Microbial symbionts of marine invertebrates: opportunities for microbial biotechnology

Marine invertebrates are sources of a diverse array of bioactive metabolites with great potential for development as drugs and research tools. In many cases, microorganisms are known or suspected to be the biosynthetic source of marine invertebrate natural products. The application of molecular microbiology to the study of these relationships will contribute to basic biological knowledge and facilitate biotechnological development of these valuable resources. The bryostatin-producing bryozoan B. neritina and its specific symbiont "Candidatus Endobugula sertula" constitute one promising model system. Another fertile subject for investigation is the listhistid sponges that contain numerous bioactive metabolites, some of which originate from bacterial symbionts.     

Antimicrobial activity in sub-Arctic marine invertebrates

In the marine environment, any living or non-living surface is exposed to bacterial colonization. Many invertebrate species in temperate, tropical and Antarctic regions have demonstrated chemical defences against the formation of microbial films. In the present study, the antimicrobial activity of sub-Arctic invertebrates was investigated for the first time. Crude extracts of abundant invertebrates belonging to several taxonomic groups were tested for their inhibitory effects on the growth of five sympatric phylogenetically diverse bacterial strains. Six out of 18 (33%) crude extracts inhibited bacterial growth at natural extract concentrations. The crude extract of the sponge Haliclona viscosa inhibited growth of all five bacterial strains, suggesting the presence of metabolites with broad-spectrum activity. Three active compounds were isolated from H. viscosa having antibacterial properties similar to those of the crude extract. Our data indicate that antibacterial secondary metabolites are present in sub-Arctic marine invertebrates but are less abundant than in temperate, tropical or Antarctic species.     

From chemical structure to environmental biosynthetic pathways: navigating marine invertebrate-bacteria associations

Many natural products from marine invertebrates are promising drugs or drug leads but the problem of supply has delayed the development of these agents. Bacteria living in complex associations with animals are often proposed to be the real producers of "invertebrate" metabolites. Recent advances in biosynthetic pathway discovery from these symbiotic bacteria herald a new era in which biosynthetic genes will be cloned rapidly to provide promising molecules from marine invertebrates.     

Highlights of marine invertebrate-derived biosynthetic products: their biomedical potential and possible production by microbial associants

Coral reefs are among the most productive marine ecosystems and are the source of a large group of structurally unique biosynthetic products. Annual reviews of marine natural products continue to illustrate that the most prolific source of bioactive compounds consist of coral reef invertebrates-sponges, ascidians, mollusks, and bryozoans. This account examines recent milestone developments pertaining to compounds from invertebrates designated as therapeutic leads for biomedical discovery. The focus is on the secondary metabolites, their inspirational structural scaffolds and the possible role of micro-organism associants in their biosynthesis. Also important are the increasing concerns regarding the collection of reef invertebrates for the discovery process. The case examples considered here will be useful to insure that future research to unearth bioactive invertebrate-derived compounds will be carried out in a sustainable and environmentally conscious fashion. Our account begins with some observations pertaining to the natural history of these organisms. Many still believe that a serious obstacle to the ultimate development of a marine natural product isolated from coral reef invertebrates is the problem of compound supply. Recent achievements through total synthesis can now be drawn on to forcefully cast this myth aside. The tools of semisynthesis of complex natural products or insights from SAR efforts to simplify an active pharmacophore are at hand and demand discussion. Equally exciting is the prospect that invertebrate-associated micro-organisms may represent the next frontier to accelerate the development of high priority therapeutic candidates. Currently in the United States there are two FDA approved marine-derived therapeutic drugs and two others that are often cited as being marine-inspired. This record will be examined first followed by an analysis of a dozen of our favorite examples of coral reef invertebrate natural products having therapeutic potential. The record of using complex scaffolds of marine invertebrate products as the starting point for development will be reviewed by considering eight case examples. The potential promise of developing invertebrate-derived micro-organisms as the starting point for further exploration of therapeutically relevant structures is considered. Also significant is the circumstance that there are some 14 sponge-derived compounds that are available to facilitate fundamental biological investigations.     

Are there adequate data to assess how well theories of rarity apply to marine invertebrates?

Understanding the biology of rare species is a very important part of conservation biology. Most of our current understanding of rarity has, however, come from studies of terrestrial plants, birds, mammals and some insects. Freshwater and marine habitats are underrepresented in published studies of rare species or conservation biology. We therefore have little knowledge about how well our understanding of what makes particular species rare and how rare species persist applies to marine invertebrates which form a major component of coastal biodiversity. In this review, I examine some theories about rarity with respect to intertidal and shallow subtidal invertebrates to identify whether there are adequate data to apply these theories to marine invertebrates and how well such theories apply. The general conclusions are that the lack of quantitative data on abundances, ranges, habitat-requirements, dispersal and connectedness among populations for marine invertebrates means that their status as rare species cannot really be assessed appropriately. It is also unlikely that, without extensive sampling programmes and considerable expense, adequate data could be obtained for these small, cryptic animals, which typically have very patchy, variable and unpredictable patterns of distribution and abundance. Intertidal and subtidal assemblages are diverse, including species with many different life-histories from many phyla, occupying the same suite of habitats. It is therefore suggested that future research on rare organisms in marine habitats should build upon the long and successful history of experimental marine studies to test specific hypotheses about processes influencing rarity in the field. Such studies would not only add a new dimension to our current understanding of rarity, but would also provide badly-needed data on the status of rare marine invertebrates. abundances, invertebrates, marine, range, rarity     

beta-cyanoalanine production by marine bacteria on cyanide-free medium and its specific inhibitory activity toward cyanobacteria

In screening the culture broth of marine bacteria collected at Yap (Micronesia), Palau (Belau), and Okinawa (the southwest islands of Japan) for antimicroalgal activity, 37 out of 2,594 bacterial isolates tested were found to produce anticyanobacterial substances against Oscillatoria amphibia NIES-361. One strain, C-979, identified as a Vibrio sp., was selected and cultured in 2.4 liters of marine broth 2216 to identify the bioactive compound produced by the strain. The purified very hydrophilic compound (16.4 mg) was determined to be beta-cyano-L-alanine (L-CNAla) by instrumental analyses and the application of the advanced Marfey method. L-CNAla did not inhibit the growth of bacteria, yeast, or eukaryotic microalgae, but some cyanobacteria were found to be sensitive to L-CNAla at a concentration of 0.4 to 25 microg/ml. The effect of L-CNAla on some other environmental organisms, including invertebrates and a macroalgae, is discussed. CNAla production in marine broth was examined by thin-layer chromatography for the 37 bacterial isolates which produced an anticyanobacterial substance. The broth of 36 of these strains contained CNAla, suggesting the wide distribution of CNAla production by marine bacteria. This is the first report on bacteria that produce CNAla without a supply of the cyanide ion in the medium.     

cDNA cloning of the lysozyme of the white shrimp Penaeus vannamei

Lysozyme, an antibacterial protein, has been implicated in innate immunity in invertebrates, but its activity in shrimp remained to be determined. We cloned the white shrimp lysozyme cDNA using a PCR strategy and detected its activity in haemocytes using a lytic-zone assay against Micrococcus luteus. The cloning was based on a reported EST (dbEST BE18831). The deduced amino acid sequence resulted in 150 amino with 46% identity to hen egg white lysozyme. RT-PCR was used to detect lysozyme mRNA in haemocytes. Analysis of the amino acid sequence of the shrimp lysozyme showed that it belongs to the C-type family of lysozymes. Furthermore, the lysozyme amino acid sequence contained extra residues at its C-terminus, which are characteristic of marine invertebrates. This information will be useful in future studies on the molecular mechanisms of immunity in marine invertebrates.     

Ocean acidification affects marine chemical communication by changing structure and function of peptide signalling molecules

Ocean acidification is a global challenge that faces marine organisms in the near future with a predicted rapid drop in pH of up to 0.4 units by the end of this century. Effects of the change in ocean carbon chemistry and pH on the development, growth and fitness of marine animals are well documented. Recent evidence also suggests that a range of chemically mediated behaviours and interactions in marine fish and invertebrates will be affected. Marine animals use chemical cues, for example, to detect predators, for settlement, homing and reproduction. But, while effects of high CO₂ conditions on these behaviours are described across many species, little is known about the underlying mechanisms, particularly in invertebrates. Here, we investigate the direct influence of future oceanic pH conditions on the structure and function of three peptide signalling molecules with an interdisciplinary combination of methods. NMR spectroscopy and quantum chemical calculations were used to assess the direct molecular influence of pH on the peptide cues, and we tested the functionality of the cues in different pH conditions using behavioural bioassays with shore crabs (Carcinus maenas) as a model system. We found that peptide signalling cues are susceptible to protonation in future pH conditions, which will alter their overall charge. We also show that structure and electrostatic properties important for receptor binding differ significantly between the peptide forms present today and the protonated signalling peptides likely to be dominating in future oceans. The bioassays suggest an impaired functionality of the signalling peptides at low pH. Physiological changes due to high CO₂ conditions were found to play a less significant role in influencing the investigated behaviour. From our results, we conclude that the change of charge, structure and consequently function of signalling molecules presents one possible mechanism to explain altered behaviour under future oceanic pH conditions.     

A decade of climate change experiments on marine organisms: procedures,patterns and problems

The first decade of the new millennium saw a flurry of experiments to establish a mechanistic understanding of how climate change might transform the global biota, including marine organisms. However, the biophysical properties of the marine environment impose challenges to experiments, which can weaken their inference space. To facilitate strengthening the experimental evidence for possible ecological consequences of climate change, we reviewed the physical, biological and procedural scope of 110 marine climate change experiments published between 2000 and 2009. We found that 65% of these experiments only tested a single climate change factor (warming or acidification), 54% targeted temperate organisms, 58% were restricted to a single species and 73% to benthic invertebrates. In addition, 49% of the reviewed experiments had issues with the experimental design, principally related to replication of the main test‐factors (temperature or pH), and only 11% included field assessments of processes or associated patterns. Guiding future research by this inventory of current strengths and weaknesses will expand the overall inference space of marine climate change experiments. Specifically, increased effort is required in five areas: (i) the combined effects of concurrent climate and non‐climate stressors; (ii) responses of a broader range of species, particularly from tropical and polar regions as well as primary producers, pelagic invertebrates, and fish; (iii) species interactions and responses of species assemblages, (iv) reducing pseudo‐replication in controlled experiments; and (v) increasing realism in experiments through broad‐scale observations and field experiments. Attention in these areas will improve the generality and accuracy of our understanding of climate change as a driver of biological change in marine ecosystems.     

Endocrine disruptors in marine organisms: approaches and perspectives

Organic pollutants exhibiting endocrine disrupting activity (Endocrine Disruptors--EDs) are prevalent over a wide range in the aquatic ecosystems; most EDs are resistant to environmental degradation and are considered ubiquitous contaminants. The actual potency of EDs is low compared to that of natural hormones, but environmental concentrations may still be sufficiently high to produce detrimental biological effects. Most information on the biological effects and mechanisms of action of EDs has been focused on vertebrates. Here we summarize recent progress in studies on selected aspects of endocrine disruption in marine organisms that are still poorly understood and that certainly deserve further research in the near future. This review, divided in four sections, focuses mainly on invertebrates (effects of EDs and mechanisms of action) and presents data on top predators (large pelagic fish and cetaceans), a group of vertebrates that are particularly at risk due to their position in the food chain. The first section deals with basic pathways of steroid biosynthesis and metabolism as a target for endocrine disruption in invertebrates. In the second section, data on the effects and alternative mechanisms of action of estrogenic compounds in mussel immunocytes are presented, addressing to the importance of investigating full range responses to estrogenic chemicals in ecologically relevant invertebrate species. In the third section we review the potential use of vitellogenin (Vtg)-like proteins as a biomarker of endocrine disruption in marine bivalve molluscs, used worldwide as sentinels in marine biomonitoring programmes. Finally, we summarize the results of a recent survey on ED accumulation and effects on marine fish and mammals, utilizing both classical biomarkers of endocrine disruption in vertebrates and non-lethal techniques, such as non-destructive biomarkers, indicating the toxicological risk for top predator species in the Mediterranean. Overall, the reviewed data underline the potential to identify specific types of responses to specific groups of chemicals such as EDs in order to develop suitable biomarkers that could be useful as diagnostic tools for endocrine disruption in marine invertebrates and vertebrates.     

Microbial antagonism: a neglected avenue of natural products research

Competition amongst microbes for space and nutrients in the marine environment is a powerful selective force which has led to the evolution of a variety of effective strategies for colonising and growing on surfaces. We are particularly interested in the chemical ecology of marine epibiotic bacteria which live on the surfaces of marine algae or invertebrates. Over 400 strains of surface-associated bacteria from various species of seaweed and invertebrate from Scottish coastal waters were isolated and 35% of them shown to produce antimicrobial compounds. This is a much higher proportion than free living marine isolates or soil bacteria. In addition, many strains which did not normally produce antibiotics could be induced to do so by exposing them to small amounts of live cells, supernatants from other bacterial cultures or other chemicals. Thus the number of strains able to produce antibiotics appears to be much higher than previously thought. Induction of antibiotic production was elicited by other marine epibionts and also by terrestrial human pathogens such as Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa. An understanding of this type of chemical induction and the factors regulating non-constitutive secretion of antimicrobial compounds will allow more effective strategies for searching for new chemotherapeutic antibiotics to be designed.     

Acquisition of fishes and aquatic invertebrates for zoological collections. Is there a future?

The majority of the freshwater fishes in the ornamental trade now originate from captive‐bred sources, as do a large proportion of the freshwater species exhibited in public aquariums. In contrast, commercial operators who also supply marine specimens to the ornamental trade remove directly from the wild approximately 98% of the marine fishes and invertebrates exhibited in public aquariums. The common perception prevails that captive propagation is inherently a better alternative to obtaining animals from the wild. Although captive propagation has been shown to have many benefits for terrestrial species, there are a number of features unique to marine species that challenge the idea that every species should be bred in captivity. Some of the key issues relating to the development of widespread conservation‐oriented captive propagation programs include: 1) the high taxonomic diversity in marine animals; 2) the resultant variety in their reproductive methods; 3) their ecological, behavioral, physiological, and nutritional needs; and 4) our general lack of knowledge on their husbandry and medical care. There are several characteristics of marine fish and invertebrate populations that make them suitable candidates for sustainable harvest. For instance, marine teleosts are “r‐selected,” meaning that they have an extremely high fecundity, and most marine teleosts have a wide distribution and the ability to disperse over long distances. In locations considered for fish collection, appropriate management techniques should be employed to ensure that fishes and invertebrates are collected with as little impact on the ecosystem as possible. The collection of marine fishes and invertebrates for public aquariums and the hobby trade should be managed like a fishery to ensure long‐term sustainability. The public aquarium community should support marine organism certification initiatives, such as the Marine Aquarium Council (MAC). Marine organism certification will create market incentives that encourage and support quality and sustainable practices by creating consumer demand and confidence for certified organisms, practices, and industry participants. The creation of refuges that supply propagules to harvested areas, the rotation of areas fished, species‐specific size limits and seasons, and standardization of collecting, handling, and transportation techniques should be used to manage these fisheries and harvest areas. Zoo Biol 22:519–527, 2003. © 2003 Wiley‐Liss, Inc.     

Natural halogenated fatty acids: their analogues and derivatives

A comprehensive survey has been made of all fatty acids containing halogen atoms covalently bonded to carbon and which are deemed as naturally occurring. Generally thought to be minor components produced by many different organisms, these interesting compounds now number more than 300. Recent research, especially in the marine area, indicates this number will increase in the future. Sources of halogenated fatty acids include microorganisms, algae, marine invertebrates, and higher plants and some animals. Their possible biological significance has also been discussed     

海洋细菌抗菌和细胞毒活性的初步研究

从不同海域的生物、海水和海泥中分离海洋细菌,利用琼脂扩散法和MTT法对细菌培养液的乙酸乙酯提取物进行了抗菌和细胞毒活性筛选。比较了活性菌株与来源的相关性．结果表明,在分离的341株海洋细菌中。42株细菌的代谢产物具有抗菌活性,7株具有细胞毒活性,其中来源于海洋无脊椎动物和海藻的活性菌株比例(22％和11％)大于来源于海水和海泥的细菌(7％和5％)．细菌分类鉴定结果显示,具有活性的细菌大部分属于假单胞菌属、发光杆菌属、梭状芽孢杆菌属、交替单胞菌属和黄杆菌属．     

Copper affects biofilm inductiveness to larval settlement of the serpulid polychaete Hydroides elegans (Haswell)

Copper (Cu) contamination is a potential threat to the marine environment due to the use of Cu-based antifouling paints. Cu stress on larval settlement of the polychaete Hydroides elegans was investigated, and this was linked to Cu stress on biofilms and on the biofilm development process. The inductiveness of young biofilms was more easily altered by Cu stress than that of old biofilms, indicating the relative vulnerability of young biofilms. This might result from changes in bacterial survival, the bacterial community composition and the chemical profiles of young biofilms. Cu also affected biofilm development and the chemical high performance liquid chromatograph fingerprint profile. The results indicate that Cu affected larval settlement mainly through its effect on the process of biofilm development in the marine environment, and the chemical profile was crucial to biofilm inductiveness. It is strongly recommended that the effects of environmentally toxic substances on biofilms are evaluated in ecotoxicity bioassays using larval settlement of invertebrates as the end point.     

Microbial diversity in Frenulata (Siboglinidae,Polychaeta) species from mud volcanoes in the Gulf of Cadiz (NE Atlantic)

Frenulates are a group of gutless marine annelids belonging to the Siboglinidae that are nutritionally dependent upon endosymbiotic bacteria. We have characterized the bacteria associated with several frenulate species from mud volcanoes in the Gulf of Cadiz by PCR-DGGE of bacterial 16S rRNA genes, coupled with analysis of 16S rRNA gene libraries. In addition to the primary symbiont, bacterial consortia (microflora) were found in all species analysed. Phylogenetic analyses indicate that the primary symbiont in most cases belongs to the Gammaproteobacteria and were related to thiotrophic and methanotrophic symbionts from other marine invertebrates, whereas members of the microflora were related to multiple bacterial phyla. This is the first molecular evidence of methanotrophic bacteria in at least one frenulate species. In addition, the occurrence of the same bacterial phylotype in different Frenulata species, from different depths and mud volcanoes suggests that there is no selection for specific symbionts and corroborates environmental acquisition as previously proposed for this group of siboglinids.     

Combined ecological factors permit classification of developmental patterns in benthic marine invertebrates: a discussion note

Traditional classifications of developmental patterns of marine benthic invertebrates are based on combinations of embryological (direct or indirect development) and ecological (such as nutritional source or habitat) characteristics. Different schemes have been proposed for different reasons, relating to ecology, evolution and/or development. However, these classifications contain interconnected characters that do not efficiently discriminate between developmental patterns and, thus, do not fully apply to either ecological or embryological studies. An ecological multifactor classification based on three independent two-state characters (pelagic/benthic, free/protected, and feeding/non-feeding) is proposed. It discriminates between eight developmental patterns and can encompass any ecological pattern of development among marine benthic invertebrates.     

Temperature and embryonic development in polar marine invertebrates

The life-history tactics of many Antarctic marine invertebrates suggest that the commonly observed slow rates of growth are adaptations to the pattern of food availability, and not due to low temperature per se. This implies that marine invertebrates have been able, over the course of evolutionary time, to compensate their rates of embryonic development for the effect of temperature. Data from north Atlantic copepods indicate that this is so. It is therefore suggested that the slow rates of embryonic development in many Antarctic marine invertebrates are the result of large egg size, and not the low temperature. Large, slowly developing eggs are part of a suite of tactics, often called K-strategies, which characterise many marine invertebrates in Antarctica.     

Trends in the Discovery of New Marine Natural Products from Invertebrates over the Last Two Decades – Where and What Are We Bioprospecting?

It is acknowledged that marine invertebrates produce bioactive natural products that may be useful for developing new drugs. By exploring untapped geographical sources and/or novel groups of organisms one can maximize the search for new marine drugs to treat human diseases. The goal of this paper is to analyse the trends associated with the discovery of new marine natural products from invertebrates (NMNPI) over the last two decades. The analysis considers different taxonomical levels and geographical approaches of bioprospected species. Additionally, this research is also directed to provide new insights into less bioprospected taxa and world regions. In order to gather the information available on NMNPI, the yearly-published reviews of Marine Natural Products covering 1990-2009 were surveyed. Information on source organisms, specifically taxonomical information and collection sites, was assembled together with additional geographical information collected from the articles originally describing the new natural product. Almost 10000 NMNPI were discovered since 1990, with a pronounced increase between decades. Porifera and Cnidaria were the two dominant sources of NMNPI worldwide. The exception was polar regions where Echinodermata dominated. The majority of species that yielded the new natural products belong to only one class of each Porifera and Cnidaria phyla (Demospongiae and Anthozoa, respectively). Increased bioprospecting efforts were observed in the Pacific Ocean, particularly in Asian countries that are associated with the Japan Biodiversity Hotspot and the Kuroshio Current. Although results show comparably less NMNPI from polar regions, the number of new natural products per species is similar to that recorded for other regions. The present study provides information to future bioprospecting efforts addressing previously unexplored taxonomic groups and/or regions. We also highlight how marine invertebrates, which in some cases have no commercial value, may become highly valuable in the ongoing search for new drugs from the sea.