceps) from Ningaloo Reef, Coral Bay, Western Australia. All fish analysed had at least five MAAs present, and results showed that fish had increased UV absorbance in mucus over the dorsal area, which receives the brunt of UV radiation. Little UV protection was found in mucus from the ventral area, which receives the lower level of UV radiation mostly via reflection of the sand and reef surfaces. Furthermore, UV absorbance per mg dry mucus versus standard fish length showed that there is a positive relationship in C. sordidus with increasing size. I examined whether there is a difference in the quantity of UV screening compounds found in the mucus of fish along a longitudinal geographical gradient from inshore reefs (Lizard Island, Great Barrier Reef) to the outer edge reefs to oceanic reefs (Osprey Reef). MAA absorbance increased with longitudinal distance from the mainland landmass of Australia to more oligotrophic outer reefs, where UV attenuation is reduced and the ocean is more transparent to UV wavelength. I determined that fish living on inshore, more turbid reefs where UV attenuation in shallow waters is high have lower levels of MAA protection than fish from clear oceanic reefs. Furthermore, there seems to be a direct relationship between light attenuation and exposure with the quantity of protective sunscreening found in the mucus of reef fish. It is know that UV irradiation decreases with water depth and that mucus from fish with deep habitats absorbs less UV than that of fish from shallow habitats. It is unknown however, whether this UV protection is variable within the same individuals and if so, how fast changes 11 occur. To test this, I relocated 9 ambon damselfish from a deep reef (18 m) to a shallow reef (1.5 m) to expose fish to increased levels of UV and relocated another 7 fish from a shallow to a deep reef to expose fish to decreased levels of UV. One week after relocation, all fish were returned to their original reef site to determine whether MAA levels would return to their initial levels. Fish relocated to a shallower depth were recovered and had a 60% (SD+/-2%) increase in mucus UV absorbance. Conversely, the fish relocated to a deeper depth were recovered and had a 41% (SD+/-1%) decrease mucus UV absorbance. No difference was found between UV absorbance of relocated and original fish at both depth. Six days after fish were returned to their original reef, mucus UV absorbance levels had returned to 67% +/- 4% of the original level. These results show that mucus UV absorbance is variable in individual ambon damselfish and that the sunscreen protection typical for a certain depth is reached in relocated fish within just a few days of relocation. The rate of MAA loss is higher than the accumulation of MAAs suggesting that diet is not the sole determining factor involved in the sequestration of MAAs to mucus. The cleaner fish Labroides dimidiatus performs a mutualistic service by removing ectoparasites such as gnathiid isopods as well other dead infected tissue from its clients. Cleaner fish however are also known to feed on client mucus. The benefits of eating mucus until recently were unclear. In this study, we analysed the mucus of several cleaner fish clients to determine whether mucus feeding has a nutritional advantage over gnathiids and whether cleaner fish obtain their own MAA protection through this dietary mucus ingestion. Results show that host fish that are infected with gnathiids of poor nutritional value, in contrast to those that harbour gnathiids with higher nutritional value, continuously exude mucus that has both high nutritional value and high MAA content. These findings support the conclusion that in a competitive market for cleaners some host fish are forced to offer more than parasites to cleaners. Ultraviolet light that is not filtered by UV absorbing compounds such as MAA may still lead to DNA damage such as the formation of cyclobutane pyrimidine dimers (CPDs) or 6-4 photoproducts (6-4 PPs). However, coral reef fish have alternative mechanisms to overcome UV induced damage via the photolyase DNA repair mechanisms. We experimentally demonstrated for the first time that a coral reef fish species, the moon wrasse Thalassoma lunare has the ability to repair DNA damage via photoreactivation. Fish both with and without MAA protection were irradiated with UVB wavelength to induce DNA lesions. Half of the experimental fish were then exposed to photoreactivating wavelength to induce DNA repair 12 while the other fish were blocked from the repair mechanisms. Fish which had undergone DNA repair had the lowest number of lesions regardless of mucus MAA protection. When fish were blocked from photoreactivation wavelengths MAA sunscreens clearly served a photoprotective role. The amount of damage was greatest in fish which both lacked MAAs and which were also blocked from photoreactivating wavelengths. Thus for the overall UV protection of fish both the MAA sunscreens as well as the DNA repair system play a significant role in counteracting UV damage. Ultraviolet protection by MAA sunscreens is ubiquitous in marine fish. To date the same 5 MAA compounds (palythine (λmax 320 nm), asterina (λmax 330 nm), palythinol (λmax 332 nm), usujirene (λmax 357 nm) and palythene (λmax 360nm) have been identified in the mucus of several different species of reef fish from Australia. Here we report the first evidence of the presence of additional UV absorbing compounds found in the mucus of fish from Indonesia. Using UV spectroscopy the mucus of four species of fish was compared between both geographical regions. The presence of an additional peak between 294-296 nm wavelengths suggests the presence of gadusol and/or deoxygadusol, which are photoprotective compounds, thought to be the precursors of MAAs. Thus, UV protecting compounds in the mucus of fish may not be as conserved between different regions as previously assumed. Our knowledge concerning the effect of UV radiation has advanced considerably in the past decade and my research findings contribute to the better understanding of protective mechanisms of marine fish. The correlations I have found between UV attenuation/exposure, depth, and longitude of sampled individuals lead me to believe that mucus UV absorbing MAA compounds are a highly efficient adaptive defence.