title: Chemical ecological insights into metabolites of the New Zealand marine sponge, Cliona celata

authors: Webb, Nicola G.

subjects: chemical ecology

abstract: Marine sponges are known to produce secondary metabolites that have roles including chemical defence against cellular challenge. An investigation into Cliona celata around the Bay of Plenty (New Zealand) was conducted in response to discovery of unusual metabolites that had the potential to be biologically active. The study was designed to determine chemical variation in the sponge over small spatial and temporal scales and to determine the possible ecological triggers for the presence of these metabolites. The hypothesis was that a chemical defensive response would be elicited 24-48 hours post a cellular challenge event such as physical damage. The hypothesis was based on the fact that the sponge heals quickly when damaged with little evidence of necrosis.

Cliona celata was chosen as there is little knowledge of the species within New Zealand, and previous work showed a single specimen to produce brominated compounds, never before reported for this genus. This project examines whether the production of brominated compounds is common throughout all C. celata within the Bay of Plenty region or is a localised response.

A preliminary study was conducted to investigate spatial chemical variability of sponges from three locations, Leisure Island (intertidal), Rabbit Island (subtidal) (both on the coast adjacent to Mount Maunganui, Tauranga) and White Island (geothermal subtidal). A damage response experiment for Leisure Island and Rabbit Island specimens was conducted to determine chemical differences in healthy versus damaged sponges, and whether a chemical defence mechanism was utilised by the sponge species. Lastly, a stratification experiment was performed to establish whether chemical changes occur throughout the sponge or are localised to the immediate damage site.

Cliona celata identification was confirmed using spicule mounts. However, some variation between intertidal and subtidal specimens was noted with differences in spicular morphology, likely due to variations in chemistry and micro-environments. 

On a small scale, extracts of each sample were prepared and analysed using Liquid Chromatography Mass Spectrometry (LCMS). Damaged and non damaged sponge specimens were subjected to an MTT assay using the human cervical cancer cell line (HeLa). But the crude extracts showed no bioactivity in this assay. 

Chromatograms obtained from LCMS, for all samples contained a common set of peaks, however, variations in intensities and some minor peak variations within the sites (intertidal, subtidal and geothermal subtidal) and individual specimens were noted, and could represent interactions with different micro-environments. A lack of chemical response to damage was not expected. It is therefore hypothesised that the lack of secondary metabolite production observed, is due to cells reverting to archaeocytes to repair and re-grow the removed sponge section, a histological response rather than a chemical one. 

It was hypothesised that specimens with increased levels of epibiont coverage had increased production of certain metabolites, as represented by increased intensities of some peaks in chromatograms, however, to a threshold. Once epibiont coverage of approximately 50% was met, novel chemicals were thought to be produced by the sponge. Two sponge samples did produce monobrominated and dibrominated compounds, however, these were localised events and could potentially be the response to an unidentified external stressor or epibiont coverage. The stratification experiment indicated that the isolation and production of these brominated compounds may be restricted to the pinacoderm of the sponge, possibly suggesting a transient chemical response as cells re-organise themselves in a healing process. However, further investigation is required with all experiments and a more comprehensive time series of samples needs to be taken post damage treatment.
This project provides a preliminary study into the chemical variation and chemical response to damage for intertidal and subtidal Bay of Plenty Cliona celata. Any hypotheses made during this thesis need to be further examined to be accepted.

text: Chemical ecological insights into metabolites of the New Zealand marine sponge, Cliona celata Marine sponges are known to produce secondary metabolites that have roles including chemical defence against cellular challenge. An investigation into Cliona celata around the Bay of Plenty (New Zealand) was conducted in response to discovery of unusual metabolites that had the potential to be biologically active. The study was designed to determine chemical variation in the sponge over small spatial and temporal scales and to determine the possible ecological triggers for the presence of these metabolites. The hypothesis was that a chemical defensive response would be elicited 24-48 hours post a cellular challenge event such as physical damage. The hypothesis was based on the fact that the sponge heals quickly when damaged with little evidence of necrosis.

Cliona celata was chosen as there is little knowledge of the species within New Zealand, and previous work showed a single specimen to produce brominated compounds, never before reported for this genus. This project examines whether the production of brominated compounds is common throughout all C. celata within the Bay of Plenty region or is a localised response.

A preliminary study was conducted to investigate spatial chemical variability of sponges from three locations, Leisure Island (intertidal), Rabbit Island (subtidal) (both on the coast adjacent to Mount Maunganui, Tauranga) and White Island (geothermal subtidal). A damage response experiment for Leisure Island and Rabbit Island specimens was conducted to determine chemical differences in healthy versus damaged sponges, and whether a chemical defence mechanism was utilised by the sponge species. Lastly, a stratification experiment was performed to establish whether chemical changes occur throughout the sponge or are localised to the immediate damage site.

Cliona celata identification was confirmed using spicule mounts. However, some variation between intertidal and subtidal specimens was noted with differences in spicular morphology, likely due to variations in chemistry and micro-environments. 

On a small scale, extracts of each sample were prepared and analysed using Liquid Chromatography Mass Spectrometry (LCMS). Damaged and non damaged sponge specimens were subjected to an MTT assay using the human cervical cancer cell line (HeLa). But the crude extracts showed no bioactivity in this assay. 

Chromatograms obtained from LCMS, for all samples contained a common set of peaks, however, variations in intensities and some minor peak variations within the sites (intertidal, subtidal and geothermal subtidal) and individual specimens were noted, and could represent interactions with different micro-environments. A lack of chemical response to damage was not expected. It is therefore hypothesised that the lack of secondary metabolite production observed, is due to cells reverting to archaeocytes to repair and re-grow the removed sponge section, a histological response rather than a chemical one. 

It was hypothesised that specimens with increased levels of epibiont coverage had increased production of certain metabolites, as represented by increased intensities of some peaks in chromatograms, however, to a threshold. Once epibiont coverage of approximately 50% was met, novel chemicals were thought to be produced by the sponge. Two sponge samples did produce monobrominated and dibrominated compounds, however, these were localised events and could potentially be the response to an unidentified external stressor or epibiont coverage. The stratification experiment indicated that the isolation and production of these brominated compounds may be restricted to the pinacoderm of the sponge, possibly suggesting a transient chemical response as cells re-organise themselves in a healing process. However, further investigation is required with all experiments and a more comprehensive time series of samples needs to be taken post damage treatment.
This project provides a preliminary study into the chemical variation and chemical response to damage for intertidal and subtidal Bay of Plenty Cliona celata. Any hypotheses made during this thesis need to be further examined to be accepted.

year: 2015

title: Enzymic approach to eurythermalism of Alvinella pompejana and its Episymbionts

authors: Lee, Charles Kai-Wu

subjects: biology

abstract: The equilibrium model, which describes the influence of temperature on enzyme activity, has been established as a valid and useful tool for characterizing enzyme eurythermalism and thermophily. By introducing Keq, a temperature-dependent equilibrium constant for the interconversion between Eact, the active form of enzyme, and Einact, a reversibly inactive form of enzyme, the equilibrium model currently provides the most complete description of the enzyme-temperature relationship; its derived parameters are intrinsic and apparently universal and, being derived under reaction conditions, potentially have physiological significance. One of these parameters, Teq, correlates with host growth temperature better than enzyme stability does. The vent-dwelling annelid Alvinella pompejana has been reported as an extremely eurythermal organism, and the symbiotic complex microbial community associated with its dorsal surface is likely to experience similar environmental thermal conditions. The A. pompejana episymbiont community, predominantly composed of epsilonproteobacteria, has been analyzed metagenomically, enabling direct retrieval of genes coding for enzymes suitable for equilibrium model applications. Two such genes, coding for isopropylmalate dehydrogenase and glutamate dehydrogenase, have been isolated from the A. pompejana episymbionts, heterologously expressed, and shown by reverse transcription-quantitative PCR to be actively expressed. The equilibrium model parameters of characterized expression products suggested that enzyme eurythermalism constitutes part of the thermal adaptation strategy employed by the episymbionts. Moreover, the enzymes' thermal characteristics correspond to their predicted physiological roles and the abundance and expression of the corresponding genes. This paper demonstrates the use of the equilibrium model as part of a top-down metagenomic approach to studying temperature adaptation of uncultured organisms.

text: Enzymic approach to eurythermalism of Alvinella pompejana and its Episymbionts The equilibrium model, which describes the influence of temperature on enzyme activity, has been established as a valid and useful tool for characterizing enzyme eurythermalism and thermophily. By introducing Keq, a temperature-dependent equilibrium constant for the interconversion between Eact, the active form of enzyme, and Einact, a reversibly inactive form of enzyme, the equilibrium model currently provides the most complete description of the enzyme-temperature relationship; its derived parameters are intrinsic and apparently universal and, being derived under reaction conditions, potentially have physiological significance. One of these parameters, Teq, correlates with host growth temperature better than enzyme stability does. The vent-dwelling annelid Alvinella pompejana has been reported as an extremely eurythermal organism, and the symbiotic complex microbial community associated with its dorsal surface is likely to experience similar environmental thermal conditions. The A. pompejana episymbiont community, predominantly composed of epsilonproteobacteria, has been analyzed metagenomically, enabling direct retrieval of genes coding for enzymes suitable for equilibrium model applications. Two such genes, coding for isopropylmalate dehydrogenase and glutamate dehydrogenase, have been isolated from the A. pompejana episymbionts, heterologously expressed, and shown by reverse transcription-quantitative PCR to be actively expressed. The equilibrium model parameters of characterized expression products suggested that enzyme eurythermalism constitutes part of the thermal adaptation strategy employed by the episymbionts. Moreover, the enzymes' thermal characteristics correspond to their predicted physiological roles and the abundance and expression of the corresponding genes. This paper demonstrates the use of the equilibrium model as part of a top-down metagenomic approach to studying temperature adaptation of uncultured organisms.

year: 2007

title: Sustainable management, a sustainable ethic?

authors: Daya-Winterbottom, Trevor

abstract: New Zealand legislated for sustainability by enacting the Resource Management Act 1991 (RMA). The RMA received the Royal Assent on 22 July 1991 and came into force as law on 1 October 1991. It restated and reformed “the law relating to the use of land, air, and water”, and is the principal statute governing the New Zealand environment. 
Despite the rhetoric of sustainable management, environmental law and governance in New Zealand continue to grapple with persistent challenges in relation to protecting biodiversity on private land, maintaining freshwater quantity and quality, and recognising Maori interests in resource management. New strategies are however emerging for promoting environmental justice and using the law to advance sustainability. My paper will therefore explore these aspects of the New Zealand experiment in legislating for sustainable management.

text: Sustainable management, a sustainable ethic? New Zealand legislated for sustainability by enacting the Resource Management Act 1991 (RMA). The RMA received the Royal Assent on 22 July 1991 and came into force as law on 1 October 1991. It restated and reformed “the law relating to the use of land, air, and water”, and is the principal statute governing the New Zealand environment. 
Despite the rhetoric of sustainable management, environmental law and governance in New Zealand continue to grapple with persistent challenges in relation to protecting biodiversity on private land, maintaining freshwater quantity and quality, and recognising Maori interests in resource management. New strategies are however emerging for promoting environmental justice and using the law to advance sustainability. My paper will therefore explore these aspects of the New Zealand experiment in legislating for sustainable management.

year: 2012