Advertisement

Immunogenetics

, Volume 68, Issue 9, pp 719–731 | Cite as

Identification of interleukin genes in Pogona vitticeps using a de novo transcriptome assembly from RNA-seq data

  • Alexandra Livernois
  • Kristine Hardy
  • Renae Domaschenz
  • Alexie Papanicolaou
  • Arthur Georges
  • Stephen D Sarre
  • Sudha Rao
  • Tariq Ezaz
  • Janine E Deakin
Original Article

Abstract

Interleukins are a group of cytokines with complex immunomodulatory functions that are important for regulating immunity in vertebrate species. Reptiles and mammals last shared a common ancestor more than 350 million years ago, so it is not surprising that low sequence identity has prevented divergent interleukin genes from being identified in the central bearded dragon lizard, Pogona vitticeps, in its genome assembly. To determine the complete nucleotide sequences of key interleukin genes, we constructed full-length transcripts, using the Trinity platform, from short paired-end read RNA sequences from stimulated spleen cells. De novo transcript reconstruction and analysis allowed us to identify interleukin genes that are missing from the published P. vitticeps assembly. Identification of key cytokines in P. vitticeps will provide insight into the essential molecular mechanisms and evolution of interleukin gene families and allow for characterization of the immune response in a lizard for comparison with mammals.

Keywords

Pogona vitticeps De novo assembly Cytokine Interleukin 

Notes

Acknowledgments

This work was supported by a University of Canberra postdoctoral fellowship (awarded to Tariq Ezaz, Sudha Rao, Stephen Sarre, Janine Deakin, Kris Hardy and Arthur Georges, and supporting Renae Domaschenz and Alexandra Livernois). Tariq Ezaz is supported by an Australian Research Council Future Fellowship (FT110100733).

Compliance with ethical standards

Wild-caught male P. vitticeps (Id #UC<Aus>:111880108089) was euthanized according to ethics approval by the Animal Ethics Committee (reference number CE-13-10) at the University of Canberra.

Supplementary material

251_2016_922_MOESM1_ESM.pdf (750 kb)
Supplemental Fig. 1 (PDF 750 kb)
251_2016_922_MOESM2_ESM.pdf (55 kb)
Supplemental Fig. 2 (PDF 54 kb)
251_2016_922_MOESM3_ESM.pdf (813 kb)
Supplemental Fig. 3 (PDF 813 kb)
251_2016_922_MOESM4_ESM.pdf (502 kb)
Supplemental Fig. 4 (PDF 501 kb)
251_2016_922_MOESM5_ESM.pdf (412 kb)
Supplemental Fig. 5 (PDF 412 kb)
251_2016_922_MOESM6_ESM.pdf (217 kb)
Supplemental Fig. 6 (PDF 216 kb)
251_2016_922_MOESM7_ESM.pdf (181 kb)
Supplemental Fig. 7 (PDF 181 kb)

References

  1. Afifi A, Mohamed ER, El Ridi R (1993) Seasonal conditions determine the manner of rejection in reptiles. J Exp Zool 265:459–468CrossRefGoogle Scholar
  2. Alexander WS (1998) Cytokines in hematopoiesis. Int Rev Immunol 16:651–682CrossRefPubMedGoogle Scholar
  3. Avery S, Rothwell L, Degen WD, Schijns VE, Young J, Kaufman J, Kaiser P (2004) Characterization of the first nonmammalian T2 cytokine gene cluster: the cluster contains functional single-copy genes for IL-3, IL-4, IL-13, and GM-CSF, a gene for IL-5 that appears to be a pseudogene, and a gene encoding another cytokinelike transcript, KK34. J Interf Cytokine Res 24:600–610CrossRefGoogle Scholar
  4. Bao K, Reinhardt RL (2015) The differential expression of IL-4 and IL-13 and its impact on type-2 immunity. Cytokine 75:25–37CrossRefPubMedGoogle Scholar
  5. Baran J, Kowalczyk D, Ozog M, Zembala M (2001) Three-color flow cytometry detection of intracellular cytokines in peripheral blood mononuclear cells: comparative analysis of phorbol myristate acetate-ionomycin and phytohemagglutinin stimulation. Clin Diagn Lab Immunol 8:303–313PubMedPubMedCentralGoogle Scholar
  6. Baxter EW, Mirabella F, Bowers SR, James SR, Bonavita AM, Bertrand E, Strogantsev R, Hawwari A, Bert AG, Gonzalez de Arce A, West AG, Bonifer C, Cockerill PN (2012) The inducible tissue-specific expression of the human IL-3/GM-CSF locus is controlled by a complex array of developmentally regulated enhancers. J Immunol 189:4459–4469CrossRefPubMedPubMedCentralGoogle Scholar
  7. Bazan JF, McKay DB (1992) Unraveling the Structure of IL-2. Science 257:410–413Google Scholar
  8. Beck G (1998) Macrokines: invertebrate cytokine-like molecules? Front Biosci 3:d559–d569CrossRefPubMedGoogle Scholar
  9. Bird S, Secombes CJ (2006) Danio rerio partial mRNA for interkeukin-4 (il-4 gene). GenBank Accession No. AM403245Google Scholar
  10. Bird S, Zou J, Kono T, et al. (2005a) Characterisation and expression analysis of interleukin 2 (IL-2) and IL-21 homologues in the Japanese pufferfish, Fugu rubripes, following their discovery by synteny. Immunogenetics 56:909–923Google Scholar
  11. Bird S, Zou J, Savan R, Kono T, Sakai M, Woo J, Secombes C (2005b) Characterisation and expression analysis of an interleukin 6 homologue in the Japanese pufferfish, Fugu rubripes. Dev Comp Immunol 29:775–789CrossRefPubMedGoogle Scholar
  12. Bird S, Zou J, Wang T, Munday B, Cunningham C, Secombes CJ (2002) Evolution of interleukin-1beta. Cytokine Growth Factor Rev 13:483–502CrossRefPubMedGoogle Scholar
  13. Bolger AM, Lohse M, Usadel B (2014) Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30:2114–2120CrossRefPubMedPubMedCentralGoogle Scholar
  14. Brocker C, Thompson D, Matsumoto A, Nebert DW, Vasiliou V (2010) Evolutionary divergence and functions of the human interleukin (IL) gene family. Hum Genomics 5:30–55CrossRefPubMedPubMedCentralGoogle Scholar
  15. Caput D, Beutler B, Hartog K, Thayer R, Brown-Shimer S, Cerami A (1986) Identification of a common nucleotide sequence in the 3'-untranslated region of mRNA molecules specifying inflammatory mediators. Proc Natl Acad Sci U S A 83:1670–1674CrossRefPubMedPubMedCentralGoogle Scholar
  16. Chomarat P, Banchereau J (1998) Interleukin-4 and interleukin-13: their similarities and discrepancies. Int Rev Immunol 17:1–52CrossRefPubMedGoogle Scholar
  17. Cockerill PN (2004) Mechanisms of transcriptional regulation of the human IL-3/GM-CSF locus by inducible tissue-specific promoters and enhancers. Crit Rev Immunol 24:385–408CrossRefPubMedGoogle Scholar
  18. Commins SP, Borish L, Steinke JW (2010) Immunologic messenger molecules: cytokines, interferons, and chemokines. J Allergy Clin Immunol 125:S53–S72CrossRefPubMedGoogle Scholar
  19. Crawford JE, Guelbeogo WM, Sanou A, Traoré A, Vernick KD, Sagnon N, Lazzaro BP (2010) De novo transcriptome sequencing in Anopheles funestus using Illumina RNA-seq technology. PLoS One 5:e14202CrossRefPubMedPubMedCentralGoogle Scholar
  20. Diaz-Rosales P, Bird S, Wang TH, Fujiki K, Davidson WS, Zou J, Secombes CJ (2009) Rainbow trout interleukin-2: cloning, expression and bioactivity analysis. Fish Shellfish Immunol 27:414–422CrossRefPubMedGoogle Scholar
  21. Dijkstra JM, Takizawa F, Fischer U, Friedrich M, Soto-Lampe V, Lefevre C, Lenk M, Karger A, Matsui T, Hashimoto K (2014) Identification of a gene for an ancient cytokine, interleukin 15-like, in mammals; interleukins 2 and 15 co-evolved with this third family member, all sharing binding motifs for IL-15Ralpha. Immunogenetics 66:93–103CrossRefPubMedGoogle Scholar
  22. Dinarello CA, Wolff SM (1993) The role of interleukin-1 in disease. N Engl J Med 328:106–113CrossRefPubMedGoogle Scholar
  23. Drozdetskiy A, Cole C, Procter J, Barton GJ (2015) JPred4: a protein secondary structure prediction server. Nucleic Acids Res 43:W389–W394CrossRefPubMedPubMedCentralGoogle Scholar
  24. Dunn E, Sims JE, Nicklin MJ, O'Neill LA (2001) Annotating genes with potential roles in the immune system: six new members of the IL-1 family. Trends Immunol 22:533–536CrossRefPubMedGoogle Scholar
  25. Eberl G, Colonna M, Di Santo JP, McKenzie AN (2015) Innate lymphoid cells. Innate lymphoid cells: a new paradigm in immunology. Science 348:aaa6566CrossRefPubMedGoogle Scholar
  26. Eisenberg SP, Brewer MT, Verderber E, Heimdal P, Brandhuber BJ, Thompson RC (1991) Interleukin-1 receptor antagonist is a member of the interleukin-1 gene family—evolution of a cytokine control mechanism. Proc Natl Acad Sci U S A 88:5232–5236CrossRefPubMedPubMedCentralGoogle Scholar
  27. Eisenmesser EZ, Horita DA, Altieri AS, Byrd RA (2001) Solution structure of interleukin-13 and insights into receptor engagement. J Mol Biol 310:231–241CrossRefPubMedGoogle Scholar
  28. El Ridi R, Badir N, El Rouby S (1981) Effect of seasonal variations on the immune system of the snake, Psammophis schokari. J Exp Zool 216:357–365CrossRefGoogle Scholar
  29. Farag MA, El Ridi R (1985) Mixed leucocyte reaction (MLR) in the snake Psammophis sibilans. Immunology 55:173–181PubMedPubMedCentralGoogle Scholar
  30. Farag MA, El Ridi R (1990) Functional markers of the major histocompatibility gene complex of snakes. Eur J Immunol 20:2029–2033CrossRefPubMedGoogle Scholar
  31. Frazer KA, Ueda Y, Zhu Y, Gifford VR, Garofalo MR, Mohandas N, Martin CH, Palazzolo MJ, Cheng JF, Rubin EM (1997) Computational and biological analysis of 680 kb of DNA sequence from the human 5q31 cytokine gene cluster region. Genome Res 7:495–512PubMedGoogle Scholar
  32. Frolova EI, Dolganov GM, Mazo IA, Smirnov DV, Copeland P, Stewart C, O'Brien SJ, Dean M (1991) Linkage mapping of the human CSF2 and IL3 genes. Proc Natl Acad Sci U S A 88:4821–4824CrossRefPubMedPubMedCentralGoogle Scholar
  33. Gaffen SL, Liu KD (2004) Overview of interleukin-2 function, production and clinical applications. Cytokine 28:109–123Google Scholar
  34. Georges A, Li Q, Lian J, O'Meally D, Deakin J, Wang Z, Zhang P, Fujita M, Patel HR, Holleley CE, Zhou Y, Zhang X, Matsurbara K, Waters P, Graves JAM, Sarre SD, Zhang G (2015) High-coverage sequencing and annotated assembly of the genome of the Australian dragon lizard Pogona vitticeps. Gigascience 4:45CrossRefPubMedPubMedCentralGoogle Scholar
  35. Gibson MS, Kaiser P, Fife M (2014) The chicken IL-1 family: evolution in the context of the studied vertebrate lineage. Immunogenetics 66:427–438CrossRefPubMedPubMedCentralGoogle Scholar
  36. Goujon M, McWilliam H, Li W, Valentin F, Squizzato S, Paern J, Lopez R (2010) A new bioinformatics analysis tools framework at EMBL-EBI. Nucleic Acids Res 38:W695–W699CrossRefPubMedPubMedCentralGoogle Scholar
  37. Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I, Adiconis X, Fan L, Raychowdhury R, Zeng Q, Chen Z, Mauceli E, Hacohen N, Gnirke A, Rhind N, di Palma F, Birren BW, Nusbaum C, Lindblad-Toh K, Friedman N, Regev A (2011) Full-length transcriptome assembly from RNA-seq data without a reference genome. Nat Biotechnol 29:644–652CrossRefPubMedPubMedCentralGoogle Scholar
  38. Haas BJ, Papanicolaou A, Yassour M, Grabherr M, Blood PD, Bowden J, Couger MB, Eccles D, Li B, Lieber M, Macmanes MD, Ott M, Orvis J, Pochet N, Strozzi F, Weeks N, Westerman R, William T, Dewey CN, Henschel R, Leduc RD, Friedman N, Regev A (2013) De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis. Nat Protoc 8:1494–1512CrossRefPubMedGoogle Scholar
  39. Harrison GA, Wedlock DN (2000) Marsupial cytokines. Structure, function and evolution. Dev Comp Immunol 24:473–484 http://www.cbs.dtu.dk/services/NetNGlyc/N
  40. Hsu E (1998) Mutation, selection, and memory in B lymphocytes of exothermic vertebrates. Immunol Rev 162:25–36Google Scholar
  41. Huising MO, Kruiswijk CP, Flik G (2006) Phylogeny and evolution of class-I helical cytokines. J Endocrinol 189:1–25CrossRefPubMedGoogle Scholar
  42. Hussein MF, Badir N, el-Ridi R, Akef M (1978) Effect of seasonal variation on lymphoid tissues of the lizards, Mabuya quinquetaeniata Licht. and Uromastyx aegyptia Forsk. Dev Comp Immunol 2:469–478CrossRefPubMedGoogle Scholar
  43. Hussein MF, Badir N, el-Ridi R, el-Deeb S (1979) Effect of seasonal variation on immune system of the lizard Scincus scincus. J Exp Zool 209:91–96CrossRefGoogle Scholar
  44. Igawa D, Sakai M, Savan R (2006) An unexpected discovery of two interferon gamma-like genes along with interleukin (IL)-22 and -26 from teleost: IL-22 and -26 genes have been described for the first time outside mammals. Mol Immunol 43:999–1009CrossRefPubMedGoogle Scholar
  45. Kotenko SV (2002) The family of IL-10-related cytokines and their receptors: related, but to what extent? Cytokine Growth Factor Rev 13:223–240CrossRefPubMedGoogle Scholar
  46. Kumagai N, Benedict SH, Mills GB, Gelfand EW (1987) Requirements for the simultaneous presence of phorbol esters and calcium ionophores in the expression of human T lymphocyte proliferation-related genes. J Immunol 139:1393–1399PubMedGoogle Scholar
  47. Langmead B, Salzberg SL (2012) Fast gapped-read alignment with Bowtie 2. Nat Methods 9:357–359CrossRefPubMedPubMedCentralGoogle Scholar
  48. Li JH, Shao JZ, Xiang LX, Wen Y (2007) Cloning, characterization and expression analysis of pufferfish interleukin-4 cDNA: the first evidence of Th2-type cytokine in fish. Mol Immunol 44:2078–2086CrossRefPubMedGoogle Scholar
  49. Milburn MV, Hassell AM, Lambert MH, Jordan SR, Proudfoot AE, Graber P, Wells TN (1993) A novel dimer configuration revealed by the crystal structure at 2.4 A resolution of human interleukin-5. Nature 363:172–176CrossRefPubMedGoogle Scholar
  50. Minty A, Chalon P, Derocq JM, Dumont X, Guillemot JC, Kaghad M, Labit C, Leplatois P, Liauzun P, Miloux B, et al. (1993) Interleukin-13 is a new human lymphokine regulating inflammatory and immune responses. Nature 362:248–250CrossRefPubMedGoogle Scholar
  51. Mirabella F, Baxter EW, Boissinot M, James SR, Cockerill PN (2010) The human IL-3/granulocyte-macrophage colony-stimulating factor locus is epigenetically silent in immature thymocytes and is progressively activated during T cell development. J Immunol 184:3043–3054CrossRefPubMedGoogle Scholar
  52. Morgan DA, Ruscetti FW, Gallo R (1976) Selective in vitro growth of T lymphocytes from normal human bone marrows. Science 193:1007–1008CrossRefPubMedGoogle Scholar
  53. Moy FJ, Diblasio E, Wilhelm J, Powers R (2001) Solution structure of human IL-13 and implication for receptor binding. J Mol Biol 310:219–230CrossRefPubMedGoogle Scholar
  54. Mulero JJ, Nelken ST, Ford JE (2000) Organization of the human interleukin-1 receptor antagonist gene IL1HY1. Immunogenetics 51:425–428CrossRefPubMedGoogle Scholar
  55. Munoz FJ, De la Fuente M (2001) The effect of the seasonal cycle on the splenic leukocyte functions in the turtle Mauremys caspica. Physiol Biochem Zool 74:660–667CrossRefPubMedGoogle Scholar
  56. Nicola NA (1989) Hemopoietic cell growth factors and their receptors. Annu Rev Biochem 58:45–77CrossRefPubMedGoogle Scholar
  57. Ogryzko NV, Renshaw SA, Wilson HL (2014) The IL-1 family in fish: swimming through the muddy waters of inflammasome evolution. Dev Comp Immunol 46:53–62CrossRefPubMedGoogle Scholar
  58. Ohtani M, Hayashi N, Hashimoto K, Nakanishi T, Dijkstra JM (2008) Comprehensive clarification of two paralogous interleukin 4/13 loci in teleost fish. Immunogenetics 60:383–397CrossRefPubMedGoogle Scholar
  59. Olsen I, Sollid LM (2013) Pitfalls in determining the cytokine profile of human T cells. J Immunol Methods 390:106–112CrossRefPubMedGoogle Scholar
  60. Petersen TN, Brunak S, von Heijne G, Nielsen H (2011) SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods 8:785–786CrossRefPubMedGoogle Scholar
  61. Rao S, Gerondakis S, Woltring D, Shannon MF (2003) c-Rel is required for chromatin remodeling across the IL-2 gene promoter. J Immunol 170:3724–3731CrossRefPubMedGoogle Scholar
  62. Rao S, Procko E, Shannon MF (2001) Chromatin remodeling, measured by a novel real-time polymerase chain reaction assay, across the proximal promoter region of the IL-2 gene. J Immunol 167:4494–4503CrossRefPubMedGoogle Scholar
  63. Roberts A, Pachter L (2013) Streaming fragment assignment for real-time analysis of sequencing experiments. Nat Methods 10:71–73CrossRefPubMedGoogle Scholar
  64. Robinson JT, Thorvaldsdottir H, Winckler W, Guttman M, Lander ES, Getz G, Mesirov JP (2011) Integrative genomics viewer. Nat Biotechnol 29:24–26CrossRefPubMedPubMedCentralGoogle Scholar
  65. Robinson MD, McCarthy DJ, Smyth GK (2010) edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26:139–140CrossRefPubMedGoogle Scholar
  66. Secombes CJ, Wang T, Bird S (2011) The interleukins of fish. Dev Comp Immunol 35:1336–1345CrossRefPubMedGoogle Scholar
  67. Sievers F, Wilm A, Dineen D, Gibson T, Karplus K, Li W, Lopez R, McWilliam H, Remmert M, Söding J, Thompson J, Higgins D (2011) Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol 7Google Scholar
  68. Takahashi A, de Andres MC, Hashimoto K, Itoi E, Oreffo RO (2015) Epigenetic regulation of interleukin-8, an inflammatory chemokine, in osteoarthritis. Osteoarthr Cartil 23:1946–1954CrossRefPubMedPubMedCentralGoogle Scholar
  69. Thorvaldsdottir H, Robinson JT, Mesirov JP (2013) Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration. Brief Bioinform 14:178–192CrossRefPubMedGoogle Scholar
  70. Wang T, Diaz-Rosales P, Martin SA, Secombes CJ (2010) Cloning of a novel interleukin (IL)-20-like gene in rainbow trout Oncorhynchus mykiss gives an insight into the evolution of the IL-10 family. Dev Comp Immunol 34:158–167CrossRefPubMedGoogle Scholar
  71. Wang T, Johansson P, Abos B, Holt A, Tafalla C, Jiang Y, Wang A, Xu Q, Qi Z, Huang W, Costa MM, Diaz-Rosales P, Holland JW, Secombes CJ (2016) First in-depth analysis of the novel Th2-type cytokines in salmonid fish reveals distinct patterns of expression and modulation but overlapping bioactivities. Oncotarget 7:10917–10946PubMedPubMedCentralGoogle Scholar
  72. Wang T, Secombes CJ (2015) The evolution of IL-4 and IL-13 and their receptor subunits. Cytokine 75:8–13CrossRefPubMedGoogle Scholar
  73. Wells TN, Graber P, Proudfoot AE, Arod CY, Jordan SR, Lambert MH, Hassel AM, Milburn MV (1994) The three-dimensional structure of human interleukin-5 at 2.4-angstroms resolution: implication for the structures of other cytokines. Ann N Y Acad Sci 725:118–127CrossRefPubMedGoogle Scholar
  74. Wenchao A, Haishan L, Song N, Lei L, Huiming C (2013) Optimal method to stimulate cytokine production and its use in immunotoxicity assessment. Int J Environ Res Public Health 10:3834–3842CrossRefGoogle Scholar
  75. Wong ES, Young LJ, Papenfuss AT, Belov K (2006) In silico identification of opossum cytokine genes suggests the complexity of the marsupial immune system rivals that of eutherian mammals. Immunome Res 2:4CrossRefPubMedPubMedCentralGoogle Scholar
  76. Wu TD, Watanabe CK (2005) GMAP: a genomic mapping and alignment program for mRNA and EST sequences. Bioinformatics 21:1859–1875CrossRefPubMedGoogle Scholar
  77. Wynn TA (2003) IL-13 effector functions. Annu Rev Immunol 21:425–456CrossRefPubMedGoogle Scholar
  78. Yamaguchi T, Takizawa F, Fischer U, Dijkstra JM (2015) Along the axis between type 1 and type 2 immunity; principles conserved in evolution from fish to mammals. Biology (Basel) 4:814–859Google Scholar
  79. Yang YC, Ciarletta AB, Temple PA, Chung MP, Kovacic S, Witek-Giannotti JS, Leary AC, Kriz R, Donahue RE, Wong GG, et al. (1986) Human IL-3 (multi-CSF): identification by expression cloning of a novel hematopoietic growth factor related to murine IL-3. Cell 47:3–10CrossRefPubMedGoogle Scholar
  80. Young PR, Sylvester D (1989) Cloning of rabbit interleukin-1 beta: differential evolution of IL-1 alpha and IL-1 beta proteins. Protein Eng 2:545–551CrossRefPubMedGoogle Scholar
  81. Zapata AG, Varas A, Torroba M (1992) Seasonal variations in the immune system of lower vertebrates. Immunol Today 13:142–147CrossRefPubMedGoogle Scholar
  82. Zelus D, Robinson-Rechavi M, Delacre M, Auriault C, Laudet V (2000) Fast evolution of interleukin-2 in mammals and positive selection in ruminants. J Mol Evol 51:234–244PubMedGoogle Scholar
  83. Zimmerman LM, Vogel LA, Bowden RM (2010) Understanding the vertebrate immune system: insights from the reptilian perspective. J Exp Biol 213:661–671Google Scholar
  84. Zurawski G, de Vries JE (1994) Interleukin 13 elicits a subset of the activities of its close relative interleukin 4. Stem Cells 12:169–174CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Alexandra Livernois
    • 1
  • Kristine Hardy
    • 2
  • Renae Domaschenz
    • 3
  • Alexie Papanicolaou
    • 4
  • Arthur Georges
    • 1
  • Stephen D Sarre
    • 1
  • Sudha Rao
    • 2
  • Tariq Ezaz
    • 1
  • Janine E Deakin
    • 1
  1. 1.Institute for Applied EcologyUniversity of CanberraCanberraAustralia
  2. 2.Discipline of Biomedical Sciences, Faculty of Education, Science, Technology and MathematicsUniversity of CanberraCanberraAustralia
  3. 3.John Curtin School of Medical ResearchAustralian National UniversityCanberraAustralia
  4. 4.The Hawkesbury Institute for the EnvironmentUniversity of Western SydneyPenrithAustralia

Personalised recommendations