İpekböceği, Bombyx mori L., 1758 (Lepidoptera: Bombycidae) orta bağırsak kök hücrelerinde ekdizonla ilişkili genlerin ekspresyon profilleri ve olası rolleri

Öz

Böcek orta bağırsağı, özellikle kök hücreler tarafından hücre düzenlemesinin kontrol edilmesiyle ilgili olarak omurgalı bağırsağıyla dikkate değer benzerliklere sahiptir. Pupa orta bağırsağı, kök hücreler tarafından oluşturulur ve ekdizon, orta bağırsağın yeniden şekillenme olaylarını düzenlerken, juvenil hormon, ekdizon salınımını baskılayarak engeller. Bu çalışma, larva-pupa metamorfozu sırasında Bombyx mori L., 1758 (Lepidoptera: Bombycidae)'nin kök hücrelerinde ekdizonla ilişkili genlerin olası işlevlerini araştırmıştır. Bu çalışma 2014-2018 yılları arasında Ege Üniversitesi ipek böceği kültür laboratuvarı ve böcek fizyolojisi araştırma laboratuvarında yapılmıştır. Juvenil hormon analoğu fenoksikarb, pupa orta bağırsak oluşumunu geciktirmek veya engellemek için 5. dönem Bombyx larvalarına uygulanmıştır. Morfolojik gözlemler hematoksilen ve eozin boyama ile gerçekleştirilmiş; kök hücrelerin proliferasyon hızı, bromodeoksiüridin hücre proliferasyon analizi ile analiz edilmiş ve ekdizon ile ilgili genlerin ekspresyon durumları kantitatif gerçek zamanlı ters transkriptaz-polimeraz zincir reaksiyonu ile tespit edilmiştir. Genlerin ekspresyon durumları ve orta bağırsaktaki gelişimsel olaylar, uygulama zamanına bağlı olarak fenoksikarb tedavisinden farklı şekilde etkilenmiştir. Sonuçlara göre, genler önce fenoksikarb duyarlılıklarına göre sınıflandırılmış, daha sonra kök hücrelerin morfolojik değerlendirmeleri ile bağlantılı olarak ekspresyon profillerine göre gruplandırılmıştır.

Anahtar Kelimeler

• Bombyx mori
• ekdizon bağlantılı genler
• orta bağırsak
• kök hücre

Expression profiles and possible functions of the ecdysone-related genes in the midgut stem cells of the silkworm, Bombyx mori L., 1758 (Lepidoptera: Bombycidae)

Abstract

The insect midgut has remarkable similarities with the vertebrate intestine especially concerning controlling cell regulation by the stem cells. While the formation of the pupal midgut from stem cells is regulated by ecdysone, it is inhibited by juvenile hormone via suppression of ecdysone release. This study investigated the possible functions of ecdysone-related genes in the stem cells of Bombyx mori L., 1758 (Lepidoptera: Bombycidae) during larval-pupal metamorphosis. The study was conducted in the Ege University silkworm culture laboratory and insect physiology research laboratory during the years between 2014-2018. Juvenile hormone analogue, fenoxycarb was applied to the fifth instar Bombyx larvae to delayed or inhibit the formation of the pupal midgut. Morphologic observations were performed by hematoxylin plus eosin staining; the proliferation rate of stem cells was analyzed by bromodeoxyuridine cell proliferation assay and expression patterns of ecdysone-related genes were detected by quantitative real-time reverse transcriptase-polymerase chain reaction. Expression states of genes and developmental events of the midgut were differently affected by fenoxycarb treatment in an application time-dependent manner. According to results, genes were firstly classified based on their sensitivity of fenoxycarb, then grouped according to their expression profile in connection with morphological evaluations of stem cells.

Keywords

• Bombyx mori
• ecdysone-related genes
• midgut
• stem cell

References

1. Ali, M. S., M. Iwanaga & H. Kawasaki, 2013. Ecdysone-responsive transcriptional regulation determines the temporal expression of cuticular protein genes in wing discs of Bombyx mori. Gene, 512 (2): 337-347.
2. Baldwin, K. M., R. S. Hakim, M. Loeb & S. Sadrud-Din, 1996. “Midgut Development, 31-54”. In: Biology of the Insect Midgut (Eds. M. J. Lehane & P. F. Billingsley). Chapman and Hall, London, England, 486 pp.
3. Bernardo, T. J. & E. B. Dubrovsky, 2012. The Drosophila juvenile hormone receptor candidates methoprene-tolerant (MET) and germ cell-expressed (GCE) utilize a conserved LIXXL motif to bind the FTZ-F1 nuclear receptor. The Journal of Biological Chemistry, 287 (10): 7821-7833.
4. Boyd, L., E. O'Toole & C. S. Thummel, 1991. Patterns of E74A RNA and protein expression at the onset of metamorphosis in Drosophila. Development, 112 (4): 981-995.
5. Cermenati, G., P. Corti, S. Caccia, B. Giordana & M. A. Casartelli, 2007. Morphological and functional characterization of Bombyx mori larval midgut cells in culture. Invertebrate Survival Journal, 4 (2): 119-126.
6. Cranna, N. & L. Quinn, 2009. Impact of steroid hormone signals on Drosophila cell cycle during development. Cell Division, 4 (3): 1-13.
7. Dubrovskaya, V. A., E. M. Berger & E. B. Dubrovsky, 2004. Juvenile hormone regulation of the E75 nuclear receptor is conserved in Diptera and Lepidoptera. Gene, 13 (2): 171-177.
8. Dubrovsky, E. B., V. A. Dubrovskaya & E. M. Berger, 2004. Hormonal regulation and functional role of Drosophila E75A orphan nuclear receptor in the juvenile hormone signaling pathway. Developmental Biology, 268 (2): 258-270.

9. Fletcher, J. C., K. C. Burtis, D. S. Hogness & C. S. Thummel, 1995. The Drosophila E74 gene is required for metamorphosis and plays a role in the polytene chromosome puffing response to ecdysone. Development, 121 (5): 1455-1465.
10. Fletcher, J. C. & C. S. Thummel, 1995. The ecdysone-inducible Broad-complex and E74 early genes interact to regulate target gene transcription and Drosophila metamorphosis. Genetics, 141 (3): 1025-1035.
11. Franzetti, E., Z. J. Huang, Y. X. Shi, K. Xie, X. J. Deng, J. P. Li, Q. R. Li, W. Y. Yang, W. N. Zeng, M. Casartelli, H. M. Deng, S. Cappellozza, A. Grimaldi, Q. Xia & Q. Feng, 2012. Autophagy precedes apoptosis during the remodeling of silkworm larval midgut. Apoptosis, 17 (3): 305-324.
12. Gibson, U. E., C. A. Heid & P. M. Williams, 1996. A novel method for real-time quantitative RT-PCR. Genome Research, 6 (10): 995-1001.
13. Goncu, E. & O. Parlak, 2011. The influence of juvenile hormone analogue, fenoxycarb on the midgut remodeling in Bombyx mori (L., 1758) (Lepidoptera: Bombycidae) during larval-pupal metamorphosis. Turkish Journal of Entomology, 35 (2): 179-194.
14. Goncu, E., R. Uranlı, G. Selek & O. Parlak, 2016. Developmental expression of ecdysone-related genes associated with metamorphic changes during midgut remodeling of silkworm Bombyx mori (Lepidoptera: Bombycidae). Journal of Insect Science, 164 (1): 1-13.
15. Hakim, R. S, K. Baldwin & G. Smagghe, 2010. Regulation of midgut growth, development, and metamorphosis. Annual Review of Entomology, 55: 593-608.
16. Hakim, R. S., M. B. Blackburn, P. Corti, D. B. Gelman, C. Goodman, K. Elsen, M. J. Loeb, D. Lynn, T. Soin & G. Smagghe, 2007. Growth and mitogenic effects of arylphorin in vivo and in vitro. Archives of Insect Biochemistry and Physiology, 64 (2): 63-73.
17. Hakim, R. S, S. Caccia, M. Loeb & G. Smagghe, 2009. Primary culture of insect midgut cells. In Vitro Cellular & Developmental Biology-Animal, 45 (3-4): 106-110.
18. Huang, J. H., J. Lozano & X. Belles, 2013. Broad-complex functions in postembryonic development of the cockroach Blattella germanica shed new light on the evolution of insect metamorphosis. Biochimica et Biophysica Acta, 1830 (1): 2178-2187.
19. Jindra, M., F. Malone, K. Hiruma & L. M. Riddiford, 1996. Developmental Profiles and Ecdysteroid Regulation of the mRNAs for Two Ecdysone Receptor Isoforms in the Epidermis and Wings of the Tobacco Hornworm, Manduca sexta. Developmental Biology, 180 (1): 258-272.
20. Kamimura, M. & M. Kiuchi, 1998. Effects of a juvenile hormone analog, fenoxycarb, on 5th stadium larvae of the silkworm, Bombyx mori (Lepidoptera: Bombycidae). Applied Entomology and Zoology, 33 (2): 333-338.
21. Keshan, B., K. Hiruma & L. M. Riddiford, 2006. Developmental expression and hormonal regulation of different isoforms of the transcription factor E75 in the tobacco hornworm Manduca sexta. Developmental Biology, 295 (2): 623-632.
22. Lee, C. Y., C. R. Simon, C. T. Woodard & E. H. Baehrecke, 2002. Genetic Mechanism for the Stage-and Tissue-Specific Regulation of Steroid Triggered Programmed Cell Death in Drosophila. Developmental Biology, 252 (1): 138-148.
23. Nishiura, J. T., K. Ray & J. Murray, 2005. Expression of nuclear receptor-transcription factor genes during Aedes aegypti midgut metamorphosis and the effect of methoprene on expression. Insect Biochemistry and Molecular Biology, 35 (6): 561-573.
24. Parthasarathy, R. & S. R. Palli, 2007. Stage-and cell-specific expression of ecdysone receptors and ecdysone-induced transcription factors during midgut remodeling in the yellow fever mosquito Aedes aegypti. Journal of Insect Physiology, 53 (3): 216-229.
25. Parthasarathy, R. & S. R. Palli, 2008. Proliferation and differentiation of intestinal stem cells during metamorphosis of the red flour beetle, Tribolium castaneum. Developmental Dynamics, 223 (4): 893-903.
26. Quinn, L., J. Lin, N. Cranna, J. Lee, N. Mitchell & R. Hannan, 2012. “Steroid Hormones in Drosophila: How Ecdysone Coordinates Developmental Signaling with Cell Growth and Division, 141-168”. In: Steroids - Basic Science (Ed. H. Abduljabbar). InTech, Rijeka, Croatia, 234pp.
27. Restifo, L. L. & K. White, 1992. Mutations in a steroid hormone-regulated gene disrupt the metamorphosis of internal tissues in Drosophila: salivary glands, muscle, and gut. Roux's Archives of Developmental Biology, 201 (4): 221-234.
28. Riddiford, L. M., P. Cherbas, & J. W. Truman, 2000. Ecdysone receptors and their biological actions. Vitamins and Hormones, 60: 1-73.
29. Sakurai, S., M. Kayav & S. I. Satake, 1998. Hemolymph ecdysteroid titer and Ecdysteroid-dependent developmental events in the last larval stadium of the silkworm, Bombyx mori: the 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.
30. Siaussat, D., F. Bozzolan, I. Queguiner, P. Porcheron & S. Debernard, 2005. Cell cycle profiles of EcR, USP, HR3 and B cyclin mRNAs associated to 20E-induced G2 arrest of Plodia interpunctella imaginal wing cells. Insect Molecular Biology, 14 (2): 151-161.
31. Smagghe, G., K. Elsen, M. J. Loeb, D. B. Gelman & M. Blackbur, 2003. Effects of a fat body extract on larval midgut cells and growth of Lepidoptera. In Vitro Cellular & Developmental Biology-Animal, 39 (1): 8-12.
32. Smagghe, G., W. Vanhassel, C. Moeremans, D. Wilde, S. Goto, M. J. Loeb, M. B. Blacburn & R. S. Hakim, 2005. Stimulation of midgut stem cell proliferation and differentiation by insect hormones and peptides. Annals of the New York Academy of Sciences, 1040 (1): 472-475.
33. Stilwell, G. E., C. A. Nelson, J. Weller, H. Cui, K. Hiruma, J. W. Truman & L. M. Riddiford, 2003. E74 exhibits stage-specific hormonal regulation in the epidermis of the tobacco hornworm, Manduca sexta. Developmental Biology, 258 (1): 76-90.
34. Talbot, W. S., E. A. Swyryd & D. S. Hogness, 1993. Drosophila tissues with different metamorphic responses to ecdysone express different ecdysone receptor isoforms. Cell, 73 (7): 1323-1337.
35. Tettamanti, G., A. Grimaldi, M. Casartelli, E. Ambrosetti, B. Ponti, T. Congiu, R. Ferrarese, M. R. Rivas-Pena, F. Pennacchio & M. De Eguileor, 2007. 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.
36. Zhou, B., K. Hiruma, T. Shinoda & L. M. Riddiford, 1998. Juvenile hormone prevents ecdysteroid-induced expression of broad complex RNAs in the epidermis of the tobacco hornworm, Manduca sexta. Developmental Biology, 203 (2): 233-244.
37. Zhou, X. & L. M. Riddiford, 2002. Broad specifies pupal development and mediates the ‘status quo’ action of juvenile hormone on the pupal-adult transformation in Drosophila and Manduca. Development, 129 (9): 2259-2269.