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Methods for Monitoring Autophagy in Silkworm Organs

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Autophagy in Differentiation and Tissue Maintenance

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1854))

Abstract

In holometabolous insects, various larval organs are remodeled by autophagy during metamorphosis. Although moths and butterflies are among the first animal models in which this self-eating process was described, only in recent years autophagy has been analyzed in detail in these insects. In particular, the silkworm Bombyx mori, which represents a well-studied model among Lepidoptera, provides a wide repertoire of cellular and molecular tools useful for studying the occurrence of autophagy and for evaluating its role in postembryonic development. Here, we describe some morphological, biochemical, and molecular methods to monitor autophagy in silkworm organs.

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References

  1. Locke M, Collins JV (1965) The structure and formation of protein granules in the fat body of an insect. J Cell Biol 26:857–884

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Locke M, Collins JV (1968) Protein uptake into multivesicular bodies and storage granules in the fat body of an insect. J Cell Biol 36:453–483

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Tian L, Ma L, Guo E, Deng X et al (2013) 20-hydroxyecdysone upregulates Atg genes to induce autophagy in the Bombyx fat body. Autophagy 9:1172–1187

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Franzetti E, Romanelli D, Caccia S, Cappellozza S, Congiu T et al (2015) The midgut of the silkmoth Bombyx mori is able to recycle molecules derived from degeneration of the larval midgut epithelium. Cell Tissue Res 361:509–528

    Article  CAS  PubMed  Google Scholar 

  5. Romanelli D, Casartelli M, Cappellozza S, de Eguileor M, Tettamanti G (2016) Roles and regulation of autophagy and apoptosis in the remodelling of the lepidopteran midgut epithelium during metamorphosis. Sci Rep 6:32939

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Klionsky DJ, Abeliovich H, Agostinis P, Agrawal DK, Aliev G et al (2008) Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes. Autophagy 4:151–175

    Article  CAS  PubMed  Google Scholar 

  7. Klionsky DJ, Abdalla FC, Abeliovich H, Abraham RT, Acevedo-Arozena A et al (2012) Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy 8:445–544

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Klionsky DJ, Abdelmohsen K, Abe A, Abedin MJ, Abeliovich H et al (2016) Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition). Autophagy 12:1–222

    Article  PubMed  PubMed Central  Google Scholar 

  9. Romanelli D, Casati B, Franzetti E, Tettamanti G (2014) A molecular view of autophagy in lepidoptera. Biomed Res Int 2014:902315

    Article  PubMed  PubMed Central  Google Scholar 

  10. Mita K, Kasahara M, Sasaki S, Nagayasu Y, Yamada T (2004) The genome sequence of silkworm, Bombyx mori. DNA Res 11:27–35

    Article  CAS  PubMed  Google Scholar 

  11. Liu X, Dai F, Guo E, Li K, Ma L et al (2015) 20-Hydroxyecdysone (20E) primary response gene E93 modulates 20E signaling to promote Bombyx larval-pupal metamorphosis. J Biol Chem 290:27370–27383

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Hu W, Liu C, Cheng T, Li W, Wang N, Xia Q (2016) Histomorphometric and transcriptomic features characterize silk glands’ development during the molt to intermolt transition process in silkworm. Insect Biochem Mol Biol 76:95–108

    Article  CAS  PubMed  Google Scholar 

  13. Ji MM, Lee JM, Mon H, Xu J, Tatsuke T, Kusakabe T (2016) Proteasome inhibitor MG132 impairs autophagic flux through compromising formation of autophagosomes in Bombyx cells. Biochem Biophys Res Commun 479:690–696

    Article  CAS  PubMed  Google Scholar 

  14. Ji MM, Lee JM, Mon H, Iiyama K, Tatsuke T et al (2017) Lipidation of BmAtg8 is required for autophagic degradation of p62 bodies containing ubiquitinated proteins in the silkworm, Bombyx mori. Insect Biochem Mol Biol 89:86–96

    Article  CAS  PubMed  Google Scholar 

  15. Xie K, Tian L, Guo X, Li K, Li J et al (2016) BmATG5 and BmATG6 mediate apoptosis following autophagy induced by 20-hydroxyecdysone or starvation. Autophagy 12:381–396

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Xia Q, Li S, Feng Q (2014) Advances in silkworm studies accelerated by the genome sequencing of Bombyx mori. Annu Rev Entomol 59:513–536

    Article  CAS  PubMed  Google Scholar 

  17. Franzetti E, Huang ZJ, Shi YX, Xie K, Deng XJ et al (2012) Autophagy precedes apoptosis during the remodeling of silkworm larval midgut. Apoptosis 17:305–324

    Article  CAS  PubMed  Google Scholar 

  18. Montali A, Romanelli D, Cappellozza S, Grimaldi A, de Eguileor M, Tettamanti G (2017) Timing of autophagy and apoptosis during posterior silk gland degeneration in Bombyx mori. Arthropod Struct Dev 46:518–528

    Article  PubMed  Google Scholar 

  19. Khoa DB, Takeda M (2012) Expression of autophagy 8 (Atg8) and its role in the midgut and other organs of the greater wax moth, Galleria mellonella, during metamorphic remodelling and under starvation. Insect Mol Biol 21:473–487

    Article  CAS  PubMed  Google Scholar 

  20. Gai Z, Zhang X, Islam M, Wang X, Li A et al (2013) Characterization of Atg8 in lepidopteran insect cells. Arch Insect Biochem Physiol 84:57–77

    PubMed  CAS  Google Scholar 

  21. Li YB, Li XR, Yang T, Wang JX, Zhao XF (2016) The steroid hormone 20-hydroxyecdysone promotes switching from autophagy to apoptosis by increasing intracellular calcium levels. Insect Biochem Mol Biol 79:73–86

    Article  CAS  PubMed  Google Scholar 

  22. Shiba H, Yabu T, Sudayama M, Mano N, Arai N et al (2016) Sequential steps of macroautophagy and chaperone-mediated autophagy are involved in the irreversible process of posterior silk gland histolysis during metamorphosis of Bombyx mori. J Exp Biol 219:1146–1153

    Article  PubMed  Google Scholar 

  23. Hu C, Zhang X, Teng YB, Hu HX, Li WF (2010) Structure of autophagy-related protein Atg8 from the silkworm Bombyx mori. Acta Crystallogr Sect F Struct Biol Cryst Commun 66:787–790

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Li Q, Deng X, Yang W, Huang Z, Tettamanti G et al (2010) Autophagy, apoptosis, and ecdysis-related gene expression in the silk gland of the silkworm (Bombyx mori) during metamorphosis. Can J Zool 88:1169–1178

    Article  CAS  Google Scholar 

  25. Denton D, Shravage B, Simin R, Mills K, Berry DL et al (2009) Autophagy, not apoptosis, is essential for midgut cell death in Drosophila. Curr Biol 19:1741–1746

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Santos DE, Azevedo DO, Campos LA, Zanuncio JC, Serrão JE (2015) Melipona quadrifasciata (Hymenoptera: Apidae) fat body persists through metamorphosis with a few apoptotic cells and an increased autophagy. Protoplasma 252:619–627

    Article  CAS  PubMed  Google Scholar 

  27. Tindwa H, Jo YH, Patnaik BB, Lee YS, Kang SS, Han YS (2015) Molecular cloning and characterization of autophagy-related gene TmATG8 in Listeria-invaded hemocytes of Tenebrio molitor. Dev Comp Immunol 51:88–98

    Article  CAS  PubMed  Google Scholar 

  28. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  CAS  Google Scholar 

  29. Moss WD (1983) Methods of enzymatic analysis. In: Bergmeyer J, Grassi M (eds) Esterases, glycosidases, lyases, ligases, vol 4. Verlag-Chemie, Weinheim, pp 92–106

    Google Scholar 

  30. Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 76:4350–4354

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

This work was partially supported by FAR 2017 (University of Insubria) to GT. Aurora Montali is a Ph.D. student of the “Life Sciences and Biotechnology” course at Università degli Studi dell’Insubria. Daniele Bruno is a Ph.D. student of the “Biotechnologies, Biosciences and Surgical Technologies” course at Università degli Studi dell’Insubria.

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Correspondence to Gianluca Tettamanti .

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Montali, A., Casartelli, M., Bruno, D., Grimaldi, A., Tettamanti, G. (2018). Methods for Monitoring Autophagy in Silkworm Organs. In: Turksen, K. (eds) Autophagy in Differentiation and Tissue Maintenance. Methods in Molecular Biology, vol 1854. Humana Press, New York, NY. https://doi.org/10.1007/7651_2018_122

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  • DOI: https://doi.org/10.1007/7651_2018_122

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8747-4

  • Online ISBN: 978-1-4939-8748-1

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