IntroductionMarine sponges are chemical factories that are sources of potential pharmaceuticals(1). The isolation by Werner Bergman of two marine sponge nucleosides in the early 1950’s led to the synthesis of the anti-HIV drug AZT and the anti-herpes drug Acyclovir (2). Since then, thousands of new compounds have been reported (1,3). Many have bioactivity against cancer, pathogenic viruses, bacteria, and fungi (1). Some are currently undergoing clinical trials (4).

The ecological function of these metabolites is poorly understood. Their role in feeding deterrence, allelopathy, and control of bacterial attachment have been described (5,6). It is speculated that some sponge metabolites may in fact be synthesized by symbiotic microorganisms that can represent up to 40% of the sponge biomass (7). Little is known of the nature of these associations.

Two species of marine sponges of the genus Halichondria are commonly found in the Gulf of Maine. Both are described as having a sulfur odor, which suggests potential biosynthesis of toxic chemicals.

A search of the Napralert database determined that among marine sponges occurring locally, species of Halichondria were the most studied, but all studies were done abroad. (8). A variety of steroids were isolated from H. bowerbanki by an Italian group (8). Extracts from both H. panicea and H. bowerbanki that were tested by Andersson et al. (Sweden) for antimicrobial activity were reported inactive (8).

Abstract

Many of the chemical substances found in marine sponges are bioactive and research on secondary metabolites is being pursued in the fields of drug discovery and chemical ecology. Specimen of Halichondria sp., a marine sponge commonly found in the waters of the Gulf of Maine, were investigated for their production and bioactivity of such compounds. Three of the purified fractions produced in this study showed to inhibit growth of the gram (-) bacterium Pseudomonas aeruginosa.

Materials and MethodsCollection of animal material

A total of 6 organisms of the sponge genus Halichondria were collected on March 20, 2007, within a 50 yard radius at a depth of 3 feet off Laighton Point, Pembroke, Maine (44o55’N;66o55’W). Water temperature was 36oF; the water salinity was 32 ppt. Area of collection Halichondria specimen

Sponge squeezeHow sanitary can a stinky sponge be?

An analysis of antimicrobial activity in Gulf of Maine specimen of the Halichondria sp. marine sponge

Françoise MorisonDepartment of Chemistry, Inco 590, University of New Hampshire, Manchester

Extraction and purification

The fresh sponge samples (760 g, wet wt) were extracted with methanol (1 L x 3). After concentration, the methanol crude extract (300 ml) was submitted to solvent-solvent partitioning into hexane, ethyl acetate, and methylene chloride phases successively. The ethyl acetate and methylene chloride extracts were chromatographed on a silica gel column using a mixture of 30% hexane 70% ethyl acetate as eluent.

Results Table 1. Three fractions inhibited the growth of P. aeruginosa.

P. aeruginosa 1 P. mirabilis S. aureus

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FM2-I-(2-3) Inhibited Inconclusive2 Negative FM2-II-(4) Inhibited Negative NegativeFM2-III-(5) Inhibited Inconclusive2 NegativeFM2-IV-(6) Negative Negative NegativeTetracycline* Resistant Resistant3 SensitiveChloramphenicol* Resistant4 Sensitive Sensitive

1-Notorious for its resistance to antibiotics 4- expected2-Inhibition ring present around solvent control3-Reported occurrence of R-plasmid in strains of P. mirabilis *Reported antimicrobial sensitivity are based on the guidelines of the Kirby-Bauer method (1995).

Three of the fractions obtained from the ethyl acetate extract showed some inhibition of Pseudomonas aeruginosa. Inhibition was observed on that bacterial species only. Some zones had an irregular shape.

These fractions were eluted from the column successively and may therefore contain the same compound.

While results of this study do not support what had been reported by Andersson et al., the group had tested different extracts (H2O, Pet. Ether, CHCl3, and methanol) (8).

Because the extract concentrations could not be calculated, due to minimal immeasurable amount of product extracted, it is not possible to assess the significance of the zone of inhibition observed. Furthermore, whether inconclusive zones of inhibition observed on some plates are related or not to possible toxicity of lingering solvent remains unclear. No inhibition was observed when solvent testing was repeated.

Bioassay

Four ethyl acetate fractions were tested. Trypticase soy agar plates were swabbed with Pseudomonas aeruginosa , Proteus mirabilis, and Staphylococcus aureus. The extracts were tested using the sterile filter paper disk method. A blank disk and two standard antibiotic disks (tetracycline 30μg and chloramphenicol 30μg) were used as control.

Fraction FM2-I-(2-3) with ring of inhibition

Fraction FM2-II-(4) showing inhibition. Note irregularity around one disk.

Tetracycline (30μg) with small ring of

inhibition (<14 mm)

Chloramphenicol (30μg) (no ring of

inhibition)

Blank disk with solvent

Conclusion

Some of the results supported the hypothesis that Halichondria sp. may produce toxic substances Further study is needed to try and resolve the uncertainties mentioned in the results, i.e. concentration of the extracts and solvent involvement in toxicity. Sufficient amount of compound needs to be extracted to evaluate its concentration. Solvent control volume and time of evaporation must systematically be measured.

NMR analysis may be pursued in an attempt to determine the structure of the active compound. Structure elucidation may however require further purification.

Extraction and culture of bacteria or separation of microalgae possibly present in the sponge may provide additional information as to the source of the active substance(s).Literature cited

1. Bhakuni D.S., Rawat D.S. Bioactive Marine Natural Products. New York: Springer; 2005. 382 p.

2. Harbor Branch Oceanographic Institution Media Lab website. The pipeline and the finish line: the first wave of marine-derived drugs. <http://www.marinebiotech.org/pipeline.html#w1>

3. Kornprobst Jean-Michel.. Les medicaments de la mer. Faculté de Pharmacie et Institut Substances et Organismes de la mer (ISOmer) Université de Nantes. 2001.

4. Simmons T. L., Andrianasolo E. , McPhail K., Flatt P. , Gerwick W. H. Marine natural products as anticancer drugs. Molecular cancer therapeutics [online] 2005; 4(2): 333-342. Avail. From: http://mct.aacrjournals.org/cgi/reprint/4/2/333.

5. Kelly S.R., Jensen P.R., Henkel T.P., Fenical W., Pawlik J.R. Effects of Caribbean sponge extracts on bacterial attachment. Aquatic microbial ecology 2003; 31:175-182.

6. Assmann M., Lichte E., Pawlik J.R., Kock M. Chemical defenses of the Caribbean sponge Agelas wiedenmayeri and Agelas conifera. Marine ecology progress series 2000; 207:255-262.

7. Guyot Michele. Intricate aspects of sponge chemistry. Zoosystema 2000; 22(2).

8. Napralert. Program of collaborative research in the pharmaceutical sciences. College of Pharmacy. University of Illinois at Chicago.<http://www.napralert.org/>

Acknowledgements

I want to thank Dr. Lorraine Doucet, Dr. Sarah Kenick, Dr. Stephen Pugh, Professor Allan Ray, and Keith Legro, for their help, advice and support. I also want to acknowledge Dr. William Sponholtz from Cushing Academy, Ma., for sharing methods used in his own research. Thank you to Dr. Richard Johnson from the Durham Chemistry department for lending the equipment. Thanks Jeremy Neal for sharing the dishwashing.

For further information

Please don’t hesitate to contact the author at frhop@comcast.net

Materials and Methods (cont’d)