Hostname: page-component-586b7cd67f-r5fsc Total loading time: 0 Render date: 2024-11-24T04:36:16.912Z Has data issue: false hasContentIssue false
  • English
  • Français

Fluorescent protein expression in temperature tolerant and susceptible reef-building corals

Published online by Cambridge University Press:  02 March 2021

Exequiel Gabriel S. Dizon Open the ORCID record for Exequiel Gabriel S. Dizon [Opens in a new window] ,
Jeric P. Da-Anoy Open the ORCID record for Jeric P. Da-Anoy [Opens in a new window] ,
Melissa S. Roth Open the ORCID record for Melissa S. Roth [Opens in a new window]  and
Cecilia Conaco Open the ORCID record for Cecilia Conaco [Opens in a new window]
Exequiel Gabriel S. Dizon
Affiliation:
Marine Science Institute, College of Science, University of the Philippines, Diliman, Quezon City, 1101, Philippines
Jeric P. Da-Anoy
Affiliation:
Marine Science Institute, College of Science, University of the Philippines, Diliman, Quezon City, 1101, Philippines
Melissa S. Roth
Affiliation:
Department of Plant and Microbial Biology, University of California, BerkeleyCA, 94720-3102, USA
Cecilia Conaco*
Affiliation:
Marine Science Institute, College of Science, University of the Philippines, Diliman, Quezon City, 1101, Philippines
*
Author for correspondence: Cecilia Conaco, E-mail: [email protected]
Get access Rights & Permissions [Opens in a new window]

Abstract

Fluorescent proteins (FPs) are reported to play an important role as photoprotectants and antioxidants in corals subjected to stressful conditions. Identifying the various FP genes expressed and FP gene expression patterns under stress in diverse coral species can provide insight into FP function. In this study, we identified 16 putative FP homologues from the transcriptomes of corals with varying susceptibility to elevated temperature, including Acropora digitifera, Favites colemani, Montipora digitata and Seriatopora caliendrum. Each coral expressed a different complement of FP transcripts, which were predicted to have distinct spectral properties. The most diverse and abundant repertoire of FP transcripts, including at least 6 green FPs, were expressed in the temperature-tolerant coral, F. colemani. In comparison, the other corals expressed fewer FP types. Specific FP transcripts exhibited variable expression profiles in coral fragments subjected to 32 ± 1 °C (treatment) or 28 ± 1 °C (control) for up to 72 h, suggesting that distinct FPs may have different roles. Further studies on the expression of the proteins encoded by these FP transcripts, their fluorescence activity, tissue localization, and possible antioxidant properties, are needed to reveal their contribution to thermal stress tolerance in certain species of corals.

Keywords

Bleaching tolerancefluorescent proteinsthermal stresstranscriptome
Type
Research Article
Information
Journal of the Marine Biological Association of the United Kingdom , Volume 101 , Issue 1 , February 2021 , pp. 71 - 80
Copyright
Copyright © The Author(s), 2021. Published by Cambridge University Press on behalf of The Marine Biological Association of the United Kingdom

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

Aihara, Y, Maruyama, S, Baird, AH, Iguchi, A, Takahashi, S and Minagawa, J (2019) Green fluorescence from cnidarian hosts attracts symbiotic algae. Proceedings of the National Academy of Sciences USA 116, 2118–2123.CrossRefGoogle ScholarPubMed
Alieva, NO, Konzen, KA, Field, SF, Meleshkevitch, EA, Hunt, ME, Beltran-Ramirez, V, Miller, DJ, Wiedenmann, J, Salih, A and Matz, MV (2008) Diversity and evolution of coral fluorescent proteins. PLoS ONE 3, e2680.CrossRefGoogle ScholarPubMed
Baird, AH, Bhagooli, R, Ralph, PJ and Takahashi, S (2009) Coral bleaching: the role of the host. Trends in Ecology and Evolution 24, 16–20.CrossRefGoogle ScholarPubMed
Barondeau, DP, Kassmann, CJ, Tainer, JA and Getzoff, ED (2006) Understanding GFP posttranslational chemistry: structures of designed variants that achieve backbone fragmentation, hydrolysis, and decarboxylation. Journal of the American Chemical Society 128, 4685–4693.CrossRefGoogle ScholarPubMed
Barshis, DJ, Ladner, JT, Oliver, TA, Seneca, FO, Traylor-Knowles, N and Palumbi, SR (2013) Genomic basis for coral resilience to climate change. Proceedings of the National Academy of Sciences USA 110, 1387–1392.CrossRefGoogle ScholarPubMed
Bay, RA and Palumbi, SR (2014) Multilocus adaptation associated with heat resistance in reef-building corals. Current Biology 24, 2952–2956.CrossRefGoogle ScholarPubMed
Bellantuono, AJ, Hoegh-Guldberg, O and Rodriguez-Lanetty, M (2011) Resistance to thermal stress in corals without changes in symbiont composition. Proceedings of the Royal Society B: Biological Sciences 279, 1100–1107.CrossRefGoogle ScholarPubMed
Bhattacharya, D, Agrawal, S, Aranda, M, Baumgarten, S, Belcaid, M, Drake, JL, Erwin, D, Foret, S, Gates, RD, Gruber, DF, Kamel, B, Lesser, MP, Levy, O, Liew, YJ, MacManes, M, Mass, T, Medina, M, Mehr, S, Meyer, E, Price, DC, Putnam, HM, Qiu, H, Shinzato, C, Shoguchi, E, Stokes, AJ, Tambutté, S, Tchernov, D, Voolstra, CR, Wagner, N, Walker, CW, Weber, AP, Weis, V, Zelzion, E, Zoccola, D and Falkowski, PG (2016) Comparative genomics explains the evolutionary success of reef-forming corals. eLife 5, e13288.CrossRefGoogle ScholarPubMed
Bou-Abdallah, F, Chasteen, ND and Lesser, MP (2006) Quenching of superoxide radicals by green fluorescent protein. Biochimica et Biophysica Acta 1760, 1690–1695.CrossRefGoogle ScholarPubMed
Castresana, J (2000) Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Molecular Biology and Evolution 17, 540–552.CrossRefGoogle ScholarPubMed
Chudakov, DM, Matz, MV, Lukyanov, S and Lukyanov, KA (2010) Fluorescent proteins and their applications in imaging living cells and tissues. Physiological Reviews 90, 1103–1163.CrossRefGoogle ScholarPubMed
Cziesielski, MJ, Liew, YJ, Cui, G, Schmidt-Roach, S, Campana, S, Marondedze, C and Aranda, M (2018) Multi-omics analysis of thermal stress response in a zooxanthellate cnidarian reveals the importance of associating with thermotolerant symbionts. Proceedings of the Royal Society B: Biological Sciences 285, 20172654.CrossRefGoogle Scholar
Da-Anoy, JP, Cabaitan, PC and Conaco, CG (2019) Species variability in the response to elevated temperature of select corals in northwestern Philippines. Journal of the Marine Biological Association of the United Kingdom 99, 1273–1279.CrossRefGoogle Scholar
Desalvo, MK, Voolstra, CR, Sunagawa, S, Schwarz, JA, Stillman, JH, Coffroth, MA, Szmant, AM and Medina, MM (2008) Differential gene expression during thermal stress and bleaching in the Caribbean coral Montastraea faveolata. Molecular Ecology 17, 3952–3971.CrossRefGoogle ScholarPubMed
Deschaseaux, ESM, Beltran, VH, Jones, GB, Deseo, MA, Swan, HB, Harrison, PL and Eyre, BD (2014) Comparative response of DMS and DMSP concentrations in Symbiodinium Clades C1 and D1 under thermal stress. Journal of Experimental Marine Biology and Ecology 459, 181–189.CrossRefGoogle Scholar
Dove, SG, Hoegh-Guldberg, O and Ranganathan, S (2001) Major colour patterns of reef-building corals are due to a family of GFP-like proteins. Coral Reefs 19, 197–204.CrossRefGoogle Scholar
Eddy, SR (1998) Profile hidden Markov models. Bioinformatics 14, 755–763.CrossRefGoogle ScholarPubMed
El-Gebali, S, Mistry, J, Bateman, A, Eddy, SR, Luciani, A, Potter, SC, Qureshi, M, Richardson, LJ, Salazar, GA, Smart, A, Sonnhammer, EL, Hirsh, L, Paladin, L, Piovesan, D, Tosatto, SCE and Finn, RD (2019) The Pfam protein families database in 2019. Nucleic Acids Research 47, D427–D432.CrossRefGoogle ScholarPubMed
Erez, J, Reynaud, S, Silverman, J, Schneider, K and Allemand, D (2011) Coral calcification under ocean acidification and global change. In Dubinsky, Z and Stambler, N (eds), Coral Reefs: An Ecosystem in Transition. Berlin: Springer, pp. 151–173.CrossRefGoogle Scholar
Follenius-Wund, A, Bourotte, M, Schmitt, M, Iyice, F, Lami, H, Bourguignon, JJ, Haiech, J and Pigault, C (2003) Fluorescent derivatives of the GFP chromophore give a new insight into the GFP fluorescence process. Biophysical Journal 85, 1839–1850.CrossRefGoogle ScholarPubMed
Fu, JL, Kanno, T, Liang, S-C, Matzke, AJM and Matzke, M (2015) GFP loss-of-function mutations in Arabidopsis thaliana. G3: Genes, Genomics, Genetics 5, 1849–1855.CrossRefGoogle ScholarPubMed
Gajigan, AP and Conaco, C (2017) A microRNA regulates the response of corals to thermal stress. Molecular Ecology 26, 3472–3483.CrossRefGoogle ScholarPubMed
Gardner, SG, Raina, J, Ralph, PJ and Petrou, K (2017) Reactive oxygen species (ROS) and dimethylated sulphur compounds in coral explants under acute thermal stress. Journal of Experimental Biology 220, 1787–1791.Google ScholarPubMed
Gittins, JR, D'Angelo, C, Oswald, F, Edwards, RJ and Wiedenmann, J (2015) Fluorescent protein-mediated colour polymorphism in reef corals: multicopy genes extend the adaptation/ acclimatization potential to variable light environments. Molecular Ecology 24, 453–465.CrossRefGoogle ScholarPubMed
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 and Regev, A (2011) Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nature Biotechnology 29, 644–652.CrossRefGoogle ScholarPubMed
Gruber, DF, DeSalle, R, Lienau, EK, Tchernov, D, Pieribone, VA and Kao, HT (2009) Novel internal regions of fluorescent proteins undergo divergent evolutionary patterns. Molecular Biology and Evolution 26, 2841–2848.CrossRefGoogle ScholarPubMed
Hall, TA (1999) Bioedit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series 41, 95–98.Google Scholar
Heim, R, Prasher, DC and Tsien, RY (1994). Wavelength mutations and posttranslational autoxidation of green fluorescent protein. Proceedings of the National Academy of Sciences USA 91: 12501–12504.CrossRefGoogle ScholarPubMed
Hoegh-Guldberg, O and Bruno, JF (2010) The impact of climate change on the world's marine ecosystems. Science (New York, N.Y.) 328, 1523–1528.CrossRefGoogle ScholarPubMed
Huelsenbeck, JP and Ronquist, F (2001) MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 17, 754–755.CrossRefGoogle ScholarPubMed
Hughes, TP, Baird, AH, Bellwood, DR, Card, M, Connolly, SR, Folke, C, Grosberg, R, Hoegh-Guldberg, O, Jackson, JBC, Kleypas, J, Lough, JM, Marshall, P, Nyström, M, Palumbi, SR, Pandolfi, JM, Rosen, B and Roughgarden, J (2003) Climate change, human impacts, and the resilience of coral reefs. Science (New York, N.Y.) 301, 929–933.CrossRefGoogle ScholarPubMed
Hume, B, D'Angelo, C, Burt, J, Baker, AC, Riegl, B and Wiedenmann, J (2013) Corals from the Persian/Arabian Gulf as models for thermotolerant reef-builders: prevalence of clade C3 Symbiodinium, host fluorescence and ex situ temperature tolerance. Marine Pollution Bulletin 72, 313–322.CrossRefGoogle ScholarPubMed
Jensen, LJ, Julien, P, Kuhn, M, von Mering, C, Muller, J, Doerks, T and Bork, P (2008). eggNOG: automated construction and annotation of orthologous groups of genes. Nucleic Acids Research 36(Database issue), D250–D254.CrossRefGoogle ScholarPubMed
Kenkel, CD, Traylor, MR, Wiedenmann, J, Salih, A and Matz, MV (2011) Fluorescence of coral larvae predicts their settlement response to crustose coralline algae and reflects stress. Proceedings of the Royal Society B: Biological Sciences 278, 2691–2697.CrossRefGoogle ScholarPubMed
Klueter, A, Loh, W, Hoegh-Guldberg, O and Dove, S (2006) Physiological and genetic properties of two fluorescent colour morphs of the coral Montipora digitata. Symbiosis 42, 123–134.Google Scholar
Kumar, S, Stecher, G and Tamura, K (2016) MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Molecular Biology and Evolution 33, 1870–1874.CrossRefGoogle ScholarPubMed
Langmead, B, Trapnell, C, Pop, M and Salzberg, SL (2009) Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biology 10, R25.CrossRefGoogle ScholarPubMed
Lazenby, D (2000) Cygwin: for Windows NT. Linux Journal 75es, art. 14.Google Scholar
Lesser, MP (2006) Oxidative stress in marine environments: biochemistry and physiological ecology. Annual Review of Physiology 68, 253–257.CrossRefGoogle ScholarPubMed
Li, B and Dewey, CN (2011) RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics 12, 323.CrossRefGoogle ScholarPubMed
Liew, YJ, Aranda, M and Voolstra, CR (2016) Reefgenomics.Org – a repository for marine genomics data. Database (Oxford) 2016, baw152.CrossRefGoogle ScholarPubMed
Lyndby, NH, Kühl, M and Wangpraseurt, D (2016) Heat generation and light scattering of green fluorescent protein-like pigments in coral tissue. Scientific Reports 6, 26599.CrossRefGoogle ScholarPubMed
Mann, HB and Whitney, DR (1947) On a test of whether one of two random variables is stochastically larger than the other. Annals of Mathematical Statistics 1, 50–60.CrossRefGoogle Scholar
Marshall, PA and Baird, AH (2000) Bleaching of corals on the Great Barrier Reef: differential susceptibilities among taxa. Coral Reefs 19, 155–163.CrossRefGoogle Scholar
Matz, MV, Labas, YA and Ugalde, J (2006) Evolution of function and color in GFP-like proteins. Methods of Biochemical Analysis 47, 139–161.Google ScholarPubMed
Mayfield, AB, Chan, PH, Putnam, HM, Chen, CS and Fan, TY (2012) The effects of a variable temperature regime on the physiology of the reef-building coral Seriatopora hystrix: results from a laboratory-based reciprocal transplant. Journal of Experimental Biology 215, 4183–4195.Google ScholarPubMed
Mazel, CH, Lesser, MP, Gorbunov, MY, Barry, TM, Farrell, JH, Wyman, KD and Falkowski, PG (2003) Green-fluorescent proteins in Caribbean corals. Limnology and Oceanography 48, 402–411.CrossRefGoogle Scholar
Ormö, M, Cubitt, AB, Kallio, K, Gross, LA, Tsien, RY and Remington, SJ (1996) Crystal structure of the Aequorea victoria green fluorescent protein. Science (New York, N.Y.) 273, 1392–1395.CrossRefGoogle ScholarPubMed
Palmer, CV, Modi, CK and Mydlarz, LD (2009) Coral fluorescent proteins as antioxidants. PLoS ONE 4, e7298.CrossRefGoogle ScholarPubMed
Parkinson, JE, Banaszak, AT, LaJeunesse, TC and Baums, IB (2015) Intraspecific diversity among partners drives functional variation in coral symbioses. Scientific Reports 5, 15667.CrossRefGoogle ScholarPubMed
Pinzon, JH, Kamel, B, Burge, CA, Harvell, CD, Medina, M, Weil, E and Mydlarz, LD (2015) Whole transcriptome analysis reveals changes in expression of immune-related genes during and after bleaching in a reef-building coral. Royal Society Open Science 2, 140214.CrossRefGoogle Scholar
R Core Team (2013) R: A Language and Environment for Statistical Computing. Vienna: R Foundation for Statistical Computing. Available at http://www.R-project.org/.Google Scholar
Robinson, MD, McCarthy, DJ and Smyth, GK (2010) Edger: a bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics (Oxford, England) 26, 139–140.CrossRefGoogle ScholarPubMed
Rodriguez-Lanetty, M, Phillips, WS and Weis, VM (2006) Transcriptome analysis of a cnidarian – dinoflagellate mutualism reveals complex modulation of host gene expression. BMC Genomics 7, 23.CrossRefGoogle ScholarPubMed
Roth, MS and Deheyn, DD (2013) Effects of cold stress and heat stress on coral fluorescence in reef-building corals. Scientific Reports 3, 1421.CrossRefGoogle ScholarPubMed
Roth, MS, Latz, MI, Goericke, R and Deheyn, DD (2010) Green fluorescent protein regulation in the coral Acropora yongei during photoacclimation. Journal of Experimental Biology 213, 3644–3655.CrossRefGoogle ScholarPubMed
Roth, MS, Fan, TY and Deheyn, DD (2013) Life history changes in coral fluorescence and the effects of light intensity on larval physiology and settlement in Seriatopora hystrix. PLoS ONE 8, e59476.CrossRefGoogle ScholarPubMed
Salih, A, Larkum, A, Cox, G, Kuhl, M and Hoegh-Guldberg, O (1998) Photoprotection of symbiotic dinoflagellates by fluorescent pigments in reef corals. In Greenwood, JG and Hall, NJ (eds), Proceedings of the Australian Coral Reef Society 75th Anniversary Conference, Heron Island, October 1997. Brisbane: School of Marine Science, The University of Queensland, pp. 217–230.Google Scholar
Salih, A, Larkum, A, Cox, G, Kühl, M and Hoegh-Guldberg, O (2000) Fluorescent pigments in corals are photoprotective. Nature 408, 850–853.CrossRefGoogle ScholarPubMed
Salih, A, Cox, G, Szymczak, R, Coles, SL, Baird, A, Dunstan, A, Mills, J and Larkum, AW (2006) The role of host-based color and fluorescent pigments in photoprotection and in reducing bleaching stress in corals. In Proceedings of the 10th International Coral Reef Symposium, Okinawa, Japan, 28 June – 2 July 2004, pp. 746–756.Google Scholar
Schlichter, D, Fricke, HW and Weber, W (1986) Light harvesting by wavelength transformation in a symbiotic coral of the Red Sea twilight zone. Marine Biology 91, 403–407.CrossRefGoogle Scholar
Seneca, FO, Foret, S, Ball, EE, Smith-Keune, C, Miller, DJ and van Oppen, MJH (2009) Patterns of gene expression in a scleractinian coral undergoing natural bleaching. Marine Biotechnology 12, 594–604.CrossRefGoogle Scholar
Shagin, DA, Barsova, EV, Yanushevich, YG, Fradkov, AF, Lukyanov, KA, Labas, YA, Semenova, TN, Ugalde, JA, Meyers, A, Nunez, JM, Widder, EA, Lukyanov, SA and Matz, MV (2004) GFP-like proteins as ubiquitous metazoan superfamily: evolution of functional features and structural complexity. Molecular Biology and Evolution 21, 841–850.CrossRefGoogle ScholarPubMed
Shimomura, O (2005) The discovery of aequorin and green fluorescent protein. Journal of Microscopy 217, 3–15.CrossRefGoogle ScholarPubMed
Smith, EG, D'Angelo, C, Salih, A and Wiedenmann, J (2013) Screening by coral green fluorescent protein (GFP)-like chromoproteins supports a role in photoprotection of zooxanthellae. Coral Reefs 32, 463–474.CrossRefGoogle Scholar
Smith-Keune, C and Dove, S (2007). Gene expression of a green fluorescent protein homolog as a host-specific biomarker of heat stress within a reef-building coral. Marine Biotechnology 10, 166–180.CrossRefGoogle Scholar
Sniegowski, JA, Lappe, JW, Patel, HN, Huffman, HA and Wachter, RM (2005). Base catalysis of chromophore formation in Arg96 and Glu222 variants of green fluorescent protein. Journal of Biological Chemistry 280, 26248–26255.CrossRefGoogle ScholarPubMed
Stepanenko, OV, Stepanenko, OV, Shcherbakova, DM, Kuznetsova, IM, Turoverov, KK and Verkhusha, VV (2011) Modern fluorescent proteins: from chromophore formation to novel intracellular applications. Biotechniques 51, 313–327.CrossRefGoogle ScholarPubMed
Stepanenko, OV, Stepanenko, OV, Kuznetsova, IM, Verkhusha, VV and Turoverov, KK (2013) Beta-barrel scaffold of fluorescent proteins: folding, stability and role in chromophore formation. International Review of Cellular and Molecular Biology 302, 221–278.CrossRefGoogle ScholarPubMed
Strader, ME, Aglyamova, GV and Matz, MV (2016) Red fluorescence in coral larvae is associated with a diapause-like state. Molecular Ecology 25, 559–569.CrossRefGoogle ScholarPubMed
Takahashi-Kariyazono, S, Satta, Y and Terai, Y (2015) Genetic diversity of fluorescent protein genes generated by gene duplication and alternative splicing in reef-building corals. Zoological Letters 1, 23.CrossRefGoogle ScholarPubMed
Takahashi-Kariyazono, S, Gojobori, J, Satta, Y, Sakai, K and Terai, Y (2016) Acropora digitifera encodes the largest known family of fluorescent proteins that has persisted during the evolution of Acropora Species. Genome Biology and Evolution 8, 3271–3283.CrossRefGoogle ScholarPubMed
Takahashi-Kariyazono, S, Sakai, K and Terai, Y (2018) Presence–absence polymorphisms of highly expressed FP sequences contribute to fluorescent polymorphisms in Acropora digitifera. Genome Biology and Evolution 10, 1715–1729.CrossRefGoogle ScholarPubMed
Tatusov, RL, Galperin, MY, Natale, DA and Koonin, EV (2000) The COG database: a tool for genome-scale analysis of protein functions and evolution. Nucleic Acids Research 28, 33–36.CrossRefGoogle ScholarPubMed
Tavare, L (1986) Some probabilistic and statistical problems of the analysis of DNA sequences. Lecture Notes on Mathematical Modelling in the Life Sciences 17, 57–86.Google Scholar
Thompson, JD, Higgins, DG and Gibson, TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position specific gap penalties and weight matrix choice. Nucleic Acids Research 22, 4673–4680.CrossRefGoogle ScholarPubMed
Verkhusha, VV and Lukyanov, KA (2004) The molecular properties and applications of Anthozoa fluorescent proteins and chromoproteins. Nature Biotechnology 22, 289–296.CrossRefGoogle Scholar
Yang, F, Moss, LG and Phillips, GN Jr. (1996) The molecular structure of green fluorescent protein. Nature Biotechnology 14, 1246–1251.CrossRefGoogle ScholarPubMed
Ye, J, Coulouris, G, Zaretskaya, I, Cutcutache, I, Rozen, S and Madden, TL (2012) Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction. BMC Bioinformatics 13, 134.CrossRefGoogle ScholarPubMed
Supplementary material: File

Dizon et al. supplementary material

Dizon et al. supplementary material 1

Download Dizon et al. supplementary material(File)
File 5.5 KB
Supplementary material: File

Dizon et al. supplementary material

Dizon et al. supplementary material 2

Download Dizon et al. supplementary material(File)
File 86.1 KB
Supplementary material: PDF

Dizon et al. supplementary material

Dizon et al. supplementary material 3

Download Dizon et al. supplementary material(PDF)
PDF 279.8 KB