Advertisement

Springer Nature is making Coronavirus research free. View research | View latest news | Sign up for updates

Activation of autophagic programmed cell death and innate immune gene expression reveals immuno-competence of integumental epithelium in Bombyx mori infected by a dipteran parasitoid

Abstract

In insects, the integument forms the primary barrier between the environment and internal milieu, but cellular and immune responses of the integumental epithelium to infection by micro- and macro-parasites are mostly unknown. We elucidated cellular and immune responses of the epithelium induced through infection by a dipteran endoparasitoid, Exorista bombycis in the economically important silkworm Bombyx mori. Degradative autophagic vacuoles, lamella-like bodies, a network of cytoplasmic channels with cellular cargo, and an RER network that opened to vacuoles were observed sequentially with increase in age after infection. This temporal sequence culminated in apoptosis, accompanied by the upregulation of the caspase gene and fragmentation of DNA. The infection significantly enhanced the tyrosine level and phenol oxidase activity in the integument. Proteomic analysis revealed enhanced expression of innate immunity components of toll and melanization pathways, cytokines, signaling molecules, chaperones, and proteolytic enzymes demonstrating diverse host responses. qPCR analysis revealed the upregulation of spatzle, BmToll, and NF kappa B transcription factors Dorsal and BmRel. NF kappa B inhibitor cactus showed diminished expression when Dorsal and BmRel were upregulated, revealing a negative correlation (R = -0.612). During melanization, prophenol oxidase 2 was expressed, a novel finding in integumental epithelium. The integument showed a low level of melanin metabolism and localized melanism in order to prevent the spreading of cytotoxic quinones. The gene-encoding proteolytic enzyme, beta-N-acetylglucosaminidase, was activated at 24 h post-infection, whereas chitinase, was activated at 96 h post-infection; however, most of the immune genes enhanced their expression in the early stages of infection. Thus the integument contributes to humoral immune responses that enhance resistance against macroparasite invasion.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

References

  1. Acharya MM, Katyare SS (2004) An improved micromethod for tyrosine estimation. Z Naturforsch 59c:897–900

  2. Amaya KE, Asgari S, Jung R, Hongskula M, Beckage NE (2005) Parasitization of Manduca sexta larvae by the parasitoid wasp Cotesia congregata induces an impaired host immune response. J Insect Physiol 51:505–512

  3. Ambrose RL, Mackenzie JM (2010) West Nile virus differentially modulates the unfolded protein response to facilitate replication and immune evasion. J Virol 85:2723–2732

  4. Asano T, Ashida M (2001) Cuticular pro-phenoloxidase of the silkworm, Bombyx mori: purification and demonstration of its transport from hemolymph. J Biol Chem 276:11100–11112

  5. Ashida M, Brey PT (1995) Role of the integument in insect defense: pro-phenol oxidase cascade in the cuticular matrix. Proc Natl Acad Sci USA 92:10698–10702

  6. Ashida M, Brey PT (1998) Recent advances in research on the insect prophenoloxidase cascade. In: Brey PT, Hultmark D (eds) Molecular mechanisms of immune responses in insects. Chapman and Hall, London, pp 135–172

  7. Biron DG, Ponton F, Marché L, Galeotti N, Renault L, Demey-Thomas E, Poncet J, Brown SP, Jouin P, Thomas F (2006) Suicide of crickets harbouring hairworms: a proteomics investigation. Insect Mol Biol 15:731–742

  8. Boltz KA, Carney GE (2008) Loss of p24 function in Drosophila melanogaster causes a stress response and increased levels of NF-κB-regulated gene products. BMC Genomics 9:212

  9. Bras M, Queenan B, Susin SA (2005) Programmed cell death via mitochondria: different modes of dying. Biochem Mosc 70:284–293

  10. Brey PT, Lee WJ, Yamakawa M, Koizumi Y, Perrot S, Francois M, Ashida M (1993) Role of the integument in insect immunity: epicuticular abrasion and induction of cecropin synthesis in cuticular epithelial cells. Proc Natl Acad Sci USA 90:6275–6279

  11. Bruchhaus I, Roeder T, Rennenberg A, Heussler VT (2007) Protozoan parasites: programmed cell death as a mechanism of parasitism. Trends Parasitol 23:376–383

  12. Bursch W, Ellinger A, Gerner CH, Fröhwein U, Schulte-Hermann R (2000) Programmed cell death (PCD): apoptosis, autophagic PCD or others? Ann N Y Acad Sci 926:1–12

  13. Chan DC (2006) Mitochondria: dynamic organelles in disease, aging, and development. Cell 125:1241–1252

  14. Chatterjee SN, Mohandas TP, Taraphdar T (2003) Molecular characterization of the gene pool of Exorista sorbillans (Diptera: Tachinidae) a parasitoid of silkworm, Bombyx mori, in India. Eur J Entomol 100:195–200

  15. Cheng TC, Zhang YL, Chun L, Xu PZ, Gao ZH, Xia QY, Xiang ZH (2008) Identification and analysis of toll-related genes in the domesticated silkworm, Bombyx mori. Dev Comp Immunol 32:464–475

  16. Christophides GK, Zdobnov E, Barillas-Mury C, Birney E, Blandin S, Blass C, et al (2002) Immunity-related genes and gene families in Anopheles gambiae. Science 298:159–165

  17. Clark TM, Hutchinson MJ, Huegel KL, Moffett SB, Moffett DF (2005) Additional morphological and physiological heterogeneity within the midgut of larval Aedes aegypti (Diptera: Culicidae) revealed by histology, electrophysiology and effects of Bacillus thuringiensis endotoxin. Tissue Cell 37:457–468

  18. De Gregorio E, Spellman PT, Tzou P, Rubin GM, Lemaitre B (2002) Toll and IMD pathways are the major regulators of the immune response in Drosophila. EMBO J 21:2568–2579

  19. Dohke K (1973) Studies on prophenoloxidase-activating enzyme from cuticle of the silkworm. Bombyx mori. II. Purification and characterization of the enzyme. Arch Biochem Biophys 157:210–221

  20. DosReis GA, Barcinski MA (2001) Apoptosis and parasitism: from the parasite to the host immune response. Adv Parasitol 49:133–161

  21. Etebari K, Palfreyman RW, Schlipalius D, Nielsen LK, Glatz RV, Asgari S (2011) Deep sequencing-based transcriptome analysis of Plutella xylostella larvae parasitized by Diadegma semiclausum. BMC Genomics 12:446

  22. Evans JD, Aronstein K, Chen YP, Hetru C, Imler JL, Jiang H et al (2006) Immune pathways and defence mechanisms in honey bees Apis mellifera. Insect Mol Biol 15:645–656

  23. Ferrandon D, Imler JL, Hetru C, Hoffmann JA (2007) The Drosophila systemic immune response: sensing and signalling during bacterial and fungal infections. Nat Rev Immunol 7:862–874

  24. Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R (1994) DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol Mar Biol Biotechnol 3:294–299

  25. Gandhe AS, John SH, Nagaraju J (2007) Noduler, a novel immune up-regulated protein mediates nodulation response in insects. J Immunol 179:6943–6951

  26. Gillespie JP, Kanost MR, Trenczek T (1997) Biological mediators of insect immunity. Annu Rev Entomol 42:611–643

  27. Goldsmith MR, Shimada T, Abe H (2005) The genetics and genomics of the silkworm, Bombyx mori. Ann Rev Entomol 50:71–100

  28. Gottlieb E, Armour SM, Harris MH, Thompson CB (2003) Mitochondrial membrane potential regulates matrix configuration and cytochrome c release during apoptosis. Cell Death Diff 10:709–717

  29. Hardt N, DeKegel D, Vanheule L, Villani G, Spadari S (1980) DNA polymerase γ, cytochrome c oxidase and mitochondrial integrity in rabbit spleen lymphocytes stimulated with concanavalin A. Exp Cell Res 127:269–276

  30. Higes M, García-Palencia P, Martín-Hernández R, Meana A (2007) Experimental infection of Apis mellifera honey bees with Nosema ceranae (Microsporidia). J Invertebr Pathol 94:211–217

  31. Hoffmann JA, Kafatos FC, Janeway CA, Ezekowitz RA (1999) Phylogenetic perspectives in innate immunity. Science 284:1313–1318

  32. Hurd H, Carter V, Nacer A (2005) Interactions between malaria and mosquitoes: the role of apoptosis in parasite establishment and vector response to infection. Curr Top Microbiol Immunol 289:185–217

  33. Ioannou YA, Chen FW (1996) Quantitation of DNA fragmentation in apoptosis. Nucleic Acids Res 24:992–993

  34. Ishii K, Hamamoto H, Kamimura M, Nakamura Y, Noda H, Imamura K, Mita K, Sekimizu K (2010) Insect cytokine paralytic peptide (PP) induces cellular and humoral immune responses in the silkworm Bombyx mori. J Biol Chem 285:28635–28642

  35. Jiang J, Cai H, Zhou Q, Levine M (1993) Conversion of a dorsal-dependent silencer into an enhancer: evidence for dorsal corepressors. EMBO J 12:3201–3209

  36. Kuraishi T, Binggeli O, Opota O, Buchon N, Lemaitre B (2011) Genetic evidence for a protective role of the peritrophic matrix against intestinal bacterial infection in Drosophila melanogaster. Proc Natl Acad Sci USA 108:15966–15971

  37. LeBlanc PM, Saleh M (2009) Caspases in inflammation and immunity. eLS, Wiley Online Library

  38. Leist M, Jaattela M (2001) Four deaths and a funeral: from caspases to alternative mechanisms. Nature Rev Mol Cell Biol 2:589–598

  39. Lemaitre B, Nicolas E, Michaut L, Reichhart JM, Hoffmann JA (1996) The dorsoventral regulatory gene cassette spatzle/Toll/cactus controls the potent antifungal response in Drosophila. EMBO J 14:536–545

  40. Liu L, Tang Q, Fu C, Peng J, Yang H, Li Y, Hong H (2007) Influence of glucose starvation on the pathway of death in insect cell line Sl: apoptosis follows autophagy. Cytotechnology 54:97–105

  41. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the folin-phenol reagent. J Biol Chem 193:265–275

  42. Martin DN, Baehrecke EH (2004) Caspases function in autophagic programmed cell death in Drosophila. Development 131:275–284

  43. Matus S, Lisbona F, Torres M, Leon C, Thielen P, Hetz C (2008) The stress rheostat: an interplay between the unfolded protein response (UPR) and autophagy in neurodegeneration. Curr Mol Med 8:157–172

  44. McBride HM, Neuspiel M, Wasiak S (2006) Mitochondria: more than just a powerhouse. Curr Biol 16:R551–R560

  45. Meister M, Govind S (2003) Hematopoietic development in Drosophila: a parallel with vertebrates. In: Godin I, Cumano A (eds) Hematopoietic stem cells. Landes Bioscience, Austin

  46. Merzendorfer H, Zimoch L (2003) Chitin metabolism in insects: structure, function and regulation of chitin synthases and chitinases. J Exp Biol 206:4393–4412

  47. Mrinal N, Tomar A, Nagaraju J (2011) Role of sequence encoded κB DNA geometry in gene regulation by Dorsal. Nucleic Acids Res 39:9574–9591

  48. Nakahara Y, Kanamori Y, Kiuchi M, Kamimura M (2003) Effects of silkworm paralytic peptide on in vitro hematopoiesis and plasmatocyte spreading. Arch Insect Biochem Physiol 52:163–174

  49. Nappi AJ, Vass E (1993) Melanogenesis and the generation of cytotoxic molecules during insect cellular immune reactions. Pigment Cell Res 6:117–126

  50. Narayanaswamy KC, Devaiah MC (1998) Silkworm uzi fly. Zen Publishers, Bangalore

  51. Noguchi H, Tsuzuki S, Tanaka K, Matsumoto H, Hiruma K, Hayakawa Y (2003) Isolation and characterization of a dopa decarboxylase cDNA and the induction of its expression by an insect cytokine, growth-blocking peptide in Pseudaletia separate. Insect Biochem Mol Biol 33:209–217

  52. Oeckinghaus A, Hayden MS, Ghosh S (2011) Crosstalk in NF-κB signaling pathways. Nat Immunol 12:695–708

  53. Pahl HL, Baeuerle PA (1997) The ER-overload response: activation of NF-κB. Trends Biochem Sci 22:63–67

  54. Palmer CA, Wittrock DD, Christensen BM (1986) Ultrastructure of Malpighian tubules of Aedes aegypti infected with Dirofilaria immitis. J Invertebr Pathol 48:310–317

  55. Perumalsamy LR, Nagala M, Sarin A (2010) Notch-activated cascade interacts with mitochondrial remodeling proteins to regulate cell survival. Proc Natl Acad Sci USA 107:6882–6887

  56. Pinheiro DO, Silva MD, Gregorio EA (2010) Mitochondria in the midgut epithelial cells of sugarcane borer parasitized by Cotesia flavipes (Cameron, 1891). Braz J Biol 70:163–169

  57. Pradeep AR, Awasthi AK, Urs RS (2008) Association of A/T rich microsatellites with responses to artificial selection for larval developmental duration in the silkworm Bombyx mori. Mol Cells 25:467–478

  58. Pradeep AR, Awasthi AK, Singh CK, Anuradha HJ, Rao CG, Vijayaprakash NB (2011) Genetic evaluation of eri silkworm Samia cynthia ricini: ISSR loci specific to high and low altitude regimes and quantitative attributes. J Appl Genet 52:345–353

  59. Prasad MD, Muthulakshmi M, Madhu M, Archak S, Mita K, Nagaraju J (2005) Survey and analysis of microsatellites in the silkworm, Bombyx mori: frequency, distribution, mutations, marker potential and their conservation in heterologous species. Genetics 169:197–214

  60. Pye AE (1974) Microbial activation of prophenoloxidase from immune insect larvae. Nature 251:610–613

  61. Reddy BK, Rao JVK (2009) Seasonal occurrence and control of silkworm diseases, grasserie, flacherie and muscardine and insect pest, uzi fly in Andhra Pradesh, India. Int J Indust Entomol 18:57–61

  62. Rosenfeld J, Capdevielle J, Guillemot C, Ferrara P (1992) In-gel digestion for internal sequence analysis after one- or two-dimensional gel electrophoresis. Anal Biochem 203:173–179

  63. Santamaría-Fríes M, Fajardo LF, Sogin ML, Olson PD, Relman DA (1996) Lethal infection by a previously unrecognised metazoan parasite. Lancet 347:1797–1801

  64. Sass M (2008) Autophagy research on insects. Autophagy 4:265–267

  65. Satake S, Kaya M, Sakurai S (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. J Insect Physiol 44:867–881

  66. Sathisha GJ, Prakash YKS, Chachadi VB, Nagaraja NN, Inamdar SR, Leonidas DD, Savithri HS, Swamy BM (2008) X-ray sequence ambiguities of Sclerotium rolfsii lectin resolved by mass spectrometry. Amino Acids 35:309–320

  67. Schaub GA, Sehnitker A (1988) Influence of Blastocrithidia triatomae (Trypanosomatidae) on the reduviid bug Triatoma infestans: alterations in the Malpighian tubules. Parasitol Res 75:88–97

  68. Schlenke TA, Morales J, Govind S, Clark AG (2007) Contrasting infection strategies in generalist and specialist wasp parasitoids of Drosophila melanogaster. PLoS Pathog 3:e158

  69. Schluns H, Sadd BM, Schmid-Hempel P, Crozier RH (2010) Infection with the trypanosome Crithidia bombi and expression of immune – related genes in the bumble bee Bombus terrestris. Dev Comp Immunol 34:705–709

  70. Schmid-Hempel P (2005) Evolutionary ecology of insect immune defenses. Ann Rev Entomol 50:529–551

  71. Schmidt O (2008) Insect immune recognition and suppression. In: Beckage NE (ed) Insect immunology. Elsevier/Academic Press, San Diego, pp 271–294

  72. Sengupta K, Kumar P, Baig M, Govindaiah M (1990) Handbook of pest and disease control in mulberry and silkworm. United Nations Economic and Social Commission for Asia and the Pacific, Bangkok

  73. Sorrentino RP, Carton Y, Govind S (2002) Cellular immune response to parasite infection in the Drosophila lymph gland is developmentally regulated. Dev Biol 243:65–80

  74. Strengerg C, Benchimol M, Linden R (2010) Caspase dependence of the death of neonatal retinal ganglion cells induced by axon damage and induction of autophagy as survival mechanism. Braz J Med Biol Res 43:950–956

  75. Suganuma I, Ushiyama T, Yamada H, Iwamoto A, Kobayashi M, Ikeda M (2011) Cloning and characterization of a dronc homologue in the silkworm, Bombyx mori. Insect Biochem Mol Biol 41:909–921

  76. Takahashi M, Kiuchi M, Kamimura M (2002) A new chitinase-related gene, BmChiR1, is induced in the Bombyx mori anterior silk gland at molt and metamorphosis by ecdysteroid. Insect Biochem Mol Biol 32:147–151

  77. Takayama S, Reed JC, Homma S (2003) Heat-shock proteins as regulators of apoptosis. Oncogene 22:9041–9047

  78. Tanaka H, Yamamoto M, Moriyama Y, Yamao M, Furukawa S, Sagisaka A et al (2005) A novel Rel protein and shortened isoform that differentially regulate antibacterial protein genes in the silkworm, Bombyx mori. Biochim Biophys Acta 1730:10–21

  79. Tanaka H, Ishibashi J, Fujita K, Nakajima Y, Sagisaka A, Tomimoto K, Suzuki N et al (2008) A genome-wide analysis of genes and gene families involved in innate immunity of Bombyx mori. Insect Biochem Mol Biol 38:1087–1110

  80. Tang H (2009) Regulation and function of the melanization reaction in Drosophila. Fly (Austin) 3:105–111

  81. Torres M, Castillo K, Armisén R, Stutzin A, Soto C, Hetz C (2010) Prion protein misfolding affects calcium homeostasis and sensitizes cells to endoplasmic reticulum stress. PLoS One 5:e15658

  82. Valanne S, Wang JH, Rämet M (2011) The Drosophila toll signaling pathway. J Immunol 186:649–656

  83. Wertheim B, Kraaijeveld AR, Schuster E, Blanc E, Hopkins M, Pletcher SD, Strand MR, Partridge L, Godfray HCJ (2005) Genome-wide gene expression in response to parasitoid attack in Drosophila. Genome Biol 6:R94

  84. Wertheim B, Kraaijeveld AR, Hopkins MG, Boer MW, Godfray HCJ (2011) Functional genomics of the evolution of increased resistance to parasitism in Drosophila. Mol Ecol 20:932–949

  85. Wirawan E, Berghe TV, Lippens S, Agostinis P, Vandenabeele P (2011) Autophagy: for better or for worse. Cell Res 22:43–61

  86. Yano T, Kurata S (2011) Intracellular recognition of pathogens and autophagy as an innate immune host defence. J Biochem 150:143–149

  87. Zeiske W, Meyer H, Wheczoreck H (2002) Insect midgut K secretion concerted run-down of apical/basolateral transporters with extra/intracellular acidity. J Exp Biol 205:463–467

  88. Zhang YM, Lu XF, Bhavnani BR (2003) Equine estrogens differentially inhibit DNA fragmentation induced by glutamate in neuronal cells by modulation of regulatory proteins involved in programmed cell death. BMC Neurosci 4:32

  89. Zhang FX, Shao HL, Wang JX, Zhao XF (2011) β-Thymosin is upregulated by the steroid hormone 20-hydroxyecdysone and microorganisms. Insect Mol Biol 20:519–527

  90. Zou Z, Shin SW, Alvarez KS, Bian G, Kokoza V, Raikhel AS (2008) Mosquito RUNX4 in the immune regulation of PPO gene expression and its effect on avian malaria parasite infection. Proc Nat Acad Sci USA 105:18454–18459

Download references

Acknowledgements

The authors thank the anonymous referees for critical comments, Mr. B. Srinivasa for uzi fly maintenance and silkworm rearing, and the Central Silk Board, Government of India, Bangalore for facilities.

Author information

Correspondence to Appukuttan Nair R. Pradeep.

Additional information

Appukuttan Nair R. Pradeep and Jayaram Anitha are equal contributors

Authors acknowledge the financial support received from Department of Biotechnology, Government of India, New Delhi, in the form of a research project to ARP (BT/PR11722/PBD/19/197/2008 dated 11/6/2009). AJ and AKH are supported by DBT junior research fellowships.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Fig. S1
figure8

a-d. Variations induced in cuticular morphology of B. mori larvae after infection by the parasitoid, E. bombycis. (a) Egg (x70) of E. bombycis is attached to the larval cuticle by a cementing substance (arrow). (b) Scanning electron micrograph of the cuticle where egg was attached showing blackening (arrow) of the area (x500) at 24 h after infection. (c) Control cuticle showing normal features such as hairs and microtrichia (x200). (d) Fifth instar larva showing translucent cuticle (bolded white arrow) adjoining the melanized cuticle ( black arrow) at 96 h after infection. (JPEG 44 kb)

Fig. S2
figure9

a-d. Variation in protein profile of integument of B. mori larvae after infection by the parasitoid, E. bombycis. (a) Total protein content showed significant reduction over ages after infection. (b) Tyrosine content showed significant increase at 24 h after infection compared to control. (c) PO activity was measured by optical density at 490 nm showed significant increase at 96 h after infection. (d) SDS-PAGE showed resolution of total protein extracted from integument at 72 h after infection. Two boxed bands at ~155 kDa and ~30 kDa showed enhanced expression after infection (arrows) and were cut and processed for mass spectrometry. Ctrl. – saline injected control; Inf. – infected; M - marker. (JPEG 37 kb)

Fig. S3
figure10

Differential expression of various host-response genes in integumental epithelium of B. mori larva after infection by the parasitoid E. bombycis. Total RNA was isolated from integument at 24 h intervals from the control and infected larvae and cDNA was synthesized by RT-PCR using oligo- d(T)23 primer. All genes were amplified as per the details given in Table 1. Number of cycles was optimized for each gene. β- actin was used as control housekeeping gene. Control – saline injected control; M- Marker. (JPEG 55 kb)

High resolution image (TIFF 5075 kb)

High resolution image (TIFF 6390 kb)

High resolution image (TIFF 4663 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Pradeep, A.R., Anitha, J., Awasthi, A.K. et al. Activation of autophagic programmed cell death and innate immune gene expression reveals immuno-competence of integumental epithelium in Bombyx mori infected by a dipteran parasitoid. Cell Tissue Res 352, 371–385 (2013). https://doi.org/10.1007/s00441-012-1520-7

Download citation

Keywords

  • Host–parasite interaction
  • Apoptosis
  • Toll pathway activation
  • PPO2 expression
  • Bombyx mori (Insecta)