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Structure Elucidation of Metabolite


Weili Chan 2016

Summary

Classification
Kingdom: Animalia 

Phylum: Porifera

Class: Demospongiae

Subclass: Heteroscleromorpha

Order: Poecilosclerida

Family: Mycalidae

Genus: Mycale

Subgenus: Mycale (Arenochalina)

Species: Mycale (Arenochalina) mirabilis



(Van Soest 2007a; Van Soest 2007b)
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Figure 1

Glossary
Anisochelae: asymmetric chelae

Choanosome: the internal flesh of a sponge

Chelae: characteristic order Poecilosclerida c-shaped microscleres 

Conules: cone-shaped projections on sponge surface caused by the underlying skeleton

Ectosome: the external, superficial region of a sponge

Megascleres: the major and large structural spicules that form the sponge skeletal framework

Microscleres: the small spicules that reinforce the framework by supporting the canal system lining

Mycalostyles: characteristic Mycalidae family rod-shaped spicules

Oscules: an aperture of the sponge through which water is expelled

Sigma: microscleres that are either C- or S- shaped

Spicule: an inorganic structural element found in most sponges that are made of either siliceous or calcareous

Spongin: fibrous, collagenous substance forming the skeletal network of Demonspongiae sponges by binding spicules together 

Subtylostyle: a tylostyle that is pointed at one end and knob-shaped at the other end

Tylostyle: megasclere spicules that are pin-shaped



(Boury-Esnault & Rützler 1997; Goudie, Norman & Finn 2013; Ruppert, Fox & Barnes 2004; Van Soest & Hajdu 2002).

Acknowledgements
I would like to thank Dr John N. A. Hooper of the Queensland Museum for the identification of Mycale (Arenochalina) mirabilis.

I am indebted to Professor Mary Garson of the School of Chemistry & Molecular Biosciences at the University of Queensland for firstly agreeing to the commencement of the project presented, and for the exceptional guidance, assistance and patience provided to me throughout its course by Professor Mary and PhD student Ariyanti Dewi.

Many thanks also to Professor Bernie Degnan of the School of Biological Sciences at the University of Queensland for the advice and assistance given throughout this course.

Physical Description

General
The specimen encrusting over a sediment plate was a bright orange red with a soft, fleshy consistency (Figure 2). Other recorded colours of this species are beige or cream to light brown (Van Soest & Hajdu 2002; Pulitzer-Finali 1980-1981) while Dendy (1896) also noted a specimen of vinaceous (dark red or purple) color. There is no definite shape associated with this species, with variation of growth forms that include lobate, massive, contorted, erect, flabellate or digitate (Hooper & Van Soest 2002; Dendy 1896).

The oscules, about 1-2 mm in diameter, are conspicuous and are situated at the base of the conules (Hooper 2014).

Similar to the specimen found by Goudie, Norman & Finn (2013), this sponge itself is also encrusted by housing tubes built by symbiotic polychaetes (Figure 3 & 4) (see Symbiosis).

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Figure 2
3
Figure 3
4
Figure 4

Spongin
Foreign debris such as filamentous algae or sand are typically incorporated into the spongin fibres of Mycale (Arenochalina) Mirabilis – a characteristic of the family Mycalidae (Van Soest & Hajdu 2002; Pulitzer-Finali 1980-1981; Dendy 1896). 

The arrangement of spongin fibres is regular and quadrangular, with only one category of mycalostyles and anisochelae (Van Soest & Hajdu 2002). Megascleres and microscleres are rare and widely dispersed throughout the sponge choanosome, while there are none in the ectosome (Van Soest & Hajdu 2002). The megasclere subtylostyles are long and thin, about 254-282 x 2-4 µm (Hooper 2014). Pulitzer-Finali (1982) and Dendy (1896) also observed tylostyles, about 0.158-0.0027 mm long with a gradual pointing at the apex, to be longitudinally-arranged in the fibres with high densities. Both the anisochelae and sigma, however, are often absent or rare (Van Soest & Hajdu 2002). The scattered anisochelae microsclere, if present, is about 22-32 µm long (Dendy 1896; Hooper 2014) and the microsclere sigmas, if present, are 48-54 x 2 µm long (Hooper 2014).


See Glossary for assistance with terminology.

Ecology

Habitat
Species of the order Poecilosclerida are widely distributed across the oceans and inhabit a wide range of habitats from shallow or intertidal to abyssal depths (Hooper & Van Soest 2002). This specimen was found encrusting on a sediment plate, similar to another record of this species encrusting over pier piles (Goudie, Norman & Finn 2013).

Other habitats of Mycale (Arenochalina) mirabilis recorded by Sorokin, Laperousaz & Drabsch (2007) range over depths of 3 to 16m and include a sloping, sandy substrate with small seagrass beds and low algal cover, a low-relief rocky reef with an average algal cover, a moderately-sloping rocky reef with a high algal cover and a limestone rocky reef with a high coralline algal cover. 

Although the depths of which this species are found typically ranges from 3 – 30m (Pulitzer-Finali 1980-1981; Sorokin, Laperousaz & Drabsch 2007), this sponge had also been found as deep as 350-400m (Barrow et al. 1996). This is the only known record of this species habituating such depths thus far.

Symbiosis

The abundant biomass and diversity of sponges contribute to their function as ecological hosts in marine habitats. These symbiotic interactions between sponges and other marine organisms are thought to be facilitated by the homogeneity and malleability of the canal systems of the sponge structure (Wulff 2006). 

Although records of symbiosis of Mycale (arenochalina) mirabilis with filamentous algae (Van Soest & Hajdu 2002) and polychaete (Goudie, Norman & Finn 2013) are known, no in-depth study of this close association has been conducted so far. It is thus uncertain if the relationship is commensal, mutualistic or parasitic.


Symbiosis with Algae:
M. mirabilis is characterised by the incorporation of filamentous algae into its skeletal fibres (Van Soest & Hajdu 2002; Sorokin, Laperousaz & Drabsch 2007). It is thought that this embedment of algae provides strength and resistance to the skeletal spongin against water movement, while the algae gains nutrients from the filter-feeding activity of the sponge, as well as protection from grazers (Wulff 2006).


Symbiosis with Polychaeta:
Polychaetes are frequently found in sponge hosts, with the main benefit being obtaining food from the sponge. The indentations and perforations of the sponge structure are also thought to provide shelter. This relationship is thought to be commensal as no known benefit is received by the sponge, although further studies need to be conducted for this to be concluded (Wulff 2006).

Structure Elucidation of Metabolite
The Porifera is an extremely important phylum for the discovery of many biologically active natural products. These are allelochemicals that are frequently secondary metabolites that function as a defence mechanism or as an anti-foulant to mitigate the competition for space (Webster & Taylor 2012). Due to the cytotoxic, anti-microbial and/or anti-tumour properties of these natural products, they possess much pharmaceutical and biomedical potential (Webster & Taylor 2012; Coello, Martin & Reyes 2009; Habener, Hooper & Carroll 2016).

Although only one study of the alkaloids of Mycale (Arenochalina) mirabilis has been studied thus far by Barrow et al. (1996), many studies have been conducted on the metabolites associated with the family Mycalidae which yielded other chemical structures such as polyketides and peptides (Coello, Martin & Reyes 2009; Habener, Hooper & Carroll 2016).

The extraction and isolation of one of the metabolites present in this specimen was conducted, and the structure elucidation was performed via spectroscopic analyses i.e. 1H NMR, 1H-1H COSY, HMQC, HMBC and low resolution mass spectrometry. The identification of the compound mycalenitrile-3, an alkaloid with a 2,5-disubstituted pyrrole derivative, was successful with the structure pictured below in Figure 5 below.

This compound was also found in another species of the Mycale genus, Mycale cecilia (Ortega et al. 2004). It has been noted that while the family Mycalidae is associated with a high chemical diversity of natural products (Habener, Hooper & Carroll 2016), nitrogenous metabolites with a pyrrole nucleus are characteristic of the genus Mycale (Ortega et al. 2004; Compagnone et al. 1999).

Most of the pyrrole derivatives isolated from this family are represented by 5-alkylpyrrole-2-carboxaldehyde derivatives that vary in the length and saturation of the alkyl chain (mycalazals) or presence of functional groups such as terminal nitriles (mycalenitriles) (Habener, Hooper & Carroll 2016). However, metabolites isolated from the same species Mycale (Arenochalina) mirabilis by Barrow et al. (1996), yielded structures of tricyclic alkaloids (mirabilins) that appear to have significantly different structural backbones. This difference in the chemical scaffolding is further discussed in the section Evolution and Systematics (Chemotaxonomy).  

5
Figure 5

Experimental
Collection, Extraction and Isolation of Metabolites:
The sponge Mycale (Arenochalina) mirabilis was collected from a sediment plate at a depth of 30m from Moreton Bay. The diced sample was extracted with 1:1 CHCl3:MeOH and the subsequent brown organic extract was removed, dried with anhydrous magnesium sulphate then filtered and evaporated under nitrogen. The compound mycalenitrile-3 was isolated upon elution with 100% DCM on a silica gel column and then purified with RP-HPLC with 100% MeOH, flow rate 1.5 mL/min.


Structure Elucidation:
Mycalenitrile-3 was identified by spectroscopic analysis i.e. 1H NMR, 1H-1H COSY, HMQC, HMBC and low resolution mass spectrometry. 1H NMR spectrum and proton positions are pictured in Figures 6 and 7 respectively.

Mycalenitrile-3. Colourless oil. 1H NMR δ 9.38 (s, 1H, H-1), 6.88 (dd, J = 3.8, 2.5 Hz, 1H, H-3), 6.07 (d, J = 3 Hz, 1H, H-4), 2.63 (t, J = 7.7 Hz, 2H, H-6), 2.33 (t, J = 7.2 Hz, 2H, H-26), 1.25 (broad signal, 32H, H-7-25). C27H46N2O (m/z 414.4).


Instrument Details:
1H NMR, 1H-1H COSY, HMQC, HMBC were recorded on Bruker Ultrashield NMR spectrometer at 500 MHz in CDCl3  solvent. Proton chemical shifts were referenced to the residual CDCl3 signal at δ 7.26. Low resolution mass spectra were recorded on an Esquire HCT mass spectrometer. Column chromatography was carried out using Merck Silica gel 60 (0.04 – 0.06 mm, 230 – 400 mesh ASTM.) Reverse phase HPLC separations were performed on an Agilent 1100 series equipped with a Phenomenex Gemini 5 μm C18 10 x 250 mm column.
6
Figure 6
7
Figure 7

Life History and Behaviour

Reproduction
Both asexual and sexual methods of reproduction are evident in the phylum Porifera. The most common method of clonal (asexual) reproduction is fragmentation whereby wave action causes a fragment to detach. This dislodged fragment then re-attaches to another substrate where it organises and re-builds itself into a functional sponge. Other forms of asexual reproduction are via budding, whereby a bud is developed and then separated from the parent sponge upon maturity to settle and grow into another individual. Gemmules, a protective and dormant reproductive structure, produced by a dying sponge can be activated to differentiate into a new sponge upon more favourable conditions (Ruppert, Fox & Barnes 2004).

Most sponges are hermaphroditic, and typically release sperm into the water column during spermcast spawning. The expelled sperm is then carried by the currents and are taken into the aquiferous system upon contact with another sponge of the same species, allowing internal fertilisation to occur (Ruppert, Fox & Barnes 2004). 

No detailed study of the reproduction of Mycale (Arenochalina) mirabilis has been conducted so far, hence the reproductive cycle and type of larva is unknown. 

Lifecycle
Poriferans are characterised by the common biphasic life cycle. This includes two distinct phases, the pelagic, free-swimming larval phase which transitions into the sessiile benthic, adult phase upon settlement of the larvae on a substate. The individual then metamorphoses and matures into a sexually-reproductive that produce gametes (Degnan & Degnan 2006).

Antimicrobial Activity
McCaffrey & Endean (1985) found a lack of anti-microbial activity of Mycale (Arenochalina) mirabilis against cyanobacteria and fouling organisms, but strong activity against Gram-positive bacteria such as Staphylococcus aureus and Bacillus subtilis. This antibacterial property is similar to that of penicillin G and streptomycin (McCaffrey & Endean 1985). It is, however, unclear at the present moment if this antibacterial metabolite is simply due to the incidental metabolic processes of the sponge symbionts (McCaffrey & Endean 1985).

Anatomy and Physiology
The anatomical design of this encrusting Mycale (Arenochalina) mirabilis was identified to be that of leuconoid, where the aquiferous system is a complex network of choanocyte-lined chambers convoluting throughout the sponge body in high densities. Unlike poriferans of asconoid and syconoid design with a single and large osculum, leuconoids have many oscules and excurrent canals instead that gradually become larger upon joining with other excurrent canals (Ruppert, Fox & Barnes 2004).

No detailed study of the internal body plan, tissue, or cell types of M. mirabilis has been conducted so far. 

Biogeographic Distribution
The family Mycalidae is known to occur throughout all the oceans and all marine habitats. The species Mycale (Arenochalina) mirabilis, however, is distributed amongst more temperate waters – mainly the north, east and south-east coasts of Australia (Van Soest & Hajdu 2002). Most recorded specimens have been collected from central and southern Great Barrier Reef (GBR), mainly Heron Island and Wistari Reef from depths of 11 - 30 m (Pulitzer-Finali 1980-1981). Specimens have also been collected at Northern GBR (Torres Strait) and Shark Bay (Van Soest & Hajdu 2002; Lendenfeld 1887). Records of M. mirabilis are also noted, though less commonly, in South India and Sri Lanka (Burton 1928). The specimen on this page was collected on a sediment plate from Moreton Bay at a depth of 30m.
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Figure 8

Evolution and Systematics

Spicule Characteristics
The genus Mycale have characteristic mycalostyle spicules (Figure 9), while asymmetric anisochelaes (Figure 10)are unique to the family Mycalidae (Van Soest & Hajdu 2002; Hooper & Van Soest 2002; Goudie, Norman & Finn 2013). Hence, the microscleres and megascleres are vital in the identification of Mycale (Arenochalina) mirabilis. The characteristic anisochelae is rare and often absent in this species, however, and is problematic for its identification (Van Soest & Hajdu 2002).
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Figure 9
10
Figure 10

Synonyms
Information regarding the listed synonymised names is provided by (Van Soest 2007b):

Esperella spongiosa (Dendy 1896)
Mycale (Arenochalina) tylostrongyla (Pulitzer-Finali, 1982)
Mycale (Naviculina) mirabilis (Lendenfeld, 1887)
Mycale fistulata (Hentschel, 1911)
Mycale mirabilis (Lendenfeld, 1887)
Mycale spongiosa (Dendy, 1896)
Mycale tylostrongyla (Pulitzer-Finali, 1982)
Naviculina mirabilis (Lendenfeld, 1887)

Chemotaxonomy
Chemotaxonomy is the biological classification of organisms based on the chemical analysis in the structural similarities of their natural biochemical products, that is the secondary metabolites in the case of poriferans (Braekman et al. 1992). Habener, Hooper & Carroll (2016) states that the largest group of compounds isolated from the family Mycalidae are those of pyrrole derivatives. This is demonstrated by the compound mycalenitrile-3 isolated from this specimen of Mycale (Arenochalina) mirabilis, as well as others of the same genus such as Mycale cecilia (Ortega et al. 2004) and Mycale Microsigmatosa (Compagnone et al. 1999). However, a study by Barrow et al. (1996) of the same species, Arenochalina mirabilis, isolated six structures of guanidine-containing tricyclic alkaloids, named mirabilins, that are of acetyl derivatives instead. 

This was the only source of mirabilins isolated from the family Mycalidae; furthermore, the specimen appeared to also possess characteristics of the family Monanchora upon re-examination. An incorrect identification of the specimen was hence suggested (Habener, Hooper & Carroll 2016).

Another possible explanation for this difference in chemical backbone was the vastly different habitat – the specimen addressed on this page was collected at Moreton Bay on the eastearn coast of Australia at a depth of 30m, while the other was collected at the Great Australian Bight on the south coast of Australia, at a depth of 350-400m. This suggests that the associated metabolite is of a habitat-dependent symbiotic origin instead. It is also possible that a new turnover of symbiotic microbials had occurred, due to their rapid regeneration time and quick evolutionary potential (Schmidt et al. 2007).

Conservation and Threats

Despite the increasing records of sponge disease outbreaks over many geographic locations, not much is known about this threat (Webster 2007). A potential reason for the lack of understanding is the difficulty in determining the causative agents of the disease, due to the considerable symbiotic associations within the tissue of sponges. There are, however, records of potential disease agents and/or environmental factors such as climate change and anthropogenic pollution that appear to be correlated to the increased prevalence of sponge disease in the Great Barrier Reef (Webster 2007). 

Owing to the lack of study on Mycale (Arenochalina) mirabilis, the specific threats imposed upon this species are unknown. The necessity of conservation measures such as management strategies required for M. mirabilis thus remains unclear. Further efforts in understanding poriferans should be conducted, due to their ecological and pharmaceutical importance.


References
Barrow, RA, Murray LM, Lim, TK & Capon, RJ 1996, ‘Mirabilins (A-F): new alkaloids from a Southern Australian marine sponge, Arenochalina mirabilis’, Australian Journal of Chemistry, vol. 49, 49-52.

Boury-Esnault, N & Rützler, K 1997, Thesaurus of Sponge Morphology, Smithsonian Institution Press, Washington.  

Braekman, J, Daloze, D, Stoller, C & Van Soest, RWM 1992, ‘Chemotaxonomy of Agelas (Porifera: Demospongiae)’, Biochemical Systematics and Ecology, vol. 20, pp. 417-431.

Burton, M, 1928, ‘Report on some Deep-Sea Sponges from the Indian Museum collected by R.I.M.S. ‘Investigator’. Part II. Tetraxonida (concluded) and Euceratosa’, Records of the Indian Museum, vol. 30, pp. 109-138.

Coello, L, Martín, MJ & Reyes, F 2009, ‘1,5-Diazacyclohenicosane, a New Cytotoxic Metabolite from the Marine Sponge Mycale sp.’, Marine Drugs, vol. 7, pp. 445-450.

Compagnone, RS, Oliveri, MC, Piña, IC, Marques, S, Rangel, HR, Dagger, F, Suárez, AI & Gómez, M 1999, ‘5-Alkylpyrrole-2-Carboxaldehydes From the Caribbean Sponges Mycale Microsigmatosa and Desmapsamma Anchorata', Natural Product Research, vol. 13, pp. 203-211.

Degnan, SM & Degnan, BM 2006, ‘The Origin of the Pelagobenthic Metazoan Life Cycle: What's Sex Got to Do with It?’, Integrative and Comparative Biology, vol. 46, pp. 683-690.

Dendy, A 1896, ‘Catalogue of Non-Calcareous Sponges collected by J. Bracebridge Wilson, Esq., M.A., in the neighbourhood of Port Phillip Heads. Part II.’, Proceedings of the Royal Society of Victoria (New Series), vol. 8, pp. 14-51.

Goudie, L, Norman, M & Finn, J 2013, Sponges, Museum Victoria, Melbourne Vic, Australia. 

Habener, LJ, Hooper, JNA & Carroll, AR 2016, ‘Chemical and biological aspects of marine sponges form the family Mycalidae’, Planta Medica.

Hooper, JNA & Van Soest, RWM 2002, ‘Order Poecilosclerida Topsent, 1928’ in Hooper JNA & Van Soest RWM, Systema Porifera: A Guide to the Classification of Sponges, Kluwer Academic/Plenum Publishers, New York, pp. 403-408.

Hooper, JNA 2014, QM0270 Mycale (Arenochalina) mirabilis(Lendenfeld, 1887), SpongeMaps: an online community for taxonomy and identification of sponges, viewed 26th May 2016, <http://www.spongemaps.org/#!search-for-sponges-public/c16m4>.

Lendenfeld, RV 1887, ‘Die Chalineen des australischen Gebietes’, Zoologische Jahrbücher, vol. 2, pp. 723-828.

McCaffrey, EJ & Endean, R 1985, ‘Antimicrobial activity of tropical and subtropical sponges’, Marine Biology, vol. 8, pp. 1-8.

Ortega, MJ, Zubía, E, Sánchez, MC, Salvá, J & Carballo, JL 2004, ‘Structure and cytotoxicity of new metabolites from the sponge Mycale cecilia’, Tetrahedron, vol. 60, pp. 2517-2524.

Pulitzer-Finali, G 1980-1981, ‘Some new or little-known sponges from the Great Barrier Reef of Australia’, Bollettino dei Musei e degli Istituti Biologici della (R) Universita di Genova, vol. 48-49, pp. 87-141.

Ruppert, EE, Fox, RS & Barnes, RD 2004, Intertebrate Zoology: A functional evolutionary approach, 7th edn, Brooks/Cole Thompson Learning, Belmont, California.

Schmidt, SK, Costello, EK, Nemergut, DR, Cleveland, CC, Reed, SC, Weintraub, MN, Meyer, AF & Martin, AM 2007, ‘Biogeochemical Consequences of Rapid Microbial Turnover and Seasonal Succession in Soil’, Ecology, vol. 88, pp. 1379-1385.

Sorokin SJ, Laperousaz TCD & Drabsch SL 2007, ‘A catalogue of shallow-water sponges from the Investigator Islands, South Australia’, Report to Nature Foundation SA Inc., vol. 258, pp. 1-37.

Van Soest, RWM & Hadju, E 2002, ‘Family Mycalidae Lundbeck, 1905’ in Hooper JNA & Van Soest RWM, Systema Porifera: A Guide to the Classification of Sponges, Kluwer Academic/Plenum Publishers, New York, pp. 669-690.


Van Soest, R 2007a, ‘Mycale (Arenochalina) mirabilis (Lendenfeld, 1887)’, World Register of Marine Species, viewed 25 May 2016, <http://www.marinespecies.org/aphia.php?p=taxdetails&id=168562>.


Van Soest, R 2007b, ‘Mycale (Arenochalina) mirabilis (Lendenfeld, 1887)’, World Porifera Database, viewed 25 May 2016, <http://www.marinespecies.org/porifera/porifera.php?p=taxdetails&id=168562>.

Webster, NS 2007, ‘Sponge disease: a global threat?’, Environmental Microbiology, vol. 9, pp. 1363-1375. 

Webster, NS & Taylor, MW 2012, ‘Marine sponges and their microbial symbionts: love and other relationships’, Environmental Microbiology, vol. 14, pp. 335-346.

Wulff, JL 2006, ‘Ecological interactions of marine sponges’, Canadian Journal of Zoology, vol. 84, 146-166.