Quality of cellulose and biostimulant extracts from Oedogonium calcareum cultivated during primary wastewater treatment.

A practical two-product cascading biorefinery was developed to extract a biostimulant and cellulose from the freshwater filamentous macroalga Oedogonium calcareum grown while treating primary wastewater. Biostimulant production provides a valuable extract with production of disinfected residual biomass for further product development. Both Escherichia coli and F-specific RNA bacteriophage, indicators of human pathogens contamination, were absent from the residual biomass. The chemical composition of the biostimulant was complex, consisting of growth-promoting substances, free amino acids, and minerals. The O. calcareum cellulose fractions yielded between 9.5% and 10.1% (w/w) with purities from 84% to 90% and closely resembled microcrystalline cellulose. Biostimulant extraction improved cellulose quality by increasing crystallinity from 59% to 62%. Biomass condition, drying process, and biostimulant production influenced the crystallinity index. This study demonstrates a two-step process of biostimulant and cellulose extraction from wastewater-grown Oedogonium, simultaneously disinfecting biomass and isolating high-quality cellulose as a sustainable alternative to conventional extraction methods.

Denigrins H-L: Sulfated Derivatives of Denigrins D and E from a New Zealand Dictyodendrilla c.f. dendyi Marine Sponge.

Five new sulfated arylpyrrole and arylpyrrolone alkaloids, denigrins H-L (1-5), along with two known compounds, dictyodendrin B and denigrin G, were isolated from an extract of a New Zealand Dictyodendrilla c.f. dendyi marine sponge. Denigrins H-L represent the first examples of sulfated denigrins, with denigrins H and I (1-2), as derivatives of denigrin D, containing a pyrrolone core, and denigrins J-L (3-5), as derivatives of denigrin E (6), containing a pyrrole core. Their structures were elucidated by interpretation of 1D and 2D NMR spectroscopic data, ESI, and HR-ESI-MS spectrometric data, as well as comparison with literature data. Compounds 1-5, along with six known compounds previously isolated from the same extract, showed minimal cytotoxicity against the HeLa cervical cancer cell line.

Characterising the Physiological Responses of Chinook Salmon (Oncorhynchus tshawytscha) Subjected to Heat and Oxygen Stress.

In New Zealand, during the hottest periods of the year, some salmon farms in the Marlborough Sounds reach water temperatures above the optimal range for Chinook salmon. High levels of mortality are recorded during these periods, emphasising the importance of understanding thermal stress in this species. In this study, the responses of Chinook salmon (Oncorhynchus tshawytscha) to chronic, long-term changes in temperature and dissolved oxygen were investigated. This is a unique investigation due to the duration of the stress events the fish were exposed to. Health and haematological parameters were analysed alongside gene expression results to determine the effects of thermal stress on Chinook salmon. Six copies of heat shock protein 90 (HSP90) were discovered and characterised: HSP90AA1.1a, HSP90AA1.2a, HSP90AA1.1b, HSP90AA1.2b, HSP90AB1a and HSP90AB1b, as well as two copies of SOD1, named SOD1a and SOD1b. The amino acid sequences contained features similar to those found in other vertebrate HSP90 and SOD1 sequences, and the phylogenetic tree and synteny analysis provided conclusive evidence of their relationship to other vertebrate HSP90 and SOD1 genes. Primers were designed for qPCR to enable the expression of all copies of HSP90 and SOD1 to be analysed. The expression studies showed that HSP90 and SOD1 were downregulated in the liver and spleen in response to longer term exposure to high temperatures and lower dissolved oxygen. HSP90 was also downregulated in the gill; however, the results for SOD1 expression in the gill were not conclusive. This study provides important insights into the physiological and genetic responses of Chinook salmon to temperature and oxygen stress, which are critical for developing sustainable fish aquaculture in an era of changing global climates.

Investigation of the Dietary Preferences of Two Dorid Nudibranchs by Feeding-Choice Experiments and Chemical Analysis.

Feeding-choice experiments were conducted under laboratory conditions with two dorid spongivorous nudibranchs, Goniobranchus aureomarginatus and Ceratosoma amoenum, collected from a sponge meadow off Tauranga, New Zealand with two sponge prey (Dysidea teawanui sp.nov. and an undescribed species from the Dictyodendrillidae family, possibly Dictyodendrilla tenella (Lendenfeld 1888). The first choice of prey, the total number of prey choices made, and the time spent on each prey target was recorded, results indicating that each nudibranch had strong preferences for specific prey species. Preferences were significant when the time spent grazing on prey was taken into consideration. Goniobranchus aureomarginatus had a strong preference for the undescribed Dictyodendrillid sponge, while Ceratosoma ameonum preferred Dysidea teawanui. The results of the feeding-choice experiments matched observations in the wild. Chemical analysis of the undescribed Dictyodendrillid sponge led to the isolation and characterisation of six known bioactive metabolites, dictyodendrin C (1), D (2) and F (3), as well as denigrin E (4), dactylpyrrole A (5) and lamellarin O1 (6). Two of the known compounds, dictyodendrins C (1) and F (3) were also isolated from G. aureomarginatus individuals. Chemical analysis of D. teawanui afforded ergosterol peroxide, 5α,8α-epidioxy-24-methylcholesta-6,22-dien-3ß-ol (7). The structures of the isolated natural products were elucidated based on extensive analysis of 1D and 2D NMR data.

Description of two new species of Dysidea (Porifera, Demospongiae, Dictyoceratida, Dysideidae) from Tauranga Harbour, Bay of Plenty, New Zealand.

Differentiation of species within the genus Dysidea Johnston, 1842 (Order Dictyoceratida Minchin, 1900, Family Dysideidae Gray, 1867) is extremely difficult as they lack spicules which are strongly diagnostic in other Demospongiae, and their primary and secondary fibres and the mesh that they form, may be irregular in shape and thickness, thus difficult to measure for comparisons. Here we review species of Dysidea known from the New Zealand Exclusive Economic Zone (EEZ), validating five species: Dysidea cristagalli Bergquist, 1961a, from the Hauraki Gulf; D. hirciniformis (Carter, 1885a) sensu Dendy (1924), from North Cape; D. navicularis Lendenfeld, 1888, from Port Lyttleton on the east coast of the South Island; D. ramsayi (Lendenfeld, 1888) from the Chatham Islands; D. spiculivora Dendy, 1924, from Cape Maria Van Diemen and the Three Kings Islands to the north of New Zealand. Dysidea fragilis (Montagu, 1818) sensu Bergquist (1961b), from Mernoo Bank on Chatham Rise, is now considered to be invalid, and D. elegans (Nardo, 1847) sensu Brøndsted (1927), from the Coromandel Peninsula, is considered unrecognisable. Several partially characterised species have also been cited in the literature. Two new species from Tauranga Harbour, on the northeast coast of the North Island, Dysidea tuapokere sp. nov. and D. teawanui sp. nov., are described. These descriptions are based on fresh material and in situ photography, facilitating clear, informative descriptions, that will enable ease of identification of these species in the future.

Phylogeny drives large scale patterns in Australian marine bioactivity and provides a new chemical ecology rationale for future biodiscovery.

Twenty-five years of Australian marine bioresources collecting and research by the Australian Institute of Marine Science (AIMS) has explored the breadth of latitudinally and longitudinally diverse marine habitats that comprise Australia's ocean territory. The resulting AIMS Bioresources Library and associated relational database integrate biodiversity with bioactivity data, and these resources were mined to retrospectively assess biogeographic, taxonomic and phylogenetic patterns in cytotoxic, antimicrobial, and central nervous system (CNS)-protective bioactivity. While the bioassays used were originally chosen to be indicative of pharmaceutically relevant bioactivity, the results have qualified ecological relevance regarding secondary metabolism. In general, metazoan phyla along the deuterostome phylogenetic pathway (eg to Chordata) and their ancestors (eg Porifera and Cnidaria) had higher percentages of bioactive samples in the assays examined. While taxonomy at the phylum level and higher-order phylogeny groupings helped account for observed trends, taxonomy to genus did not resolve the trends any further. In addition, the results did not identify any biogeographic bioactivity hotspots that correlated with biodiversity hotspots. We conclude with a hypothesis that high-level phylogeny, and therefore the metabolic machinery available to an organism, is a major determinant of bioactivity, while habitat diversity and ecological circumstance are possible drivers in the activation of this machinery and bioactive secondary metabolism. This study supports the strategy of targeting phyla from the deuterostome lineage (including ancestral phyla) from biodiverse marine habitats and ecological niches, in future biodiscovery, at least that which is focused on vertebrate (including human) health.

Bacterial community dynamics in the marine sponge Rhopaloeides odorabile under in situ and ex situ cultivation.

Cultivation of sponges is being explored to supply biomaterial for the pharmaceutical and cosmetics industries. This study assesses the impact of various cultivation methods on the microbial community within the sponge Rhopaloeides odorabile during: (1) in situ cultivation under natural environmental conditions, (2) ex situ cultivation in small flow-through aquaria and (3) ex situ cultivation in large mesocosm systems. Principal components analysis of denaturing gradient gel electrophoresis profiles indicated a stable microbial community in sponges cultured in situ (grown in the wild) and in sponges cultured ex situ in small flow-through aquaria over 12 weeks. In contrast, a shift in the microbial community was detected in sponges cultivated ex situ in large mesocosm aquaria for 12 months. This shift included (1) a loss of some stable microbial inhabitants, including members of the Poribacteria, Chloroflexi and Acidobacteria and (2) the addition of new microbes not detected in the wild sponges. Many of these acquired bacteria had highest similarity to known sponge-associated microbes, indicating that the sponge may be capable of actively selecting its microbial community. Alternatively, long-term ex situ cultivation may cause a shift in the dominant microbes that facilitates the growth of the more rare species. The microbial community composition varied between sponges cultivated in mesocosm aquaria with different nutrient concentrations and seawater chemistry, suggesting that these variables play a role in structuring the sponge-associated microbes. The high growth and symbiont stability in R. odorabile cultured in situ confirm that this is the preferred method of aquaculture for this species at this time.

Therapeutic agents from the sea: biodiversity, chemo-evolutionary insight and advances to the end of Darwin's 200th year.

Drugs from the sea? Darwin may not have considered this concept when he was thinking about mechanisms that drove diversification of life on earth. In recognition of his 200th year, and celebration of the publication in 1859 of his "On the origin of species", we review the global status of marine biodiscovery in medicinal fields, with a focus on the South Pacific. Furthermore, in the Darwinian spirit, we touch on putative evolutionary drivers and the chemical ecology of the successful leads. We argue that, for the relatively limited investment in effort to date, the success of marine leads as therapeutics promotes enhanced focus on marine biodiversity as a source of useful medicinal agents. The simple prime argument in support of this is the fact that we can exploit over four billion years of evolution in combinatorial chemistry in marine organisms, directed at relevant and effective biological activity.

Production of manoalide and its analogues by the sponge Luffariella variabilis Is hardwired.

The Great Barrier Reef sponge Luffariella variabilis (Poléjaeff 1884) produces a range of potent anti-inflammatory compounds as its major metabolites. These major metabolites-manoalide monoacetate, manoalide, luffariellin A and seco-manoalide-were monitored temporally and spatially to quantify the potential yield from wild harvest or aquaculture. Production of the major metabolites was hardwired at the population level with little variation in space and time over meters to tens of kilometers in the Palm Islands, Queensland, Australia. Manoalide monoacetate (35 to 70 mg g(-1) dry weight of sponge) was consistently the most abundant compound followed by manoalide (15 to 20 mg g(-1) dry weight). Luffariellin A and seco-manoalide were 10 to 70 times less abundant and varied between 0 and 3 mg g(-1) dry weight. On a larger spatial scale, L. variabilis from Davies Reef and Magnetic Island contained the same rank order and yields of compounds as the Palm Islands, indicating a generality of pattern over at least 100 km. The "hardwiring" of metabolite production at the population level by L. variabilis was also reflected in the lack of any inductive effect on metabolite production. In addition, individually monitored sponges produced fixed ratios of the major metabolites over time (years). However, these ratios varied between individuals, with some individuals consistently producing high levels of manoalide and manoalide monoacetate, providing the potential for selection of high-yielding stocks.

Biomedicinals from the phytosymbionts of marine invertebrates: a molecular approach.

Marine invertebrate animals such as sponges, gorgonians, tunicates and bryozoans are sources of biomedicinally relevant natural products, a small but growing number of which are advancing through clinical trials. Most metazoan and anthozoan species harbour commensal microorganisms that include prokaryotic bacteria, cyanobacteria (blue-green algae), eukaryotic microalgae, and fungi within host tissues where they reside as extra- and intra-cellular symbionts. In some sponges these associated microbes may constitute as much as 40% of the holobiont volume. There is now abundant evidence to suggest that a significant portion of the bioactive metabolites thought originally to be products of the source animal are often synthesized by their symbiotic microbiota. Several anti-cancer metabolites from marine sponges that have progressed to pre-clinical or clinical-trial phases, such as discodermolide, halichondrin B and bryostatin 1, are thought to be products derived from their microbiotic consortia. Freshwater and marine cyanobacteria are well recognised for producing numerous and structurally diverse bioactive and cytotoxic secondary metabolites suited to drug discovery. Sea sponges often contain dominant taxa-specific populations of cyanobacteria, and it is these phytosymbionts (= photosymbionts) that are considered to be the true biogenic source of a number of pharmacologically active polyketides and nonribosomally synthesized peptides produced within the sponge. Accordingly, new collections can be pre-screened in the field for the presence of phytobionts and, together with metagenomic screening using degenerate PCR primers to identify key polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) genes, afford a biodiscovery rationale based on the therapeutic prospects of phytochemical selection. Additionally, new cloning and biosynthetic expression strategies may provide a sustainable method for the supply of new pharmaceuticals derived from the uncultured phytosymbionts of marine organisms.

Acetylated sesterterpenes from the Great Barrier Reef sponge Luffariella variabilis.

Chemical investigation of the sponge Luffariella variabilis collected from the Palm Island group of the Great Barrier Reef, Australia, yielded three new acetylated compounds, 25-acetoxyluffariellin A (1), 25-acetoxyluffariellin B (2), and 25-acetoxyseco-manoalide (3). The structures of the new compounds were elucidated on the basis of interpretation of their spectroscopic data. The known metabolites manoalide (4), seco-manoalide (5), luffariellin A (8), and manoalide monoacetate (10) were also isolated. The new acetylated compounds (1-3) were labile in the sponge tissue when samples were allowed to thaw prior to extraction, but were stable once isolated. Sponge samples that were completely thawed contained only hydroxylated compounds (alcohols). This finding supported the deduction that the acetylated compounds are being enzymatically transformed and/or degraded.

New methods for medicinal chemistry--universal gene cloning and expression systems for production of marine bioactive metabolites.

Natural products from symbiotic or commensal associations between marine invertebrate and microbial organisms show exceptional promise as pharmaceuticals in many therapeutic areas. An economic and sustainable global market supply due to difficulty of synthesis is cited as the main obstacle for exploitation of these otherwise exciting marine bioactive compounds. Different strategies have been evoked to overcome this impediment as long-term harvesting of wild stocks from the environment is considered unsound, and other modes of production based on biosynthesis, such as aquaculture, have not yet been proven as reliable. One option is to clone the genes encoding the biosynthetic expression of a lead metabolite into a surrogate host suitable for industrial-scale fermentation. To facilitate this goal we are developing a universal system to clone and express genes responsible for biosynthesis of natural products from both eukaryotic and prokaryotic partners of marine symbioses. The ability to harness the complete meta-transcriptome of entire biosynthetic pathways is particularly valuable where the biogenesis of a target natural product occurring within a complex symbiotic association is unclear.

Diverse microbial communities inhabit Antarctic sponges.

Genetic techniques were employed to investigate the archaeal, bacterial and eukaryotic communities associated with the Antarctic sponges Kirkpatrickia varialosa, Latrunculia apicalis, Homaxinella balfourensis, Mycale acerata and Sphaerotylus antarcticus. The phylogenetic affiliation of sponge-derived bacteria was assessed by 16S rRNA sequencing of cloned DNA fragments. Denaturing gradient gel electrophoresis (DGGE) was used to determine the stability of bacterial associations within each sponge species and across spatial scales. Of the 150 archaeal clones from L. apicalis, K. varialosa and M. acerata screened by restriction fragment length polymorphism (RFLP) analysis, four unique operational taxonomic units (OTUs) were observed and all clustered closely together within the Crenarchaeota. Of the 250 sponge-derived bacterial clones screened by RFLP analysis, 61 were unique OTUs that were not detected during examination of 160 seawater-derived clones. Rarefaction analysis indicated that the clone libraries represented between 44 and 83% of the total estimated diversity. Phylogenetic analysis of sequence data revealed that the bacterial communities present in Antarctic sponges primarily clustered within the Gamma and Alpha proteobacteria and the Cytophaga/Flavobacterium of Bacteroidetes group. Bacterial DGGE analysis for replicate sponge and seawater samples at each Antarctic site revealed that bacterial communities were consistently detected within a particular species regardless of the collection site, with six bacterial bands exclusively associated with a single sponge species. Phylogenetic analysis of sequence data from eukaryotic DGGE analysis revealed that the communities present in Antarctic sponges fell into diatom and dinoflagellate clusters with many sequences having no known close relatives. In addition, seven eukaryotic sequences that were not detected in seawater samples or other sponge species were observed in K. varialosa.