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Coregulation of host–response genes in integument: switchover of gene expression correlation pattern and impaired immune responses induced by dipteran parasite infection in the silkworm, Bombyx mori

  • Animal Genetics • Original Paper
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Abstract

The activation of host response proteins against parasitic infection is dependent on the coregulation of immune gene expression. The infection of commercially important silkworm Bombyx mori through endoparasite Exorista bombycis enhanced host–response gene expression in integument early in the infection and was lowered asymptotically. Principal component analysis (PCA) showed heterogeneity while explaining ∼80 % variance among expression timings. PCA showed positive and negative correlation with gene expression and differentiated transcriptional timings, and revealed cross talk within the immune system. Pearson correlation analysis showed significant linear correlation (mean R 2 = >0.7; P < 0.004) between the expression of 16 pairs of genes in control, while the relation switched over to curvilinear due to parasitism. The genes showed pleiotropic interaction among them, with four genes each for prophenoloxidase activating enzyme (PPAE) and caspase. Besides, after parasitism, exclusive correlation of five gene pairs including PPAE–Spatzle pair (R 2 = 0.9; P < 0.011) was observed in the integument. In integument, the phenol oxidase (PO) activity showed a positive correlation with the tyrosine level (R 2 = 0.410; P < 0.002) and a curvilinear relation (R 2 = 0.745; P < 0.0002) with the expanding lysis area. The PO activity was positively correlated with BmToll expression and negatively correlated with paralytical peptide expression, revealing polygenic influence. Caspase expression was tightly regulated by signal genes in control integument, whereas they were deregulated after infection. Switchover from linear to curvilinear correlation and the appearance of new gene correlations in parasitized integument revealed deviation from gene coregulation, leading to impaired immune responses, characterized by lowered gene expression and varied phenotypic consequences.

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References

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

    PubMed  CAS  Google Scholar 

  • Adhikari BN, Lin C-Y, Bai X, Ciche TA, Grewal PS, Dillman AR, Chaston JM, Shapiro-Ilan DI, Bilgrami AL, Gaugler R, Sternberg PW, Adams BJ (2009) Transcriptional profiling of trait deterioration in the insect pathogenic nematode Heterorhabditis bacteriophora. BMC Genomics 10:609

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • Arvas M, Pakula T, Smit B, Rautio J, Koivistoinen H, Jouhten P, Lindfors E, Wiebe M, Penttilä M, Saloheimo M (2011) Correlation of gene expression and protein production rate—a system wide study. BMC Genomics 12:616

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Ashida M, Dohke K (1980) Activation of prophenoloxidase by the activating enzyme of the silkworm Bombyx mori. Insect Biochem 10:37–47

    Article  CAS  Google Scholar 

  • Aykanat T, Heath JW, Dixon B, Heath DD (2012) Additive, non-additive and maternal effects of cytokine transcription in response to immunostimulation with Vibrio vaccine in Chinook salmon (Oncorhynchus tshawytscha). Immunogenetics 64:691–703

    Article  PubMed  CAS  Google Scholar 

  • Barton NH, Keightley PD (2002) Understanding quantitative genetic variation. Nat Rev Genet 3:11–21

    Article  PubMed  CAS  Google Scholar 

  • Bidla G, Hauling T, Dushay MS, Theopold U (2009) Activation of insect phenoloxidase after injury: endogenous versus foreign elicitors. J Innate Immun 1:301–308

    Article  PubMed  CAS  Google Scholar 

  • Brey PT, Lee WJ, Yamakawa M, Koizumi Y, Perrot S, François 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 U S A 90:6275–6279

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Carlborg O, Haley CS (2004) Epistasis: too often neglected in complex trait studies? Nat Rev Genet 5:618–625

    Article  PubMed  CAS  Google Scholar 

  • Casso DJ, Biehs B, Kornberg TB (2011) A novel interaction between hedgehog and notch promotes proliferation at the anterior–posterior organizer of the Drosophila wing. Genetics 187:485–499

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • Cordell HJ (2009) Detecting gene–gene interactions that underlie human diseases. Nat Rev Genet 10:392–404

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • de Celis JF, Bray SJ (2000) The Abruptex domain of Notch regulates negative interactions between Notch, its ligands and Fringe. Development 127:1291–1302

    PubMed  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  • Erler S, Popp M, Lattorff HM (2011) Dynamics of immune system gene expression upon bacterial challenge and wounding in a social insect (Bombus terrestris). PLoS One 6(3):e18126

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Evans JD, Aronstein K, Chen YP, Hetru C, Imler JL, Jiang H, Kanost M, Thompson GJ, Zou Z, Hultmark D (2006) Immune pathways and defence mechanisms in honey bees Apis mellifera. Insect Mol Biol 15:645–656

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Fiumera AC, Dumont BL, Clark AG (2005) Sperm competitive ability in Drosophila melanogaster associated with variation in male reproductive proteins. Genetics 169:243–257

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Flint J, Mott R (2001) Finding the molecular basis of quantitative traits: successes and pitfalls. Nat Rev Genet 2:437–445

    Article  PubMed  CAS  Google Scholar 

  • Glazier AM, Nadeau JH, Aitman TJ (2002) Finding genes that underlie complex traits. Science 298:2345–2349

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  • Hansen BB, Bowers J (2008) Covariate balance in simple, stratified and clustered comparative studies. Stat Sci 23:219–236

    Article  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  • Hozo I, Djulbegovic B, Clark O, Lyman GH (2005) Use of re-randomized data in meta-analysis. BMC Med Res Methodol 5:17. doi:10.1186/1471-2288-5-17

    Article  PubMed Central  PubMed  Google Scholar 

  • Kroymann J, Mitchell-Olds T (2005) Epistasis and balanced polymorphism influencing complex trait variation. Nature 435:95–98

    Article  PubMed  CAS  Google Scholar 

  • Kwon TH, Kim MS, Choi HW, Joo CH, Cho MY, Lee BL (2000) A masquerade-like serine proteinase homologue is necessary for phenoloxidase activity in the coleopteran insect, Holotrichia diomphalia larvae. Eur J Biochem 267:6188–6196

    Article  PubMed  CAS  Google Scholar 

  • LeBlanc PM, Saleh M (2009) Caspases in inflammation and immunity. eLS, Wiley Online Library. doi:10.1002/9780470015902.a0021990

  • Lemaitre B, Nicolas E, Michaut L, Reichhart JM, Hoffmann JA (1996) The dorsoventral regulatory gene cassette spätzle/Toll/cactus controls the potent antifungal response in Drosophila adults. Cell 86:973–983

    Article  PubMed  CAS  Google Scholar 

  • Li D, Guo Y, Shao H, Tellier LC, Wang J, Xiang Z, Xia Q (2010) Genetic diversity, molecular phylogeny and selection evidence of the silkworm mitochondria implicated by complete resequencing of 41 genomes. BMC Evol Biol 10:81

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Ligoxygakis P, Pelte N, Ji C, Leclerc V, Duvic B, Belvin M, Jiang H, Hoffmann JA, Reichhart J-M (2002) A serpin mutant links Toll activation to melanization in the host defence of Drosophila. EMBO J 21:6330–6337

    Article  PubMed Central  PubMed  CAS  Google Scholar 

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

    PubMed  CAS  Google Scholar 

  • Mackay TF (2001) The genetic architecture of quantitative traits. Annu Rev Genet 35:303–339

    Article  PubMed  CAS  Google Scholar 

  • Månsson R, Tsapogas P, Akerlund M, Lagergren A, Gisler R, Sigvardsson M (2004) Pearson correlation analysis of microarray data allows for the identification of genetic targets for early B-cell factor. J Biol Chem 279:17905–17913

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  • Michalopoulos I, Pavlopoulos GA, Malatras A, Karelas A, Kostadima M-A, Schneider R, Kossida S (2012) Human gene correlation analysis (HGCA): a tool for the identification of transcriptionally co-expressed genes. BMC Res Notes 5:265

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Mutz D, Pemantle R (2011) The perils of randomization checks in the analysis of experiments. Available online at: http://www.math.upenn.edu/∼pemantle/papers/Preprints/perils.pdf

  • Nappi AJ, Christensen BM (2005) Melanogenesis and associated cytotoxic reactions: applications to insect innate immunity. Insect Biochem Mol Biol 35:443–459

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

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

    Google Scholar 

  • Naylor MG, Lin X, Weiss ST, Raby BA, Lange C (2010) Using canonical correlation analysis to discover genetic regulatory variants. PLoS One 5:e10395

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Paddibhatla I, Lee MJ, Kalamarz ME, Ferrarese R, Govind S (2010) Role for sumoylation in systemic inflammation and immune homeostasis in Drosophila larvae. PLoS Pathog 6(12):e1001234. doi:10.1371/journal.ppat.1001234

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • 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

    PubMed  CAS  Google Scholar 

  • Pradeep AR, Awasthi AK, Singh CK, Anuradha HJ, Rao CGP, 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

    Article  PubMed  Google Scholar 

  • Pradeep AN, Anitha J, Awasthi AK, Babu MA, Geetha MN, Arun HK, Chandrashekhar S, Rao GC, Vijayaprakash NB (2013) 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. doi:10.1007/s00441-012-1520-7

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  • Reid AJ, Berriman M (2013) Genes involved in host–parasite interactions can be revealed by their correlated expression. Nucleic Acids Res 41:1508–1518. doi:10.1093/nar/gks1340

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Reynier F, Petit F, Paye M, Turrel-Davin F, Imbert P-E, Hot A, Mougin B, Miossec P (2011) Importance of correlation between gene expression levels: application to the type I interferon signature in rheumatoid arthritis. PLoS One 6:e24828. doi:10.1371/journal.pone.0024828

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Sackton TB, Lazzaro BP, Clark AG (2010) Genotype and gene expression associations with immune function in Drosophila. PLoS Genet 6:e1000797

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Schlenke TA, Morales J, Govind S, Clark AG (2007) Contrasting infection strategies in generalist and specialist wasp parasitoids of Drosophila melanogaster. PLoS Pathog 3:1486–1501

    Article  PubMed  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  • 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

    Google Scholar 

  • Sinha H, Nicholson BP, Steinmetz LM, McCusker JH (2006) Complex genetic interactions in a quantitative trait locus. PLoS Genet 2:e13

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Söderhäll K, Cerenius L (1998) Role of the prophenoloxidase-activating system in invertebrate immunity. Curr Opin Immunol 10:23–28

    Article  PubMed  Google Scholar 

  • Sorrentino RP, Melk JP, Govind S (2004) Genetic analysis of contributions of dorsal group and JAK-Stat92E pathway genes to larval hemocyte concentration and the egg encapsulation response in Drosophila. Genetics 166:1343–1356

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Southworth LK, Owen AB, Kim SK (2009) Aging mice show a decreasing correlation of gene expression within genetic modules. PLoS Genet 5(12):e1000776

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Steinmetz LM, Sinha H, Richards DR, Spiegelman JI, Oefner PJ, McCusker JH, Davis RW (2002) Dissecting the architecture of a quantitative trait locus in yeast. Nature 416:326–330

    Article  PubMed  CAS  Google Scholar 

  • Tanaka H, Ishibashi J, Fujita K, Nakajima Y, Sagisaka A, Tomimoto K, Suzuki N, Yoshiyama M, Kaneko Y, Iwasaki T, Sunagawa T, Yamaji K, Asaoka A, Mita K, Yamakawa M (2008) A genome-wide analysis of genes and gene families involved in innate immunity of Bombyx mori. Insect Biochem Mol Biol 38:1087–1110

    Article  PubMed  CAS  Google Scholar 

  • Ting L, Cowley MJ, Hoon SL, Guilhaus M, Raftery MJ, Cavicchioli R (2009) Normalization and statistical analysis of quantitative proteomics data generated by metabolic labeling. Mol Cell Proteomics 8:2227–2242

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • VanderSluis B, Bellay J, Musso G, Costanzo M, Papp B, Vizeacoumar FJ, Baryshnikova A, Andrews B, Boone C, Myers CL (2010) Genetic interactions reveal the evolutionary trajectories of duplicate genes. Mol Syst Biol 6:429

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Vijayan K, Anuradha HJ, Nair CV, Pradeep AR, Awasthi AK, Saratchandra B, Rahman SAS, Singh KC, Chakraborti R, Urs SR (2006) Genetic diversity and differentiation among populations of the Indian eri silkworm, Samia cynthia ricini, revealed by ISSR markers. J Insect Sci 6:1–11. Available online at: http://insectscience.org/6.30/

    Article  PubMed  CAS  Google Scholar 

  • Wang H, Azuaje F, Bodenreider O, Dopazo J (2004) Gene expression correlation and gene ontology-based similarity: an assessment of quantitative relationships. In: Proceedings of the 2004 I.E. Symposium on Computational Intelligence in Bioinformatics and Computational Biology (CIBCB ’04), La Jolla, San Diego, California, October 2004, pp 25–31

  • Wentzell AM, Rowe HC, Hansen BG, Ticconi C, Halkier BA, Kliebenstein DJ (2007) Linking metabolic QTLs with network and cis-eQTLs controlling biosynthetic pathways. PLoS Genet 3:1687–1701. doi:10.1371/journal.pgen.0030162

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Zeeberg BR, Kohn KW, Kahn A, Larionov V, Weinstein JN, Reinhold W, Pommier Y (2012) Concordance of gene expression and functional correlation patterns across the NCI-60 cell lines and the cancer genome atlas glioblastoma samples. PLoS One 7(7):e40062

    Article  PubMed Central  PubMed  CAS  Google Scholar 

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Acknowledgments

The authors express their sincere thanks to the anonymous reviewers for their critical reading and wish to thank Central Silk Board, Government of India, Bangalore, for the use of the facilities. The authors acknowledge the financial support received from the Department of Biotechnology (DBT), Government of India, New Delhi, in the form of a research project to ANRP (BT/PR11722/PBD/19/197/2008 dated 11/6/2009). AJ and SS were supported by DBT junior research fellowships.

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Authors and Affiliations

  1. Proteomics Division, Seribiotech Research Laboratory, Central Silk Board, Carmelaram. P.O., CSB—Kodathi Campus, Bangalore, 560035, Karnataka, India

    Anitha Jayaram, Appukuttan Nair R. Pradeep, Arvind K. Awasthi, Geetha N. Murthy & Guruprasad C. Rao

  2. Genomics Division, Seribiotech Research Laboratory, Central Silk Board, Carmelaram. P.O., CSB—Kodathi Campus, Bangalore, 560035, Karnataka, India

    Kangayam M. Ponnuvel & Sirigineedi Sasibhushan

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  1. Anitha Jayaram
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Correspondence to Appukuttan Nair R. Pradeep.

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Anitha Jayaram and Appukuttan Nair R. Pradeep are equal contributors.

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Supplementary Figure 1

Effect of infection of B. mori larvae by E. bombycis on integument: scatter plot (a) showing a close linear correlation with high significance (R 2 = 0.633; P < 1.93 × 10−16) between the original fold change values and its normalized values of gene expression at different time points after the infection of B. mori larvae. (JPEG 40 kb)

High resolution image (TIFF 161 kb)

Supplementary Figure 2

Effect of infection of B. mori larvae by E. bombycis on integument: fold change in the expression of different host–response genes (a–d) in the integument. Each point represents the mean of two independent analyses performed at five time points (age in hours) after infection. The transcript level was normalized to that of β-actin. Abbreviations of gene names are expanded and given in the text. (JPEG 78 kb)

High resolution image (TIFF 268 kb)

Supplementary Figure 3

Effect of infection of B. mori larvae by E. bombycis on integument: relative expression of caspase 3 gene (mean ± SD) showed low and suboptimal level of expression in control and upregulation in the early and late stages of parasitism (with copyright permission from Springer-Verlag GmbH, Germany, to reuse the data on the relative expression of caspase published in Cell and Tissue Research 2013 May;352(2):371–85; online 2012). (JPEG 43 kb)

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Jayaram, A., Pradeep, A.N.R., Awasthi, A.K. et al. Coregulation of host–response genes in integument: switchover of gene expression correlation pattern and impaired immune responses induced by dipteran parasite infection in the silkworm, Bombyx mori . J Appl Genetics 55, 209–221 (2014). https://doi.org/10.1007/s13353-013-0183-8

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