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Impact Factor : 1. 9508(UIF) ISSN No :2231-5063

International Multidisciplinary Research Journal

Golden Research Thoughts

Chief EditorDr.Tukaram Narayan Shinde

PublisherMrs.Laxmi Ashok Yakkaldevi

Associate EditorDr.Rajani Dalvi

HonoraryMr.Ashok Yakkaldevi

Vol III Issue VII Jan 2014

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International Advisory Board

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Welcome to GRTISSN No.2231-5063

Golden Research Thoughts Journal is a multidisciplinary research journal, published monthly in English, Hindi & Marathi Language. All research papers submitted to the journal will be double - blind peer reviewed referred by members of the editorial board.Readers will include investigator in universities, research institutes government and industry with research interest in the general subjects.

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Golden Research ThoughtsISSN 2231-5063Impact Factor : 1.9508(UIF)

Volume-3 | Issue-7 | Jan-2014Available online at www.aygrt.isrj.net

EXPRESSION OF VARIOUS PROTEINS IN HOUSEFLY MAGGOTS

Abstract:-Houseflies are insects which live in an environment surrounded by millions of pathogens and bacteria. However they escape from the infection due to their innate immunity.

They produce an effective self defensive mechanism against various microorganisms. Proteins play an important role in immune system. It is essential to know the protein structure and function at molecular level. By studying 2D gel electrophoresis and mass spectroscopy of homogenate of housefly maggots, a series of proteins were identified. For further analysis of protein MALDI-TOF- MS technique has been used. It has also been observed that various proteins might have major role to play in their defense mechanism.

Keywords:Maggots, Proteomics, MALDI-TOF-MS.

INTRODUCTION

Housefly is an important medical insect which has high immune defense mechanism and is rarely infected (Rahuma et al., 2005).They are able to survive successfully in different abrasive conditions. Insect's growth is constrained by the exoskeleton and the development involves a series of moults. The life cycle of insects mostly hatch from eggs The immature stages are different from the adult. The habit and habitat can include passive pupal stages in those groups which undergo metamorphosis. Incomplete metamorphosis lack a pupal stage and adult develops through nymphal stage ( Mc Graw Hill Encyclopedia 2007). Insects are used in medicine. The housefly larvae are called maggots, which were formerly used to treat wounds and prevent infection. Maggots would only consume dead flesh . This treatment is finding modern usage in some hospitals (Sherman 2009).

Dipterans produce Defensins and Diptericin which act as two antibacterial peptides. The haemolymph of housefly larva contains a variety of antibacterial and antifungal peptides (Bai et al., 2002). Insects are particularly resistant to microorganism. Their host defense mechanism relies on several innate reactions. Upon injury, immediate action of proteiolytic cascade leading to localized blood clotting and melanisation occurs .The later process involves production of cytotoxic molecules. The phagocytosis of bacteria and the encapsulation of larger parasites by blood cells, the induced synthesis of fat body by battery of potential antimicrobial peptides which are secreted into the haemolymph where they act synergistically to kill the invading microorganism ( Hoffman, 1995). It is believed that antimicrobial peptides play a significant role in the defense system of housefly (Dang et al., 2010). The 2D gel electrophoresis combined with MS can be used to study the immune response of housefly larvae at the protein level.

Proteomics is nothing but the molecular studies of proteins. They are large polymers of amino acids which are involved in various metabolic reactions of cells. Application of proteomics to insect immunity results in prediction of new roles in immunity for proteins. It helps in diagnosis of various diseased treatments. Marc Wilkins (1994) first explained the term proteome and described as a complete set of proteins expressed and modified according to their genome sequence. 2D gel electrophoresis is one of the best methods used for proteomic studies (Wasinger et al., 1995). Francine et al., (2004) studied the peptidomic and proteomic analysis of Drosophila melanogaster. They have taken advantage of MS and 2D gel electrophoresis. From this study they have identified a series of proteins with respect to immune response of Drosophila.

Shi and Paskawetz (2006) reviewed the proteomic studies in relation to insect immunity. They have stated that this study is useful in prediction of new roles of immunity for proteins and useful to the target specific members of multigene groups respond to different pathogens. Evy Vlerstrate et al., (2004) carried out the experiment by using the fruit fly , Drosophila melanogaster haemolymph to study the proteomic approach for the analysis of instant wound and immune

*Shagalolu V.V , ** Rao K.R and ***E Bhikshapati , “EXPRESSION OF VARIOUS PROTEINS IN HOUSEFLY MAGGOTS”, Golden Research Thoughts | Volume 3 | Issue 7 | Jan 2014 | Online & Print

*Shagalolu V.V , ** Rao K.R and ***E Bhikshapati

*D.B.F. Dayanand College of Arts and Science Solapur , M S India.** Walchand college of Arts and Science Solapur , M S India.

*** Centre for Cellular and Molecular Biology Hyderabad A P India.

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GRT

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response proteins. They have identified certain predicted immune response protein which includes alcohol dehydrogenase, retinoid and fatty acid binding proteins from Drosophila. Hans et al., (2012) studied genomic and proteomic studies in beetle species Tribolium castaneum due to the effect of insect growth regulator. The present study is mainly focused on expression of proteins in housefly maggots.

MATERIAL AND METHODS:

To study the various proteins Housefly maggots were obtained from different colonies and maintained in sterile agar media containing larval food.

Preparation of Housefly larval extract and its Proteomic Analysis:

By maintaining aseptic conditions in laboratory, newly reared housefly maggots were carefully collected. With standardized technique, larval homogenate were prepared using Rehydration buffer and preserved for further analysis in controlled conditions. After obtaining sufficient quantity of larval extract, this extract was centrifuged (10,000 rpm) to obtain purified homogenate. The homogenized extract was collected and studied for 2D gel electrophoresis analysis for various protein separations. After analysis, the proteomic identification has been done with the help of MALDI TOF/ MS. The protein identification of these results was done with help of MASCOT database.

RESULTS & DISCUSSION:

The proteins were identified by MALDI and the m/z valve is subjected to MASCOT data base for their identification. This reveals the protein ID, Accession number score and molecular weight.

From our results, it was observed that significant proteins were expressed and they were analyzed using MALDI TOF MS. The protein analysis, revealed 18 such different proteins. Few prominent proteins to be mentioned were RNA directed RNA polymerase, (Accession No:81945898), P74 (Accession No: 26209049), Hypothetical protein (Accession No 187384766), Staufen, (Accession No:7229534), Caspase I (Accession No 194459444), Cytochrome P450( Accession No 15738242) ,Inhibitor of apoptosis protein (Accession No 187384764) and Attacin. (Accession Number 225904226). The identification of these proteins of 2D gel was carried out where protein spot gel are digested with trypsin and processed for MALDI TOF MS analysis and MASCOT data base. Webster et al.,(2012).

To understand proteomic technique, gel electrophoresis was used where proteins are separated initially. The separated proteins are isolated with help of 2D standard polyacrylamide gel electrophoresis, resulting in 2D display of proteins. By using software programming 2D gels were analyzed. During analysis, the software automates the alignment of spots and produces the data for analysis. From our results it has also been observed that defensin protein is an antimicrobial peptide inhibitor of epidermal growth factor receptor. Similarly an Hypothetical protein was reported by Gracia Marunaik (2008).

Our results are in support of the earlier work. Attacin is also antibacterial peptide (Wang et al.,2008). Defensin are members of large family of antimicrobial peptides (Gang, 2003). Staufen is a double stranded RNA motif which is widely found in variety of protein including double stranded RNA dependent protein kinase, RNA helicase, Drosophila Staufen protein, Ecoli RNAase III Micklem et al.,(2009). RNA directed RNA polymerase which was reported by Rodrignez et al.,(2002). P74 was reported by Prompiboon et al.,(2002). Cytochrome P450 was reported by Zhu et al. (2008).

From our study it can be summarized that from housefly maggot extract, it was clearly noted that various proteins were expressed which play a major role in the immunity against different pathogen. This is because the houseflies live and survive in abrasive conditions and survive successfully without infection.

Expression Of Various Proteins In Housefly Maggots

2Golden Research Thoughts | Volume 3 | Issue 7 | Jan 2014

Site for collection of housefly larvae Laboratory rearing of Housefly larvae

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ACKNOWLEDGEMENT:

The authors wish to thank Dr. A. H. Manikshete, Principal Walchand college of Arts and Science and Dr. S. K. Vadagbalkar , Principal D. B. F Dayanand College of Arts and Science Solapur for providing laboratory facilities .

REFERENCES:

1.Bai, M., Zhong, Guo Sheng., Wu, Hua Xue Yu Fen Zi Sheng Wu Xue Bao.(2002) Some structure information and biological activities of an antibacterial protein from Musca domestica (house fly). 18:633-637. 2.Dang, X.L., Wang , Y.S., Haung, Y.D., Yu, X.Q and zhang WQ. (2010). Purification and Charecterisation of an Antimicrobial peptide, Insect Defensin from Immunised Housefly.Journal of Medical Entomology. 47(6): 1145-1145. 3. Evy, V., Peter, V. , Geert, B., Wannes, D. H., Gert, Van den B., Lutgarde, Arnold D,and Lillane S.(2004). A proteomic approach of analysis of instantlyr eleased wound and immune proteins in Drosophila melanogaster hemolymph PNAS.101: 470-475.4.Francine , L., David, R., Maurice, C., ,Philippe, B., Jules,A,H. ,and Laurence, E .S (2004). Peptidomic and proteiomic analysis of the systemic response of Drosophila. Biochime 86: 607-616.5. Ganz T. 2003). Defensins :antimicrobial peptides of Innate Immunity.Nat Rev. Immunol 3(9):710-120. 6. Garacia, M. A., James, E M., and Drion G .B (2008). Sequence analysis of non classified non occluded DNA virus that causes salivary gland hypertrophy of Musca domestica. MdSGHV. Virology 377 (1): 184-196.7..Hans, M., Hee S K., Sujata, S,, Meera, K., Charles A,, Stephen, B Robert M., Richard W B., and Karl, J. K.(2012). Genomic and proteomic studies on the effect of the insect growth regulatordiflubenzuron in the model beeyle species Tribolium castaneum. Insect Biochemistry and Molecular Biology 42:264-276.8. Hoffman J.A.(1995) Innate Immunity in higher Insects. Current Opnion in Immunol.7(1):4-10.9. Micklem, D., Andres, R., Stefan, G., Daniel J and Gabriel. (2000)RNA recognition by a staufen double stranded RNA binding domain . The EMBOJournal 19:(5)997-100910. Mc Graw Hill Encyclopedia of Science and Technology. (2007). Insect Physiology Chapter 9: 233.11. Prompiboon, P., Verena, U.L., John, S.S., Denton., Christopher J., Genden., Tore ., Sad Drion G.B (2010). Musca domestica salivary gland hypertrophy virus a globally distributed insect virus that infects and sterilizes female houseflies. Appl.Environ.Microbiol. 76 (4):994-998.12. Rodrignez, E B., Lawerence, W D., Ruben, G C., Catlina, F. and Marcelino C. (2002).Inhibitors of glycosphingolipid synthesis reduce transepithelial electrical resistance in MDCK-1 and FRT cells Am. J. Physiol Cell Physiology.284:1021-1031.13. Rahuma, K.S.G., R.B. Aissa., and A. Elamaari. (2005). Carriage by the housefly (Musca domestica) of multiple-antibiotic-resistant bacteria that are potentially pathogenic to humans, in hospital and other urban environments in Misurata, Libya. Ann. Trop. Med. Parasitol. 72: 896-903.14. Sherman, R A (2009). Maggot therapy takes us back to the future of wound care; New and Improved maggot therapy for the 21stCentury. J.Diabetic Sc.Technol 3(2): 336-344.15. Webster, J. and Oxley, D (2012). Protein Identification by MALDI TOF MS Methods Mol Bio 800;227-240.16. Wasinger, VC, Cordwell, S J., Cerpa P. A., Yan, J X., Gooley A A., William, K. L, and Humphery S I (1995). Progress with gene product mapping of the Mollicutes: Mycoplasm gentalium Electrophoresis 16-1090-1094.17...Zhu, F., Feng, J N., Zhang, L and Liu N (2008). Charecterisation of two novel cytochrome P450 genes in insecticide resistant houseflies.Insect Mol. Biol. 17 (1): 27-37.

3Golden Research Thoughts | Volume 3 | Issue 7 | Jan 2014

Expression Of Various Proteins In Housefly Maggots

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