Analysis of silicatein gene expression and spicule formation in the demosponge amphimedon queenslandica

Analysis of silicatein gene expression and spicule formation in the demosponge Amphimedon queenslandica Aude Gauthier BMarSt (Hons) A thesis submitted for the degree of Master of Philosophy at The University of Queensland in 2014 School of Biological Sciences Abstract The skeletal elements in most sponges are siliceous spicules These are fabricated into species-specific sizes and shapes Demosponges, in particular, have specialised cells called sclerocytes that possess the unique ability to synthesise biosilica and these spicules Underlying the diversity of demosponge spicules morphology is a conserved protein, called silicatein This thesis aims to investigate the process of spiculogenesis in the different developmental stages of the demosponge Amphimedon queenslandica, and the evolution and developmental expression of the silicatein gene family in relation to spicule formation A queenslandica is the only sponge species to have its genome fully sequenced, assembled and annotated, and currently is one of the best models to study sponge development (Srivastava et al 2010) This species broods embryos year-round, facilitating the access to embryological and larval material (Leys and Degnan 2001) This combination of logistical advantages means that I was able to trace the expression of silicatein genes through A queenslandica embryonic, larval and postlarval development Spicule formation starts in the early embryogenesis in A queenslandica during, gastrulation or the brown stage Spicule number increases throughout embryonic development until the pre-hatching larval stage, with the emerging larvae having about 1000 spicules No detectable increase in spicule number was recorded during larval and postlarval development Spicule number varied remarkably between different individual embryos and larvae of the same stage of development I initially identified six silicatein β like genes in the genome of A queenslandica, among which four can be categorised as non-conventional by the absence of the serine in the catalytic triad of the protein These genes not have direct orthologues in other sponge species and appear to have evolved by a lineage-specific gene duplication A comprehensive phylogenetic analysis of this gene family in sponge indicated that silicatein α arose from silicatein β by gene duplication and that silicatein β gene share traits with both cathepsin L and silicatein Conservation of gene structure and exon length in silicatein and cathepsin L genes suggests that these genes have preserved an ancestral gene structure common to both gene families in both marine and freshwater sponges Using in situ hybridisation, I demonstrated that silicatein genes are expressed during A queenslandica early embryonic development, with genes being expressed exclusively in sclerocytes Analysis of gene expression levels through embryogenesis and metamorphosis, using RNA-Seq performed on a pool of same stage individuals, revealed that all silicatein-like genes are differentially expressed throughout development, and the expression of silicatein genes occurs prior to spicule formation However, some silicatein-like gene expression levels and spicule number not appear to be tightly correlated Declaration by author This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, and any other original research work used or reported in my thesis The content of my thesis is the result of work I have carried out since the commencement of my research higher degree candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis Publication during candidature No publications Publication included in this thesis No publications included Contributions by others to the thesis My supervisor, Professor Bernard Degnan, contributed to the conception and design of the research and critically revised and proofread all sections of this thesis My co-supervisor, Dr Nagayasu Nakanishi performed technical work required for the RNA-Seq data Statement of parts of the thesis submitted to quality for the award of another degree None Acknowledgements I am extremely grateful to my supervisor Bernie Degnan for accepting me into his group and introducing me to the world of sponges and research, and for his continuous support and invaluable advice through this project Additionally, I would like to thank my committee members, Dr Nagayasu Nakanishi and Dr David Merritt, for their encouragement and interest in my work I would also like to thank everyone in the Degnan lab, both past and present members, for their help, support, coffee breaks, stimulating discussions, and fun times in and out of the office throughout the years This includes in no particular order, Carmel McDougall, Andrew Calcino, Laura Grice, Kerry Roper, Maely Gauthier, Jo Bayes, Jaret Bilewitch, Nobuo Ueda, Felipe Aguilera, Jabin Watson, Simone Higgie, Federico Gaiti, Katia Jindrich, Kevin Kocot, Sunsuke Sogabe, Selene Fernandez Valverde, Ben Yuen, Tahsha Say, Rebecca Fieth and William Hatleberg I am most particularly thankful to Carmel McDougall and Kerry Roper for their invaluable help and guidance in the laboratory A special thanks to Sandie Degnan for her support and encouragement through the different milestones of this project, Nagayasu Nakanishi for providing help and suggestions with my in situ hybridization approach, and Katia Jindrich and Ben Yuen for going through some troubleshooting with me in the lab I also wish to thank Maely Gauthier, Kevin Kocot, Laura Grice, Simone Higgie and William Hatleberg for taking the time to proofread parts of my thesis During the course of this project, I was lucky to participate in field trips to Heron Island So, I would like to acknowledge staff members of Heron Island Research Station, which are always available and understanding of our last minute experimental set-up, for their technical help and great working environment atmosphere Thanks to the sponge people who made those trips worth remembering for their assistance in the field, the great times and sunset drinks, as well as for going through those endless night with me fixing material and dissecting brood chambers I would like to thank friends and family who supported me during my time here Above all, my parents for supporting my choice and giving me the opportunity to undertake my studies in Australia A special thanks ‘au Nain’ (Arnault Gauthier) for listening to my complaints when things were not working to plan and checking for the numerous grammatical mistakes Keywords Porifera, silicatein, silicatein gene expression, spicules Australian and New Zealand Standard Research Classifications (ANZSRC) ANZSRC code: 060309 Phylogeny and comparative analysis 50% ANZSRC code: 060808 Invertebrate biology 50% Fields of Research (FoR) Classification FoR code: 0603 Evolutionary biology 50% FoR code: 0608 Zoology 50% Table of Contents Chapter 1: General Introduction 1.1 Sponge biosilica structure…………………………………………………………………………………………….2 1.1.1 Spicule morphology………………………………………………………………………………………………3 1.1.2 Spicule formation…………………………………………………………………………………………………4 1.2 Silicatein……………………………………………………………………………………………………………………….7 1.2.1 Catalytic mechanism of silicatein………………………………………………………………………….7 1.3 Amphimedon queenslandica as a study model………………………………………………………………8 1.4 Aims of this study………………………………………………………………………………………………………….9 Chapter 2: Identification of silicatein genes in the demosponge Amphimedon queenslandica and their evolutionary relationship to other sponge silicatein and cathepsin genes……………………………………………… 11 2.1 Abstract…………………………………………………………………………………………………………………… 11 2.2 Introduction……………………………………………………………………………………………………………….12 2.3 Materials and Methods………………………………………………………………………………………………14 2.3.1 Identification of silicatein genes in Amphimedon queenslandica……………………… 14 2.3.2 Gene architecture analyses……………………………………………………………………………… 15 2.3.3 Molecular phylogenetic analyses……………………………………………………………………… 15 2.4 Results……………………………………………………………………………………………………………………… 16 2.4.1 Identification and genomic organisation of A queenslandica silicatein genes…… 16 2.4.2 Conservation in the genomic structure of A queenslandica silicatein genes……… 20 2.4.3 Phylogenetic relationship of sponge silicateins……………………………………………………23 2.4.4 More detailed analysis of silicatein protein sequences……………………………………… 27 2.4.5 Presence of the CY motif in other metazoan cathepsin L sequences……………………28 2.5 Discussion………………………………………………………………………………………………………………… 33 Chapter 3: Analysis of spicule formation and silicatein gene expression in the demosponge Amphimedon queenslandica……………………………………………38 3.1 Abstract…………………………………………………………………………………………………………………… 38 3.2 Introduction……………………………………………………………………………………………………………….38 3.3 Materials and Methods………………………………………………………………………………………………41 3.3.1 Sponge collection and fixation of biological materials…………………………………………41 3.3.1.1 Brooded embryos………………………………………………………………………………………41 3.3.1.2 Larval and postlarval stages……………………………………………………………………….42 3.3.2 Spicule preparation…………………………………………………………………………………………….43 3.3.3 Gene expression analysis – RNA-Seq protocol…………………………………………………….43 3.3.3.1 RNA extraction and cDNA synthesis………………………………………………………… 44 3.3.3.2 Samples processing………………………………………………………………………………… 44 3.3.4 Gene expression analysis – qRT-PCR………………………………………………………………… 44 3.3.4.1 Primer design for genes of interest…………………………………………………………….45 3.3.4.4 qRT-PCR analyses………………………………………………………………………………………46 3.3.5 Whole-mount in situ hybridisation…………………………………………………………………… 47 3.3.5.1 Probe synthesis…………………………………………………………………………………………48 3.3.6 Statistical analyses…………………………………………………………………………………………… 48 3.4 Results……………………………………………………………………………………………………………………… 49 3.4.1 Spicule number increases during embryogenesis but not in larvae and early postlarvae………………………………………………………………………………………………….49 3.4.2 Temporal expression of A queenslandica silicatein genes during development…50 3.4.3 Expression pattern of A queenslandica silicatein genes, Aqu2.41046, Aqu2.41047 and Aqu2.42494…………………………………………………………………………… 54 3.4.4 Localised expression of Aqu2.42494 during development………………………………… 54 3.4.5 Individual variation in gene expression and spicule number……………………………….57 3.5 Discussion………………………………………………………………………………………………………………… 60 Chapter 4: General Discussion…………………………………………………………………… 64 4.1 Does silicatein expression correlate with spicule number? …………………………………………65 4.2 Why does variation in silicatein expression and spicule number occur? .66 4.3 Conclusions and future directions……………………………………………………………………………….67 References………………………………………………………………………………………………….69 Appendices…………………………………………………………………………………………………81 …A1 GeneBank (NCBI) accession numbers of protein sequences of demosponges and hexactinellid (asterix) used in this study……………………………………………………………….81 …A2 Protein sequences alignment of the conserved domain, Inhibitor I29 and Peptidase C1A, of Amphimedon queenslandica silicatein and cathepsin L genes…………………………83 …A3 Protein sequences alignment of the conserved domain, Inhibitor I29 and Peptidase C1A, of silicatein-like sequences in different animals ……………………………………………… 84 Berquist PR 1978 Sponges UC Press, Berkeley Berti PJ, and Storer AC 1995 Alignment/Phylogeny of the papain superfamily of cysteine proteases J Mol Biol 246: 273-283 Bond C 1992 Continuous cell movements rearrange anatomical structures in intact sponges J Exp Zool 263: 284-302 Borojevic RWG, Fry WC, Jones C, Levi R, Rasmont M, and Vacelet J 1968 Mise au point actuelle de la terminologie des éponges Bulletin du Museum National d’Histoire Naturelle (Paris) 39: 1224-1235 Boury-Esnault N, and Rützler K 1997 Thesaurus of sponge morphology Smithsonian Contributions to Zoology 596: 1-55 Cao X, Fu W, Yu X, and Zhang W 2007 Dynamics of spicule production in the marine sponge Hymeniacidon perlevis during in vitro cell culture and seasonal development in the field Cell Tissue Res 329: 595-608 Cha JN, Shimizu K, Zhou Y, Christiansen SC, Chmelka BF, Stucky GD, and Morse DE 1999 Silicatein filaments and subunits from a marine sponge direct the polymerization of silica and silicones in vitro Proc Natl Acad Sci 96: 361-365 Cha JN, Stucky GD, Morse DE, and Deming TJ 2000 Biomimetic synthesis of ordered silica structures mediated by block copolypeptides Nature 403: 289-292 Chanas B, and Pawlik JR 1995 Defenses of Caribbean sponges against predatory reef fish II Spicules, tissue toughness, and nutritional quality Mar Ecol Prog Ser 127: 195-211 Chombard C, Boury-Esnault N, and Tillier S 1998 Reassessment of homology of morphological characters in tetractinellid sponges based on molecular data Sys Biol 47: 351–366 Cusack M, and Freer A 2008 Biomineralization: elemental and organic influence in carbonate systems Chem Rev 108: 4433-4454 David R, and Wedlich D 2001 PCR-based RNA probes, a quick and sensitive method to improve whole mount embryo in situ hybridizations Biotechniques 30: 769-772 Degnan BM, Leys SP, and Larroux C 2005 Sponge development and antiquity of animal pattern formation Integr Comp Biol 45: 335-341 Degnan BM, Adamska M, Craigie A, Degnan SM, Fahey B, Gauthier M, Hooper JNA, Larroux C, Leys SP, Lovas E, and Richards GS 2008a The Demosponge Amphimedon queenslandica: Reconstructing the ancestral metazoan genome and deciphering the origin of animal multicellularity Cold Spring Harb.Protoc.doi:10.1101/pdb.emo108 70 Degnan SM, Craigie A, and Degnan BM 2008b Genotyping individual Amphimedon embryos, larvae and adults Cold Spring Harb.Protoc.doi:10.1101/pdb.prot5098 Degnan SM, and Degnan BM 2010 The initiation of metamorphosis as an ancient polyphonic trait and its role in metazoan life-cycle evolution Phil Trans R Soc 365: 641-651 Delage Y 1892 Embryogénie des éponges Archives De Zoologie Experimental 10: 345–498 DeMaster DJ 1981 The supply and accumulation of silica in the marine environment Geochimica and Cosmochimica Acta 45: 1715-1732 Donati V 1753 Auszug einer Natur-Geschichte des Adriatischen Meers CP Franckens, Halle Dunn CW, Hejnol A, Matus DQ, Pang K, Browne WE, Smith SA, Seaver E, Rouse GW, Obst M, Edgecombe GD, Sørensen MV, Haddock SHD, Schmidt-Rhaesa A, Okusu A, Kristensen RM, Wheeler WC, Martindale MQ, and Giribet G 2008 Broad phylogenomic sampling improves resolution of the animal tree of life Nature 452: 745–749 Eckert C Schröder HC, Brandt D, Perovic-Ottstadt S, and Müller WEG 2006 Histochemical and electron microscopic analysis of spiculogenesis in the demosponge Suberites domuncula J Histochem Cytochem 54: 1031-1040 Elliott JC 2002 Calcium phosphate biominerals Reviews in Mineralogy and Geochemistry 48: 427-453 Epstein E 1994 The anomaly of silicon in plant biology Proc Natl Acad Sci 91: 11-17 Erwin DH 2011 The Cambrian conundrum: early divergence and later ecological success in the early history of amimals Science 334: 1091-1097 Exley C 2009 Silicon in life: whither biological silicification? Prog Mol Subcell Biol 47: 173184 Fairhead M, Kowatz T, McMahon SA, Carter LG, Oke M, Johnson KA, Liu H, Naismith JH, and Van der Walle CF 2008 Crystal structure and silica condensing activities of silicatein α / cathepsin L chimeras Chem Commun 15: 1765-1767 Fell PE 1976 The reproduction of Haliclona loosanofi and its apparent relationship to water temperature Bioliogical Bulletin 150: 200-210 Funayama N, Nakatsukasa M, Kuraku S, Takechi K, Dohi M, Iwabe N, Miyata T, and Agata K 2005 Isolation of Ef silicatein and Ef lectin as molecular markers for sclerocytes and cells involved in innate immunity in the freshwater sponge Ephydatia fluviatilis Zoological Science 22: 1113-1122 Gavaze E, Lapébie P, Ereskovsky A, Vacelet J, Renard E, Cárdenas P, and Borchiellini C 2012 No longer Demospongiae: Homoscleromorpha formal nomination as a fourth class of Porifera Hydrobiologia 687:3-10 71 Grant RE 1826 Observations and experiments on the structure and functions of the sponges Edinb Philos J 14:336-341 Guindon S and Gascuel O 2003 A simple, fast and accurate algorithm to estimate large phylogenies by maximum likelihood Systematic Biology 52: 696-704 Haeckel E 1972 Die Kalkschwämme Verlag Georg Reimer, Berlin Hamm CE, Merkel R, Springer O, Jurkoic P, Maier C, Prechtel K, and Smetacek V 2003 Architecture and material properties of diatom shells provide effective mechanical protection Nature 421: 841-843 Hartman WD 1981 Form and distribution of silica in sponges In: Simpson TL, Volcani BE, editors Silicon and siliceous structures in biological systems New York: Springer P453-493 Hellemans J, Mortier G, De Paepe A, Speleman F, and Vandesompele J 2007 qBase relative quantification framework and software for management and automated analysis of realtime quantitative PCR data Genome Biol 8, R19 Hildebrand M 2008 Diatoms, biomineralization processes, and genomics Chem Rev 108: 4855-4874 Hoffman PF, and Schrag DP 2002 The snowball earth hypothesis: testing the limits of global change Terra Nova 14: 129-155 Review Hooper JNA, and Van Soest R 2006 A new species of Amphimedon (Porifera, Demospongiae, Haplosclerida, Niphatidae) from the Capricorn-Bunker Group of Islands, Great Barrier Reef, Australia: target species for the ‘sponge genome project Zootaxa 1314: 31-39 Huang J, Zhang C, Ma Z, Xie L, and Zhang R 2007 A novel extracellular EF-hand protein involved in the shell formation of pearl oyster Biochim Biophys Acto 1770: 1037-1044 Huelsenbeck JP, and Ronquist F 2001 MrBayes: Bayesian inference of phylogeny Bioinformatics 17: 754-755 Imsiecke G, Steffen R, Custodio M, Borojevic R, and Müller WEG 1995 Formation of spicules by sclerocytes from the freshwater sponge Ephydatia muelleri in short term cultures in vitro In vitro cellular & developmental Biology Animal 31: 528-535 Kaluzhnaya OV, Belikov SI, Schröder HC, Rothenberger M, Zapf S, Kaandorp JA, Borejko A, Müller IM, and Müller WEG 2005 Dynamics of skeleton formation in the lake Baikal sponge Lubomirskia baicalensis Part I biological and biochemical studies Naturwissenschaften 92: 128-133 72 Kaluzhnaya OV, Krasko AG, Grebenyuk VA, Itskovich VB, Semiturkina NA, Solovarov IS, Müller WEG, and Belikov SI 2011 Freshwater sponge silicateins: comparison of gene sequences and exon-intron structure Molecular Biology 45: 567-575 Katoh K, Misawa K, Kuma KI, and Miyata T 2002 MAFFT: a novel method for rapid multiple sequence based on fast Fourier transform Nucleic Acid Research 30: 3059-3066 Ki MR, and Pack SP 2013 Expression and characterization of nematostella cathepsin as biosilifiction-inducing agent Proceedings of the 2013 International Conference on Biology and Biomedicine Konstantinova MI 1966 Characteristics of movement of pelagic larvae of marine invertebrates Dokl Acad Nauk SSSR 170t726-729 Kozhemyako V, Veremeichik GN, Shkryl YN, Kovalchuk SN, Krasokhin VB, Rasskazov VA, Zhuravlev YN, Bulgakov VP, and Kulchin YN 2009 Silicatein genes in spicule-forming and nonspicule-forming Pacific demosponges Mar Biotech 12: 403-409 Krasko A, Lorenz B, Batel R, Schröder HC, Müller IM, and Müller WEG 2000 Expression of silicatein and collagen genes in the marine sponge Suberites domuncula is controlled by silicate and myotrophin Eur J Biochem 267: 4878-4887 Krasko A, Schröder HC, Batel R, Grebenjuk VA, Steffen R, Müller IM, and Müller WEG 2002 Iron induces proliferation and morphogenesis in primmorphs from the marine sponge Suberites domuncula DNA and Cell Biology 21: 67-80 Kröger N, Deutzmann R, and Sumper M 1999 Polycationic peptides from diatom biosilica that direct silica nanosphere formation Science 286: 1129-1132 Kugimiya F, Kawaguchi H, Kamekura S, Chikuda H, Ohba S, Yano F, Ogata N, Katagiri T, Harada Y, Azuma Y, Nakamura K, and Chung UI 2005 Involvement of endogenous bone morphogenic protein (BMP) and BMP6 in bone formation J Biol Chem 280: 35704-35712 Larroux C, Fahey B, Adamska M, Richards G, Gauthier M, Green K, Lovas E, and Degnan BM 2008 Whole-Mount in situ hybridization in Amphimedon Cold Spring Harb Protoc doi:10.1101/pdb.prot5096 Lavrov DV, Wang X, and Kelly M 2008 Reconstructing ordinal relationships in the Demospongiae using mitonchondrial genomic data Molecular Phylogenetics and Evolution 49: 111-124 Leadbeater BSC and Jones WC 1984 Silicification of cell walls of certain protistan flagellates Phil Trans R Soc.Lond 304:529-536 Le Pennec G, Perovic S, Ammar MS, Grebenjuk VA, Steffen R, Brummer F, and Müller WEG 2003 Cultivation of primmorphs from the marine sponge Suberites domuncola: morphogenetic potential of silicon and iron: a review J Biotechnol 100: 93-108 73 Lévi C 1973 Systématique de la classe des Demospongiaria (Démosponges) In: Grassé P, editor Spongiaires Traité de zoologie 3(1) Paris: Masson P.577-632 Leys SP, and Degnan BM 2001 Cytological Basis of Photoresponsive Behavior in a Sponge Larva Biological Bulletin 201: 323-338 Leys SP 2003 Comparative study of spiculogenesis in demosponge and hexactinellid larvae Microscopy Research and Technique 62: 300-311 Leys SP, Larroux C, Gauthier M, Adamska M, Fahey B, Richards GS, Degnan SM, and Degnan BM 2008 Isolation of Amphimedon developmental material Cold Spring Harb Protoc doi:1101/pdb.prot5095 Long M, Rosenberg C, and Gilbert W 1995 Intron phase correlations and the evolution of the intron/exon structure of genes Proc Natl Acad Sci 92: 12495-12499 Lowenstam HA 1971 Opal precipitation by marine gastropods (Mollusca) Science 171: 487490 Maldonado M, George SB, Young CM, and Vaquerizo E 1997 Depth regulation in parenchymella larvae of a demosponge: relative roles of skeletogenesis, biochemical changes and behavior Mar Ecol Prog Ser 148: 115-124 Maldonado M, Carmona MC, Uriz MJ, and Cruzado A 1999 Decline in Mesozoic reefbuilding sponges explained by silicon limitation Nature 401: 785-788 Maldonado M, and Riesgo A 2007 Intra-epithelial spicules in a homosclerophorid sponge Cell Tissue Res 328:639-650 Maldonado M, Navarro L, Grasa A, Gonzalez A, and Vaquerizo I 2011 Silicon uptake by sponges: a twist to understanding nutrient cycling on continental margins Scientific reports 4:1-8 Maldonado M, Ribes M, and Van Duyl FC 2012 Nutrient fluxes through sponges: biology, budgets, and ecological implications Advances in Marine Biology 62:113-182 Miller CB, Nelson DM, Weiss C, and Soeldner AH 1990 Morphogenesis of opal teeth in calanoid copepods Marine Biology 106: 91-101 Mohri K, Nakatsukasa M, Masuda Y, Agata K, and Funayama N 2008 Toward understanding the morphogenesis of siliceous spicules in freshwater sponge: Differential mRNA expression of spicule-type specific silicatein genes in Ephydatia Fluviatilis Developmental Dynamics 237: 3024-3039 Monniot F, Martoja R and Monniot C 1992 Silica distribution in ascidian ovaries, a tool for systematics Biochem System Ecol 20: 541-552 74 Morse DE 1999 Silicon biotechnology: harnessing biological silica production to construct new materials Trends in Biotechnology 17: 230–232 Müller WEG 1995 Molecular phylogeny of metazoa (Animals): monophyletic origin Naturwissenschaften 82: 321-329 Müller WEG 1998 Origin of metazoan: sponges as living fossils Naturwissenschaften 85: 11-25 Müller WEG, Wiens M, Batel R, Steffen R, Schröder HC, Borojevic R, and Custodio MR 1999 Establishment of a primary cell culture from a sponge: primmorphs from Suberites domuncula Mar Ecol Prog Ser 178: 205-219 Müller WEG, Krasko A, Le Pennec G, and Schröder HC 2003 Biochemistry and cell biology of silica formation in sponges Microscopy Research and Technique 62: 368-377 Müller WEG, Rothenberger M, Boreiko A, Tremel W, Reiber A, and Schröder HC 2005a Formation of siliceous spicules in the marine demosponge Suberites domuncula Cell Tissue Res 321: 285-297 Müller WEG, Borejko A, Brandt D, Osinga R, Ushijima H, Hamer B, Krasko A, Xupeng C, Müller IM & Schröder HC 2005b Selenium affects biosilica formation in the demosponge Suberites domuncula: effect on gene expression and spicule formation FEBS Journal : 38383852 Müller WEG, Belikov SI, Tremel W, Perry CC, Gieskes WWC, Boreiko A, and Schröder HC 2006 Siliceous spicules in marine demosponges (example Suberites domuncula) Micron 37: 107-120 Review Müller WEG, Eckert C, Kropf K, Wang X, Schlobmacher U, Seckert C, Wolf S, Tremal W, and Schröder H 2007a Formation of giant spicules in the deep-sea hexactinellid Monorhaphis chuni ( Schulze 1904): electron-microscopic and biochemical studies Cell Tissue Res 329: 363-378 Müller WEG, Boreiko A, Wang X, Belikov S, Wiens M, Grebenjuk VA, Schloβmacher U, and Schröder HC 2007b Silicateins, the major biosilica forming enzymes present in demosponges: protein analysis and phylogenetic relationship Gene 395: 62-71 Müller WEG, Schloβmacher U, Eckert C, Krasko A, Boreiko A, Ushijima H, Wolf SE, Tremel W, Muller IM, and Schröder HC 2007c Analysis of the axial filament in spicules of the demosponge Geodia cydonium: different silicatein composition in microscleres (asters) and megascleres (oxeas and triaenes) Eur J Cell Biol 86: 473-487 Müller WEG, Schloβmacher U, Wang X, Boreiko A, Brandt D, Wolf SE, Tremel W, and Schröder HC 2008a Poly(silicate)-metabolizing silicatein in siliceous spicules and silicasomes of demosponges comprises dual enzymatic activities (Silica polymerase and silica esterase) FEBS Journal 275: 362-370 75 Müller WEG, Wang X, Kropf K, Boreiko A, Schloβmacher U, Brandt D, Schröder HC, and Wiens M 2008b Silicatein expression in the hexactinellid Crateromorpha meyeri: the lead marker gene restricted to siliceous sponges Cell Tissue Res 333: 339-351 Müller WEG, Wang X, Cui F-Z, Jochum KP, Tremel W, Bill J, Shröder HC, Natalio F, Schloβmacher U, and Wiens M 2009 Sponge spicules as blueprints for the biofabrication of inorganic-organic composites and biomaterials Appl Microbiol Biotechnol 83: 397-413 Müller WEG, Link T, Schröder HC, Korzhev M, Neufurth M, Brandt D, and Wang X 2014 Dissection of the structure-forming activity from the structure-guiding activity of silicatein: a biomimetic molecular approach to print optical fibers J Mater Chem 2: 5368-5377 Palumbi SR 1986 How body plans limit acclimation: responses of a demosponge to wave force Ecology 67: 208-214 Philippe H, Derelle R, Lopez P, Pick K, Borchiellini C, Boury-Esnault N, Vacelet J, Deniel E, Houliston E, Quéinnec E, Da Silva C, Wincker P, Le Guyader H, Leys S, Jackson DJ, Schreiber F, Erpenbeck D, Morgenstern B, Wörheide G, and Manuel M 2009 Phylogenomics revives traditional views on deep animal relationships Current Biology 19: 706–712 Pickett-Heaps J, Schmid AMM, and Edgar LA 1990 The cell biology of diatom valve formation Progress in Phycological Research (Round, F E., and Chapman, D J., eds) pp.1168, Biopress Ltd., Bristol, UK Pisera A 2003 Some aspects of silica deposition in lithistid demosponge desmas Microsc Res Tech 62: 312-326 Pohnert G 2002 Biomineralization in diatoms mediated through peptide and polyamine assisted condensation of silica Angew Chem Int 41: 3167-3169 Pozzolini M, Sturla L, Cerrano C, Bavestrello G, Camardella L, Parodi AM, Raheli F, Benatti U , Müller WEG, and Giovine M 2004 Molecular cloning of silicatein gene from marine sponge Petrosia ficiformis (Porifera, Demospongiae) and development of primmorphs as a model for biosilicification studies Mar Biotechnol 6: 594-603 Preisig HR 1994 Siliceous structures and silicification in flagellated protists Protoplasma 184:29-42 Rambaldi D, and Ciccarelli FD 2009 FancyGene: dynamic visualization of gene structure and protein architectures on genomic loci Bioinformatics 25: 2281-2282 Richards GS 2010 The origins of cell communication in the animal kingdom: Notch signalling during embryogenesis and metamorphosis of the demosponge Amphimedon queenslandica PhD University of Queensland 76 Round FE, Crawford RM, and Mann DG 1990 The diatoms: biology and morphology of the genera Cambridge University Press, Cambridge 747pp Rozen S, and Skaletsky HJ 2000 Primer3 on the WWW for general users and for biologist programmers Humana Press, Totowa, NJ Ryan JF, PangK, NISC Comparative Sequencing Program, Mullikin JC, Martindale MQ, and Baxevanis AD 2010 The homeodomaine complement of the ctenophore Mnemiopsis leidyi suggests that Ctenophora and Porifera diverged prior to the ParaHoxozoa EvoDevo 1: 1-18 Ryan JF, Pang K, Schnitzler CE, Nguyen AD, Moreland T, Simmons DK, Koch BJ, Francis WR, Havlak P, NISC Comparative Sequencing Program, Smith SA, Putman NH, Haddock SHD, Dunn CW, Wolfsberg TG, Mullikin JC, Martindale MQ, and Baxevanis AD 2013 The genome of the ctenophore Mnemiopsis leidyi and its implications for cell type evolution Science 342 Schierwater B, Eitel M, Jakob W, Osigus HJ, Hadrys H, Dellaporta SL, Kolokotronis SO, and DeSalle R 2009 Concatenated analysis shedslight on early metazoan evolution and fuels a modern “Urmetazoon”hypothesis PLoS Biology 9: 0036-0044 Schloβmacher U, Wiens M, Schröder HC, Wang XH, Jochum KP, and Müller WEG 2011 Silintaphin-1: interaction with silicatein during structure guiding biosilica formation FEBS Journal 278: 1145-55 Schönberg CHL, and Barthel D 1997 Inorganic skeleton of the demosponge Halichondria panacea Seasonality in spicule production in the Baltic Sea Marine Biology 130: 133-140 Schröder HC, Perovic-Ottstadt S, Rothenberger M, Wiens M, Schwertner H, Batel R, Korzhev M, Müller IM, and Müller WEG 2004 Silica transport in the demosponge Suberites domuncula: fluorescence emission analysis using the PDMPO probe and cloning of a potential transporter Biochem J 381: 665-673 Schröder HC, Perovic-Ottstadt S, Grebenjuk VA, Engel S, Müller IM, and Müller WEG 2005 Biosilica formation in spicules of the sponges Suberites domuncula: synchronous expression of a gene cluster Genomics 85: 666-678 Schröder HC, Boreiko A, Korzhev M, Tahir MN, Tremel W, Eckert C, Ushijima H, Müller IM, and Müller WEG 2006 Co-expression and functional interaction of silicatein with galectin J Biol Chem 281: 12001-12009 Schröder HC, Natalio F, Shukoor I, Tremel W, Schloβmacher U, Wang X, and Müller WEG 2007a Apposition of silica lamellae during growth of spicules in the demosponge Suberites domuncula: biological/biochemical studies and chemical/biomimetical confirmation Journal of Structural Biology 159: 325-334 Schröder HC, Brandt D, Schloβmacher U, Wang X, Tahir MN, Tremel W, Belikov S and Müller WEG 2007b Enzymatic production of biosilica glass using enzymes from sponges: basic 77 aspects and application in nanobiotechnology (material sciences and medicine) Naturwissenschaften 94: 339-359 Schröder HC, Wang X, Tremel W, Ushijima H, and Müller WEG 2008 Biofabrication of biosilica-glass by living organisms Natural Product Report 25: 455-474 Review Schulze FE 1904 Hexactinellida In: Chun, C (Ed.), Wissenschaftliche Ergebnisse der Deutschen Tiefsee-Expedition auf dem Dampfer “Valdivia” 1898-1899 Gustav Fischer, Jena Seilacher A 1994 Early multicellular life: late Proterozoic fossils and the Cambrian explosion In Early Life on Earth (Bengtson, S.,ed.), pp 389±400 Columbia University Press, NY Sharp P 1981 Speculations on RNA Splicing Cell 23: 643-646 Shimizu K, Cha J, Stucky GD, and Morse DE 1998 Silicatein α: Cathepsin L-like protein in sponge biosilica Proc Natl Acad Sci 95: 6234-6238 Shore RE 1972 Axial filament of siliceious sponge spicules, its organic components and synthesis Biological Bulletin 143: 689-698 Simpson TL, and Volcani BE 1981 Silicon and siliceous structures in biological systems New York: Springer Simpson TL 1984 The cell biology of sponges New York: Springer-Verlag Simpson TL, Gil M, Connes R, Diaz JP, and Paris J 1985 Effects of germanium (Ge) on the silica spicules of the marine sponge Suberites domuncula: transformation of spicule type J Morphol 183: 117-128 Srivastava M, Simakov O, Chapman J, Fahey B, Gauthier MEA, Mitros T, Richards GS, Conaco C, Dacre M, Hellsten U, Larroux C, Putnam NH, Stanke M, Adamska M, Darling A, Degnan SM, Oakley TH, Plachetzki DC, Zhai Y, Adamski M, Calcino A, Cummins SF, Goodstein DM, Harris C, Jackson DJ, Leys SP, Shu S, Woodcroft BJ, Vervoort M, Kosik KS, Manning G, Degnan BM, and Rokhsar DS 2010 The Amphimedon queenslandica genome and the evolution of animal complexity Nature 466: 720-727 Swift NB 2012 Biomineral Structure and Strength of Barnacle Exoskeletons Colgate Academic Review Available at: http://commons.colgate.edu/car/vol8/iss1/10 Tréguer P, Nelson DM, Van Bennekom AJ, DeMaster DJ, Leynaert A, and Quéguiner B 1995 The silica balance in the world ocean: a reestimate Science 268: 375-379 Uriz MJ, Turon X, and Becerro MA 2000 Silica deposition in Demosponges: spiculogenesis in Crambe crambe Cell Tissue Res 301: 299-309 78 Uriz MJ, Turon X, Becerro MA, and Agell G 2003 Siliceous spicules and skeleton frameworks in sponges: origin, diversity, ultrastructural patterns, and biological functions Microsc Res Tech 62: 279-299 Uriz MJ 2006 Mineral skeletogenesis in sponges Canadian Journal of Zoology 84: 322-356 Review Valisano L, Pozzolini M, Giovine M, and Cerrano C 2012 Biosilica deposition in the marine sponge Petrosia ficiformis (Poiret, 1789): the model of primmorphs reveals time dependence of spiculogenesis Hydrobiologia 687: 259-273 Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A et al 2002 Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes Genome Biol., 3, 0034.01-0034.11 Van Soest RWM, Boury-Esnault N, Vacelet J, Dohrmann M, Erpenbeck D, De Voogd NJ, Santodomingo N, Vanhoorne B, Kelly M, and Hooper JNA 2012 Global diversity of sponges (Porifera) PLoS ONE 7:e35105 doi:10.1371/journal.pone.0035105 Veremeichik GN, Shkryl YN, Bulgakov VP, Shedko SV, Kozhemyako B, Kovalchuk SV, Krasokhin VB, Zhuravlev YN, and Kulchin YN 2011 Occurrence of a silicatein gene in glass sponges (Hexactinellida: Porifera) Mar Biotechnol 13: 810-819 Wang J, Xia Q, Zhou H, Sun P, Li B, Ding D, and Chen T 2007 Radiolaria-like silica with radial spines fabricated by a dynamic self-organiszation J Phys Chem C 111: 16544-16548 Wang X, Wiens M, Schröder HC, Hu S, Mugnaiolo E, Kolb U, Tremel W, Isignano D, and Müller WEG 2010 Morphology of sponge spicules: silicatein a structural protein for biosilica formation Advanced engineering materials 12: B422-B437 Wang X, Wiens M, Schröder HC, Schloβmacher U, Pisignano D, Jochum KP, and Müller WEG 2011a Evagination of cells controls bio-silica formation and maturation during spicule formation in sponges PLoS ONE 6:e20523 Wang X, and Müller WEG 2011b Complex structures-smart solutions, formation of siliceous spicules Commun integr Biol 4: 684-688 Wang X, Schröder HC, Wiens M, Schloβmacher U, and Müller WEG 2012 Biosilica: molecular biology, biochemistry and function in demosponges as well as its applied aspects for tissue engineering Adv Mar Biol 62: 231-271 Wealthall RJ, Brooker LR, Macey DJ, and Griffin BJ 2005 Fine structure of the mineralized teeth of the chiton Acanthopleura echinata (Mollusca: Polyplacophora) J Morphol 265: 165–175 Weaver JC, Milliron GW, Allen P, Miserez A, Rawal A, Garay J, Thurner PJ, Seto J, Mayzel B, Friesen LJ, Chmelka BF, Fratzl P, Aizenberg J, Dauphin Y, Kisailus D, and Morse DE 2010 79 Unifying design strategies in demosponge and hexactinellid skeletal systems The Journal of Adhesion 86: 72-95 Wiens M, Bausen M, Natalio F, Link T, Schloβmacher U, and Müller WEG 2009 The role of the silicatein-α interactor silintaphin-1 in biomimetic biomineralization Biomaterials 30: 1648-1656 Wiens M, Schröder HC, Wang XH, Link T, Steindorf D and Müller WEG 2011 Isolation of the silicatein-a interactor silintaphin-2 by a novel solid-phase pull-down assay Biochemistry 50: 1981-1990 Wilkinson CR, and Garrone R 1980 Ultrastructure of siliceous spicules and microsclerocytes in the marine sponge Neofibularia irata N.SP J Morphol 166: 51-64 Woollacott RM 1990 Structure and swimming behaviour of the larva of Halichondria melanadocia (Porifera: Demospongiae) J Morphol 205: 135-145 Woollacott RM 1993 Structure and swimming behavior of the larva of Haliclona tubifera (Porifera: Demospongiae) J Morphol 218: 301-321 Woollacott RM 2003 Spicule content in larvae of two species of demosponge (Porifera) Species Diversity 8: 203-217 Wörheide G, Dohrmann M, Erpenbeck D, Larroux C, Maldonada M, Voigt O, Borchiellini C, and Lavrov DV 2012 Deep phylogeny and evolution of sponges (phylum Porifera) Adv Mar Biol 61: 1-78 Zhou Y, Shimizu K, Cha JN, Stucky GD, and Morse DE 1999 Efficient catalysis of polysiloxane synthesis by silicatein α requires specific hydroxyl and imidazole functionalities Angewandte Chemie International Edition 38: 779-782 80 Appendices A1 GeneBank (NCBI) accession numbers of protein sequences of demosponges and hexactinellid (asterix) used in this study Species Acanthodendrilla.sp Vietnam Aphrocallistes vastus Aulosaccus sp GV-2009 * Baikalospongia fungiformis Baikalospongia intermedia Bathydorus sp GV-2009 Crateromorpha meyeri * Discodermia japonica Ephydatia fluviatilis Ephydatia muelleri Ephydatia sp n.1 PW-2008 Ephydatia sp n.2 PW-2008 Euplectella aspergillum * Geodia cydonium Halichondria okadai Hymeniacidon perlevis Silicatein isoforms/cathepsin sil-a sil-b cath-L cath-L Sil-like cath-L1 cath-L2 sil-a1 sil-a2 sil-a3 sil-a4 sil-a1 sil-a1’ sil-a4 sil-a4’ cath-L1 cath-L2 sil cath-like1 cath-like2 cath-like3 cath-like4 cath-like5 sil sil sil-2 sil-a4 sil-G1 sil-G2 sil-M1 sil-M2 sil-M3 sil-M4 sil-a2 sil-a3 sil-a4 sil-a4 sil-a2 sil sil-a cath sil sil-a sil-1 81 GenBank accession numbers ACH92669.1 ACH92668.1 ACJ02498.1 CAI91577.1 ACU86976.1 ACU86972.1 ACU86974.1 AEO36958.1 AEO36959.1 AEO36960.1 ADQ74580.1 ACO51493.1 CAQ54043.1 ACO51494.1 CAQ54044.1 ACU86973.1 ACU86975.1 CAP49202.2 CAP17584 CAP17585.1 CAP17586.1 CAP17587.1 CAP17588.1 CBY80151.1 CAJ44453.1 CAJ44454.1 ACO51487.1 BAG74346.1 BAG74347.1 BAE54434.1 BAG74343.1 BAG74344.1 BAG74345.1 CAQ54046.1 CAQ54047.1 CAQ54048.1 CAQ54050.1 CAQ54049.1 CBY80150.1 CAM57981.1 CAA71554.1 BAB86343.1 ABC94586.1 ABM47424.1 Comments Partial sequence “ Partial sequence “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ Partial sequence “ “ “ Partial sequence Partial sequence Latrunculia oparinae Lubomirskia baicalensis Lubomirskia incrustans Monorhaphis chuni Petrocia ficiformis Pheromena raphanus Spongilla lacustris Suberites domuncula Swartschewskia papyracea Tethya aurantium Tethya aurantium red variant Tethya aurantium yellow variant sil-2 cath-L sil-a1 sil-a1’ sil-a2 sil-a3 sil-b cath sil sil-a sil-a2 sil-a2’ sil-a3 sil-a4 cath-L cath-L’ cath-L2 sil-a1 sil-a4 sil sil-b cath-L1 cath-L2 sil-a3 sil-a4 sil-a4’ sil-a sil-b sil-b’ cath-L sil-a4 sil-a sil-b sil sil ABM47425.2 ABM47423.1 ACG63793.1 ACH47999.1 ACH48000.1 ACH48001.1 ACH48002.1 ACH48003.1 CAH10753.1 CAI43319.1 ADQ74585.1 CAI91571.1 CAI91572.1 CAI91573.1 CAI43320.1 CAH10752.1 CAI91575.1 ACO51492.1 ACO51491.1 CAZ04880.1 AAO23671.1 ACU82389.1 ACU82390.1 CAQ54051.1 ACO51489.1 CAQ54052.1 CAI46305.1 CAI46304.1 CAH04635.1 CAH04632.1 CAQ54053.1 AAC23951.1 AAF21819.1 CBY80148.1 CBY80149.1 82 “ “ Partial sequence Partial sequence “ Partial sequence Only amino acid differ between a2 and a2’ Partial sequence Partial sequence “ “ Partial sequence Only amino acids differ between sil-b and sil-b’ Partial sequence A2 Protein sequences alignment of the conserved domain, Inhibitor I29 and Peptidase C1A, of Amphimedon queenslandica silicatein and cathepsin L genes Conserved amino acid are highlighted in black (100% similarity) and similar amino acid (>60%) are highlighted in grey Arrows indicate the catalytic amino acids, Cysteine (Cys/C) or Serine (Ser/S), Histidine (His/H) and Asparagine (Asn/N) Key conserved silicateins residues are underlined The Cys residues involved in the formation of disulphide bonds are represented by dots and the serine cluster by a box 83 A3 Protein sequences alignment of the conserved domain, Inhibitor I29 and Peptidase C1A, of silicatein-like sequences in different animals 84 ... Identification of silicatein genes in the demosponge Amphimedon queenslandica and their evolutionary relationship to other sponge silicatein and cathepsin genes 2.1 Abstract Silicatein genes are involved in. .. investigate the process of spiculogenesis in the different developmental stages of the demosponge Amphimedon queenslandica, and the evolution and developmental expression of the silicatein gene family in. .. identify and verify the conserved region position of domains in proteins and genes 2.3.2 Gene architecture analyses Silicatein and cathepsin L genes in A queenslandica, S domuncula (silicatein α and