Variation in the bacterial biota of the snakelocks anemone Anemonia viridis - natural vs. Abnormal shifts induced by global climate change and ist relevance for drug discovery

Variation in the bacterial biota of the snakelocks anemone Anemonia viridis – natural vs. abnormal shifts induced by global climate change and its relevance for drug discovery JOANA CATARINA MONIZ DA ROCHA Tese de doutoramento em Ciências do Mar e do Ambiente 2015 JOANA CATARINA MONIZ DA ROCHA Variation in the bacterial biota of the snakelocks anemone Anemonia viridis – natural vs. abnormal shifts induced by global climate change and its relevance for drug discovery Tese de Candidatura ao grau de Doutor em Ciências do Mar e do Ambiente Especialidade em: Planeamento e Gestão Ambiental. Programa Doutoral da Universidade do Porto (Instituto de Ciências Biomédicas de Abel Salazar e Faculdade de Ciências) e da Universidade de Aveiro. Orientador – Doutor Ricardo Jorge Guerra Calado Categoria – Investigador Principal Afiliação – Departamento de Biologia & CESAM, Universidade de Aveiro Co-orientador – Doutor Newton Carlos Marcial Gomes Categoria – Investigador Principal Afiliação – Departamento de Biologia & CESAM, Universidade de Aveiro Co-orientadora – Doutora Luísa Maria Sobreira Vieira Peixe Categoria – Professora Associada com agregação Afiliação – REQUIMTE, Laboratório de Microbiologia, Faculdade de Farmácia, Universidade do Porto LEGAL DETAILS In compliance with what is stated in Decree-Law nº 216/92 of October 13th, it is hereby declared that the author of this thesis participated in the creation and execution of the experimental work leading to the results here stated, as well as in their interpretation and writing of the respective manuscripts. This thesis includes two scientific papers published in international journals ranked on ISI Web of Science and two articles in preparation, originate from the results obtained in the experimental work referenced to as: Rocha J., Peixe L., Gomes N.C.M., Calado R. Cnidarians as a source of new marine bioactive compounds - an overview of the last decade and future steps for bioprospecting. Marine drugs, 2011. 9(10): 1860-1886. Rocha J., Coelho F.J.R.C., Peixe L., Gomes N.C.M., Calado R. Optimization of preservation and processing of sea anemones for microbial community analysis using molecular tools. Scientific Reports, 2014. 4: 6986. Rocha J., Rocha R., Peixe L., Gomes N.C.M. Calado R. Bacterial communities associated with snakelocks anemone Anemonia viridis - natural variability and its relevance for experimental studies (in preparation). Rocha, J., Peixe, L., Gomes N.C.M. and Calado, R., Use of an experimental life support system to predict the effects of temperature and depth in the bacterial communities associated with Anemonia viridis (in preparation). TABLE OF CONTENTS Acknowledgements i Figures Index iii Tables Index vii Summary ix Resumo xi CHAPTER 1 - General Introduction 1.1. Phylum Cnidaria 5 1.1.1. Class Anthozoa 8 1.2. Sea Anemones 8 1.3. Microbial communities associated with Anthozoans - a glint of these communities in sea anemones 12 1.4. Global climate change and its potential impact in cnidarians 15 1.5. Overview of natural product discovery from cnidarians 17 1.6. References 22 CHAPTER 2 - Optimization of preservation and processing of sea anemones for microbial community analysis using molecular tools 2.1. Abstract 35 2.2. Introduction 35 2.3. Methods 37 2.4. Results 41 2.5. Discussion 43 2.6. References 45 CHAPTER 3 - Bacterial communities associated with snakelocks anemone Anemonia viridis - natural variability and its relevance for experimental studies 3.1. Introduction 51 3.2. Characterization of Anemonia viridis (Forskål, 1775) 51 3.3. Similarity of bacterial populations associated with the whole body of snakelocks anemone Anemonia viridis and solely its tentacles 52 3.3.1. Methods 52 3.3.2. Results 54 3.3.3. Discussion 55 3.4. Similarity of bacterial populations associated with the two morphotypes of the snakelocks anemone Anemonia viridis 55 3.4.1. Methods 55 3.4.2. Results 56 3.4.3. Discussion 56 3.5. Micro-spatial variation analysis of bacterial populations associated with the snakelocks anemone Anemonia viridis 57 3.5.1. Methods 57 3.5.2. Results 58 3.5.3. Discussion 58 3.6. Seasonal variation of bacterial populations associated with the snakelocks anemone Anemonia viridis 59 3.6.1. Methods 59 3.6.2. Results 60 3.6.3. Discussion 61 3.7. Conclusions 61 3.8. References 62 CHAPTER 4 - Use of an experimental life support system to predict the effects of temperature and depth in the bacterial communities associated with Anemonia viridis 4.1. Introduction 67 4.2. Methods 68 4.3. Results 72 4.4. Discussion 77 4.5. Conclusions 78 4.6. References 79 CHAPTER 5 - Marine natural products in Cnidarians 5.1. Marine ecosystems, holobionts, environmental stressors and the bioprospecting of marine natural products 89 5.2. Cnidarians as a source of new marine bioactive compounds - an overview of the last decade and future steps for bioprospecting 91 5.2.1. Abstract 91 5.2.2. Introduction 92 5.2.3. Methodology 93 5.2.4. Class Anthozoa 94 5.2.4.1. Order Alcyonacea (Soft Corals) 95 5.2.4.2. Order Gorgonacea (Sea Fans) 102 5.2.4.3. Other Orders 105 5.2.5. Class Hydrozoa 106 5.2.6. Class Scyphozoa 107 5.2.7. Other Classes 107 5.2.8. Exploring the unexplored and being creative: future perspectives for the bioprospecting of cnidarians 107 5.2.9. Conclusions 109 5.3. References 111 CHAPTER 6 - Conclusions and future perspectives 6.1. Conclusions and future perspectives 131 6.2. References 133 Supplementary Information (Chapter 2) vi vii TABLES INDEX CHAPTER 1 - General Introduction Table 1.1. Classes and orders in the phylum Cnidaria (according to the classification proposed in the World Register of Marine Species (WoRMS) 5 Table 1.2. Number of new compounds discovered in the most representative taxa of phylum Cnidaria in the 1990s and 2000s decades 19 CHAPTER 3 - Bacterial communities associated with snakelocks anemone Anemonia viridis - natural variability and its relevance for experimental studies Table 3.1. Bacterial community analysis from PCR-DGGE fingerprints of A. viridis from four tide pools 59 CHAPTER 4 - Use of an experimental life support system to predict the effects of temperature and depth in the bacterial communities associated with Anemonia viridis Table 4.1. List of most abundant OTUs (≥100 sequences) including OTU numbers, number of sequences (reads) for each treatment (R50T28, R50T15, R100T28 and R100T15) and the sum of all the reads (Total), their taxonomic affiliation, GenBank GenInfo sequence identifiers (GI) of closely related organisms identified using BLAST and sequence identity (Sq ident) of these organisms with our representative OTU sequences 83 CHAPTER 5 - Marine natural products in Cnidarians Table 5.1. Classes and orders in the phylum Cnidaria followed in this paper 95 Table 5.2. Most promising compounds studied in the last decade from cnidarian species in order Alcyonacea (soft corals), class Anthozoa 96 Table 5.3. Most promising compounds studied in the last decade from cnidarian species in order Gorgonacea (sea fans), class Anthozoa 102 viii ix SUMMARY Cnidarians, namely the snakelocks anemone Anemonia viridis (Forskål, 1775), are good model organisms for ecological studies and important elements in marine communities. These invertebrates display a microbial biota capable of providing food and protection by the production of metabolites and other natural products. Some of these substances may be bioactive metabolites with biotechnological interest. Given the lack of studies on the natural dynamics of the microbial biota associated with sea anemones in temperate regions, one of the main goals of this thesis is to provide detailed information on how environmental drivers affect the microbial biota of Anemonia viridis, in south-west Europe. By acquiring this knowledge we expect to be able to understand how potential global climate change in the marine environment may disrupt the symbiotic relations between sea anemones and its bacterial populations, as well as determine if bacterial symbionts of anemones are capable of biosynthesizing bioactive compounds with biotechnological interest. The studies presented in this thesis provide detailed information on the bacterial communities colonizing A. viridis and fluctuations induced by seasonal, and spatial variation, as well as physical stressors (temperature and depth) in controlled environments mimicking their natural habitat. Additionally, evidences show that environmental stressors (temperature and depth) affect not only the sea anemone – zooxanthellae symbiosis, but also associated bacterial populations. The abundance of some bacterial taxa can change significantly, with some group’s even disappearing from their host anemone. Moreover, the same study reveals that bacteria in symbiosis with A. viridis can produce natural products and this type of biosynthesis is most active under abnormally high temperatures and full solar radiation. These observations provide important insights for future studies targeting the production of bioactive compounds by these bacteria. x xi RESUMO Os Cnidários, nomeadamente a anémona-do-mar Anemonia viridis, são importantes elementos das comunidades marinhas sendo considerados bons organismos modelo em estudos ecológicos. Estes invertebrados exibem um biota microbiano capaz de proporcionar alimento e proteção pela produção de metabolitos e outros compostos naturais. Podendo alguns destes metabolitos ter propriedades bioativas com interesse biotecnológico. Dada a falta de estudos sobre a dinâmica natural do biota microbiano associado a anémonas-do-mar em regiões temperadas, um dos principais objectivos desta tese é fornecer informações detalhadas sobre a forma como os factores ambientais afetam o biota bacteriano da Anemonia viridis, no sudoeste europeu. Ao se adquirir este conhecimento, pretende ser-se capaz de entender como potenciais alterações climáticas globais no ambiente marinho, podem perturbar as relações simbióticas entre anémonas do mar e suas populações bacterianas; bem como determinar se os simbiontes bacterianos das anémonas são capazes de biosintetizar compostos bioativos com interesse biotecnológico. Os estudos apresentados nesta tese fornecem informações detalhadas sobre as comunidades bacterianas que colonizam a A. viridis e flutuações induzidas pela variação sazonal e espacial; bem como por condições físicas (temperatura e profundidade) capazes de provocar stress em ambientes controlados e que simulam o habitat natural. As evidências demostram que estas condições físicas afectam não só a simbiose anémona-zooxanthellae, mas também as populações bacterianas associadas. A abundância de alguns taxa bacterianos podem alterar significativamente, com alguns grupos bacterianos desaparecendo mesmo da anémona hospedeira. Adicionalmente, o mesmo estudo revela que as bactérias em simbiose com A. viridis podem produzir produtos naturais e que este tipo de biossíntese é mais ativa sob temperaturas anormalmente elevadas e radiação solar total. Estas observações fornecem entendimentos importantes a serem usados em futuros estudos que visem a produção de compostos bioativos por estas bactérias. xii CHAPTER 1 General Introduction Parts of this chapter include excerpts from the chapter book: Rocha J., Leal M., Calado R. (2014) Marine Bioactive Compounds from Cnidarians; SeKwon, K. Springer Handbook of Marine Biotechnology. Springer ISBN 978-3-642-53970-1 10 2) raptorial capture, where zooplankters and motile benthic prey are caught by the tentacles (Sebens, 1981), and 3) passive capture, where sessile prey are dislodged by foraging predators or wave action and carried onto the tentacles (Sebens, 1981). Feeding behaviour in many zoantharians is under chemical control. Some respond to reduced glutathione but in certain others two compounds are involved: asparagine (the feeding activator) that causes a bending of tentacles toward the mouth, and reduced glutathione that then induces swallowing of food (Hickman et al., 2007). Sea anemones have no central brain, but a simple network of nerves in the body wall communicates between different parts of the anemone's body. Also muscles are well developed in sea anemones and several kinds of them can be found in their tissues. They are used to contract and bend the column in various directions. These include longitudinal fibers, which occur only in the tentacles and oral disc of most species and run perpendicular to the base and serve to contract the column vertically, and circular muscles, that run around the column parallel to the base and serve to reduce the diameter of the column (Cavendish, 2010, McCloskey, 2012). The muscles are also used to expand the column and tentacles by the use of hydrostatic pressure. That is, by using the muscles to apply pressure to the water in one part of the closed coelenteron, this water is forced into other parts which subsequently appear extended and more rigid, being termed as hydrostatic skeleton (McCloskey, 2012). Most sea anemones can glide along slowly on their pedal discs and can even crawl on their side or by using their tentacles. They can expand and stretch their tentacles in search of small vertebrates and invertebrates, which they overpower with tentacles and nematocysts and carry to the mouth. As referred, sea anemones are, for the most part, sessile creatures remaining attached to one spot. However, when disturbed or if conditions grow inhospitable, sea anemones can detach from their home and swim off in search of a more suitable location. Disturbance can be, for e.g., the touch or exposure to toxic and irritating extracts of a predatory sea star and nudibranchs (Hickman et al., 2007, Springer & Holley, 2012). The sexes in sea anemones are separate in some species (like Anemonia viridis; Forskål, 1775), while most species, like the brooding anemone, are protandric hermaphrodites. Nonetheless usually only one type of gamete is reproduced at any time. The gametes develop in gastrodermal bands just behind the free edge of the mesenteries. Both sexual and asexual reproduction can occur. In sexual reproduction males release sperm to stimulate females to release eggs, and fertilization occurs. Anemones eject eggs and sperm through the mouth. Fertilization and early development may occur externally in the sea water or within the gastrovascular cavity. The fertilized egg develops into a planula larva and then into a ciliated planktonic polypoid larva in which mesentries and pharynx 11 appears. This polyp soon settles and becomes attached as a proper young sea anemone (Springer & Holley, 2012). Asexual reproduction is also common in sea anemones. One such method occurs by pedal laceration, in which small pieces of the pedal disc are left behind as the animal moves with the remnants forming new anemones. Many reproduce asexually by longitudinal (lengthwise) fission, and few by transverse (crosswise) fission or by budding (Hickman et al., 2007, Springer & Holley, 2012). Anemones form some interesting mutualistic relationships with other organisms. Many species harbour symbiotic algae (zooxanthellae and zoochlorellae) within their tissues, and anemones profit from the product of algal photosynthesis (Bergschneider & MullerParker, 2008, Davy et al., 2012, Muller-Parker & Davy, 2001, Yellowlees et al., 2008). Hermit crabs also have developed associations with sea anemones (Brooks, 1988, Brooks, 1991, McLean, 1983, McLean & Mariscal, 1973, Ross, 1971). When hermit finds the right sea anemone, he encourages the relationship and attachment happens upon the active, tactile behaviour of one or both partners (Brooks & Mariscal, 1986, Ross, 1974). Although little information exists about how or if partners might locate each other at a distance, it has been demonstrated that chemoreception play an important role in this interaction (Brooks, 1991). It is interesting to observe that some young hermit crabs often pick up a young sea anemone to attach to their shell and they become partners for life, growing even roughly at the same rate. The crab derives some protection against predators by the anemone. The anemone gets free transportation and particles of food dropped by the hermit crab (Hickman et al., 2007). Also, certain anemonefishes (family Pomacentridae) form associations with anemones, especially in tropical Indo-Pacific waters (Cleveland et al., 2011, Elliot & Mariscal, 1997, Fautin & Allen, 1992, Holbrook & Schmitt, 2005, Mariscal, 1970, Porat & Chadwick-Furman, 2004). These fish have evolved the ability to live among the tentacles of sea anemones, even though these tentacles could quickly paralyze other fishes that touched them (Raven & Johnson, 2002). In the process of adaptation to the host, the anemonefishes chemically modify the skin mucus to prevent the anemone nematocysts from discharging (Yoshiyama et al., 1996). The anemone fishes feed on the detritus left from the meals of the host anemone, remaining uninjured under remarkable circumstances (Raven & Johnson, 2002). The anemone obviously provides shelter for the anemonefish, and the fish may help ventilate the anemone by its movements, keep the anemone free of sediment, and even lure an unwary victim to seek the same shelter (Hickman et al., 2007, Roopin et al., 2008, Szczebak et al., 2013). 12 1.3. Microbial communities associated with Anthozoans - a glint of these communities in sea anemones Most eukaryotes are believed to associate with a diverse assemblage of microbial symbionts that aid in their development and health (Morrow et al., 2012). Cnidarians are simple animals with no physical barriers between the host tissue and microorganisms. Because of this they occupy an important evolutionary position for understanding direct host–microbe relations and their role in aquatic (mostly marine) ecosystems (Egan et al., 2008, Fraune & Bosch, 2007). Anthozoan cnidarians, especially sessile animals like corals, are known to host rich and diverse populations of associated bacteria. Corals have received great attention and are the most studied anthozoans in communities associations (Di Camillo et al., 2012, Egan et al., 2008). In fact it has been commonly reported the fascinating interactions between bacteria and corals in marine habitats, although most of their ecological functions remain enigmatic (Schuett et al., 2007). It is believed that coral microorganisms contribute to the overall health of the coral host (Ducklow & Mitchell, 1979, Medina, 2011, Rosenberg et al., 2007a, Rosenberg et al., 2007b). In addition, these microorganisms may be ecologically important once they appear to contribute to the ability of reef building corals to adapt to and evolve under changing environmental conditions (Reshef et al., 2006, Rosenberg et al., 2007a). It is hypothesized that corals probably coevolved with their microbial symbionts, which likely fill a critical and beneficial role in coral colony immune function. Microorganisms are found throughout the coral holobiont and appear to be regulated in part by the coral host (Morrow et al., 2012). Recently it has been suggested that diversity and types of coral-associated bacteria may also be tightly coupled with the clade of Symbiodinium spp. found within the coral host tissues (Littman et al., 2010, Medina, 2011). Coral microorganisms are thought to benefit the host by providing nutritional by-products, protein, and nitrogenous compounds and also by synthesizing essential vitamins (Croft et al., 2005, Lesser et al., 2007, Lesser et al., 2004, Xu & Gordon, 2003). Microbial symbionts may also protect corals from disease by preventing opportunistic infections through the occupation of otherwise available niches and by producing antibacterial agents (Koh, 1997, Ritchie & Smith, 2004, Rypien et al., 2010, Shashar et al., 1994) and other metabolites (Rocha et al., 2011). Shifts in coral microbial assemblages have been linked to bleaching (Bourne et al., 2008, Pantos et al., 2003), thermal stress (Littman et al., 2010, Thurber et al., 2008), irradiance (Muller & van Woesik, 2009), disease (Bourne et al., 2008), changes in dissolved organic nutrients (Dinsdale et al., 2008, Kline et al., 2006, Smith et al., 2006, Wang et al., 2007), and shifting pH (Medina, 2011). Shifts in both microbial diversity and metabolism have also been related to the proximity of coral to human populations (Dinsdale et al., 2008), 13 demonstrating that geographic location may indirectly influence coral reef health through microbial mediation. Although evidence suggests that congeneric coral species associate with similar microorganisms (Medina, 2011, Rohwer et al., 2002), the metabolic functions and specificity of coral-microbe associations are less predictable than initially hypothesized (Daniels et al., 2011, Hansson et al., 2009, Kvennefors et al., 2010, Sunagawa et al., 2010) and cannot be said that in these animals the microbial communities are species specific as deemed in Hydra (Fraune & Bosch, 2007). While symbioses between microorganisms and some anthozoans have been widely investigated (mainly in corals), the interactions between bacteria and sea anemones are still largely unknown. In fact to date only a limited number of studies have examined the diversity of bacterial communities isolated from sea anemones (Du et al., 2010, Meron et al., 2013, Palincsar, 1989, Schuett et al., 2007, Xiao et al., 2009) and key questions concerning the phylogenetic relationship of bacterial symbionts, their ecological and physiological function, their secondary metabolites, their chemical structure and the effects of these compounds on host tissue, are still poorly known. As an example, understanding these communities and their interactions is paramount to perform an accurate estimate on the diversity of natural products that may be yielded from the sea (popularly termed as “the blue gold”). Numerous natural products primarily thought to be produced by marine invertebrates show striking structural similarities to known metabolites of microbial origin, suggesting that microorganisms (e.g., bacteria) are at least involved in their biosynthesis or are in fact the true sources of these metabolites (Proksch et al., 2002). While both culture-dependent and independent methodologies are currently used for describing microbial diversity, the application of molecular approaches has greatly enhanced the knowledge of species diversity and population structure in natural microbial communities (Du et al., 2010). Furthermore, the development of high-throughput pyrosequencing technology has revolutionized the traditional cloning and capillary Sanger sequencing technique (Binladen et al., 2007, Margulies et al., 2005, Ronaghi et al., 1998, Rothberg & Leamon, 2008). Using simple techniques, light and electron microscope and fatty acids profiles, Palincsar et al. (Palincsar et al., 1988, Palincsar, 1989) made the first attempt published to determine bacterial symbionts in sea anemones. They described Gram-negative bacteria (suggested to belong to the genus Vibrio) in aggregates on the epidermis of Aiptasia pallida. In 2007, Schuett et al. (Schuett et al., 2007) provided information on organ-like bacterial aggregates in caverns of the tentacles epidermis of the sea anemone Metridium senile. In this study sequence analysis revealed three different subgroups of intra-tentacular proteobacteria. It was strongly suggested that the bacteria embedded in the aggregates were closely related to Gram-negative Endozoicimonas 14 elysicola. Additionally two bacteria were detected and assigned to be Pseudomonas saccherophilia and Ralstonia pickettii. Other three papers were published using conventional culture-dependent methods (Du et al., 2010, Williams et al., 2007, Xiao et al., 2009). The first one from Williams et al. (Williams et al., 2007) used classical methodologies to identify isolates of associated bacteria from tentacles and body tissues of Stichodactyla haddoni. They were able to identify eight genera: Alcaligenes, Corynebacterium, Aeromonas, Sporosarcina, Renibacterium, Camobacterium, Oarnobacterium and Salinococcus. In the other two articles the isolates were characterized by 16S rRNA gene sequence analysis. In the case of sea anemone Anthopleura midori (Du et al., 2010) the analysis revealed that all the strains isolated belonged to twenty two genera: Colwellia, Pseudoalteromonas, Vibrio, Acinetobacter, Pseudomonas, Endozoicomonas, Roseovarius, Paracoccus, Loktanella, Leisingera, Sulfitobacter, Bacillus, Staphylococcus, Plantibacter, Microbacterium, Micrococcus, Joostella, Psychroserpens, Cellulophaga, Krokinobacter, Polaribacter and Psychrobacter (of eight clusters: Alteromonadales, Vibrionates, Peseudomonadales, Oceanospirillales, Rhodobacterales, Bacillales, Actinomycetales, Flavobacteriales). Pseudoalteromonas was the dominant genus of the bacterial flora associated with the surface of this sea anemone. For the non-identified sea anemone from coast of the Naozhou island in Zhanjiang, China (Xiao et al., 2009), the sequence analysis revealed that the isolates were members of eighteen genera (Alteromonas, Bacillus, Brachybacterium, Brevibacterium, Halobacillus, Halomonas, Nocardiopsis, Oceanobacillus, Piscibacillus, Planococcus, Pontibacillus, Pseudoalteromonas, Pseudonocardia, Salinicoccus, Salinivibrio, Staphylococcus, Vibrio, Virgibacillus) belonging to three major phylogenetic groups (Actinobacteria, Firmicutes, Gammaproteobacteria). The most abundant and diverse isolates were within the phylum Firmicutes and the class Gammaproteobacteria. Finally, in 2013 a paper was published by Meron et al. (Meron et al., 2013) describing the microbial communities associated with the sea anemone Anemonia viridis using pyrosequencing 16S rRNA gene clone libraries. Thirteen main bacterial groups were identified: Actinobacteria, Alphaproteobacteria, Bacteroidetes, Betaproteobacteria, Candidate_division_TM, Cyanobacteria, Deltaproteobacteria, Epsilonproteobacteria, Firmicutes, Fusobacteria, Gammaproteobacteria, Planctomycetes, Verrucomicrobia. The dominant groups in the case of A. viridis were Gammaproteobacteria, Firmicutes and Actinobacteria, the same major phylogenetic groups determined in a non-identified sea anemone from China (Xiao et al., 2009). Although there is a long way to run to determine bacterial communities in sea anemones and their functional role in these associations, it is interesting to note that, along with the identification of associated bacterial communities, is frequent the search of antimicrobial activity. This may be due to the assumption that the production of these 15 metabolites may play an important role in bacteria-host interactions (Du et al., 2010); even though the cellular basis of antibacterial activity still remains largely unknown. Moreover, there is an urgent need to discover new compounds of this type to cure new and old diseases that cannot be treated with the existing drugs. As some species of sea anemones are currently economically important, not only as marine ornamentals (Chapman et al., 1997) but also as a cuisine delicacy (Schwabe, 1979) and as producers of biotechnologically important natural products (Rocha et al., 2011), additional research efforts in these matters are necessary to gain an in depth knowledge on the true bioprospecting potential of sea anemones and their associated bacterial biota. 1.4. Global climate change and its potential impact in cnidarians Marine ecosystems harbor a substantial fraction of the earth´s biodiversity and are maintained by the flow of energy through the food webs (Doney et al., 2012, Halpern et al., 2012). Consequently, marine communities are biological networks in which the success of species is linked, directly or indirectly through various biological interactions (e.g., predator-prey relationships, competition, facilitation, mutualism), to the performance of other species in the community. The combined effect of these interactions constitutes ecosystem function (e.g., nutrient cycling, primary and secondary productivity), through which ocean and coastal ecosystems provide the wealth of free natural benefits (Doney et al., 2012). Anthropogenic disturbances, including climate change, are however having profound and varied consequences in marine ecosystems. The rising of atmospheric carbon dioxide (CO 2 ) is one of the major critical problems nowadays since its effects are globally pervasive and irreversible on ecological timescales (Natl. Res. Counc., 2011). Primary direct consequences are increasing ocean temperatures and acidity (Doney et al., 2012, Donner, 2009). Rising temperature creates an horde of additional changes, such as rising sea level, increased ocean stratification, decreased sea-ice extent and altered patterns of ocean circulation, precipitation, and freshwater input. Additionally, both warming and altered ocean circulation act to reduce subsurface oxygen (O 2 ) concentrations (Keeling et al., 2010). In recent decades, the rates of change have been hasty and may exceed the current and potential future tolerances of many organisms to adapt. The direct and indirect effects of physical and chemical changes can be observed by alterations in the physiological functioning, behavior, and demographic traits (e.g., productivity) of organisms, leading to shifts in the size structure, spatial range, and 16 seasonal abundance of populations. These modifications, in turn, lead to altered or disrupted species interactions and trophic pathways with climate signals thereby propagating through ecosystems in both bottom-up and top-down directions. Cnidarian species have a crucial role in marine diversity as they form both the trophic and structural foundation of coral reefs ecosystems, as well as in diverse temperate habitats (Moya et al., 2012). Coral reefs are among the most biologically rich and productive ecosystems on earth as one-quarter of all marine species associate with this specific ecosystem (Burke et al., 2012, Doney et al., 2012). The ecological impacts of changing climates and chemistry on overall marine biodiversity are potentially severe and widespread. Changes in cnidarian-dinoflagellate due to environmental stressors, such as high light and ultraviolet light radiation (Moya et al., 2012), increased temperature (Brown, 1997, Glynn, 1991), pathogen infection, pollution and changes in salinity (Ainsworth & Hoegh-Guldberg, 2008, Brown, 2000); seem to promote symbiosis dysfunction and breakdown, ultimately leading to the loss of zooxanthellae (bleaching) (Hoegh-Guldberg, 1999). Alongside, it is rare to witness the action of only one stressor, being more often observed a combination of stressors. Compounded and together they impact and reduce the abundance or impair the growth of the cnidarian and its hosts (Hughes et al., 2003). Genetic diversity in hosts and symbionts leads to a diversity of responses to mild temperature increases, however severe temperature anomalies almost always lead to widespread bleaching and death. In the future, reefs are likely to become dominated by symbiotic associations with warm-tolerant zooxanthellae as well as bacterial populations more adapted to the new conditions, allowing some animals to survive moderate temperature increases. Nonetheless, it is necessary to perceive that potential new symbiosis may be less suitable as partners when considering other aspects of coral health, such as growth (Jones & Berkelmans, 2010). Along with corals it is well known that sea anemones are key components in some temperate communities (Muller-Parker & Davy, 2001). They are also holobionts (with symbioses with zooxanthellae and bacterial populations) known to be delicately balanced in their symbiosis and surprisingly intolerant to stresses. For this, sea anemones are recognized to be important sentinel species (Winston & Heffernan, 1999). These organisms can be used by researchers to monitor potential environmental shifts in seawaters, triggered by global climate changes. Extreme bleaching events of Anemonia in the Mediterranean Sea under abnormally warm water conditions (Leutenegger et al., 2007) are a good example on the suitability of these anthozoans as sentinel species. Alongside, with the disruption monitoring of the photosynthetic symbiont or bleaching effect, it is also important to monitor potential shifts in the microorganisms associated with these sea anemones to understand how environmental disturbances may shape holobionts and ultimately ecosystems. 17 1.5. Overview of natural product discovery from cnidarians Research on marine natural products began in the 1950s (Bergmann & Burke, 1955), at a time when important breakthroughs on the taxonomy of marine animals took place (Blunt & Munro, 2008). This research field expanded during the 1970s and 1980s and only by the end of the 1980s and beginning of the 1990s an economically appealing activity started to take shape (Avila et al., 2008, Faulkner, 2000). Since the beginning of marine natural product research that sponges (phylum Porifera) have been recognized as the most interesting group of marine invertebrates (Osinga & Tramper, 1998). However, with the growing bioprospecting efforts and the screening of previously unexplored marine habitats and organisms, the biotechnological potential of other groups of marine invertebrates has also started to become appealing for researchers. The phylum Cnidaria is one of these groups, which is renowned by their ability to produce powerful toxins and venoms (Turk & Kem, 2009). A total of 3244 marine natural products have been described from this phylum alone since 1990 (and until 2011), which notes the importance of cnidarians for marine natural product research. Since the early 1990s that the number of new compounds from marine cnidarians has been higher than the discovery of compounds from sponges (Leal et al., 2012b), and the trend that we currently observe is still a continuous increase of natural product discovery (Figure 1.3). This shows that the bioprospecting efforts on these organisms have been continuously increasing. The quest for new MNP from cnidarians has benefited from a renaissance since 2005, namely due to the development of new methods in analytical technology, spectroscopy and high-throughput screening (Molinski et al., 2009). It has also benefited from the failure to deliver new drug leads in significant numbers by competing technologies, such as chemical synthesis. These two different reasons may support the continuous growth of natural product discovery from cnidarians in the last decade. Figure 1.3. Number of new marine natural products from cnidarians discovered between 1990 and 2011 (Figure from Rocha et al. (2014)). 0 50 100 150 200 250 300 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 Number of new natural products Year 18 Bioprospecting efforts have not been evenly distributed among cnidarian taxa. From the 3244 new compounds yielded by marine cnidarian species since 1990, 99% were discovered in organisms from class Anthozoa. The remaining 1% is associated with species from class Hydrozoa. Anthozoans display a higher biodiversity, with a higher number of orders (Table 1.1). Nonetheless, 94% of the 3244 compounds were discovered in organisms from a single anthozoan order: the Alcyonacea. Only through the analysis of the taxonomic level below order, e.g., the family level is it possible to observe a more even distribution of new compounds among taxa. Figure 1.4 shows the cumulative number of natural products discovered from alcyonaceans according to family level. It is important to underline family Alcyoniidae due to the large number of new compounds discovered from species in this group, as well as the continuous increase of new compounds relatively to other Aclyonacea families. The overall increase of new compounds associated with different Cnidaria taxa is displayed in Table 1.2. Of the most representative families from order Alcyonacea, only family Briareidae showed a small decrease on the number of new compounds discovered between the last two decades. All other families represented in Table 1.2 showed an increase between decades, which in some particular cases was relatively high (e.g., family Clavulariidae and Neptheidae). These results recorded by Leal et al. (2012b) show that the popularity of cnidarians in bioprospecting efforts continues to increase and with large numbers of new compounds being discovered every year. Figure 1.4. Cumulative number of new marine natural products from cnidarians according to the taxonomical level “Family”. Group “Other” refers to the families Acanthogorgiidae, Anthothelidae, Coelogorgiidae, Isididae, Melithaeidae, Nidaliidae, Paragorgiidae, Paralcyoniidae, Primnoidae, Subergorgiidae and Tubiporidae (Figure from Rocha et al. (2014)). 0 500 1000 1500 2000 2500 3000 3500 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 Cumulative number of new natural products Year Other Ellisellidae Clavulariidae Xeniidae Plexauridae Gorgoniidae Nephtheidae Briareidae Alcyoniidae 19 The high chemical diversity associated with cnidarians may be related to the high biodiversity displayed by this group. While about 11,000 cnidarian species are currently known (Appeltans et al., 2012), new compounds have only been recorded from 337 species (distributed over 117 genera). This means that only ~3.1% of cnidarian biodiversity has yielded new chemical compounds. This does not necessarily mean that the remaining ~97% of cnidarian species do not display any different compounds. Table 1.2. Number of new compounds discovered in the most representative taxa of phylum Cnidaria in the 1990s and 2000s decades (adapted from Leal et al. (2012b)). Taxon New compounds in the 1990s New compounds in the 2000s Decade variation of new compounds (%) Phylum Cnidaria 1031 1773 +72% Class Anthozoa 1017 1758 +73% Sub-class Octocorallia 963 1715 +78% Order Alcyonacea 934 1694 +84% Family Alcyoniidae 293 489 +67% Family Briareidae 158 156 - 1% Family Clavulariidae 41 150 +266% Family Gorgoniidae 109 165 +51% Family Nephtheidae 58 227 +291% Family Plexauridae 97 99 +2% Family Xeniidae 72 147 +107% Most likely, this is a result of the preference of scientists to search for new chemical entities that have been focused on a relatively low number of species. For instance, the most popular species among the Alcyoniidae are Clavularia viridis, Briareum excavatum and Antillogorgia elisabethae (Figure 1.5), which have been important cnidarians in the history of marine natural products research (Leal et al., 2012b). Diversification of bioprospected species has been relatively low, as it is possible to observe in Figure 1.5. This figure plot the number of new compounds discovered in cnidarian species since 1990 and sort that information according to the number of new compounds discovered in each species. The uneven result among bioprospected species is clearly observed. As most cnidarian species displaying a high number of new compounds inhabit tropical areas, this may suggests that bioprospecting efforts have been biased toward these particular species, probably driven by previous studies showing the high chemical diversity displayed by such taxa (Leal et al., 2012b). 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Journal of the Marine Biological Association of the United Kingdom, 76(3), 793-809. Zwenger S. and Basu C. (2008) Plant terpenoids: applications and future potentials. Biotechnology and Molecular Biology Reviews, 3(1), 001-007. CHAPTER 2 Optimization of preservation and processing of sea anemones for microbial community analysis using molecular tools Optimization of preservation and processing of sea anemones for microbial community analysis using molecular tools Joana Rocha 1,2,* , Francisco J.R.C. Coelho 1 , Luísa Peixe 3 , Newton C.M. Gomes 1 , Ricardo Calado 1,* 1 Departamento de Biologia & CESAM, Universidade de Aveiro, Campus Universitário de Santiago, 3810-193 Aveiro, Portugal. 2 Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, Rua de Jorge Viterbo Ferreira n.º 228, 4050-313 Porto, Portugal. 3 REQUIMTE, Laboratório de Microbiologia, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira n.º 228, 4050-313 Porto, Portugal. Rocha J., Coelho F.J.R.C., Peixe L., Gomes N.C.M., Calado R. Optimization of preservation and processing of sea anemones for microbial community analysis using molecular tools. Scientific Reports, 2014. 4: 6986. 35 2.1. Abstract For several years, knowledge on the microbiome associated with marine invertebrates was impaired by the challenges associated with the characterization of bacterial communities. With the advent of culture independent molecular tools it is possible to gain new insights on the diversity and richness of microorganisms associated with marine invertebrates. In the present study, we evaluated if different preservation and processing methodologies (prior to DNA extraction) can affect the bacterial diversity retrieved from snakelocks anemone Anemonia viridis. Denaturing gradient gel electrophoresis (DGGE) community fingerprints were used as proxy to determine the bacterial diversity retrieved (H’). Statistical analyses indicated that preservation significantly affects H’. The best approach to preserve and process A. viridis biomass for bacterial community fingerprint analysis was flash freezing in liquid nitrogen (preservation) followed by the use of a mechanical homogenizer (process), as it consistently yielded higher H’. Alternatively, biomass samples can be processed fresh followed by cell lyses using a mechanical homogenizer or mortar and pestle. The suitability of employing these two alternative procedures was further reinforced by the quantification of the 16S rRNA gene; no significant differences were recorded when comparing these two approaches and the use of liquid nitrogen followed by processing with a mechanical homogenizer. 2.2. Introduction Phylum Cnidaria is a large, diverse and ecologically important group of relatively simple organisms that is widely distributed in marine environments (Daly et al., 2007). Research on marine cnidarians experienced a significant advance over the last decades with the growing awareness on the vulnerability of certain key ecosystems (e.g. coral reefs) driven by direct or indirect anthropogenic actions (Carpenter et al., 2008, Hoegh-Guldberg et al., 2007, Hughes et al., 2003). Additionally, with the intensification of bioprospecting of marine invertebrates for drug discovery, as well as other biotechnological applications, researchers have started to target cnidarians in their quest for marine bioactive compounds (Leal et al., 2012, Rocha et al., 2011). Alongside with this new trend, there are growing evidences that microbes associated with marine invertebrates may be the true producers of such bioactive compounds or, at least, be partially involved in the process of biosynthesis of some of these molecules (Shnit-Orland & Kushmaro, 2009). Several of these compounds are secondary metabolites produced by symbiotic microorganisms in chemical mediation and/or defense of interaction among marine 42 Experimental treatments employing freezing at -80 ºC differed significantly from those processing fresh samples or samples flash frozen with liquid nitrogen (P = 0.017 and P = 0.025, respectively). The highest average H’ value (± s.d.) was displayed by LN_H (H’ = 2.72 ± 0.17), while the lowest was that of F-80_H (H’ = 1.68 ± 0.79) (Figure 2.2). The bacterial fingerprints recorded in the DGGE of the three experimental treatments promoting the highest H’ (in descending order, LN_H, Fr_H and Fr_MP) are illustrated in Figure 2.3. Figure 2.3. Denaturing gradient gel electrophoresis (DGGE) based analysis of bacterial community composition in the snakelocks anemone Anemonia viridis (cropped image, fulllength gel is presented in Supplementary Fig. S1). The DGGE gel presented compares community fingerprints of 16S rRNA gene fragments amplified from DNA for the three experimental procedures displaying the highest H’ values calculated from DGGE community profiles of Bacteria detected in samples of snakelocks anemone Anemonia viridis: fresh samples processed with homogenizer (Fr_H); samples frozen with liquid nitrogen followed by processing with homogenizer (LN_H); and fresh samples processed with mortar & pestle (Fr_MP). Equal numbers represent samples originating from the same anemone. The first two axis of the PCO explained 65.1% of the variability recorded in the bacterial fingerprints of the three experimental treatments yielding the highest H’ (Figure 2.4). Samples from treatment LN_H are clearly clustered apart from those where samples were processed fresh (Fr). Real-time PCR quantification of 16S rRNA gene did not reveal the existence of any significant differences between the three procedures yielding the highest H’ (P = 0.008). 43 Figure 2.4. PCO of the three experimental procedures displaying the highest H’ values calculated from DGGE community profiles of Bacteria detected in samples of snakelocks anemone Anemonia viridis: fresh samples processed with homogenizer (Fr_H); samples frozen with liquid nitrogen followed by processing with homogenizer (LN_H); and fresh samples processed with mortar & pestle (Fr_MP). 2.5. Discussion The present study reveals that the use of community fingerprinting approaches such as PCR-DGGE is a robust technique to assess and/or optimize processing and preservation methodologies of biological samples destined for microbial communities analysis using molecular tools (Cleary et al., 2012, Lai et al., 2006). While it is true that PCR-DGGE only detects the more abundant taxa present in the sample being analyzed it also provides an excellent high-throughput tool for comparative community structure analysis, it allows researchers to determine and compare the relative abundance of different bacterial populations, and therefore compare procedures, without the need to use more expensive and labor intensive techniques (Cleary et al., 2012, Liu, 2010, Neilson et al., 2013). Indeed, by using this approach it was possible to verify that there are no significant interactions between preservation and processing procedures employed for samples of A. viridis meant to be used in bacterial diversity analysis using molecular techniques. 44 Preservation was recorded to significantly affect H’ of bacterial communities retrieved from sea anemones, as already recorded for sponges (Simister et al., 2011). It is now recognized by researchers that the preservation technique employed for marine invertebrate samples is a key point for molecular analysis of microbial communities (Dawson et al., 1998, Gray et al., 2013). In the present study it was possible to show that flash freezing and homogenizing (LN_H) collected samples consistently yielded the highest bacterial diversity from snakelocks anemones (Figure 2.2). It was also possible to verify that sea anemones tissue can also be processed fresh (e.g. Fr_H and Fr_MP) with satisfactory results if researchers have the constraint of not being able to flash freeze samples with liquid nitrogen. According to the 16S rRNA gene quantification results from Real-time PCR, any of the three procedures was considered a suitable option to obtain bacterial DNA for molecular studies of bacterial communities from sea anemones. The best results achieved in our study through the flash freezing of collected samples are in line with the fact that at such extremely low temperatures no DNA degradation occurs through enzymatic activity (Abad et al., 2008, Nagy, 2010, Post et al., 1993, Reiss et al., 1995); in this way the bacterial diversity retained is close to that present at sampling time. Liquid nitrogen can be difficult to obtain and transport in remote locations and keeping samples frozen while in transit can at times be a challenging task. However, our results support the fact that flash freezing is indeed the most efficient approach when aiming to preserve biological samples from invertebrates for molecular analysis of their microbial communities (Dawson et al., 1998, Gray et al., 2013, Reiss et al., 1995). Successfully retrieving microbial communities associated with these marine animals can be of paramount importance for biotechnological (Leal et al., 2013) and/or ecological (Meron et al., 2013) purposes. The extraction method can affect the diversity of microorganisms retrieved from sea anemones (Simister et al., 2011). Nonetheless, as the extraction method employed in the present study displays a good compromise between the quantity and quality of extracted DNA, processing costs and processing time per sample, we recommend researchers to use our methodology. In the future, the use of standardized procedures for processing and preserving collected samples of sea anemones will allow researchers to perform reliable comparisons by ensuring homogeneity between studies. Moreover, it also makes possible the use of less expensive approaches (e.g. DGGE) to compare shifts in the relative abundance of the microbiome associated with these marine invertebrates. 45 2.6. 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(1989) Temperature-gradient gel electrophoresis of nucleic acids: analysis of conformational transitions, sequence variations, and protein-nucleic acid interactions. Electrophoresis, 10(5-6), 377-389. Rocha J., Peixe L., Gomes N.C.M. and Calado R. (2011) Cnidarians as a source of new marine bioactive compounds — An overview of the last decade and future steps for bioprospecting. Marine Drugs, 9(10), 1860-1886. Rosenberg E., Koren O., Reshef L., Efrony R. and Zilber-Rosenberg I. (2007) The role of microorganisms in coral health, disease and evolution. Nature Reviews Microbiology, 5(5), 355-362. Shnit-Orland M. and Kushmaro A. (2009) Coral mucus-associated bacteria: a possible first line of defense. FEMS Microbiology Ecology, 67(3), 371-380. Simister R.L., Schmitt S. and Taylor M.W. (2011) Evaluating methods for the preservation and extraction of DNA and RNA for analysis of microbial communities in marine sponges. Journal of Experimental Marine Biology and Ecology, 397(1), 38-43. Weisburg W.G., Barns S.M., Pelletier D.A. and Lane D.J. (1991) 16S ribosomal DNA amplification for phylogenetic study. Journal of Bacteriology, 173(2), 697-703. White J.R., Patel J., Ottesen A., Arce G., Blackwelder P. and Lopez J.V. (2012) Pyrosequencing of bacterial symbionts within Axinella corrugata sponges: diversity and seasonal variability. PLoS One, 7(6). Winston G.W. and Heffernan L.M. (1999) Development and characterization of sea anemones as bioindicators of offshore resource exploitation and environmental impact. In Institute C.M. (ed.), Louisiana: U .S. Department of the Interior, pp 82. 48 Acknowledgments Joana Rocha is supported by the Portuguese Science Foundation (FCT) through a PhD Grant (SFRH/BD/33476/2008) from PhD Programme in Marine and Environmental Sciences. This work was supported by European Funds through COMPETE and by National Funds through the Portuguese Science Foundation (FCT) within project PEstC/MAR/LA0017/2013. The authors are grateful to Ana Pires and Rita Polónia for technical support in DGGE. Author Contributions Conceived and designed the experiments: JR, NCMG, RC. Performed the experiments: JR. Analyzed the data: JR, NCMG, RC. Contributed reagents/materials/analysis tools: RC. Wrote the paper: JR, FJRCC, LP, NCMG, RC. Additional Information The authors declare no competing financial interests. CHAPTER 3 Bacterial communities associated with snakelocks anemone Anemonia viridis - natural variability and its relevance for experimental studies 51 3.1. Introduction Marine ecosystems harbor a substantial fraction of the Earth’s biodiversity and comprise invertebrate animals known to host a considerable abundance and diversity microorganisms (e.g., Olson & Kellogg, 2010, Shnit-Orland & Kushmaro, 2009, Sweet et al., 2011). In fact, the microbiome of certain marine invertebrates may represent a remarkable proportion of the holobiont biomass (Di Camillo et al., 2012). Marine organisms are known to have specific associations with a range of microorganisms (Thakur & Muller, 2005); however little is known about the microbial affiliation and diversity associated with such marine organisms (Menezes et al., 2010). Their mode of association (mutualism, commensalism or parasitism) and specific physiological functions are often unidentified (Schuett & Doepke, 2010). Nonetheless, at present, our knowledge on this topic goes little beyond the recognition that these complex microbial communities are extremely important components for functionally healthy ecosystems (Ainsworth et al., 2010, Menezes et al., 2010). Sea anemones (phylum Cnidaria) can be found globally in a multitude of environmental conditions and all exhibit mutual relationships with numerous microorganisms. These invertebrate animals possess endobiotic bacteria harbored within the column, tentacles and the mucus surface layer (Schuett & Doepke, 2010). Microbial diversity surveys in sea anemones indicate a wide variety of heterotrophic bacteria (Du et al., 2010), with a high percentage probably being host-specific species or groups (Schuett & Doepke, 2010). Sea anemones, specifically those hosting endosymbiotic photosynthetic dinoflagellates, are recognized to be important sentinel species (Winston & Heffernan, 1999). These organisms may help researchers to monitor potential environmental shifts in temperate coastal waters triggered by global climate changes. 3.2. Characterization of Anemonia viridis (Forskål, 1775) The use of snakelocks anemone Anemonia viridis (Forskål, 1775) as a sentinel species is documented (Leutenegger et al., 2007). With the growing awareness of its importance there is still a gap in knowledge concerning this species as a holobiont, namely its bacterial communities. This chapter in particular aims to answer questions that may be raised when designing an experimental procedure with this species. Snakelocks anemones, A. viridis, comprises two morphotypes: one with green coloration due to the photosynthetic zooxanthellae algae growing in the tentacle tissues, sometimes with the tentacle tips purple; and other with creamy-brown coloration. These animals have 58 Processing and extraction of nucleic acid of the twenty animals, was conducted as described in section “Sample collection, processing and extraction of nucleic acid” from 3.3.1. Methods. Bacterial community diversity Bacterial community composition was evaluated according to described in section “Bacterial community diversity” from 3.3.1. Methods. Statistical Analysis Bacterial fingerprints of each denaturing gradient gel were normalized using the GelCompar 4.0 software (Applied Maths, Belgium), as described by Smalla et al. (2001). Total species (S), Pielou’s evenness index (J’) and Shannon’s index of diversity (H’) were determined. The raw data matrix was log (x +1) transformed prior to the statistical analysis in order to place more emphasis on compositional differences among samples rather than on quantitative differences (Anderson et al., 2008). A one-way analysis of similarity (ANOSIM) and a Bray-Curtis similarity matrix were used to represent relative similarities between tide pools. 3.5.2. Results Five replicates from each tide pool were processed for PCR-DGGE analysis. Table 3.1 summarizes the results obtained for total species (S), Pielou’s evenness (J’) and Shannon-Weaver diversity indexes. Shannon-Weaver diversity index shows that diversity is similar between the four tide pools and Pielou’s evenness index indicates that community samples are quite even in each tide pool. Global R from ANOSIM (R = 0.194, P = 0.023) showed there are significant differences between the compositions of bacterial communities retrieved between tide pools. 3.5.3. Discussion Although results do not support the null hypothesis postulated and indicate there are differences in bacterial community composition of A. viridis between tide pools from the same geographic site, they are due to natural variability. Snakelocks anemones from each tide pool may have been originated by asexual reproduction, characteristic of this species, and thus transfer its communities to the next generation of sea anemones allowing 59 continuity and possible expansion of the populations. This fact can be of extreme importance when considering bacteria as the true producers of bioactive compounds (Shnit-Orland & Kushmaro, 2009). Table 3.1. Bacterial community analysis from PCR-DGGE fingerprints of A. viridis from four tide pools. Tide Pool S J' H' ANOSIM R-value P-value A 7,8 0,86 ± 0,06 1,94 ±0,21 B 7,6 0,84 ± 0,09 1,65 ± 0,28 C 8,2 0,85 ± 0,06 1,72 ± 0,44 D 8,0 0,88 ± 0,02 1,78 ± 0,29 A,B 0,188 0,0103 A,C 0,124 0,0143 A,D 0,36 0,0024 B,C -0,076 0,0722 B,D 0,284 0,0056 C,D 0,18 0,0143 3.6. Seasonal variation of bacterial populations associated with the snakelocks anemone Anemonia viridis Determining and understanding temporal stability (namely between seasons) is as important as to fully comprehend how microorganisms vary within the organisms and between geographical locations. In this study the composition of the bacterial community of A. viridis was analyzed in two different seasons: spring vs fall. The null hypothesis formulated is as fallows: there are no differences between bacterial communities composition retrieved from the two seasons. 3.6.1. Methods Sample collection, processing and extraction of nucleic acid Four snakelocks anemones of A. viridis were collected at low tide, in the intertidal region of Praia da Aguda (41°02'51.06''N; 8°39'14.20''W), Arcozelo, Portugal, in the spring and 60 fall of 2011 (n = 4 per season). Samples were individually stocked in sterile plastic bags for immediate transportation to the laboratory. Processing and extraction of nucleic acid of the eight animals, was conducted as described in section “Sample collection, processing and extraction of nucleic acid” from 3.3.1. Methods. Bacterial community diversity Bacterial community composition was evaluated according to described in section “Bacterial community diversity” from 3.3.1. Methods. Statistical Analysis Bacterial fingerprints of each denaturing gradient gel were normalized using the GelCompar 4.0 software (Applied Maths, Belgium), as described by Smalla et al. (2001). Shannon’s index of diversity (H’), was determined as H’ = - ∑ pi ln pi, where pi is often the proportion of individuals belonging to the each species in the dataset of interest. The raw data matrix was log (x + 1) transformed prior to the statistical analysis in order to place more emphasis on compositional differences among samples rather than on quantitative differences (Anderson et al., 2008). A one-way analysis of similarity (ANOSIM) and a Bray-Curtis similarity matrix were used to represent relative similarities by comparing the variation in species abundance and composition among treatments. A dendogram of PCR-DGGE fingerprints (Bray-Curtis Similarity) was constructed. 3.6.2. Results Four replicates from season (spring and fall) were processed for PCR-DGGE analysis. Shannon-Weaver diversity index shows that diversity of the bacterial communities in the snakelocks anemone A. viridis is higher in the spring (H’ spring = 2.09 ± 0.10; H’ fall = 1.74 ± 0.18). ANOSIM from the PCR-DGGE profiles showed that there are significant differences between bacterial communities composition retrieved from the two seasons (R = 0.896, P = 0.029). The dendogram of PCR-DGGE fingerprints two clusters, one for each season (Figure 3.3) 61 3.6.3. Discussion Studies in the past decade concluded that bacterial communities composition associated with marine organisms were spatially and seasonally stable (Anderson et al., 2010, Hentschel et al., 2002, Rohwer et al., 2002). However, our results reveal that changes in the composition of bacterial communities occur during seasons, even in those where differences in environmental parameters are less extreme, (e.g. spring and fall, in opposition to summer and winter). The results obtained in this study are in turn, in accordance with several studies that demonstrate shifts in bacterial communities when extreme events or changes in physical factors (e.g., temperature and radiation) occur (Higuchi et al., 2013, Ravindran et al., 2013, Simmons et al., 2008). Figure 3.3. Dendogram of PCR-DGGE fingerprints (Bray-Curtis Similarity) showing samples from the two seasons tested. 3.7. Conclusions The four studies presented in this chapter were performed considering there are almost no reports regarding bacterial communities in snakelocks anemones A. viridis (Meron et al., 2013). Although bacterial symbionts communities of A. viridis were not determined, these new data brings new insights on how future studies and experiences should be conducted to better understand the bacterial communities composition and their role with the host symbiont and the environment. Thereby in future studies samples should include tentacles and body of the sea anemone. Bacterial communities are species specific (morphotype independent) and stable enough 62 to be transferred to the next generations of sea anemones. Moreover, environmental parameters affect the bacterial communities populations and so the interactions between host-symbionts and the production of bioactive compounds. 3.8. References Ainsworth T.D., Thurber R.V. and Gates R.D. (2010) The future of coral reefs: a microbial perspective. Trends in Ecology & Evolution, 25(4), 233-240. Anderson M., Gorley R. and Clarke K. (2008) PERMANOVA+ for PRIMER: Guide to software and statistical methods. Plymouth, United Kingdom: Primer-E Ltd. Anderson S.A., Northcote P.T. and Page M.J. (2010) Spatial and temporal variability of the bacterial community in different chemotypes of the New Zealand marine sponge Mycale hentscheli. FEMS Microbiology Ecology, 72(3), 328-342. Caparkaya D., Cengiz S., Dincel B., Demir S. and Cavas L. 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Applied and Environmental Microbiology, 63(8), 3233-3241. Higuchi T., et al. (2013) Bacterial enhancement of bleaching and physiological impacts on the coral Montipora digitata. Journal of Experimental Marine Biology and Ecology, 440, 54-60. Holstein T.W., Hobmayer E. and Technau U. (2003) Cnidarians: An evolutionarily conserved model system for regeneration? Developmental Dynamics, 226(2), 257-267. 63 Leal M.C., Madeira C., Brandão C.A., Puga J. and Calado R. (2012) Bioprospecting of marine invertebrates for new natural products - A zoogeographical and chemical perspective. Molecules, 17, 9842-9854. Leutenegger A., Kredel S., Gundel S., D'Angelo C., Salih A. and Wiedenmann J. (2007) Analysis of fluorescent and non-fluorescent sea anemones from the Mediterranean Sea during a bleaching event. Journal of Experimental Marine Biology and Ecology, 353(2), 221-234. Menezes C.B.A., et al. 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(2011) Cnidarians as a source of new marine bioactive compounds — An overview of the last decade and future steps for bioprospecting. Marine Drugs, 9(10), 1860-1886. Rohwer F., Seguritan V., Azam F. and Knowlton N. (2002) Diversity and distribution of coral-associated bacteria. Marine Ecology Progress Series, 243, 1-10. Schuett C. and Doepke H. (2010) Endobiotic bacteria and their pathogenic potential in cnidarian tentacles. Helgoland Marine Research, 64(3), 205-212. Schuett C., Doepke H., Grathoff A. and Gedde M. (2007) Bacterial aggregates in the tentacles of the sea anemone Metridium senile. Helgoland Marine Research, 61(3), 211-216. Shnit-Orland M. and Kushmaro A. (2009) Coral mucus-associated bacteria: a possible first line of defense. FEMS Microbiology Ecology, 67(3), 371-380. Simmons T.L., et al. (2008) Biosynthetic origin of natural products isolated from marine microorganism-invertebrate assemblages. Proceedings of the National Academy of Sciences of the United States of America, 105(12), 4587-4594. Sweet M.J., Croquer A. and Bythell J.C. (2011) Bacterial assemblages differ between compartments within the coral holobiont. Coral Reefs, 30(1), 39-52. 64 Thakur N.L. and Muller W.E.G. (2005) Sponge-bacteria association: A useful model to explore symbiosis in marine invertebrates. Symbiosis, 39(3), 109-116. Weisburg W.G., Barns S.M., Pelletier D.A. and Lane D.J. (1991) 16S ribosomal DNA amplification for phylogenetic study. Journal of Bacteriology, 173(2), 697-703. Winston G.W. and Heffernan L.M. (1999) Development and characterization of sea anemones as bioindicators of offshore resource exploitation and environmental impact. In Institute C.M. (ed.), Louisiana: U .S. Department of the Interior, pp 82. CHAPTER 4 Use of an experimental life support system to predict the effects of temperature and depth in the bacterial communities associated with Anemonia viridis 67 4.1. Introduction Extreme environmental events, such those associated to global climate change, are often associated to the disruption of symbiotic associations of cnidarians with a range of micro and macroorganisms. Cnidarian–dinoflagellate endosymbiosis are common in the marine environment and have a key role in marine biodiversity: they form both the trophic and structural foundation of coral reefs ecosystems and are also a key component in some temperate communities (Muller-Parker & Davy, 2001). Nonetheless, these symbioses appear to be delicately balanced on the edge of dysfunction and surprisingly intolerant to stresses as high light and ultraviolet light radiation (Moya et al., 2012), increased temperature (Brown, 1997, Glynn, 1991), pathogen infection, pollution and changes in salinity (Ainsworth & Hoegh-Guldberg, 2008, Brown, 2000). Alone or combined, these factors seem to promote symbiosis dysfunction and breakdown and, ultimately, lead to the loss of zooxanthellae or so-called bleaching (Hoegh-Guldberg, 1999). In corals, ultraviolet radiation (UVR) has been shown to be an agent leading to bleaching, either directly (Gleason & Wellington, 1993) or in synergy with temperature (Lesser, 2010, Lesser & Shick, 1989), and thus contributing to the worldwide degradation of coral reef ecosystems. In sea anemones, ultraviolet radiation exposure alone holds little impact and induces limited transcriptomic response. On the other hand thermal stress combined or not with UVR, induces symbiosis disruption and bleaching (Moya et al., 2012). In this case the transcriptomic response to thermal stress is immediate, transient and potentiated by ultraviolet radiation in the sea anemone. Considering the above, sea anemones (namely those hosting endosymbiotic photosynthetic dinoflagellates) are recognized to be important sentinel species (Winston & Heffernan, 1999). These organisms can be used by researchers to monitor potential environmental shifts in temperate coastal waters triggered by global climate changes. Extreme bleaching events of Anemonia in the Mediterranean under abnormally warm water conditions (Leutenegger et al., 2007) are a good example on the suitability of these anthozoans as sentinel species. Additionally, these anemones should also be considered as holobionts (Margulis & Fester, 1991), due to its complex symbiosis between the cnidarian animal, its photosynthetic microalgae (e.g., zooxanthellae) and its intricate community of associated microorganisms that play a key role on the overall health, defense and nutrition of the cnidarian host (Paul & Puglisi, 2004). Alongside, with the disruption monitoring of the photosynthetic simbiont or bleaching effect, it is important to monitor potential shifts in the microorganisms associated with these sea anemones to understand how environmental disturbances may shape anemone individuals and/or populations. 74 treatments, respectively. In this phylum, Alphaproteobacteria is the most abundant class, mainly in R50T28 treatment with 78% of relative abundance and 10 OTUs. In this class, order Kiloniellales goes from inexistent in normal conditions (R100T15) to dominant in extreme conditions (R50T28). Spirochaetes appears only in treatments at 15ºC. Class Clostridia (Firmicutes phylum) exhibit reduced relative abundance to almost non-existent in R50T15 (0.1%). The phylogenetic tree (Figure 4.4) displays two main clusters where OTUs belonging to different classes and phyla are arranged. Each main cluster encompasses a smaller but distinct clusters consisting of OTUs from class Alphaproteobacteria, Epsilonproteobacteria or class Gammaproteobacteria. Using PiCrust, four pathways were selected to be studied: nitrogen metabolism, terpenoid backbone biosynthesis, pyruvate metabolism and lipopolysaccharide biosynthesis (Figure 4.5). In the nitrogen metabolism the percentage of total genes is greater in treatments with Figure 4.3. Relative abundance of the most abundant bacterial phyla classes and orders for the four treatments used (R100T15, R100T28, R50T15, R50T28). 75 Figure 4.4. Phylogenetic tree of the bacterial 16S rRNA gene sequences recovered from the studied treatments; bootstrap values lower than 50 % were omitted. The number of each OTU is indicated with the GenBank GenInfo sequence identifiers of the bacterial sequences. Numbers 1, 2, 3 and 4 refer to orders Alteromonadales, Vibrionales, Rhodobacterales and Campylobacteriales, respectively. higher temperature (T = 28ºC); being superior when this effect is combined with the reduction of radiation (R50T28). Terpenoid backbone biosynthesis and lipopolysaccharide 76 biosynthesis showed that the percentage of total genes is higher and equivalent at 100% of radiation. In pyruvate metabolism the percentage of genes is identical in the four treatments. Figure 4.5. Percentage of total genes for four pathways selected to be studied in PiCrust: nitrogen metabolism, terpenoid backbone biosynthesis, pyruvate metabolism and lipopolysaccharide biosynthesis. Within each pathway a Kegg ortholog was selected: glutamate synthase (NADPH/NADH) small chain (nitrogen metabolism), acetyl-CoA C-acetyltransferase (terpenoid backbone biosynthesis), 2-isopropylmalate synthase (pyruvate metabolism) and UDP-3-O-[3hydroxymyristoyl] glucosamine N-acyltransferase (lipopolysaccharide biosynthesis). Figure 4.6 presents the distribution of the total genes (%) by selected orders. Glutamate synthase (NADPH/NADH) small chain and acetyl-CoA C-acetyltransferase have a higher number of total genes when compared to 2-isopropylmalate synthase and UDP-3-O-[3hydroxymyristoyl] glucosamine N-acyltransferase. With the exception of percentage of total genes, the taxa profiles of the four treatments are very similar between the four pathways. For Kiloniellales the number of total genes increases with temperature and reduction of radiation. When the two factors are combined (R50T28) the increase of total genes is exacerbated. Contrary, the percentage of genes for Rhodobacterales, Rhizobiales, Flavobacteriales, Alteromonadales and other, decreases for the same treatment. Percentage of total genes for Alteromonadales is lower at high temperatures (T100T28 and R50T28). In treatment R100T28, acetyl-CoA C-acetyltransferase for the terpenoid backbone biosynthesis, presents higher percentage of total genes. R00T15 R00T28 R50T15 R50T28 0.0 0.2 0.4 0.6 0.8 1.0 a − Nitrogen metabolism R00T15 R00T28 R50T15 R50T28 0.0 0.1 0.2 0.3 0.4 a − Terpenoid backbone biosynthesis R00T15 R00T28 R50T15 R50T28 0.0 0.2 0.4 0.6 0.8 1.0 a − Pyruvate metabolism R00T15 R00T28 R50T15 R50T28 0.0 0.1 0.2 0.3 0.4 a − Lipopolysaccharide biosynthesis T o t a l g e n e s ( % ) 77 Figure 4.6. Distribution of total genes (%) by selected orders for the Kegg ortholog of each pathway. K00266 - Glutamate synthase (NADPH/NADH) small chain (nitrogen metabolism), K00626 - acetyl-CoA C-acetyltransferase (terpenoid backbone biosynthesis), K01649 - 2-isopropylmalate synthase (pyruvate metabolism), and K02536 - UDP-3-O-[3-hydroxymyristoyl] glucosamine N-acyltransferase (lipopolysaccharide biosynthesis). 4.4. Discussion In this study it was observed that depth (simulated through a reduction in radiation) does not affect the bacterial populations existing in the snakelocks anemone A. viridis in temperate costal waters. This result is consistent with the knowledge that short wavelengths (ultraviolet) are absorbed rapidly and in 1 meter of depth of coastal water 63% of UVR is absorbed (http://www.britannica.com/EBchecked/topic/531121/seawater/ 301669/Optical-properties, accessed 08.08.2014). However, in treatments with increased temperatures (R50T28 and R100T28) differences in bacterial populations were observed, thus agreeing with previous publications (Leutenegger et al., 2007, Moya et al., 2012, Richier et al., 2008) referring to the effects of increasing seawater temperatures. Thermal stress combined (or not) with UVR has been reported to induce the disruption of K00266 K00626 K01649 K02536 0.00 0.05 0.10 0.15 0.00 0.05 0.10 0.15 R00T15 R00T28 R50T15 R50T28 R00T15 R00T28 R50T15 R50T28 Total genes (%) Taxon Alteromonadales Campylobacterales Clostridiales Desulfobacterales Flavobacteriales Kiloniellales Oceanospirillales Other Rhizobiales Rhodobacterales Thiotrichales Vibrionales 78 Symbiodinium - A. viridis symbiosis and the bleaching of the cnidarian host (Moya et al., 2012). Our data revealed that, when combining these two factors, they cause a reduction in zooxantellae (data not shown) although not enough to trigger a bleaching effect; but induce changes in the composition bacterial populations. In fact bacterial communities in R50T28 are clearly different from other treatments (Figure 4.2). Pyrosequencing reveals that class Alphaproteobacteria growths significantly with the increase of temperature and depth. Pyrosequencing also revealed that the most abundant bacterial sequence in R50T28 belonged to Kiloniella laminariae gen. nov., sp. nov. (Wiese et al., 2009) first isolated from a marine macroalga. This alphaproteobacteria is a chemoheterotrophic aerobe with the potential for denitrification that only appears (≥100 sequences) in treatments where radiation was reduced to half. This information may indicate that, when changes in host-zooxantellae occur (for e.g. due to depth), bacterial populations can act to maintain the holobiont equilibrium. Crossing this information with results obtained for important metabolic pathways in living organisms, it was observed an increase in gene expression of enzyme glutamate synthase (NADPH/NADH) small chain (Nitrogen metabolism), responsible for the synthesis of L-Glutame (non-essential amino acid and key compound in cellular metabolisms), in R50T28. Although higher at elevated temperatures, production of natural products, such as terpenes, sterols and sisqueterpenes (Terpenoid backbone biosynthesis and pyruvate metabolism) is favored at 100% of radiation; hence major production in tide pools and shallow coastal waters. Finally, environmental shifts reduces gene expression in Lipopolysaccharide biosynthesis (UDP-3-O-[3-hydroxymyristoyl] glucosamine N-acyltransferase). 4.5. Conclusions Extreme environments affect not only the sea anemone–zooxanthellae symbiosis but also associated bacterial populations. 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(2007) Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Applied and Environmental Microbiology, 73(16), 5261-5267. Weisburg W.G., Barns S.M., Pelletier D.A. and Lane D.J. (1991) 16S ribosomal DNA amplification for phylogenetic study. Journal of Bacteriology, 173(2), 697-703. Wiese J., Thiel V., Gartner A., Schmaljohann R. and Imhoff J.F. (2009) Kiloniella laminariae gen. nov., sp nov., an alphaproteobacterium from the marine macroalga 81 Laminaria saccharina. International Journal of Systematic and Evolutionary Microbiology, 59, 350-356. Winston G.W. and Heffernan L.M. (1999) Development and characterization of sea anemones as bioindicators of offshore resource exploitation and environmental impact. In Institute C.M. (ed.), Louisiana: U .S. Department of the Interior, pp 82. Zhang Z., Schwartz S., Wagner L. and Miller W. (2000) A greedy algorithm for aligning DNA sequences. Journal of Computational Biology, 7(1-2), 203-214. 82 83 Table 4.1. List of most abundant OTUs (≥100 sequences) including OTU numbers, number of sequences (reads) for each treatment (R50T28, R50T15, R100T28 and R100T15) and the sum of all the reads (Total), their taxonomic affiliation, GenBank GenInfo sequence identifiers (GI) of closely related organisms identified using BLAST and sequence identity (Sq ident) of these organisms with our representative OTU sequences. 90 interesting for drug discovery (Molinski et al., 2009, Piel, 2004). However, as most compounds produced occur as secondary metabolites they are naturally produced in low quantities. This alone imposes critical challenges to the drug discovery pipeline, particularly during the early phases of discovery and the selection of which should advance to the next step (Li & Vederas, 2009, Radjasa et al., 2011). The production of marine natural products at a commercial level is still a remarkable challenge. Large-scale production of a given compound can be possible either through chemical synthesis or through its extraction from the source organism. Unfortunately, the first option is not always possible, as several complex molecules are simply impossible to produce or attain production costs unaffordable for commercial applications (Proksch et al., 2003, Qian et al., 2009). The harvest of the source animal (holobiont) from the wild for the extraction of a compound is invariably an unsustainable practice and hardly a long term option (Arrieta et al., 2010). Additionally, the dependence on natural samples may still entail replicability issues (Li & Vederas, 2009) as wild marine organisms collected for bioprospecting are exposed to environmental variability (including some more extreme environmental conditions associated with global climate change), as well as changes at the community level, which may significantly affect their chemical ecology (Hay & Fenical, 1996). Individuals of the same species sampled in different geographical areas, or time frames, may not display the same symbionts and thus exhibit the same chemical composition (Aratake et al., 2012) and, therefore, fail to guarantee the supply of a target metabolite (a pitfall commonly termed “loss of the source”). This may also be a potential limitation for the initial detection of bioactive metabolites, as environmental and individual variability in the chemical composition of target organisms may bias bioprospecting (Li & Vederas, 2009). Also associated with replicability issues is the potential loss of the source through extinction of target species. This issue is particularly relevant in the oceans of today and tomorrow, as vulnerability to extinction in marine ecosystems is predicted to be higher (Burke et al., 2012) due to climate changes and other external inputs. The production of source organism’s biomass (either in situ or ex situ) has been considered as a potential alternative to the collection of wild specimens (Leal et al., 2014b, Schippers et al., 2012). Additionally, the production of source organisms under controlled conditions may help to control ecophysiological diversity promoted by environmental interactions and maximize the production of target marine molecules. Unfortunately, the culture of most organisms has revealed to be more technically challenging and significantly more expensive than initially assumed (Mendola, 2003, Page et al., 2011). As seen, critical challenges are being address in order to find a constant and reliable supply to fuel the marine drug discovery (Leal et al., 2014b). Although it is consensual that 91 the use of wild marine organisms is not the best approach, studies with these animals allow researchers to better direct their lines of investigations in drug discovery. Simple knowledge’s as described in chapter 3 or more complex ones as seen in chapter 4, support future choices when addressing bioprospecting of marine natural products. In these chapters we observe that environmental or physical parameters impact the holobiont by changes on the microbial populations associated with the cnidarian host and thus affect the production of chemicals with potential to have biotechnological interest. Environmental stressors may inhibit, reduce or even enhance the production of some chemicals. They can also activate dormant genes not needed in previous conditions, or allow the incorporation of new ones enabling adaptation and evolution. This variety of possibilities can and should be driven to leverage the biotechnological potential of marine natural products as precursors of new drugs. 5.2. Cnidarians as a source of new marine bioactive compounds - an overview of the last decade and future steps for bioprospecting 5.2.1. Abstract Marine invertebrates are rich sources of bioactive compounds and their biotechnological potential attracts scientific and economic interest worldwide. Although sponges are the foremost providers of marine bioactive compounds, cnidarians are also being studied with promising results. This diverse group of marine invertebrates includes over 11,000 species, 7500 of them belonging to the class Anthozoa. We present an overview of some of the most promising marine bioactive compounds from a therapeutic point of view isolated from cnidarians in the first decade of the 21 st century. Anthozoan orders Alcyonacea and Gorgonacea exhibit by far the highest number of species yielding promising compounds. Antitumor activity has been the major area of interest in the screening of cnidarian compounds, the most promising ones being terpenoids (monoterpenoids, diterpenoids, sesquiterpenoids). We also discuss the future of bioprospecting for new marine bioactive compounds produced by cnidarians. 92 5.2.2. Introduction In terms of biodiversity, marine environments are among the richest and most complex ecosystems. Harsh chemical and physical conditions in the environment have been important drivers for the production of a variety of molecules with unique structural features. These marine molecules exhibit various types of biological activities (Jain et al., 2008), with compounds of high economic interest having potential applications in the pharmaceutical and medical sectors. Although nearly 20,000 compounds have been discovered since the field of marine bioactive compound biochemistry began in the mid1960s, only a very limited number have seen industrial application. It has been clear since marine bioprospecting began that the world’s oceans and their diverse biota represent a significant resource, perhaps the greatest resource on Earth, for the discovery of new bioactive compounds. Early National Cancer Institute (NCI) programs in the USA demonstrated that marine invertebrates were a superb source of potential lead molecules. The decisive boost to this new age of bioprospecting was provided by the NCI when it was found that bioassays with marine organism extracts were far more likely to yield anticancer drugs than terrestrial sources (Fenical et al., 2009). In this way, it is not surprising that over the past 40 years major advances in the discovery of marine drugs have been recorded in clinical trials for cancer (Hill & Fenical, 2010). Apart from anticancer activity, these compounds have proven to be an abundant source of pharmacologically active agents for the production of therapeutic entities (Glaser & Mayer, 2009) against AIDS, inflammatory conditions and microbial diseases. Marine bioactive compounds display varied potential applications, namely as molecular tools, in cosmetics, as fine chemicals, as nutraceuticals and in agrochemical industries (Fusetani, 2010). Although only a few marine-derived products are currently on the market (e.g., Prialt ® and Yondelis ® ), several new compounds are now in the clinical pipeline and several more are in clinical development. The few approvals so far for the commercialization of drugs from the sea have not been due to a lack of discovery of novel marine bioactive compounds, but because of the complexity of issues raised upon the development of these products (Glaser & Mayer, 2009). Faulkner (Faulkner, 1986, Faulkner, 1987, Faulkner, 1988, Faulkner, 1990, Faulkner, 1991, Faulkner, 1993, Faulkner, 1994, Faulkner, 1995, Faulkner, 1996, Faulkner, 1997, Faulkner, 1998, Faulkner, 1999, Faulkner, 2000, Faulkner, 2001, Faulkner, 2002), Blunt et al. (Blunt et al., 2003, Blunt et al., 2004, Blunt et al., 2005, Blunt et al., 2006, Blunt et al., 2007, Blunt et al., 2008, Blunt et al., 2009, Blunt et al., 2010, Blunt et al., 2011), and Mayer (Mayer & Gustafson, 2004, Mayer & Gustafson, 2006, Mayer & Gustafson, 2008, Mayer & Hamann, 2002, Mayer & Hamann, 93 2004, Mayer & Hamann, 2005, Mayer et al., 2011, Mayer et al., 2009, Mayer et al., 2007) have provided extensive reviews on the total number of marine natural products (MNPs) discovered over the last 25 years, the most promising ones being produced by marine invertebrates. Sponges (phylum Porifera) have long been recognized as the most interesting group of marine invertebrates for the discovery of new drugs (Fusetani, 2010, Newman & Cragg, 2004, Sipkema et al., 2005). However, with growing bioprospecting efforts and the screening of previously unexplored marine habitats, the biotechnological potential of other groups of marine invertebrates has also started to attract the attention of researchers. The ability of cnidarians (such as jellyfish, sea anemones and corals) to produce powerful toxins and venoms (Turk & Kem, 2009) has been well documented. However, further research has demonstrated that MNPs produced by cnidarians are more than toxins and venoms. The phylum Cnidaria is a large, diverse and ecologically important group of marine invertebrates that includes over 11,000 extant species (Daly et al., 2007). Over 3000 MNPs have been described from this phylum alone, mostly in the last decade. In this work, we present an overview of the most promising marine bioactive compounds isolated from cnidarians in the first decade of the 21st century, which may have applications in the therapy of human diseases. The present study also discusses future perspectives for the bioprospecting of new MNPs produced by this speciose group of marine invertebrates. 5.2.3. Methodology The most relevant peer reviewed literature published during the first decade of the 21 st century covering MNPs was surveyed for the present work (Blunt et al., 2003, Blunt et al., 2004, Blunt et al., 2005, Blunt et al., 2006, Blunt et al., 2007, Blunt et al., 2008, Blunt et al., 2009, Blunt et al., 2010, Blunt et al., 2011, Faulkner, 2001, Faulkner, 2002, Mayer & Gustafson, 2004, Mayer & Gustafson, 2006, Mayer & Gustafson, 2008, Mayer & Hamann, 2004, Mayer & Hamann, 2005, Mayer et al., 2011, Mayer et al., 2009, Mayer et al., 2007). During this period alone, over 2000 molecules from cnidarians were described. In order to focus our study and address only those compounds displaying a high potential for industrial applications, we have decided to use as guidelines the values of IC 50 (half maximal inhibitory concentration). IC 50 is a quantitative measure, which indicates how much of a particular substance (inhibitor) is needed to inhibit a given biological process or component of a process by half. It is important to highlight that the NCI has renamed the 94 IC 50 to GI 50 (Boyd et al., 1992) in order to emphasize the correction for cell count at time zero in cancer cells; in this way, some results on this quantitative measure are now also presented under these directives. Additionally, the ED 50 (the median dose that produces the desired effect of a drug in half the test population) was also used to identify promising marine bioactive compounds produced by cnidarians. Only the compounds displaying an IC 50 ≤ 10.0 µg/mL or µM (except where stated otherwise) and ED 50 ≤ 4.0 µg/mL were considered for the present study, as these values are commonly used in the surveyed literature to ascertain relevant bioactivity (e.g., Duh et al., 2002b, El-Gamal et al., 2005). In the few cases were neither IC 50 nor ED 50 values were described for a MNP in a manuscript, that compound was selected to be part of the present survey only if either the authors of that manuscript, or those citing that manuscript, clearly stated that the results recorded were highly promising for industrial applications. All species producing the compounds selected for the present work were grouped into classes and orders of phylum Cnidaria (Table 5.1) (according to the classification proposed in the World Register of Marine Species (WoRMS)) (Appeltans et al., 2010). This approach allowed us to identify which taxonomic groups of cnidarians screened so far display the highest potential to yield new drugs or pharmacological products derived from marine bioactive compounds. Nonetheless, it is important to highlight that cnidarian species identification is a challenging task and it is possible that some of the species (or even genera) referred to in the scientific literature may not be correct (Fautin, 2011). In this way, it is of paramount importance that in future works the authors addressing marine bioactive compounds produced by cnidarians provide a detailed description on how target species have been identified. 5.2.4. Class Anthozoa Class Anthozoa currently includes 10 orders and over 7,500 valid species (about 2/3 of all known cnidarian species) (Table 5.1). Within the Anthozoa, the order Alcyonacea (soft corals) and Gorgonacea (sea fans) are the ones which have contributed with the highest number of promising bioactive marine compounds, although other orders, such as Actiniaria (sea anemones) and Scleractinia (hard corals), have also yielded relevant compounds (Fontana et al., 1998, Meyer et al., 2009, Miyaoka et al., 2006, Strukelj et al., 2000). 95 Table 5.1. Classes and orders in the phylum Cnidaria followed in this paper. Phylum Class Order Cnidaria (≈11,287 species) Anthozoa (≈7500 species) Actiniaria Antipatharia Ceriantharia Corallimorpharia Scleractinia Zoanthidea Alcyonacea Gorgonacea Helioporacea Pennatulacea Cubozoa (≈36 species) Carybdeida Chirodropida Hydrozoa (≈3500 species) Anthoathecata Leptothecata Siphonophorae Actinulida Limnomedusae Narcomedusae Trachymedusae Polypodiozoa (1 species) Polypodiidea Scyphozoa (≈200 species) Coronatae Rhizostomeae Semaeostomeae Staurozoa (≈50 species) Stauromedusae 5.2.4.1. Order Alcyonacea (Soft Corals) Soft corals are generally brightly colored and rich in nutritionally important substances. However, the incidence of predation in the majority of these organisms is low due to the toxic compounds they produce to deter predators (Hooper & Davies-Coleman, 1995). Several biosynthetic studies have been carried out on the metabolites of soft corals (Bhakuni & Rawat, 2005) and some of those compounds have already shown to have great potential for the development of new pharmaceuticals and antifoulants. Table 5.2 summarizes the most promising compounds from order Alcyonacea (class Anthozoa) described in the present review. Soft corals are rich sources of secondary metabolites such as diterpenes, sesquiterpenes, furanoditerpenes, terpenoids, capnellenes and steroids (e.g., Lobophytum, Sinularia (Figure 5.1A), Sarcophyton (Konig & Wright, 1998) (Figure 5.1C), Capnella (Chang et al., 2008), Dendronephthya (Grote et al., 2008)), that have shown to display HIV-inhibitory (Rashid et al., 2000), cytotoxic (Duh & Hou, 1996, Su et al., 2006), anti-inflammatory (Norton & Kazlauskas, 1980, Williams & Faulkner, 1996), anticancer (Li et al., 2005, 96 Weinheimer et al., 1977) and antimicrobial activity (Aceret et al., 1998), as well as cardiac and vascular responses (Aceret et al., 1996). Soft corals of the family Nephtheidae are known for their content of sesquiterpenes and particularly capnellenes (Blunt et al., 2010). Some sesquiterpenes isolated from Capnella imbricate (Chang et al., 2008, Kaisin et al., 1985, Kaisin et al., 1974, Sheikh et al., 1976) showed anti-inflammatory activity and a dihydroxycapnellene (capnell-9(12)-ene-8β,10α-diol) from Dendronephthya rubeola demonstrated a good antiproliferative activity against murine fibroblasts cell line (L-929, GI 50 6.8 µM/L) and a good cytotoxicity against cancer cell lines implicated in human leukemia (K-562, IC 50 0.7 µM) and human cervix carcinoma (HeLa, IC 50 7.6 µM) (Grote et al., 2008). Capnell-9(12)-ene-8β,10α-diol strongly inhibits the interaction of the oncogenic transcription factor Myc with its partner protein Max (Hermeking, 2003, Peukert et al., 1997), making it a therapeutically interesting compound in oncology (Grote et al., 2008). Table 5.2. Most promising compounds studied in the last decade from cnidarian species in order Alcyonacea (soft corals), class Anthozoa. Family and Species Drug Class Compound Chemistry Country Ref. Alcyoniidae Klyxum simplex Anti-inflammatory Simplexin E Diterpenoid TAIW (Wu et al., 2009) Klyxum simplex Antitumor Klysimplexin B and H Diterpenoid TAIW (Chen et al., 2009) Lobophytum sp. Antitumor Lobophytene Diterpenoid VN (Nguyen et al., 2010) Lobophytum sp. Anti-HIV Lobohedleolide Diterpenoid PHL (Rashid et al., 2000) Lobophytum sp. Anti-HIV (7Z)-lobohedleolide, Diterpenoid PHL (Rashid et al., 2000) Lobophytum sp. Anti-HIV 17-dimethylamino lobohedleolide Diterpenoid PHL (Rashid et al., 2000) Lobophytum crassum Anti-inflammatory Crassumolides A and C Terpenoid TAIW (Chao et al., 2008) Lobophytum cristagalli Antitumor Cembranolide diterpene Diterpenoid RSC (Coval et al., 1996) Lobophytum durum Anti-inflammatory Durumolides A–C Terpenoid TAIW (Cheng et al., 2008) Lobophytum durum Anti-inflammatory Durumhemiketalolide A–C Cembranoid TAIW (Cheng et al., 2009b) Sarcophyton crassocaule Antitumor Crassocolides H–M Cembranoid TAIW (Huang et al., 2009) Sinularia sp. Antiulcer Sinulide Spermine (Fusetani, 1990) Sinularia sp. Antimicrobial Lipids Polyketide RUS (Dmitrenok et al., 2003) Sinularia flexibilis Antitumor Flexilarin D Cembranoid TAIW (Lin et al., 2009b) Sinularia flexibilis Antifoulant 11-episinulariolide Diterpenoid AUS (Michalek & Bowden, 1997) Sinularia gibberosa Anti-inflammatory Gibberoketosterol Steroid TAIW (Ahmed et al., 2006) Sinularia querciformis Anti-inflammatory Querciformolide C Terpenoid TAIW (Lu et al., 2008) Clavulariidae Clavularia sp. Nervous system Stolonidiol Diterpenoid JPN (Yabe et al., 2000) Clavularia koellikeri Antitumor Cembrane-type diterpenoid Diterpenoid JPN (Iwashima et al., 2000) Clavularia viridis Antitumor Claviridic acid Prostanoid TAIW (Lin et al., 2008) Clavularia viridis Antitumor Clavulones Prostanoid TAIW (Lin et al., 2008) Clavularia viridis Antitumor Claviridenone Prostanoid TAIW (Duh et al., 2002b) 97 Table 5 .2. Cont. Clavularia viridis Antitumor Halogenated prostanoids Prostanoid JPN (Watanabe et al., 2001) Clavularia viridis Antitumor Bromovulone III Prostanoid TAIW (Chiang et al., 2005, Shen et al., 2004) Clavularia viridis Antitumor Yonarasterols Steroid JPN (Iwashima et al., 2001) Clavularia viridis Antitumor Stoloniferone E Steroid TAIW (Duh et al., 2002b) Telesto riisei Antitumor Punaglandins Prostaglandin USA (Verbitski et al., 2004) Nephtheidae Dendronephthya sp. Antifoulant Isogosterones A–D Steroid JPN (Tomono et al., 1999) Dendronephthya rubeola Antitumour Capnell-9(12)-ene-8β,10α-diol Sesquiterpenoid DE (Grote et al., 2008, Hermeking, 2003, Peukert et al., 1997) Nephthea chabroli Antitumor Chabranol Terpenoid TAIW (Cheng et al., 2009c) Nephthea erecta Anti-inflammatory Ergostanoids 1 and 3 Ergostanoid TAIW (Cheng et al., 2009a) Xeniidae Asterospicularia laurae Antitumor Asterolaurin A Diterpenoid TAIW (Lin et al., 2009a) Cespitularia hypotentaculata Antitumor Cespitularin C Diterpenoid TAIW (Duh et al., 2002a) Xenia novaebritanniae Antibacterial Xeniolide I Diterpenoid ISR (Bishara et al., 2006) Xenia plicata Antitumor Blumiolide C Diterpenoid TAIW (El-Gamal et al., 2005) AUS: Australia; DE: Germany; ISR: Israel; JPN: Japan; PHL: Philippines; RSC: Republic of Seychelles; RUS: Russia; TAIW: Taiwan; VN: Vietnam. Nephthea chabroli also produces a nor-sisquiterpene compound, chabranol, which displays moderate cytotoxicity against P-388 (mouse lymphocytic leukemia cells) with an ED 50 1.81 µg/mL (Cheng et al., 2009c). Nephthea erecta produces two proteins in mediated inflammatory responses, the oxygenated ergostanoids 1 and 3. These compounds at a concentration of 10 µM significantly reduced the levels of the iNOS (inducible nitric oxide synthase) (45.8 ± 9.9 and 33.6 ± 20.6%, respectively) and COX-2 (cyclooxygenase-2) protein (68.1 ± 2.3 and 10.3 ± 6.2%, respectively), when compared with the control cells stimulated with lipopolysaccharides (LPS) (Cheng et al., 2009a). Species in the genus Xenia (family Xeniidae) (Figure 5.1B) are a rich source of diterpenoids. Xeniolides I, isolated from Xenia novaebrittanniae demonstrated antibacterial activity at a concentration of 1.25 mg/mL in Escherichia coli ATCC and Bacillus subtilis (Bishara et al., 2006). Blumiolide C, a diterpenoid from the Xenia blumi (presently accepted as Xenia plicata), exhibited potent cytotoxicity against mouse lymphocytic leukemia (P-388, ED 50 0.2 µg/mL) and human colon adenocarcinoma (HT-29, ED 50 0.5 µg/mL) cells (El-Gamal et al., 2005). 98 Figure 5.1. Some cnidarians addressed in this review (all images by Ricardo Calado). (A) Sinularia sp.; (B) Xenia sp.; (C) Sarcophyton sp.; (D) Briareum sp. Polyoxygenated cembranoids, crassocolides H–M from Sarcophyton crassocaule, demonstrated cytotoxicity against cancer cell lines of human medulloblastoma (Daoy cells) where crassocolides I and M were found to be more active (IC 50 0.8 and 1.1 µg/mL, respectively). Crassocolide H was also found to inhibit the growth of human oral epidermoid carcinoma (KB) cells (IC 50 5.3 µg/mL) and crassocolide L active against human cervical epitheloid carcinoma (HeLa) cells (IC 50 8.0 µg/mL) (Huang et al., 2009). Another example of a potential new therapeutic anticancer agent is a cembranolide diterpene from Lobophytum cristagalli, which has shown a potent inhibitory activity (IC 50 0.15 µM) (Coval et al., 1996) over farnesyl protein transferase (FPT, an important protein in signal transduction and regulation of cell differentiation and proliferation (Nakao & Fusetani, 2007)). This type of FPT inhibition enhanced interest in this group of metabolites (Konig & Wright, 1998). Other species of this genus also showed cembranolide diterpenes (lobophytene) with significant cytotoxic activity against human lung adenocarcinoma (A549) and human colon adenocarcinoma (HT-29) cell lines (Nguyen et al., 2010). Lobophytum durum and Lobophytum crassum produce durumolides A–C (Cheng et al., 2008), durumhemiketalolide A–C (Cheng et al., 2009b) and crassumolides A and C (Chao et al., 2008), with anti-inflammatory effects. They have been shown to inhibit up-regulation of the pro-inflammatory iNOS and COX-2 proteins in LPS-stimulated murine macrophage cells at IC 50 < 10 µM (Chao et al., 2008, Cheng et al., 2008). The diterpenoids, lobohedleolide, (7Z)-lobohedleolide, and 17-dimethylaminolobohedleolide, were isolated from the aqueous extract of Lobophytum species and exhibited moderate HIV-inhibitory B D A C 99 activity (IC 50 approximately 7–10 µg/mL) in a cell-based in vitro anti-HIV assay (Rashid et al., 2000). Klyxum simplex produces diterpene compounds, such as simplexin E, which at a concentration of 10 µM was found to considerably reduce the levels of iNOS and COX-2 proteins to 4.8 ± 1.8% and 37.7 ± 4.7%, respectively. These results have shown that this compound significantly inhibits the accumulation of the pro-inflammatory iNOS and COX-2 proteins in LPS-stimulated RAW264.7 macrophage cells (Wu et al., 2009). This species also produces two diterpene compounds, klysimplexins B and H, exhibiting moderate cytotoxicity towards human carcinoma cell lines. Klysimplexin B exhibits cytotoxicity toward human hepatocellular carcinoma (Hep G2 and Hep 3B), human breast carcinoma (MDA-MB-231 and MCF-7), human lung carcinoma (A549) and human gingival carcinoma (Ca9-22) cell lines with IC 50 ’s of 3.0, 3.6, 6.9, 3.0, 2.0, and 1.8 µg/mL, respectively. Metabolite klysimplexin H demonstrated cytotoxicity (IC 50 ’s 5.6, 6.9, 4.4, 5.6, 2.8 and 6.1 µg/mL) toward human hepatocellular carcinoma (Hep G2 and Hep 3B), human breast carcinoma (MDA-MB-231 and MCF-7), human lung carcinoma (A549) and human gingival carcinoma (Ca9-22) cell lines, respectively (Chen et al., 2009). In Sinularia sp. (Figure 5.1A), a tetraprenylated spermine derivative has been isolated - sinulamide - which revealed an H,K-ATPase inhibitory activity. H,K-ATPase is a gastric proton pump of stomach and is the enzyme primarily responsible for the acidification of the stomach contents. Its inhibition is a very common clinical intervention used in diseases including dyspepsia, peptic ulcer, and gastroesophageal reflux (GORD/GERD). Sinulide is a potential antiulcer drug, as it inhibits production of gastric acid by H,K-ATPase (IC 50 5.5 µM) (Fusetani, 1990). Although it has been synthesized (Sata et al., 1999), no clinical trials seem to have been reported. The steroid gibberoketosterol (Ahmed et al., 2006), isolated from Sinularia gibberosa, and the diterpenoid querciformolide C (Lu et al., 2008) from Sinularia querciformis, showed significant inhibition of the up-regulation of the proinflammatory iNOS and COX-2 proteins in LPS-stimulated murine macrophages at concentration <10 µM (Ahmed et al., 2006, Lu et al., 2008). Paralemnalia thyrsoides showed significant inhibition of pro-inflammatory iNOS protein expression (70% at IC 50 10 µM) (Huang et al., 2006). Sinularia species produce significant molecules: lipids from Sinularia grandilobata and another unspecified species of Sinularia possesses antibacterial and antifungal activity (Dmitrenok et al., 2003). The diterpene 11episinulariolide from Sinularia flexibilis is an interesting antifoulant exhibiting strong algacidal properties (Michalek & Bowden, 1997). This species also produces cembrenoids, named flexilarins, which evidence cytotoxic activity in cancer cell lines. Flexilarin D exhibited potent cytotoxicity in human hepatocarcinoma (Hep2) cells with IC 50 0.07 µg/mL, and moderate cytotoxic activity against human cervical epitheloid carcinoma 106 sensitive (F32/Tanzania) and other chloroquine-resistant (FcB1/Colombia) with IC 50 1.48 and 1.2 µg/mL, respectively (Meyer et al., 2009). Cladocorans A and B, isolated from Cladocora caespitosa (order Scleractinia) (Fontana et al., 1998), are marine sesterterpenoids which possess a γ-hydroxybutenolide moiety, which is thought to be responsible for the biological activity of these compounds. The potent anti-inflammatory activity of these natural metabolites was attributed to the inhibition of secretory phospholipase A 2 (sPLA 2 , IC 50 0.8–1.9 µM). Given the general role of inflammation in diseases that include bronchial asthma and rheumatoid arthritis, identifying and developing potent inhibitors of sPLA2 continues to be of great importance for the pharmaceutical industry, with this type of metabolite being of paramount importance for future research (Miyaoka et al., 2006). 5.2.5. Class Hydrozoa Class Hydrozoa includes seven orders and nearly 3,500 valid species (Table 5.1), some of which are solitary, some of which are colonial. Among the most emblematic species are probably hydroids and the Portuguese man-o-war (Physalia physalis). Despite the large number of species in class Hydrozoa, only a few of them have yielded interesting MNPs in the last decade. Immune escape plays an important role in cancer progression and, although not completely understood, it has been proposed that indoleamine 2,3-dioxygenase (IDO) plays a central role in evasion of T-cell-mediated immune rejection (Muller et al., 2005a). IDO catalyzes the oxidative cleavage of the 2,3 bond of tryptophan, which is the first and rate-limiting step in the kynurenine pathway of tryptophan catabolism in mammalian cells (Grohmann et al., 2003). The polyketides annulins A, B, and C, purified from the marine hydroid Garveia annulata (order Anthoathecata), potently inhibited IDO in vitro (K i 0.12– 0.69 µM) (Pereira et al., 2006). These annulins are more powerful than most tryptophan analogues known to be IDO inhibitors. These compounds are active at concentrations higher than ~10 µM and therefore more effective than 1-methyltryptophan (K i 6.6 µM), one of the most potent IDO inhibitors currently available (Muller et al., 2005b). Solandelactones C, D, and G are cyclopropyl oxylipins isolated from the hydroid Solanderia secunda (order Anthoathecata) and exhibit moderate inhibitory activity against farnesyl protein transferase (FPT, 69, 89, and 61% inhibition, respectively) at a concentration of 100 µg/mL (Seo et al., 1996). Note that FPT is associated with cell differentiation and proliferation and its inhibition may be a target for novel anticancer agents (as already referred above for the soft coral L. cristagalli). 107 5.2.6. Class Scyphozoa Approximately 200 species are currently classified in three orders in class Scyphozoa (Table 5.1). However, in the last decade, only a single MNP purified from the mesoglea of the jellyfish Aurelia aurita (order Semaeostomeae) was considered to be promising enough to be included in the present work. This compound is a novel endogenous antibacterial peptide, aurelin, which exhibited activity against Gram-positive and Gramnegative bacteria. As an example, aurelin displayed an IC 50 of 7.7 µg/mL for Esherichia coli (Gram negative bacteria) (Ovchinnikova et al., 2006). 5.2.7. Other Classes The classes Staurozoa, Cubozoa and Polypodiozoa are the least speciose in the phylum Cnidaria (Table 5.1). This fact may explain the current lack of data on secondary metabolites produced by these organisms. It is possible that with growing bioprospecting new MNPs may be revealed once these cnidarian species are screened. Cubozoa (box jellies), for example, produce some of the most harmful cnidarian toxins for humans (Brinkman & Burnell, 2009). 5.2.8. Exploring the Unexplored and Being Creative: Future Perspectives for the Bioprospecting of Cnidarians For several years, the bioprospecting of cnidarians was commonly limited to habitats that could be readily sampled by researchers, such as shallow coral reefs and the intertidal region. However, with improvements in SCUBA gear, researchers are now able to dive deeper and longer, allowing them to collect a wider range of cnidarian species for the screening of MNPs. The growing efforts to explore Earth’s last frontier, the deep sea, made it possible to start bioprospecting several unique marine ecosystems that had remained either previously unrecorded or inaccessible to researchers (Synnes, 2007). New cnidarian species (some of them belonging to new genera and probably even to new families) (e.g., Moura et al., 2007, Rodriguez et al., 2009a) are currently being sampled from the deep sea. These findings suggest that many new species are yet to be discovered along deep continental margins and open good perspectives for the discovery of new MNPs with ongoing surveys of deep sea fauna. Cnidarians are known to colonize unique deep sea biotopes, namely chemosynthetic sites (such as hydrothermal vents, cold seeps and whale falls (Fautin, 2009)), as well as seamounts (Clark et al., 2006). 108 Some of these organisms are endemic to these habitats and display remarkable adaptations to extreme environments (e.g., chemosynthetic sea anemones) (Rodríguez & Daly, 2010). These species are certainly interesting candidates for the discovery of new MNPs (Skropeta, 2008). However, some of these remarkable biotopes, namely deep sea coral reefs, are already facing serious threats to their conservation (Clark et al., 2006) and thus, the bioprospecting of these and other endangered habitats must be carefully addressed (Kingston, 2011, Synnes, 2007). Another interesting source of cnidarian species for bioprospecting is the marine aquarium industry. Over 200 species of hard and soft corals, along with several other anemone, zoanthid and corallimorph species, are harvested every year from coral reefs to supply the marine aquarium trade (Wabnitz et al., 2003). However, researchers using these organisms in the bioprospecting of new MNPs must be aware that it is not commonly possible to get reliable information on either the place of origin or the scientific name of most traded specimens. With the advent of high-throughput screening (HTS) (White, 2000), it will be possible to rapidly survey these organisms for interesting MNPs, although HTS of natural sources may present several challenges (see Koehn & Carter, 2005, Li & Vederas, 2009). If necessary, additional biomass of target organisms producing interesting MNPs can be achieved using inexpensive techniques (Sella & Benayahu, 2010, Shafir et al., 2006) and eliminate problems commonly faced by researchers screening marine organisms for MNPs - the loss of the source and reproducibility (Li & Vederas, 2009). The discovery of a new compound commonly requires only small amounts of biomass. However the production of these compounds at a scale large enough to fulfill commercial applications is still nearly impossible (Qian et al., 2010). In theory, large-scale production of bioactive compounds can be achieved by chemical synthesis or through extraction from marine animals, either harvested from the sea or maricultured. The existence of ecophysiological diversity (e.g., differences between individuals often due to differences in environmental interactions) can interfere with the production of MNPs and must be carefully addressed in future efforts for large-scale production of these compounds. The harvest of target animals from the wild for the production of chemical compounds is commonly an unsustainable solution, while mariculture has proven to be more technically challenging and expensive than previously assumed (Mendola, 2003). In other considerations, chemical synthesis is not yet developed to synthesize complex molecules at the kilogram scale and, in cases where this may already be technically possible, most of the compounds cannot be synthesized at a price affordable for commercial applications (Qian et al., 2010). Potential solutions for such bottlenecks may be the use of diverted 109 total synthesis (Paterson & Anderson, 2005) and/or metabolic engineering (Khosla & Keasling, 2003). There is growing evidence that microbes associated with marine invertebrates may be the true producers of some of the bioactive compounds isolated from these animals (Qian et al., 2010). Whether this is the case of bioactive compounds currently assumed to be produced by cnidarians remains unanswered (Piel, 2004, Piel, 2009). If so, we face another constraint for the commercial use of these compounds, as the culture of symbiotic microorganisms is generally not possible using classic/standardized methodologies. 5.2.9. Conclusions The intense pressure to find and develop more profitable molecules for all sorts of industries continues to fuel the bioprospecting of marine invertebrates. Although the phylum Cnidaria is not the most significantly bioprospected at present, this review shows that some cnidarian species are promising sources of marine bioactive compounds of medical, economic and scientific interest. Green fluorescent protein (GFP), GPF-like proteins, red fluorescent and orange fluorescent protein (OPF) are good examples of biotechnological metabolites currently employed as molecular biomarkers. They were first purified from a fluorescent hydrozoan medusa (Shimomura et al., 1962) and since then have been recorded in other cnidarian species (Ai et al., 2006, Chan et al., 2006, Goulding et al., 2008, Ip et al., 2007, Schnitzler et al., 2008, Tu et al., 2003). In the present survey, only about 0.31% of extant cnidarian species are represented, with class Anthozoa displaying by far the highest number of promising MNPs (Figure 5.2). This result is probably due to the fact that this class is the most speciose in the phylum (Table 5.1). Additionally, many anthozoans occupy marine habitats which can be readily accessed for the collection of biomass (e.g., coral reefs and intertidal regions), which facilitates bioprospecting. Of all the compounds presented in this review, 84% were detected in cnidarians collected from tropical waters (mostly from Southeast Asia and the Caribbean Sea) and the remaining 16% were recorded from species mostly occupying temperate waters (e.g., European countries and Japan). Antitumor drugs are the main area of interest in the screening of MNPs from cnidarians (41%, Figure 5.3). This is not surprising, as the major financial effort for the screening of new marine compounds is made in cancer research (Pomponi, 2001). Terpenoids (terpenoid, diterpenoid, sesquiterpenoid, sesterterpenoid, cembranoid) (Blunt & Munro, 2008) (Figure 5.4) are the main chemistry group within the MNPs analyzed in this survey. 110 Even though most pharmaceutical industries abandoned their natural product-based discovery programs over a decade ago, the lack of new compounds in their pipelines in some strategic areas (e.g., antibiotics) suggests that renewed interest in this field is imminent. The establishment of small biotech companies can play a decisive role in the initial discovery of promising marine bioactive compounds, as these enterprises will work closely together with academics and governmental agencies performing the initial steps in the discovery of new MNPs. Collaboration between private companies and public institutions can be of paramount importance for financial support in the discovery process. Figure 5.2. Marine bioactive compounds with high biotechnological potential studied from the phylum Cnidaria in the last decade. Figure 5.3. Distribution in drug classes of marine bioactive compounds with high biotechnological potential studied from cnidarian species in the last decade. 0 10 20 30 40 50 60 Alcyonacea Gorgonacea Actiniaria Scleractinia Class Anthozoa Class Class Scyphozoa Antitumor 41% Antimicrobial 24% Antiinflammatory 22% Antifoulant 10% Nervous system 2% Antiulcer 1% 111 Figure 5.4. Distribution of chemistry classes of marine bioactive compounds with high biotechnological potential studied from cnidarian species in the last decade. On the other side, crude extracts and pure compounds produced by academic laboratories may be screened by diverse bioassays as a part of broader collaboration programs, nationally and internationally, with private biotech companies. One challenge for universities is to devise mechanisms that protect intellectual property and simultaneously encourage partnerships with the private sector, by recognizing that the chances of a major commercial pay-off are small if drug discovery is pursued by a single institution (Hill & Fenical, 2010). The commercial use of some promising marine bioactive compounds isolated from cnidarians may be several years away. New compounds other than toxins and venoms produced by members of this highly diverse group of marine invertebrates may be discovered in the quest for new marine products. 5.3. 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Acknowledgements Joana Rocha is supported by a Fundação para a Ciência e Tecnologia PhD Grant (SFRH/BD/33476/2008) by PhD Program in Marine and Environmental Sciences. This research was partially financed by project LUSOEXTRACT (project no. 13107, QREN-SI I&DT, co-promotion and co-financed by POR Lisboa, AdI). The authors would like to acknowledge one anonymous reviewer and Daphne Fautin for their valuable comments on the manuscript. 128 CHAPTER 6 Conclusions and future perspectives 131 6.1. Conclusions and future perspectives Interactions between microorganisms and invertebrates are common in the marine environment. However, most of their ecological functions are still enigmatic. Current literature describes endobiotic bacteria in various eukaryotic host organisms such as protozoans, sponges, cnidarians, annelids, echinoderms, and ascidians (Althoff et al., 1998, Burnett & McKenzie, 1997, Cary et al., 1997, Deming & Colwell, 1982, Schuett et al., 2007, Schuett et al., 2005). Nonetheless, the key questions concerning the phylogenetic relationship of host-microbial symbionts, their ecological and physiological function, their secondary metabolites, chemical structure and the effects of these compounds are poorly understood (Schuett et al., 2007). Cnidarians were the dominant marine organisms late in the Precambrian period and they remain important components of marine ecological communities today Sea anemones, namely those hosting endosymbiotic photosynthetic dinoflagellates, are recognized to be important sentinel species (Winston & Heffernan, 1999). These organisms may help researchers to monitor potential environmental shifts in temperate coastal waters triggered by global climate changes. Extreme bleaching events of Anemonia in the Mediterranean under abnormally warm water conditions (Leutenegger et al., 2007) are a good example on the suitability of these anthozoans as sentinel species and an undeniable sign of ongoing global climate changes. Nonetheless there is still a deficiency of studies on the natural dynamics (either temporal and/or spatial) of the microbial biota associated with sea anemones impairing researchers to understand how potential shifts in the marine environment may affect the function of these symbioses. In light of the hologenome theory (Rosenberg et al., 2007), these anemones should be considered as holobionts (Margulis & Fester, 1991), a complex symbiosis between the cnidarian animal, its photosynthetic microalgae (e.g., zooxanthellae) and its complex community of associated microorganisms (other than microalgae) that play a key role on the overall health of the cnidarian host. Consequently, it is also important to monitor potential shifts in bacteria associated with these sea anemones to understand how environmental disturbances may shape anemone individuals and populations. The studies presented in this thesis provide detailed information on bacterial communities associating with the snakelocks anemone Anemonia viridis and fluctuations occurring in the bacterial biota of this species induced by seasonal and spatial variation, as well as physical stressors (temperature and radiation). Studies addressing bleaching events of A. viridis occurring in the wild were also performed in the frame of the present thesis. However, preliminary results from PCRDGGE and pyrosequencing were not conclusive and these and additional experiments