Investigation of the Effects of Starvation Stress in the Midgut of the Silkworm Bombyx mori

Yıl 2023, Cilt: 7 Sayı: 1, 12 - 23, 30.06.2023

Tuğçe Ergin Ordu , Ebru Göncü

https://doi.org/10.31594/commagene.1225101

Öz

During their evolution, organisms have developed various mechanisms to adapt to changing nutritional conditions such as mobilization of storage molecules and activation of autophagy. In this study, the mechanism of adaptive responses in the midgut of the silkworm Bombyx mori L., 1758 (Lepidoptera: Bombycidae) larvae, which were starved for different days, was investigated. The study was carried out at the Insect Physiology Research Laboratory and Silkworm Culture Laboratory at Ege University between 2018 and 2020. For this purpose, the histological structure of the midgut was examined using hematoxylin&eosin staining and its protein, sugar, glycogen, and lipid contents were determined. As autophagy markers, lysosomal enzyme activities were measured and expressions of autophagy-related genes (mTOR, ATG8, and ATG12) were analyzed by qRT-PCR. The results showed that, depending on the time of onset of starvation stress, autophagy plays no role as an adaptive response under starvation conditions or occurs at a much more moderate level than autophagy which happens as part of cell death during larval-pupal metamorphosis.

Anahtar Kelimeler

Lepidoptera, digestive canal, programmed cell death, ATG genes

Destekleyen Kurum

Ege University Scientific Research Projects Coordination Unit

Proje Numarası

FYL-2019-20470

Teşekkür

This study was supported by Ege University Scientific Research Projects Coordination Unit (Project number: FYL-2019-20470).

İpek Böceği Bombyx mori’ nin Orta Bağırsağında Açlık Stresinin Etkilerinin Araştırılması

Öz

Organizmalar, evrimleri sırasında değişen beslenme koşullarına uyum sağlamak için depo moleküllerinin mobilizasyonu ve otofajinin aktivasyonu gibi çeşitli mekanizmalar geliştirmiştir. Bu çalışmada, farklı günlerde aç bırakılan ipekböceği Bombyx mori L., 1758 (Lepidoptera: Bombycidae) larvalarının orta bağırsağındaki adaptif tepkilerin mekanizması araştırılmıştır. Çalışma 2018-2020 yılları arasında Ege Üniversitesi böcek fizyolojisi araştırma laboratuvarı ve ipekböceği kültür laboratuvarında yapılmıştır. Bu amaçla hematoksilen&eozin boyama ile orta bağırsağın histolojik yapısı incelenmiş ve protein, şeker, glikojen ve lipid içerikleri belirlenmiştir. Otofaji belirteçleri olarak lizozomal enzim aktiviteleri ölçülmüş ve otofaji ile ilgili genlerin (mTOR, ATG8 ve ATG12) ifadeleri qRT-PCR ile analiz edilmiştir. Sonuçlar, açlık stresinin başlama zamanına bağlı olarak, otofajinin, açlık koşulları altında adaptif bir yanıt olarak hiçbir rolü olmadığını veya larva-pupa metamorfozu sırasında hücre ölümünün bir parçası olarak meydana gelen otofajiden çok daha ılımlı bir seviyede meydana geldiğini göstermiştir.

Anahtar Kelimeler

Pul kanatlılar, Sindirim kanalı, Programlı hücre ölümü, ATG genleri

Proje Numarası

FYL-2019-20470

Kaynakça

• Arrese, E.L., & Soulages, J.L. (2010). Insect fat body: energy, metabolism, and regulation. Annual review of entomology, 55, 207. https://doi.org/10.1146/annurev-ento-112408-085356
• Bradford, M.M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical biochemistry, 72(1-2), 248-254. https://doi.org/10.1016/0003-2697(76)90527-3
• Bergmeyer, H.U. (1974). Enzymes as biochemical reagents. "Methods of Enzymatic Analysis", Academic Press, New York, 428 pp.
• Caro-Maldonado, A., & Muñoz-Pinedo, C. (2011). Dying for something to eat: how cells respond to starvation. The Open Cell Signalling Journal, 3(1), 42-51. https://doi.org/10.2174/1876390101103010042
• Chan, E.Y., & Tooze, S.A. (2009). Evolution of Atg1 function and regulation. Autophagy, 5(6), 758-765. https://doi.org/10.4161/auto.8709
• Franzetti, E., Huang, Z.J., Shi, Y.X., Xie, K., Deng, X.J., Li, J.P., ... & Feng, Q. (2012). Autophagy precedes apoptosis during the remodelling of silkworm larval midgut. Apoptosis, 17(3), 305-324. https://doi.org/10.1007/s10495-011-0675-0
• Franzetti, E., Romanelli, D., Caccia, S., Cappellozza, S., Congiu, T., Rajagopalan, M., ... & Tettamanti, G. (2015). The midgut of the silkmoth Bombyx mori is able to recycle molecules derived from degeneration of the larval midgut epithelium. Cell and tissue research, 361(2), 509-528. https://doi.org/10.1007/s00441-014-2081-8
• Franzetti, E., Casartelli, M., D'Antona, P., Montali, A., Romanelli, D., Cappellozza, S., ... & Tettamanti, G. (2016). Midgut epithelium in molting silkworm: a fine balance among cell growth, differentiation, and survival. Arthropod Structure & Development, 45(4), 368-379. https://doi.org/10.1016/j.asd.2016.06.002
• Fu, X. (2014). Chaperone function and mechanism of small heat-shock proteins. Acta Biochimica et Biophysica Sinica, 46(5), 347-356. https://doi.org/10.1093/abbs/gmt152
• Goncu, E., & Parlak, O. (2008). Some autophagic and apoptotic features of programmed cell death in the anterior silk glands of the silkworm, Bombyx mori. Autophagy, 4(8), 1069-1072. https://doi.org/10.4161/auto.6953
• Goncu, E., Uranlı, R., Selek, G., & Parlak, O. (2016). Developmental expression of ecdysone-related genes associated with metamorphic changes during midgut remodeling of silkworm Bombyx mori (Lepidoptera: Bombycidae). Journal of Insect Science, 16(1), 86. https://doi.org/10.1093/jisesa/iew061
• Goncu, E., Uranli, R., & Parlak, O. (2017). Juvenile hormone analogue, fenoxycarb, modulates ecdysone-triggered transcriptional hierarchy during programmed cell death of midgut in silkworm, Bombyx mori (Lepidoptera: Bombycidae). European Journal of Entomology, 114, 235. https://doi.org/10.14411/eje.2017.029
• Hietakangas, V., & Cohen, S.M. (2009). Regulation of tissue growth through nutrient sensing. Annual review of genetics, 43, 389-410. https://doi.org/10.1146/annurev-genet-102108-134815
• Jung, C.H., Ro, S.H., Cao, J., Otto, N.M., & Kim, D.H. (2010). mTOR regulation of autophagy. FEBS letters, 584(7), 1287-1295. https://doi.org/10.1016/j.febslet.2010.01.017
• Kakei, M., Iwami, M., & Sakurai, S. (2005). Death commitment in the anterior silk gland of the silkworm, Bombyx mori. Journal of insect physiology, 51(1), 17-25. https://doi.org/10.1016/j.jinsphys.2004.10.012
• Kamada, Y., Funakoshi, T., Shintani, T., Nagano, K., Ohsumi, M., & Ohsumi, Y. (2000). Tor-mediated induction of autophagy via an Apg1 protein kinase complex. The Journal of cell biology, 150(6), 1507-1513. https://doi.org/10.1083/jcb.150.6.1507
• Kaufmann, C., & Brown, M.R. (2008). Regulation of carbohydrate metabolism and flight performance by a hypertrehalosaemic hormone in the mosquito Anopheles gambiae. Journal of insect physiology, 54(2), 367-377. https://doi.org/10.1016/j.jinsphys.2007.10.007
• Kaufmann, C., & Brown, M. (2014). Determination of lipid, glycogen and sugars in mosquitoes. MR4 methods in Anopheles research, 4th ed. BEI Resources, Manassas, VA.
• Kaushik, S., & Cuervo, A.M. (2012). Chaperone-mediated autophagy: a unique way to enter the lysosome world. Trends in cell biology, 22(8), 407-417. https://doi.org/10.1016/j.tcb.2012.05.006
• Keshan, B., Thounaojam, B., & Kh, S.D. (2015). A comprehensive study of the changes in ecdysteroid levels during the feeding phase of fifth instar larvae of the silkworm, Bombyx mori (Lepidoptera: Bombycidae). European Journal of Entomology, 112(4), 632. https://doi.org/10.14411/eje.2015.088
• Klionsky, D.J., & Codogno, P. (2013). The mechanism and physiological function of macroautophagy. Journal of innate immunity, 5(5), 427-433. https://doi.org/10.1159/000351979
• Klowden, M.J., (2007), Physiological Systems in Insects, 298-395 pp.
• Kuma, A., Hatano, M., Matsui, M., Yamamoto, A., Nakaya, H., Yoshimori, T., ... & Mizushima, N. (2004). The role of autophagy during the early neonatal starvation period. Nature, 432(7020), 1032-1036. https://doi.org/10.1038/nature03029
• Li, W.W., Li, J., & Bao, J.K. (2012). Microautophagy: lesser-known self-eating. Cellular and molecular life sciences, 69(7), 1125-1136. https://doi.org/10.1007/s00018-011-0865-5
• Liu, Y., Liu, H., Liu, S., Wang, S., Jiang, R. J., & Li, S. (2009). Hormonal and nutritional regulation of insect fat body development and function. Archives of Insect Biochemistry and Physiology: Published in Collaboration with the Entomological Society of America, 71(1), 16-30. https://doi.org/10.1002/arch.20290
• Lum, J.J., Bauer, D.E., Kong, M., Harris, M.H., Li, C., Lindsten, T., & Thompson, C.B. (2005). Growth factor regulation of autophagy and cell survival in the absence of apoptosis. Cell, 120(2), 237-248. https://doi.org/10.1016/j.cell.2004.11.046
• Malagoli, D., Abdalla, F.C., Cao, Y., Feng, Q., Fujisaki, K., Gregorc, A., ... & Umemiya-Shirafuji, R. (2010). Autophagy and its physiological relevance in arthropods: current knowledge and perspectives. Autophagy, 6(5), 575-588. https://doi.org/10.4161/auto.6.5.11962
• Meléndez, A., Tallóczy, Z., Seaman, M., Eskelinen, E.L., Hall, D.H., & Levine, B. (2003). Autophagy genes are essential for dauer development and life-span extension in C. elegans. Science, 301(5638), 1387-1391. https://doi.org/10.1126/science.1087782
• Mizoguchi, A., Dedos, S.G., Fugo, H., & Kataoka, H. (2002). Basic pattern of fluctuation in hemolymph PTTH titers during larval–pupal and pupal–adult development of the silkworm, Bombyx mori. General and comparative endocrinology, 127(2), 181-189. https://doi.org/10.1016/S0016-6480(02)00043-6
• Mukherjee, A., Patel, B., Koga, H., Cuervo, A.M., & Jenny, A. (2016). Selective endosomal microautophagy is starvation-inducible in Drosophila. Autophagy, 12(11), 1984-1999. https://doi.org/10.1080/15548627.2016.1208887
• Nestel, D., Tolmasky, D., Rabossi, A., & Quesada-Allué, L.A. (2003). Lipid, carbohydrates and protein patterns during metamorphosis of the Mediterranean fruit fly, Ceratitis capitata (Diptera: Tephritidae). Annals of the Entomological Society of America, 96(3), 237-244. https://doi.org/10.1603/0013-8746(2003)096[0237:LCAPPD]2.0.CO;2
• Park, M.S., Park, P., & Takeda, M. (2009). Starvation induces apoptosis in the midgut nidi of Periplaneta americana: a histochemical and ultrastructural study. Cell and tissue research, 335(3), 631-638. https://doi.org/10.1007/s00441-008-0737-y
• Rabinowitz, J.D., & White, E. (2010). Autophagy and metabolism. Science, 330(6009), 1344-1348. https://doi.org/10.1126/science.1193497
• Romanelli, D., Casati, B., Franzetti, E., & Tettamanti, G. (2014). A molecular view of autophagy in Lepidoptera. BioMed research international, 2014. https://doi.org/10.1155/2014/902315
• 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. Scientific reports, 6(1), 1-15. https://doi.org/10.1038/srep32939
• Sakurai, S., & Kaya, M. (1998). Hemolymph ecdysteroid titer and ecdysteroid-dependent developmental events in the last-larval stadium of the silkworm, Bombyx mori: role of low ecdysteroid titer in larval–pupal metamorphosis and a reappraisal of the head critical period. Journal of Insect Physiology, 44(10), 867-881. https://doi.org/10.1016/S0022-1910(98)00075-4
• Scott, R.C., Schuldiner, O., & Neufeld, T.P. (2004). Role and regulation of starvation-induced autophagy in the Drosophila fat body. Developmental cell, 7(2), 167-178. https://doi.org/10.1016/j.devcel.2004.07.009
• Selek, G., Göncü, E., & Parlak, O. (2016). Programmed Cell Death in the Digestive Canal of Bombyx mori (Lepidoptera: Bombycidae) during Prepupal Period. European Journal of Biology, 73(2), 1-29.
• Sõti, C., Sreedhar, A.S., & Csermely, P. (2003). Apoptosis, necrosis and cellular senescence: chaperone occupancy as a potential switch. Aging cell, 2(1), 39-45. https://doi.org/10.1046/j.1474-9728.2003.00031.x
• Sun, X., Siri, S., Hurst, A., & Qiu, H. (2021). Heat Shock Protein 22 in Physiological and Pathological Hearts: Small Molecule, Large Potentials. Cells, 11(1), 114. https://doi.org/10.3390/cells11010114
• Tettamanti, G., Grimaldi, A., Pennacchio, F., & de Eguileor, M. (2007a). Lepidopteran larval midgut during prepupal instar: digestion or self-digestion?. Autophagy, 3(6), 630-631. https://doi.org/10.4161/auto.4908
• Tettamanti, G., Grimaldi, A., Casartelli, M., Ambrosetti, E., Ponti, B., Congiu, T., ... & Eguileor, M.D. (2007b). Programmed cell death and stem cell differentiation are responsible for midgut replacement in Heliothis virescens during prepupal instar. Cell and tissue research, 330(2), 345-359. https://doi.org/10.1007/s00441-007-0449-8
• Tettamanti, G., & Casartelli, M. (2019). Cell death during complete metamorphosis. Philosophical Transactions of the Royal Society B, 374(1783), 20190065. https://doi.org/10.1098/rstb.2019.0065
• Tian, L., Ma, L., Guo, E., Deng, X., Ma, S., Xia, Q., ... & Li, S. (2013). 20-Hydroxyecdysone upregulates Atg genes to induce autophagy in the Bombyx fat body. Autophagy, 9(8), 1172-1187. https://doi.org/10.4161/auto.24731
• Tsukada, M., & Ohsumi, Y. (1993). Isolation and characterization of autophagy-defective mutants of Saccharomyces cerevisiae. FEBS letters, 333(1-2), 169-174. https://doi.org/10.1016/0014-5793(93)80398-E
• Van Handel, E. (1985a). Rapid determination of glycogen and sugars in mosquitoes. Journal of the American Mosquito Control Association, 1(3), 299-304.
• Van Handel, E. (1985b). Rapid determination of total lipids in mosquitoes. Journal of the American Mosquito Control Association, 1(3), 302-304.
• Van Handel, E., & Day, J. (1988). Assay of lipids, glycogen and sugars in individual mosquitoes: correlations with wing length in field-collected Aedes vexans. Journal of the American Mosquito Control Association, 4(4), 549-550.
• Wang, X., & Proud, C.G. (2009). Nutrient control of TORC1, a cell-cycle regulator. Trends in cell biology, 19(6), 260-267. https://doi.org/10.1016/j.tcb.2009.03.005
• Wu, W., Wei, W., Ablimit, M., Ma, Y., Fu, T., Liu, K., ... & Hong, H. (2011). Responses of two insect cell lines to starvation: autophagy prevents them from undergoing apoptosis and necrosis, respectively. Journal of insect physiology, 57(6), 723-734. https://doi.org/10.1016/j.jinsphys.2011.02.008
• Xie, Z., & Klionsky, D.J. (2007). Autophagosome formation: core machinery and adaptations. Nature cell biology, 9(10), 1102-1109. https://doi.org/10.1038/ncb1007-1102
• Xie, K., Tian, L., Guo, X., Li, K., Li, J., Deng, X., ... & Cao, Y. (2016). BmATG5 and BmATG6 mediate apoptosis following autophagy induced by 20-hydroxyecdysone or starvation. Autophagy, 12(2), 381-396. https://doi.org/10.1080/15548627.2015.1134079
• Zhou, S., Zhou, Q., Liu, Y., Wang, S., Wen, D., He, Q., ... & Li, S. (2010). Two Tor genes in the silkworm Bombyx mori. Insect molecular biology, 19(6), 727-735. https://doi.org/10.1111/j.1365-2583.2010.01026.x