Larval Peptides – A Novel Source of Antibiotics from Lucilia cuprina Maggots

1. Larval Peptides – A Novel Source of Antibiotics from Lucilia cuprina Maggots By: Tan Sheng En Troy (HCI) Choo Jian Kai Darren (HCI) Joanne Guidry (AOS) Robbie Daitzman (AOS)

2. Introduction • Insects possess a different immune system from Man • Lack immunoglobulin • Possess peptides with antibacterial activities • Appear in insects after they are injured or challenged by bacteria (Boman. et al, 1974)

3. Introduction • Maggots thrive in bacteria filled environments. It is natural that they have an insect immunity system to defend themselves against pathogens • Disrupts cytoplasmic cell membrane of bacteria – loss of K+ ions (Boman. et al, 1974)

4. Objectives To study the antibacterial Properties of Peptides isolated from Lucilia cuprina maggots To identify the specific peptides which exhibits the antibacterial properties

5. Rationale • Antibacterial peptides found in Lucilia cuprina can be used on humans • Not just natural but also presents a wider range of activity • Wide range of peptides to use to counter all kinds of diseases and bacteria. (Nguyenn et al, 2007)

6. Hypothesis • The hemolymph of the Lucilia cuprina maggots contains peptides that have antibacterial properties

7. Variables Independent Dependant • Controlled • Species of Maggot used Types of bacteria tested Concentration of peptides Presence of antibacterial Peptides Extent of Bacteria Inhibition Sequences of peptides

8. Materials Required • Maggots • Peptide Extraction Kit • SDS-PAGE Gel • Liver Desiccate • Pig’s Heart • Gram Positive/Gram Negative bacteria • Escherichia coli • Pseudomonas Fluorescens • Staphylococcus epidermidis • Micrococcus luteus

9. Methodology Extraction of Hemolymph Anti-bacterial Screening Peptide extraction HPLC SDS-PAGE

10. Hemolymph Extraction Capillary tube inserted through thoracic pleura of maggot Hemolymph collected from 500 maggots Store at - 20oC till use Sample diluted with buffer

11. Peptide Purification 500 -1000μl maggot hemolymph collected Solid-phase extraction with CAT-SEC X cartridges Concentrate with Spin Vac Concentrator and carry out antibacterial assays Peptides with a molecular weight below 20kDa were eluted HPLC Analysis and SDS-PAGE

12. SDS-PAGE

14. Antibacterial Assay • Well Diffusion Assay • Colony Forming Units Assay

15. Colony Forming Unit Assay Culture Plates of Staphylococcus epidermidis with Water and Eluate

16. Culture Plates of Escherichia Coli with Water and Eluate

17. P-Value (E.c) = 0.007554182 P-Value (S.e) = 0.002284057

19. Well-diffusion Assay

20. Conclusion • Coinciding peaks were obtained for the Cecropin standard and the peptide extact obtained from the hemolymph of the Lucilia Cuprina maggots. • SDS-PAGE indicate that our extracts contain peptides with sizes similar to that of Cecropins (3-5kDa). • Peptide extract is likely to contain Cecropins, the antibacterial peptides commonly found in insects.

21. Conclusion • Antibacterial tests have shown that the hemolymph of the Lucilia cuprina maggots have anti bacterial properties against Escherichia coli andStaphylococcus epidermidis. • However, more conclusive tests will have to be performed to further back up this hypothesis

22. Application Antibiotic resistance of bacteria is increasing (e.g. MRSA) Novel antibiotics isolated from maggots may provide new insights on insect peptides as a method to combat these bacteria

23. Further Work • Screen greater variety of bacteria to study the antibacterial activity of maggot peptides. • Modify the extraction method to ensure a higher concentration of peptides obtained.

24. Further Work • To determine whether there is a difference in immunity responses between that of maggots bred in sterile and non-sterile environments. • Identify, isolate and characterize the peptides responsible for the antibacterial activities.

25. Acknowledgements We thank our mentor, SRC lab staff and our project consultant.

26. References Lopez, L., Morales, G., Ursic, R., Wolff, M., & Lowenberger, C. (2003). Isolation and characterization of a novel insect defensin from Rhodnius prolixus, a vector of Chagas disease. Insect Biochemistry and Molecular Biology. 33, 439-447.  Boman, H.G., Nilsson-Faye, I., Paul, K., & Rasmuson, T. (1974). Insect Immunity I. Characteristics of an Inducible Cell-Free Antibacterial Reaction in Hemolymph of Samia cynthia Pupae. Infection and Immunity. 10, 136-145. Wyatt, G.R., Loughheed, T.C., & Wyatt, S.S. (1956). The Chemistry of Insect Hemolymph: Organic components of the hemolymph of the silkworm, Bombyx mori, and two other species. The Journal of General Physiology. 39, 853-868.

27. References Jenssen, H., Hamill, P., & Hancock, R.E.W. (2006). Peptide antibacterial Agents. Clinical Microbiology Reviews. 19, 491-511. Levashina, E.A., Ohresser, S., Bulet, P., Reichhart, J.M., Hetru, C., & Hoffman, J.A. (1995). Metchnikowin, a novel immune-inducible proline-rich peptide from Drosophila with antibacterial and antifungal properties. 233, 694-700. Smith, J.B. (2001).Peptide Sequencing by Edman Degradation. Encyclopedia of Life Sciences.

28. Thank you for your kind attention theEND