Sex, Molecules, and Gene Control

Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy 288

Sex, Molecules, and Gene control

Ecophysiological and evolutionary aspects of key sponge species from Antarctic shallow waters and the deep sea

VASILIKI KOUTSOUVELI

ACTA UNIVERSITATIS UPSALIENSIS ISSN 1651-6192 ISBN 978-91-513-1059-6 UPPSALA urn:nbn:se:uu:diva-423108 2020 Dissertation presented at Uppsala University to be publicly examined in BMC, room A1, 107a, Husargatan 3, Uppsala, Thursday, 17 December 2020 at 13:00 for the degree of Doctor of Philosophy (Faculty of Pharmacy). The examination will be conducted in English. Faculty examiner: Professor Cassandra G. Extavour (Harvard University, Department of Organismic and Evolutionary Biology; Department of Molecular and Cellular Biology). Abstract Koutsouveli, V. 2020. Sex, Molecules, and Gene control. Ecophysiological and evolutionary aspects of key sponge species from Antarctic shallow waters and the deep sea. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy 288. 114 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-513-1059-6. Very little is known about the ecophysiological aspects of Porifera (sponges) from Antarctica and North Atlantic, even though they are keystone components of these habitats. Being the earliest diverging metazoan lineage, sponges also play a fundamental role in our understanding of animal evolution. The main focus of this thesis was to study several aspects of the reproduction of sponges from the Antarctic shallow waters and the North Atlantic deep-sea sponge grounds and to describe the molecular toolkit that regulates their gametogenesis from an evolutionary perspective. In paper I, the reproductive strategy of six demosponge species commonly found in the shallow waters of Antarctica was examined with histological analyses. All species were brooders and although they reproduced during similar periods of the year, differences in their reproductive strategies might have allowed their coexistence in a habitat with annual food limitation events and low temperature. In paper II, the reproductive strategy of five species of the genus Geodia, a keystone genus of boreo-arctic sponge grounds, was assessed with histological analyses. All species were gonochoristic and oviparous, reproducing during similar periods (1-2 cycles annually) and with a high reproductive effort. The abundant lipid yolk and bacterial symbionts in their oocytes might enhance embryonic survival in the water column. Slight differences in reproductive strategies among species indicate specific adaptations for their successful colonization. This is the most detailed description of the reproductive biology of deep-sea Geodia sponges, providing essential information for the design of adequate conservation strategies in these vulnerable areas. In paper III, the genes and proteins regulating the oogenesis and spermatogenesis of the same five Geodia spp. were identified with RNA-seq and proteomic analyses and it was concluded that the molecular toolkit behind the main stages of gametogenesis is conserved across Metazoa. This is the most comprehensive molecular study on the gametogenesis of sponges and has profound implications for understanding the evolution of sexual reproduction in animals. In Manuscript IV, the reproductive features, the lipid signals and the accompanying gene expression patterns during oogenesis of the keystone deep-sea sponge Phakellia ventilabrum were assessed with histological, lipidomic and RNA-seq analyses. In this oviparous species, most of the triacylglycerides showed a tendency for signal increase during oogenesis, correlated with significant overexpression of genes related to their biosynthesis. This might suggest that triacylglyceride-rich yolk is the main lipid storage for the future embryo. This study unveils lipid metabolism patterns associated with female reproduction in sponges for the first time, setting the basis for a better understanding of the chemical ecology of this species and for future comparative analyses across species. Keywords: Porifera, Demospongiae,Geodia spp.,Phakellia ventilabrum , reproduction, gametogenesis, Antarctica, deep sea, sponge grounds, histology, molecular machinery, electron microscopy, RNA-seq, proteomics, lipidomics Vasiliki Koutsouveli, Department of Medicinal Chemistry, Farmakognosi, Box 574, Uppsala University, SE-751 23 Uppsala, Sweden. © Vasiliki Koutsouveli 2020 ISSN 1651-6192 ISBN 978-91-513-1059-6 urn:nbn:se:uu:diva-423108 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-423108)

“Keep Ithaca always in your mind. Arriving there is what you are des- tined for. But do not hurry the journey at all. Better if it lasts for years, so you are old by the time you reach the island, wealthy with all you have gained on the way. Ithaca gave you the marvellous journey”

Κ. Π. Καβάφης

List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Koutsouveli, V., Taboada, S., Moles, J., Cristobo, J., Ríos, P., Bertran, A., Solà, J., Avila, C., Riesgo, A. (2018). Insights into the reproduction of some Antarctic dendroceratid, poecilosclerid, and haplosclerid demosponges. PLoS One. 13(2): e0192267. (Published)

II Koutsouveli, V., Cárdenas, P., Conejero, M., Rapp, H.T., Riesgo A. Exploring the reproductive biology of the genus Geodia from boreo-arctic North-Atlantic deep-sea sponge grounds. Frontiers Marine Science. (Manuscript submitted)

III Koutsouveli, V., Cárdenas, P., Santodomingo, N., Marina, A., Morato, E., Rapp, H.T., Riesgo, A. (2020). The molecular machinery of gametogenesis in Geodia demosponges (Porifera): evolutionary origins of a conserved toolkit across animals, Mo- lecular Biology and Evolution, msaa183, doi:10.1093/molbev/msaa183. (Published)

IV Koutsouveli, V., Balgoma, D., Checa, A., Díez-Vives, C., Riesgo, A., Cárdenas, P. Seasonality, reproductive strategy and lipid metabolism during oogenesis in the deep-sea sponge Phakellia ventilabrum: a histological, lipidomic and transcriptomic approach. (Manuscript)

All reprints are open access.

Contents

Background information ...... 11 Introduction ...... 13 Porifera, a “simple” but rather successful animal phylum ...... 13 Sexual reproduction in sponges: biological and ecological features ...... 17 Sexual reproduction in sponges: evolutionary and molecular insights .... 19 Sponge research in the last century: techniques and approaches ...... 20 Ecological success of sponges in “extreme” habitats ...... 22 The necessity for conservation of Antarctic areas and deep-sea sponge grounds ...... 24 Aims and Significance ...... 25 Material & Methods ...... 27 Collection of the studying material ...... 27 Histological Analysis ...... 28 Light Microscopy...... 28 TEM ...... 28 Transcriptomic Analysis ...... 29 RNA extraction and library preparation ...... 29 Assembly and Annotation ...... 29 DGE analysis ...... 30 GO Enrichment analysis and KEGG annotation ...... 30 Proteomic analysis ...... 30 Phylogenetic Analysis ...... 31 Lipidomic Analysis ...... 31 Lipid extraction and separation ...... 31 Oxylipin extraction and separation ...... 32 Data pre-treatment ...... 32 Statistical Analyses ...... 33 Chapter I. Insights into the reproduction of some Antarctic dendroceratid, poecilosclerid, and haplosclerid demosponges (Paper I) ...... 34 Results ...... 34 Oogenesis ...... 34 Embryogenesis ...... 35 Yolk formation ...... 38

Discussion ...... 40 Key results and conclusions from Chapter I: ...... 42 Chapter II: Exploring the reproductive biology of the genus Geodia from boreo-arctic North-Atlantic deep-sea sponge grounds (Paper II) ...... 43 Results ...... 43 Histological observations of gametogenesis in Geodia spp...... 43 Seasonality of gametogenesis ...... 48 Discussion ...... 50 Key results and conclusions from Chapter II: ...... 51 Chapter III: The molecular machinery of gametogenesis in Geodia demosponges (Porifera): evolutionary origins of a conserved toolkit across animals (Paper III) ...... 52 Results ...... 52 DGE analysis ...... 53 Discussion ...... 58 Key results and conclusions from Chapter III: ...... 61 Chapter IV: Oogenesis and lipid metabolism in the deep-sea sponge Phakellia ventilabrum: a histological, lipidomic and transcriptomic approach (Manuscript IV) ...... 62 Results ...... 62 Seasonality and cytological features of oogenesis ...... 62 Lipidomic analysis ...... 64 Transcriptomic analysis ...... 68 Discussion ...... 69 Key results and conclusions from Chapter IV:...... 71 General Discussion and future directions ...... 72 Reproductive strategies in extreme environments ...... 72 Yolk, its role in survival and dispersal of the propagule ...... 75 Lipid content and chemical ecology in Phakellia ventilabrum ...... 76 Molecular studies on the reproductive toolkit of sponges from the deep sea ...... 77 Future studies ...... 78 Acknowledgments...... 81 References ...... 84

Abbreviations

AA Arachidonic Acid ALA A-Linolenic Acid ANOVA Analysis of Variance BUSCO Benchmarking Universal Single-Copy Orthologs CCAMLR Commission for the Conservation of Antarctic Marine Living Resources CE-MS Capillary Electrophoresis–Mass Spectrometry DGE Differential Gene Expression DHA Docosahexaenoic acid DiHETEs Dihydroxyeicosatetraenoic acids DiHETrEs Dihydroxyeicosatrienoic acids DOM Dissolved Organic Matter EBSAs Ecologically or Biologically Sensitive Areas EPA Eicosapentaenoic acid EpDPEs Epoxydocosapentaenoic acids EpETE Epoxyeicosatetraenoic acids EpETrEs Epoxyeicosatrienoic acids FAO Food and Agriculture Organization FDR False Discovery Rate FFAs Free Fatty Acids GC-MS Gas chromatography–mass spectrometry GO Gene Ontology HDoHE Hydroxydocosahexaenoic acids HEPEs Hydroxyeicosapentaenoic acids HETEs Hydroxyeicosatetraenoic acids HETrEs Hydroxyeicosatrienoic acids HHTrE Hydroxyheptadecatrienoic acid HMA High Microbial Abundant HODE Hydroxy-octadecadienoic acid HOTrEs Hydroxyoctadecatrienoic acids ICES International Council for the Exploration of the Sea KEGG Kyoto Encyclopedia of Genes and Genomes KETEs Oxoeicosatetraenoic acids KODE Oxo-octadecadienoic acid LA Linoleic Acid LC-MS Liquid chromatography–mass spectrometry LMA Low Microbial Abundant LPC Lysophosphatidylcholines LPE Lysophosphatidylethanolamine

LPG Phosphatidylglycerol LXA4 Lipoxin A4 LXA5 Lipoxin A5 MAFFT Multiple Alignment using Fast Fourier Transform MFA Mono-unsaturated Fatty Acid MPA Marine Protected Area NAFO Northwest Atlantic Fisheries Organization NMR Nuclear Magnetic Resonance NR Nonreproductive Ospar Convention for the Protection of the Marine Environment of the North-East Atlantic PBS Phosphate-buffered saline PC Phosphatidylcholine PCA Principal component Analysis PE Phosphatidylethanolamine PG Phosphatidylglycerols PGD2 Prostaglandin D2 PGE2 Prostaglandin E2 PGE3 Prostaglandin E3 PGF2α Prostaglandin F2 PGF3α Prostaglandin F3α POM Particular Organic Matter PUFA Poly-Unsaturated Fatty Acid PV Previtellogenic Q-ToF Quadrupole Time-of-Flight RP-LC-MS Reverse phase-liquid chromatography Mass Spectrometry ROV Remotely Operated Vehicle RSEM RNA-Seq by Expectation-Maximization SFA Saturated Fatty Acid SPE Solid Phase Extraction SP_I Spermatic stage I SP_II Spermatic stage II TEM Transmission Electron Microscopy TGs Triacylglycerides TQ-S Triple Quadrupole Mass Spectrometer UNGA United Nations General Assembly UPLC-MS Ultra Performance Liquid Chromatography Mass Spectros- copy Vi Vitellogenic Vi_I Vitellogenic stage I Vi_II Vitellogenic stage II VI_III Vitellogenic stage III VMA Vulnerable Marine Area

Background information

This PhD project was 100% funded by the H2020 EU Framework Programme for Research and Innovation Project SponGES (Deep-sea Sponge Grounds Ecosystems of the North Atlantic: an integrated approach towards their preservation and sustainable exploitation) (Grant Agreement no. 679849) which had a duration of 5 years (March 2016 – December 2020). Under the umbrella of SponGES, there was a collaboration among more than 25 universities, research institutes, environmental non-governmental and intergovernmental organizations between EU, US and Canada. The focus of the SponGES project was the multidisciplinary research on the deep-sea sponge grounds of North Atlantic, which are considered as vulnerable marine areas and despite their high biodiversity and their ecological and biological importance, very little research has been conducted and no appropriate attention has been paid to their conservation so far. Within this context, the consortium had as main goal to accumulate basic knowledge of these ecosystems, such as their abundance, their distribution, and their function, by investigating their role in biochemical cycles, the reproduction of these populations, their dispersal capacity, the connectivity of the populations, and finally their potential for resilience. Acquiring this information, the development of models to predict the impact of anthropogenic activities such as fishing and mining on these environments is possible. Another goal was to further understand their biotechnological potential in biomechanics and biomedicine, highlighting their importance. The ultimate goal of the SponGES project was to develop adequate conservation strategies and influencing major strategic instruments such as EU Mar- itime Strategy for the Atlantic Ocean Area, the Galway Statement on Atlantic Ocean Cooperation, international agreements established to conserve VMAs and EBSAs.

Introduction

The oceans cover more than 70% of the Earth, holding the highest biodiversity, including most types of living forms, from prokaryotes to eukaryotes and from unicellular to multicellular organisms. Whether life began on land or in the oceans is still debated, with a recent hypothesis stating that life appeared in deep-sea hydrothermal vents 3.5–4 billion years ago 1,2. Either way, it is certain that life in the oceans has a long evolutionary history. The approximately 236,000 marine species identified to date 3 are estimated to represent only 50% of the extant marine creatures 4. While this diversity continues to be explored, it has also become clear that marine life provides essential global ecosystem services, for instance by supplying more than the 70% of the at- mosphere’s oxygen, maintaining biogeochemical balances, absorbing vast 5–7 amounts of CO2, regulating temperature, and thus sustaining life on Earth . Furthermore, over 12,000 bioactive compounds with biotechnological potential have been isolated from marine organisms 8–12. All the great mysteries hidden behind even the tiniest marine creatures make the exploration of marine life an attractive and exciting endeavour.

Porifera, a “simple” but rather successful animal phylum A noteworthy component of marine life is the phylum Porifera (sponges), which has attracted scientific attention for almost two centuries because of its complex morphological variability, its ecological importance, its high biotechnological potential and its key position in the Tree of Life 13. Sponges are the foremost representatives of Metazoa (multicellular animals), 14–19, dating back to 500-700 million years ago 20–22. They comprise more than 9300 species to date 23 belonging to four different classes: Hexacti- nellida 24, Calcarea 25, Demospongiae 26, and Homoscleromorpha 27. Among the four classes, Demospongiae holds the highest species richness (~7500 described species), inhabiting both freshwater and marine environments.

13 Sponges are sessile, filter-feeding organisms (apart from carnivorous sponges) with a very simple apical-basal body plan. They possess only two epithelial layers, the outer layer or pinacoderm, and the inner layer or cho- anoderm, which is made up of flagellated choanocytes 27, while other cell types, skeletal structures, and microbial symbionts fill the mesohyl between those two layers 28–30 (Fig. 1). Among the mesohyl cells, archaeocytes are the main totipotent cells of sponges 28–34, being able to differentiate into all other cell types 31,35,36. In addition, choanocytes also retain some pluripotent capacity 32,33. Water is circulated, filtered and expelled through connected water channels, known as the aquiferous system (Fig. 1).

Figure 1. Simplified depiction of the sponge body plan. Water enters from inhalant canals, the ostia, is filtered by the choanocytes, the flagellated cells which form chambers, to absorb any DOM and POM for feeding, and goes out through the central ex- halant canal, the osculum. Several types of cells are spread within the mesohyl. Cop- yright © 2013 John Wiley & Sons and reused after permission (license number: 4938730028387).

Most sponges feed on the filtered bacteria from the water (as well as on DOM and other POM 37, but they also have an intimate relationship with their microbial symbionts (prokaryotic or eukaryotic, heterotrophic or autotrophic), which occupy up to 50% of the sponge volume in some species 38–40. In particular, depending on the amount of the bacterial symbionts within their mesohyl, sponges are categorized as HMA or LMA respectively (Fig. 2) 38–40. Some of the bacterial symbionts are acquired from the water column, during the larval or juvenile stages, but in other cases symbionts are inherited from the maternal sponge through vertical transmission during either female gametogenesis or embryogenesis 39,41–45.

Figure 2. TEM picture of sponge tissue. A. High Microbial Abundant species with mesohyl full of bacteria. B. Low Microbial Abundant species with mesohyl occupied by few bacteria.

Although sponges appeared early in evolution, these simple and sessile organisms have been successful enough to withstand powerful evolutionary forces, mass extinction events and vast abiotic changes, colonizing all aquatic and freshwater ecosystems, reaching even the most remote habitats such as the poles and the deep sea 46,47. The answer on how sponges have managed to not only survive, but thrive in such variable ecological niches is hidden behind several interconnected ecological, chemical and evolutionary features of sponges. Sponges are characterized by high morphological and structural plasticity and adaptability 48. Their microbial community plays a crucial role on these successful traits and reasonably sponges are also called holobionts 38,49–51. First of all, sponges have shown a relative tolerance to abiotic changes, such as warming and acidification 51–55, being able to thrive against other more vulnerable benthic organisms, e.g. corals, in the affected areas and so expand their colonization 56. After all, the totipotent/pluripotent potential of some sponge cell types gives them the ability to regenerate their tissues quite fast in case of damage or infection by pathogens, with even generation of another individual- clone from a small piece of tissue 57–60. Some sponges can also produce asexual cysts, named gemmules, which consist of tightly packed stem cells that can survive in a dormant state during harsh conditions, like very low temperature, anoxia or pollution and other abiotic stressors 61,62. In addition, sponges can explore a range of feeding modes, enabling autotrophy (photosyn- thetic/chemosynthetic in the deep sea), through their microbial symbionts alongside heterotrophy 13. Some species have also developed carnivory as a mode of adaptation, especially in the deep sea 63,64.

15 Part of their survival success may be due to the large diversity of specialized metabolites that they hold. As they are sessile, usually long-living organisms, sponges together with their microbial symbionts produce an incredible array of specialized metabolites which are used for chemical communication, avoidance of their predators, prevention of bacterial infection, inhibition of epibionts, space competition and competition against other animals 13,65,66. Among others, a great variety of lipids 67–73, often of unique structure 68,71,74,75, are found in sponges (Fig. 3), contributing to defense mechanisms, antioxidant activity, membrane fluidity and adaptation to the environment 71,76–80. Several of those lipids and other compounds also exhibit anti-malarial, anti-mycobac- terial, anti-fungal, anti-tumoral, and anti-inflammatory properties with promising biotechnological potential 74,81–85.

Figure 3. Very long chain polyunsaturated fatty acids isolated by the sponge Phakellia ventilabrum (Blumenberg and Michaelis, 2007) A. 6-Bromo-5,9-hexacosadienoic acid (br-26:2) B. 5,9-Hexacosadienoic acid (26:2).

Furthermore, although sponges lack organs and neural systems, recent genomes and transcriptomes from sponge species of all four classes have revealed that they possess a much higher molecular complexity than previously thought. They are equipped with a molecular toolkit that is conserved in other metazoans for processes such as signaling, cell to cell communication, cell type specialization, cell cycling and growth, programmed cell death and innate immunity, and this toolkit can exhibit similar functions in sponges as well 86– 92. Interestingly, the molecular markers of a developed neural system, such as genes coding for post-synaptic densities, pro-neural regulatory proteins and signalling via neurotransmitters, have also been identified in Porifera 86,87,93,94, where they possibly have a role in contraction movements during filter feeding processes 95.

16 Sexual reproduction in sponges: biological and ecological features Despite their apparent simplicity, sponges have developed a panoply of reproductive modes, with both asexual and sexual reproduction allowing them to colonize every habitat even when harsh conditions occur. Some species are hermaphroditic, with simultaneous or sequential hermaphroditism, while others are gonochoristic (separate sexes). Some sponges are oviparous releasing the unfertilized gametes into the water column for external fertilization and others are viviparous, with internal fertilization and brooding of their embryos (Fig. 4) 29,96–98. Gonochoristic sponges are generally oviparous and hermaphroditic ones tend to be viviparous 29,99–101, with only a few exceptions of hermaphroditic, oviparous species 102. Although all the different modes of sexual reproduction are found among the various sponge groups, there is usually one dominant strategy observed within a single order, indicating that the reproductive modes are driven by phylogenetic constraints 99,101.

17 Figure 4. Simplified scheme with the main stages of the sexual reproductive cycle of viviparous hermaphrodite (1-5) and oviparous gonochoric (a-e) species. In the first case, sperm released (1) from a specimen enters to another specimen, through the canals, and fertilizes the oocytes (2) within the tissue. Embryos are developed (3) and brooded inside the sponge until the larva stage. Larvae are released to- and drifted in the water column (4) until settlement to the bottom and development of a juvenile sponge (5). In oviparous species, sperm and oocytes are released in the water column (a) where external fertilization occurs (b) followed by development of embryo (c) and larvae (d) stages. Larvae are drifted in the water column until settlement and development of a juvenile sponge (e). Few viviparous and oviparous species have direct development (6, f) in which the fertilized embryo is directly transformed to juvenile sponge without passing from the larva stage.

The majority of sponges reproduce sexually (either by brooding their embryos or by external fertilization of the gametes), while asexual reproduction occurs occasionally during their nonreproductive period 103–108. Only a number of species of the families Spongillidae, Clionaidae, Suberitidae, Polymastiidae and Tethyidae follow obligatory asexual reproduction 98,109. Embryonic development is also diverse in sponges, resulting in a large variety of exclusively lecithotrophic larval forms: amphiblastula and calciblastula in Calcarea, parenchymella, coeloblastula, dispherula and hoplitomella in Demospongiae, cinctoblastula in Homoscleromorpha, and trichimella in Hexactinellida 110,111. All propagules (embryos/larvae) are lecithotrophic, which means that before their release in the water column they are already provided with all the necessary nutrients for their development up until their benthic settlement. When the nutrient reserves are consumed, sponges settle on the substrate where they transform into adults. These nutrient reserves or yolk of sponge oocytes/embryos can be of lipid, protein and glycogen origin, formed either by homogenous droplets or by a mixture of all these different origins, packed in heterogenous platelets 29,96,97,112. The yolk can be produced by homosynthesis (by the oocyte itself) via the Golgi and endoplasmic reticulum apparatus 29,96,97,112, it can be acquired by phagocytosis directly from the mesohyl, or it can be provided by accessory cells that synthesize the yolk (heterosynthesis). These accessory cells are then either entirely phagocytized by the gamete/embryo, or they dispatch the yolk to them 29,96,97,112. The amount and type of nutrients observed within the sponge female gametes, embryos and larvae is indicative of the dispersal duration and survival potential. Lipids in particular not only provide the propagule with important amounts of energy for active swimming 113–120, but they also serve for buoyancy 121,122. Even though the reproductive ecology of sponges has been significantly investigated 98,123, most of the studies have focused on species from tropical and temperate habitats 103,124–135. In the tropics, most of the studied species are viviparous (brooders), either hermaphrodites or gonochoric, producing large

18 embryos and spermatic cysts in high densities 126,136,137, and a high number of the population is engaged in reproduction. A particular characteristic of tropical sponges is either a continuous or a very long (8-9 months) brooding period, but in the few tropical sponges with a short reproductive period (e.g. Neofibularia nolitangere), more than 50% of the tissue was producing gametes 138. Both oviparity 108,134,135 and viviparity 130,132,139 are commonly found in sponge species of temperate waters, with a more distinct seasonality in their reproductive cycles, lasting on average from 2 to 5 months. The reproductive ecology of sponges from extreme habitats (cold and deep-sea waters) is poorly understood. Very few sponge species from Antarctica 140,141, the deep sea 142,143, or cold waters 144 have been studied to date. This lack of information hinders the appropriate assessment of sponge ecological patterns of reproduction in harsh conditions as well as the understanding of specific adaptations developed by the sponges in these particular habitats.

Sexual reproduction in sponges: evolutionary and molecular insights Sexual reproduction is the ancestral mode of reproduction in eukaryotes 145, and the asexual mode appeared later in some early lineages, which have adopted both modes and alternate between them 146–149. However, lineages with exclusive asexual reproduction are very uncommon in nature 150. Find- ings of meiotic genes in protists, including those involved in recombination 151–153, and their expression during these processes support this statement 154. The advantages of sexual reproduction, e.g. higher variety of genotypes and potential elimination of deleterious mutations resulting in greater overall fitness of a population 155,156, surmount the costs 157, making the sexual mode prevalent in nature. Especially for long-lived species in environments with limited resources, sexual reproduction represents an ecological advantage, since the resulting genetically diverse populations of the same species may exploit different niches or resources within the same habitat, reducing competition among individuals and allowing their coexistence 158. Increased genetic variation obtained by sexual reproduction also aids adaptation to heterogeneous and changing environments 159,160. Sexual reproduction in Metazoa (with the exception of the Myxozoa 161) occurs with anisogamous i.e. female and male gametes 162,163. The process resulting in female gametes is termed oogenesis, while spermatogenesis produces male gametes. Both gametic cells are generated by meiosis 164. Meiosis and genetic recombination are the very essence of sexual reproduction, and they are conserved across the animal kingdom, as well as in unicellular eukaryotes and fungi 153,165,166. During meiosis, the homologous chromosomes in the nuclei of the germ cells duplicate before undergoing recombination, and

19 after two nuclear divisions the haploid gametes are generated 167. The main stages which define the progress of oogenesis in Metazoa are 1) germ cell formation, 2) meiotic arrest, and 3) maturation, growth and vitellogenesis 4) meiosis completion 168,169, while the stages of spermatogenesis are 1) germ cell formation 2) mitosis, 3) two meiotic cycles, 4) chromatin condensation, and 5) flagellum formation and capacitation 170. The germ cell population in more recently evolved Metazoa (e.g. Ecdyso- zoa and Chordata) is found exclusively in the gonads and its only purpose is to self-renew and generate the gametes 171–174. In contrast, in early-diverging Metazoa (e.g. Porifera, Ctenophora and Cnidaria), primordial stem cells give rise either to somatic stem cells or to germ cells during all adulthood 32,33,175– 178. Cytological observations in several sponge species have demonstrated that somatic totipotent and pluripotent cells (archaeocytes and choanocytes respectively) are transformed into germ cells and subsequent female or male gametic cells via transdifferentiation and differentiation processes 29,97,98,125,179–181. His- tological sections furthermore indicate that gametogenesis and embryogenesis in sponges are morphologically very similar to more recently evolved metazoans 98,125,182,183, with the most essential difference being the lack of gonads (i.e. the reproductive organs in which the gametes are generated) in sponges, where the gametes and their accessory cells are surrounded by a relatively complex follicle layer in most animals 184–186. Furthermore, genes involved in determining the germline in many metazoans 187 have been identified in all four sponge classes 32,87,88,188, and they were expressed both in gametes and in totipotent/pluripotent somatic cells (archaeocytes and choanocytes) 88,188, indicating their potential role in germline formation and gametogenesis in Po- rifera. In many Metazoa, common germline genes, such as piwi, vasa, pl10 and nanos, are mostly involved in transcriptional repression of somatic pro- grams, in chromatin reorganization, in meiosis control and in post-transcriptional regulation of several genes during gametogenesis 173. However, information on the molecular regulation of gametogenesis besides germline determination in sponges is not available thus far, making it impossible to evaluate whether sexual reproduction is driven by a conserved molecular toolkit in metazoans, or has undergone specific evolutionary routes in the different metazoan groups.

Sponge research in the last century: techniques and approaches Histological techniques have been used to study sponges for over 150 years, and they still remain an essential tool in the research of these animals, making possible the study of morphological features regarding their taxomony and physiology (including reproduction). Without these techniques it would not

20 be possible to understand the importance of sponges as biological entities, place them in the Tree of Life and trace their evolutionary significance. Regarding their reproduction, the mode, strategies and annual cycles from a large number of sponge species, inhabiting both marine and freshwater environments, have been described 97,98,123, revealing important ecological and evolutionary implications. Some cytological specificities of reproduction have also been studied under the microscope in sponges, including the somatic cell origin of gametes, the nuage, which flags germ cells in other metazoans, and the whole progress of spermatogenesis until the formation of the sperm flagellum. In the last decade, “omics” techniques have revolutionized the life sciences, as the generation of large amounts of data from any organism of interest has enhanced the understanding of various molecular processes and provided new insights into the fields of molecular biology, pharmacognosy and medicine 189. Prior to the broad use of these techniques, sponges used to be excluded from evolutionary and functional studies, and only very scarce molecular data was available for them. However, Next Generation Sequencing (NGS) opened the door to construction of de novo genome or transcriptome reference datasets from previously unexplored, non-model organisms. Since the advent of NGS, several genomes and transcriptomes generated from sponge species belonging to all four classes have revealed the molecular complexity of this phylum and have reinforced the evolutionary link of sponges with other Metazoa 90,190. For instance, genomic data have uncovered that many major metazoan gene families are conserved in sponges, providing a better understanding of the origin of those genes 86,88,191–193, while transcriptomic studies have attempted to define the presence and functional role of these conserved genes, thus correlating them with specific anatomical features and biological processes 87. Further comparative transcriptomic analyses have focused on understanding the molecular responses of sponges under environmental changes, thus providing important ecological insights 55,194–196. Large scale proteomic and metabolomic analyses, using GC-MS, LC-MS, CE-MS, and NMR techniques, have also been applied to sponge research. Proteomes and metabolomes/lipidomes give a snapshot of the peptides, proteins or small molecules present in a biological system, allowing their identification and quantification, and they reflect the instant response of the biological system to various physiological and environmental changes 189,197. Examples of proteome studies in sponges are limited but include a study that identified proteins responsible for biomineralization (Germer et al., 2015) and a comparative analysis under exposure to heavy metal stress 198,199. Comparative metabolomic studies have been conducted to understand the overall metabolic composition and variability of sponges in different physiological stages and under exposure to different temporal and spatial conditions, further leading to the discovery of new substances with pharmaceutical interest 200,201. Lipid analysis with GC-MSD has been

21 previously conducted, in combination to available genomic data, on the sponge Amphimedon queenslandica 202 to explore potential chemical biomarkers that are synthesised or modified by the sponge 203. Other recent lipid studies have targeted specific lipid groups (e.g. very long chain fatty acids or phospholipid-based fatty acids), to understand the interaction between bacterial lipid precursors and the construction of sponge lipids in cold water, deep-sea species 204,205. Further comparative lipidomic studies have been previously performed in sponges and have focused on linking fatty acid content and other lipid classes to the physiological state, energetic demands and variation on metabolic rates between deep-sea and shallow water sponges 72,206. Although several evolutionary, biochemical and ecological questions have been explored in sponges during recent years, information regarding several aspects of one the most primitive biological processes in sponges, namely sexual reproduction, is missing. Only very scarce data available describing common reproduction-related genes (germline and sex determination genes) in sponges 32,87,188 are available and biochemical approaches studying the metabolic changes and energetic demands during their reproduction are completely lacking.

Ecological success of sponges in “extreme” habitats Sponges, with a cocktail of beneficial characteristics (complex chemical ecology, variant reproductive strategies, acquired molecular mechanisms for response, complex symbiotic relationships with microbes, and high morphological plasticity, to name a few), have been adapted to almost every aquatic habitat (as well as occasionally to survival outside water for short or longer periods of time). Sponges are the main representatives of the Antarctic benthos, dominating both shallow and deep habitats 46,207–212 (Fig. 5A). Their dominance could be attributed to hydrographic (oceanic currents) and geological conditions (appropriate substrates for colonization) but also to the high amount of silica in the substrate 213, the main skeletal component of sponges of the classes Hex- actinellida and Demospongiae. Those two classes represent a much higher diversity and abundance around the Antarctic peninsula than Calcarea and Ho- moscleromorpha. Hexactinellida are found in the deeper Antarctic habitats 214, while Demospongiae are the dominant class in terms of species richness and abundance (~81%), being present in both deep and shallow regions of Antarc- tica 46. Roughly half of Antarctic Demospongiae belong to the order of Poeci- losclerida and 17% to the order Haplosclerida, while Dendroceratida, Dicty- oceratida and Verongida are scarce, comprising in total 2.5% of the demosponges present 46.

22 Sponges also dominate the deep-sea habitats of the North Atlantic. Large sponge aggregations, also known as ‘sponge grounds’ (Fig. 5B), can be found up to depths of 3000 m on continental shelves and along continental slopes of the Northwest Atlantic (Grand Banks, Flemish Cap, Labrador Slope and Davis Strait) 215,216 and the Northeast Atlantic (Norwegian Sea, Greenland Sea and Western Barents Sea) 47,217–222, with Hexactinellida and Demospongiae being the dominant classes in these areas. On sponge grounds, sponges can represent up to 95% of the total invertebrate biomass 215 and they reach densities of up to 20 individuals per m2 223. Recently, it was discovered that such massive aggregations of sponges in the North Atlantic could be driven by the large availability of silica to make spicules, coming from silicic acid deposited at the sediment from past dead sponges 224. Among the most common demosponges on sponge grounds are species of the genus Geodia, order Te- tractinellida, which are present in great biomass both in the Northwest 215,216 and the Northeast Atlantic 47,217–219,221,222. Fan-shaped sponges of the genus Phakellia are also key species of the boreal deep-sea sponge grounds of the North Atlantic 47,219,225.

Figure 5. A. shallow-water sponge assemblage in Deception Island, Antarctica. B. deep-sea sponge ground with Geodia spp. in Flemish Cap.

Sponges are essential for numerous ecosystem services by providing food, providing shelter for the reproduction of other animals, enhancing biodiversity 219,226–237 and participating in biogeochemical cycles 238–244 among others. The role of sponges in the stability and health of these habitats is so crucial that sponges are used as biomarkers for the assessment of VMAs both in Antarctic waters and other deep-sea habitats 229,245. Some Antarctic and sponge ground species have been explored in terms of their chemical ecology. The Antarctic sponges Kirkpatrickia variolosa and Latrunculia apicalis produce the alkaloids variolin A and discorhabdin G, which alienate their sea star predators 246,247. Dendrilla membranosa produces an isoquinoline pigment 248 that is an anti-fouling agent 247, while the yellow pigment erebusinone produced by Isodictya erinacea is a tryptophan catabo-

23 lite causing mortality to spongivorous amphipods 249. Ianthelline has been isolated from the boreo-arctic sponge ground two-sponge association Stryphnus fortis 250,251 and its epibiont Hexadella dedritifera 252, and has cytotoxic/antitumor and antifouling activity 250,251. Furthermore, brominated diketopipera- zines, or barretins, and disulfide peptides, or barrettides have been isolated from the boreo-arctic deep-sea sponge Geodia barretti, showing antifouling activity (both) and further antioxidant and anti-inflammatory properties (bar- retin) 253–255, making them promising drug leads.

The necessity for conservation of Antarctic areas and deep-sea sponge grounds The importance of Antarctic habitats and the deep-sea sponge grounds from both an ecological and biotechnological perspective combined with the presence of intensive anthropogenic activities that disturb these areas, make their conservation a priority for all international organizations. The first MPA was established in the Ross Sea in Antarctica in 2016 by CCAMLR and more MPAs are planned for East Antarctica, the Weddell Sea, and the Antarctic Peninsula, including areas that are high in sponge biomass. In the North At- lantic, the sponge grounds have been specifically designated as ecosystems in need of high protection, and since 2007, many of these areas have been de- clared as vulnerable, named VMAs 256–258. In most areas with dense sponge biomass in the Northwest Atlantic, bottom trawling has already been banned by NAFO 259–261 and there is a continuous interest for better understanding of these ecosystems’ dynamics in order to predict the impacts of possible habitat disturbances 261,262. However, there are still many areas lacking protection 263. Essential information is missing regarding the biology of Antarctic and North Atlantic sponges in order to develop proper and more adequate conservation strategies. The various dominant species, representing different sponge grounds, have distinct resilience and recruitment capacities. Basic research on the dispersal capacity, gene flow and connectivity patterns of the different populations is necessary. The reproductive strategy of a species can provide a starting point for the development of predictive models addressing these research gaps. For instance, the spawning period and the fecundity rates of a species, combined with oceanographic currents, are essential inputs to predict propagule dispersal.

24 Aims and Significance

As part of the EU SponGES project, the overarching goal of this thesis is to explore the reproductive biology of several demosponge species from the shallow waters of Antarctica, and the deep-sea sponge grounds of the North At- lantic Ocean, using a multidisciplinary array of techniques: light and electron microscopy, transcriptomics, proteomics, and lipidomics. Indeed, an important aim of this thesis is to generate molecular tools that can be used for a further ecological, molecular and chemical exploration of these sponge species, which have become attractive candidates to study because of their successful strategies to thrive in these “extreme” habitats.

The specific aims of this thesis are: • To describe the reproductive biology of six species from the shallow waters of Antarctica and compare their reproductive strategies in order to assess patterns of adaptation to- and coexistence in- these environments (Chapter I). • To study the seasonality and reproductive activity of five North Atlantic deep-sea species from the genus Geodia, with a focus on several aspects of their reproductive strategy such as reproductive mode, gamete features, type of yolk, and fecundity rates (Chapter II). • To understand the molecular regulation of the gametogenesis in the same five Geodia species, by applying a comparative transcriptomic and proteomic analysis (Chapter III). • To assess the reproductive biology and its molecular regulation in the deep-sea species Phakellia ventilabrum, with a special focus on the comparative lipidomic analysis and the relevant activated pathways in female individuals (Chapter IV).

By analyzing the reproductive timing and the reproductive strategies of the above species, this study can provide essential information for understanding the adaptation of these species to their habitats, the survival rates and fitness of the species in these vulnerable areas, with the potential to extrapolate information for the conservation of these marine habitats. Moreover, sponges also represent a key lineage in the evolutionary tree of life. Even though sponges are among the first animals on Earth, little is known about the molecular mechanisms that regulate patterns of their sexual reproduction. The comparative

25 transcriptomic analysis in specimens under gametogenesis can reveal the molecular toolkit that is activated during this process in sponges, and thus can give substantial information about the origin of the molecular regulation of gametogenesis in multicellular animals. Furthermore, the generation of transcriptomic data from several deep-sea sponge species, can enrich the availability of resources that can inform a broad range of phylogenetic, metabolomic, and molecular biology studies. Finally, the comparative lipidomic analysis in female sponges under oogenesis can give a clue on the type of lipids used for yolk, which have further ecological implications on understanding the dispersal capacity and the survival of the propagules, but it is also crucial for understanding the lipid metabolism of sponges during these energetically high demanding processes. The lipidomic data, describing the whole lipid range found in P. ventilabrum, can also be used in further studies related to the chemical ecology of this species, with a potential for biomarker exploration.

26 Material & Methods

Collection of the studying material The collection of fresh sponge material and the proper and immediate fixation of the samples was an important part of this thesis as it was essential to avoid any possible changes in cellular osmolarity, RNA degradation or lipid oxidation, in order to obtain high quality electron microscopy images and extract good quality RNA, and lipids for the further analyses. Sponge samples for the analyses of the first chapter (Paper I) had been previously collected by SCUBA diving on rocky outcrops at 15 m depth during several expeditions (January 2011, February 2013, and January 2016) in Deception Island, South Shetland Islands, Antarctica. Sponge samples from five species of genus Geodia and the species Phakel- lia ventilabrum 264, used for the analyses in the rest of the chapters (Paper II- IV), were collected during several expeditions, on the Swedish (May 2016 and end of March-beginning April 2019) and Norwegian coasts (September, 2016), and also in the boreo-arctic region of the North Atlantic (Sula reef in Norwegian Sea, Schulz Bank in Greenland Sea and Tromsøflaket in western Barents Sea), all supported by the SponGES project. Samples were collected at various depths, from 80 to 3000 m, with either an ROV or an Agassiz trawl. On board, for each specimen collected, six cubes of sponge tissue (~5 mm3 each) from three different parts of the sponge were fixed in either 4% formalin buffered in seawater for light microscopy (Paper I) and/or in 2.5% glutaral- dehyde solution in 0.4M PBS and 0.34M NaCl for light and electron microscopy (Papers I-IV), and stored at 4°C. For transcriptomics, another three pieces from each specimen were collected and placed in 0.2 mL RNAse-free Eppendorf tubes with RNAlater™ Stabilization Solution (Thermo Fisher Sci- entific), stored for one day at 4°C and then placed at -20°C on board and at - 80°C in the lab until further processing (for the samples of the Papers III, IV). Furthermore, for the proteomic analysis one small piece (~5 mm3) and for lipidomic analysis, one large piece from each specimen were flash frozen in liquid nitrogen, transported on dry ice and stored at -80°C (Papers III, IV). Additionally, more samples from Geodia spp., collected in the Hordaland coast Norway (May, 2017), in Svalbard (September, 2011) and Rockall Trough, Rosemary Bank Seamount, Scotland (September, 2011/2012/2015/ 2016) from other scientific expeditions and years and fixed in 4% formalin buffered in seawater or 96% ethanol, were sent to the Natural History Museum

27 and further processed for the histological study included in the second chapter (Paper II).

Histological Analysis Histological analysis was conducted mainly in the first, second and fourth chapters (Papers I, II, and IV), using the protocols described below in order to identify the gametes in the sponge tissue, quantify the reproductive output, and to describe in detail the characteristics of the reproductive elements in Antarctic species, Geodia spp. and Phakelia ventilabrum. In the laboratory, small fractions from different parts of the fixed sponge tissue were rinsed in buffer (0.4M PBS + 0.6M salt) to remove the fixative. A crucial step for the histological preparation was the overnight incubation in hydrofluoric acid 4% in order to remove spicules of silica origin from their skeleton and ease the further sectioning.

Light Microscopy After the hydrofluoric acid incubation, samples were subjected to an ethanol series dehydration (50-70-96-100%) and then embedded in paraffin overnight at 60ºC. Sections of 5 μm in thickness were obtained with a HM325 micro- tome (ThermoFisher Scientific) from the paraffin-embedded blocks. Two staining protocols were used, the standard Harris’ Hematoxylin and Eosin and the Methylene blue staining protocols, and images of the sections were obtained with Olympus microscope (BX43) with a UC50 camera. Calculations of the reproductive features (size, area, abundance) were conducted with the CellSens image analysis software (Olympus). More than 40 sections were to- tally obtained for each individual to screen for the presence of gametes/embryos in the sponge tissue and confirm some basic reproductive characteristics for each studied species (synchronous/asynchronous development; gonochorism/hermaphroditism; oviparity/viviparity).

TEM After the specification of the reproductive status for each specimen, previously fixed tissue fractions from the reproductive specimens, were prepared for TEM. During this preparation process, a post-fixation step in 2% osmium tetroxide, for two hours at 4º C, is necessary to for the tissue to obtain a heavy metal staining. LR White resin (agar Scientific) was used for the embedding of the tissue and ultrathin sections of approximately 60 nm were obtained with an Ultracut Reichert-Jung ultramicrotome and then stained with 2% uranyl acetate/lead citrate 265. TEM images were taken with a Hitachi transmission electron microscope (H-7650).

28 Transcriptomic Analysis Transcriptomic analysis was conducted in the third and fourth chapters (Pa- pers III, IV) to identify the genes expressed in female and male sponges of the genus Geodia and females of the P. ventilabrum species during gametogenesis, when compared to nonreproductive sponge specimens. The workflow was exactly the same for all species and samples, and can be followed below:

RNA extraction and library preparation The mRNA was isolated by an initial extraction of total RNA with standard TRIzol™ Reagent (ThermoFisher Scientific) protocol and a further purifica- tion with the Dynabeads mRNA kit (ThermoFisher Scientific). The quality of the mRNA was assessed with NanoDrop 2000 (ThermoFisher Scientific) and the accurate quantity with Qubit™ RNA Assay kit (ThermoFisher Scientific). The cDNA libraries were constructed with either the kits TruSeq v2 or Scriptseq v2 RNA Library Prep kit (Illumina), according to the manufacturer’s instructions, using the maximum quantity of mRNA allowed, which was 50 ng of starting material. The quality of the libraries was checked with an Ag- ilent TapeStation 2200 system (Agilent Technologies) and the quantity with Qubit™ dsDNA HS Assay kit (ThermoFisher Scientific). The sequencing was conducted with an Illumina NextSeq 500 platform at the Sequencing Facilities of the Natural History Museum of London (Research Core Labs), using a paired-end read strategy (bp length: 2x150 bp).

Assembly and Annotation A quality control check with FASTQC was conducted on the raw reads (http://www.bioinformatics.babraham.ac.uk/services.html) and then those were trimmed with Trimmomatic 266 to remove the adaptors and any bad quality short reads. After the filtering, the paired reads of all the individuals of a given species were pooled together and used for the construction of the transcriptome assembly. A de novo assembly for each species was conducted with the Trinity package v2.4 or v2.8.4 267. The completeness of the assembly was checked with BUSCO V2/3 against Metazoa 268. Some common statistics of the assembly were also assessed, such as the Nx length, GC content, the number of assembled bases, and the percentage of the alignment of paired-end reads to the assembly with bowtie2 269. Quantification of the transcripts was further conducted with the alignment-based method RSEM 270. For the annotation of the transcripts, the most recent Swissprot database for Metazoa was used to obtain the blast hits of the transcripts, with the help of Diamond 271 and using a cut-off e-value of 1e-5. The sequences with blast hit were then subjected to further annotation with Gene Ontology (GO) terms using the functional annotation pipeline in Blast2GOPRO 272.

29 DGE analysis The edgeR package 273,274 in R (R Core Team 2017) was used for the DGE analysis. Pairwise comparisons were conducted between individuals belonging to different reproductive groups (female, male, nonreproductive) as identified previously with histological analyses. The parameters for the selection of differentially expressed genes were FDR ≤ 0.01 and 2-fold change differences. In some cases, no biological replicates were available for some reproductive groups and so dispersion 0.1 was used for the conduction of DGE analysis with edgeR, as indicated by the Trinity package.

GO Enrichment analysis and KEGG annotation The upregulated genes from the pairwise comparisons with their associated GO terms were subjected to GO enrichment analysis with the Fisher’s Exact Test in Blast2GOPRO platform using a p-value 0.05 (Paper III, Paper IV). With this analysis, genes were grouped according to their function into bigger functional categories, which represent the main underlying processes. Infor- mation on the molecular function, cellular component and biological processes of those genes was retrieved. In the same platform, and based on the upregulated genes, a KEGG annotation analysis was conducted for the chapter IV (Paper IV) in order to detect the regulated lipid metabolism biochemical pathways during oogenesis in the sponge P. ventilabrum.

Proteomic analysis A proteomic analysis was performed in the third chapter (Paper III) to compare the presence of peptides expressed in individuals during oogenesis and spermatogenesis vs nonreproductive specimens of Geodia spp. Proteins were extracted from freeze-dried sponge material (~100mg wet weight), following the protocol described in Viarengo and coworkers 275, and previously tested on sponges 199. After the extraction, protein bands were observed with Coomassie staining and then they were cut, and digested 276. For the digestion, the solution based on Tris-HCl and CaCl2 with the enzymes of trypsin or chymotrypsin (100 mM Tris-HCl pH 8, with 10mM CaCl2 and 60 ng/μl trypsin or chymotrypsin at 5:1 protein enzyme (w/w) ratio) was used. First, the dried gel bands in Eppendorf tubes stayed within the solution in ice for 2h. Afterwards, they were incubated at 37°C (trypsin) or 25ºC (chymotrypsin) for 12 h. Desalination of the digested protein material occurred with a OMIX Pipette tips C18 (Agilent Technologies) previous to protein separation. Separation of proteins was performed with RP-LC-MS/MS analysis (dy- namic exclusion mode) in an Easy-nLC II system coupled to a Linear Trap Quadripole-Orbitrap-Velos-Pro hybrid mass spectrometer (ThermoFisher

30 Scientific). Peptides were eluted using a 180-min gradient (Solvent A: 0,1% formic acid in water, solvent B: 0,1% formic acid, 80% acetonitrile in water). The isolated peptides were first identified with the software PEAKS Studio 10 search engine (Bioinformatics Solutions Inc.) 277,278. The FDR for peptide spectrum matches was set to 0.01. The peptide sequences were mapped against the transcriptome assembly of each species, which was used as reference database in order to link the proteomic with the transcriptomic data.

Phylogenetic Analysis A phylogenetic analysis was conducted in the third chapter (Paper III) for several target genes important for reproduction: Sox genes and fibrillar and non–fibrillar collagen sequences. Since many different sequences were found in the transcriptomes of Geodia spp., some of them upregulated in one or another condition, it was necessary to understand if there was homology of those with the sequences of other Metazoa to further understand whether they may be involved in the same functions in sponges. Other sequences for the alignment were retrieved from previous studies and the NCBI databases and the alignment was done with MAFFT v7 279. Phylogenetic analyses were performed with Maximum Likelihood analysis 280. Further details of the analysis are described in Paper III.

Lipidomic Analysis Lipidomic analysis was performed in the fourth chapter (Paper IV) to analyse the lipid structures playing a role during oogenesis in the sponge Phakellia ventilabrum.

Lipid extraction and separation A quantity of 52 ± 2 mg of powder from previously freeze-dried sponge tissue was used for the lipid extraction from thirteen samples (see Paper IV for details on the samples extracted). The standard Bligh and Dyer protocol with chloroform/methanol 1:2 (CHCl3/MeOH) was used for the extraction. Two consecutive overnight extractions were conducted and the total amount of organic phases (1 mL) were pooled together and dried in a vacuum for ~ 1 hour. Finally, the pellet was resuspended in 50:50 ACN/IPA in a volume of 200 µL. For the lipid separation, an Acquity- UPLC (Waters) coupled with Synapt G2S Q-ToF (Waters) operated in both positive and negative mode. A BEH (Eth- ylene Bridged Hybrid) C18 column (1.7 µm, 2.1x150 mm) was used and solvents of water/acetonitrile/isopropanol in ratio 40:30:30 (v/v/v) and acetonitrile/isopropanol in ratio of 40:60 (v/v), both including 5 mM of ammonium

31 formate. The gradient started with 99% solvent A: 1% solvent B and changed linearly every minute reaching the 100% of solvent B in min 20.1. The flow rate was 0.275 mL min-1. A quality control (QC) was also prepared by mixing 10 µL from the resuspended organic phase of each sample and several injections of the QC occurred at the beginning and at the end (four injections each time) as also during every five sample injections in the machine in order to monitor the progress of the isolation and the performance of the machine.

Oxylipin extraction and separation The protocol of oxylipin extraction was adjusted by the protocol of Kolmert and co-workers 281. A 31 ± 1 mg of powder from freeze -dried sponge tissue, was used for the oxylipin extraction. This quantity was previously tested and considered an appropriate for signal detection, avoiding in parallel any signal saturation. The extraction of oxylipins was conducted using methanol as a solvent. At the beginning, samples in 2 mL methanol were sonicated for half an hour to facilitate the extraction, and then the extract was further subjected to solid phase extraction to reduce any background signals from the analysis. In detail, the solvent, from the supernatant (1.5 mL) obtained after centrifugation (15 min at 3,000 g), was first evaporated with nitrogen (TurboVap® LV) to concentrate the volume to 300 μL and then 2.7 mL of Solid Phase Extraction buffer (pH 5.6) was added to the extract in Pyrex extraction tubes. During the SPE, the extract was first washed with 10% of methanol and then eluted with 100% methanol, to obtain the eluent of interest. The solid phase extraction was performed automatically by ExtraheraTM. The eluent was evaporated again with nitrogen and diluted in 90:15 MeOH/H2O (v/v) and the final extract was filtered through a 0.1 μm nylon filters (Amicon Ultrafree-MC) by centri- fuging at 5,000 g for 3.5 min at 4oC. The final recuperated volume after filtra- tion was 80 μL. The lipid profile was detected by injection of 8 μL of the reconstituted extract onto a BEH C18 column, 2.1x150 mm, 1.7 μm (Waters, Milford, USA) and the chromatographic separation and lipid quantification were acquired by Acquity UPLC coupled to TQ-S instrument (Waters, Mil- ford, USA), operated in negative mode. The column used was again a BEH column (C18, 2.1x150 mm, 1.7μm, Waters) and the different solvents of the mobile phases were water with 0.1% acetic acid and 90:10 acetonitrile/isopropanol. The gradient started with the first solvent to 90% and changed linearly, to reach the 35% in min 15 (65% in min 3.5, 60% in min 5.5, 58% in min 7, 50% in min 9). The column temperature was 60ºC and the flow rate was 0.5 mL min-1.

Data pre-treatment For the identification of lipids, a database of lipids from Lipid Maps was first used 282. Afterwards, an in-house database based on the molecular formulae of

32 the adducts was used for comparisons of the m/z values from the fragments and the parent ions using the package Rdisop in R 283 (R Core Team, 2017). Regarding the quantification, the lipid signals were considered as the areas of the chromatographic curve of the peaks and their normalization was extracted by dividing the signals of the studied samples with those of the quality control sample. Further normalization of the signal for each specimen was held, based on the quantity of the extracted sample. Precise quantification of the oxylipins was performed. For that, a calibration curve was used with 11 external points, spiked with internal standards, with 1/X weighting, signal-to-noise >5 and <25% residuals at limit of quantification. TargetLynx (Waters, Milford, USA) was used for calculating the concentration and the quantity of the oxylipins was normalized according to the initial amount of sponge tissue used for the oxylipin extraction.

Statistical Analyses First, the factor of the location/month was assessed to understand if it significantly affected the signal of the identified lipids. Multiple t-tests were performed for each lipid group between individuals of the two locations/months with an adjusted p-value (FDR) ≤ 0.05, independently of the reproductive status, and the results were visualized with PCA and boxplots. Secondly, an ANOVA was conducted in order to test the variations of each lipid group among individuals with different reproductive status (NR, PV, Vi), consider- ing the factor location in the analysis. For the analyses, each lipid group from the lipidomic extraction was separated according to the number of bond satu- rations (SFA, MFA, PUFA). All the statistical and graphical analyses were conducted in R (R Core Team 2017).

33 Chapter I. Insights into the reproduction of some Antarctic dendroceratid, poecilosclerid, and haplosclerid demosponges (Paper I)

Results The reproductive activity and strategy of six demosponge species belonging to the orders Dendroceratida (Dendrilla antarctica 284), Poecilosclerida (Phor- bas areolatus 285, Kirkpatrickia variolosa 286, Isodictya kerguelenensis 287), and Haplosclerida (Hemigellius pilosus 286, Haliclona penicillata 288), commonly found in shallow waters of Antarctica, were examined with histological techniques. All studied species were reproductive during the Austral summer, in particular from January through to the end of February. Unfortunately, no sampling was performed in the rest of the months of the year, due to logistic and weather constraints (the Spanish Antarctic bases, which were primarily the collection points, are only operational in summer). In all the studied species, only female reproductive features were identified, and in most of them, both oogenesis and embryogenesis were studied, with the exception of P. areolatus and I. kerguelenensis where only embryos were found.

Oogenesis Variation in the developmental stage of the oocytes was detected among species from the different orders or even within the same order. Vitellogenic oocytes (~34 µm in diameter) (Fig. 1), with a thin follicle layer (Fig. 2A) were close to the canals in the dendroceratid D. antarctica. In the poecilosclerid K. variolosa, previtellogenic oocytes (~21 μm in diameter), without any follicle layer, were also found close to the canals (Figs. 1, 2B). Within the order Hap- losclerida, in H. pilosus only previtellogenic oocytes were found close to the canals (Figs. 1, 2C), and these were larger (~80 μm in diameter) than the vitellogenic oocytes of H. penicillata (~20 μm in diameter) that were located within the mesohyl (Figs. 1, 2D). In both species, oocytes lacked a follicle layer.

34 Figure 1. Average size (μm) and standard deviation of oocytes (squares) and embryos (circles) found in the studied species.

Figure 2. Light microscopy observations of oocytes in the studied species. A. Oo- cyte of D. antarctica with cellular follicle (white arrow). B. Previtellogenic oocyte of K. variolosa close to a canal (white arrow). C. Previtellogenic oocyte of H. pilosus close to a canal, phagocytosing nurse cells (white arrow). D. Vitellogenic oocytes of H. penicillata.

Embryogenesis Embryos were found in specimens of all six studied species (Fig. 3). D. antarctica had synchronous development, as all the embryos in the sponge mesohyl were at the same stage (140 µm in maximum diameter). The cleavage was equal and the resulting blastomeres had a size of 20–30 µm in diameter (Figs. 1, 3A). Embryos were surrounded by a cellular follicle (single layer, 4 µm in

35 width) (Fig. 3A). Regarding the order Poecilosclerida, in the species P. aerolatus and I. kerguelenensis only late stage embryos were detected, indicating synchronous embryonic development (Figs. 1, 3B). On the other hand, in K. variolosa embryos of early (4–8 cells), mid (200 μm) and late (300 μm) stages were observed either within the mesohyl or close to the canals (Fig. 3C and insert). The embryos of P. aerolatus measured ~180 μm in diameter (Figs. 1, 3B), while the embryos of I. kerguelenensis were the largest found in all studied species and measured 1 mm in largest diameter (Fig. 1). Embryos of all the three species of Poecilosclerida had unequal cleavage with posterior micromeres of approximately 5–10 μm (Fig. 3B, D). The size of anterior macromeres, however, varied from 35 μm in P. areolatus (Fig. 3B), to 20–50 μm in mid and late stage embryos of K. variolosa (Fig. 3E) and 30–60 μm in I. kerguelensis (Fig. 3F). A follicle cellular layer surrounded the embryos of P. areolatus and K. variolosa (Fig. 3B, D), while no follicle was observed in the embryos of I. kerguelenensis (Fig. 3F). Both haploscerid species had asynchronous development of gametes in their tissues. In H. pilosus, embryos in different stages were observed with early stage embryos measuring on average 200 μm (Fig. 3G) while later stage embryos grew to 340 μm (Fig. 3H). The cleavage of the embryos was equal, and blastomeres ranged from 15 μm to 50 μm in largest diameter in the late embryos (Fig. 3G–H). In H. penicillata, both oocytes and embryos were observed within the tissue. The embryos (75–100 μm in diameter -Figs. 1, 3I), clustered in brooding chambers of 30–60 embryos each (Fig. 1 in Paper I) and each embryo had micro (~2 μm in diameter) and macromere cells (~20–30 μm in diameter) (Fig. 3I). In both species of Haplo- sclerida, a thin layer of follicular cells was found around the embryos (Fig. 3G–I).

37 Figure 3. Light microscopy and ultrastructural observations of the embryos in the studied species. A. Embryo of D. antarctica below the pinacoderm with an obvious follicle membrane (white arrow). B. Em- bryo of P. aerolatus with micromeres (mi) in the posterior and macromeres (ma) in the anterior part. The follicle layer is also depicted (white arrow). C. Mid- and late-stage embryos (e) of K. variolosa located close to the canals, while early-stage embryos found in the mesohyl (Insert). D. Micromeres (bl) from the posterior part of a K. variolosa embryo, filled with lipids (li) and protein yolk (pp), also indicated in a close-up of the blastomere (Insert). E. Anterior macromeres (bl) of a K. variolosa embryo, with heterogeneous yolk platelets (y). F. Embryo of I. kerguelenensis showing large macromeres (ma) and micromeres (mi), containing yolk of lipid (li), protein (pp), and glycogen origin. G. Early embryo of H. pilosus and its follicle layer (white arrow). H. Late embryo of H. pilosus, and its follicle layer (white arrow). I. H. penicillata embryo showing blastomeres (bl) and engulfed amoeboid cells inside (ec). Follicle is depicted (white arrow).

Yolk formation The yolk in the oocytes and embryos of the studied species was always heterogeneous, mainly composed by protein and lipid droplets but also glycogen (Fig. 4A–F). In all species, the yolk was formed by heterosynthesis, with the help of nurse cells which were found in close proximity to the oocytes and embryos (Fig. 4F–J). These nurse cells contained heterogenous yolk and extended pseudopodia to capture nutritional elements (Fig. 4G). In Poeciloscle- rida and Haplosclerida, nurse cells were ingesting bacteria and diatoms that were processed and transformed into heterogeneous yolk (Fig. 4G–H). In Hap- losclerida, nurse cells were also found within the oocytes and embryos after phagocytosis (Figs. 2C, 4F). In some species, homosynthesis of yolk formation was also observed. For instance, the blastomeres of embryos from D. antarctica and P. areolatus had well-developed Golgi and endoplasmic reticulum apparatus forming lipid yolk (Fig. 4B–C).

38 39 Figure 4. Yolk formation. A. Blastomere (bl) of D. antarctica embryo with obvious nucleus (n), and with heterogeneous yolk platelets (y) of protein (pp) and lipid (li) content. B. Nucleus (n) of a blastomere from D. antarctica embryo, with the double membrane (nm) visible. Mitochondria (m), the Golgi apparatus (g) and lipid yolk platelets (li) around the nucleus. C. Blastomere of P. areolatus embryo with nucleolated (nu) nucleus (n), and a well-developed rough endoplasmic reticulum (rer) and Golgi apparatus (g). Around these structures newly formed lipids and larger lipid droplets (li) and glycogen (gl) are observed. D. Micromeres (mi) and macromeres (ma) within the embryo of I. kerguelenensis, filled with lipids (li), protein (pp) yolk, and glycogen (gl). E. Blastomere (bl) of the embryon of H. pilosus with electron-dense cytoplasm (white arrow) fully composed by a great amount of lipid droplets (li) and protein yolk granules (pp). F. Oocyte (oc) of H. penicillata with a distinct nucleus (n), yolk platelets (pp) and lipid droplets (li) in its mesohyl. Nurse cells (nc) are observed in near proximity, with some bacteria (b) and a collagenous layer (co) surrounding the oocytes. G. Close up of nurse cells (nc) in mesohyl of P. areolatus, showing multiple pseudopodia (p), lipid (li) droplets, and glycogen (gl). Different stages of heterogeneous yolk formation (1, engulfment of bacteria (b), 2 digestion and formation of heterogenous yolk). H. Nurse cell in mesohyl of K. variolosa, with engulfed (or produced) material of different sources: unknown material, which could be interpreted as phagocytosed bacteria (b), yolk (y) and glycogen (gl). I. Phagocytosed diatoms (d) within nurse cells of I. kerguelenensis. Bacteria (b) are seen within the mesohyl and outside the nurse cell. J. Nurse cell (nc) of H. penicillata with heterogeneous yolk granules (y), protein platelets (pp) and lipid droplets (li). Glycogen (gl) and numerous mitochondria (m) within the cytoplasm of the nurse cell.

Discussion In this study, all six Antarctic sponge species examined were brooders (embryos developing within the parent tissue) as other members of the same families 96,103,126,127,179,289–296. The fact that only individuals with female reproductive features (oocytes and embryos) were found could be explained by the limited number of specimens collected. However, other members of Den- droceratida 98,297–299, of the Hymedesmiidae family within the Poecilosclerida, and of the congeneric Mediterranean species Phorbas tenacior 103,127,130,179,291,293 have been previously reported as (simultaneous) hermaphrodites, developing male and female gametes within the same tissue and even at the same time on occasion. Given that embryos were already observed within the tissue of all studied species, the absence of male gametes is suggestive of successive hermaphroditism with protandry. Many of the reproductive features and strategies described here are novel not only for Antarctic species, but also for the entire orders in question. For instance, the only other detailed description of oogenesis and embryogenesis of a dendroceratid was conducted in Aplysilla sulfurea 98. Most of the charac-

40 teristics of the oocytes and embryos observed for D. antarctica were previously found in A. sulfurea (presence of follicle around the oocytes and embryos, oocytes formed close to the canals, heterogenous yolk, equal cleavage), the main difference being the synchronous development of oocytes and embryos in D. antarctica as opposed to the asynchronous development in A. sulfurea 98. The synchronous development indicates a single spawning event that can be an adaptation to short favourable conditions in polar areas. Synchro- nous development was also detected in two out of the three poecilosclerid species of this study, P. areolatus and I. kerguelensis, which both had embryos only of late developmental stage within their tissues, suggesting a similar adaptation to D. antarctica. This comes in accordance with observations of sequential development of the different stages (oocytes, embryos and larvae) within the same individual in the congeneric Mediterranean species P. tenacior 130, indicating rather a phylogenetic constraint at least for this genus. Another potential adaptation to cold waters was related to the embryos of I. kerguelenensis, which have an unusually large size, one of the largest ever reported in sponges, and very similar to the embryos of the Antarctic species Isodyctia setifera 289 and Stylocordyla chupachups 140. These observations can be due to phylogenetic constraints 289,300 but also due to gigantism, a common phenomenon in the eggs of marine invertebrates in polar areas, possibly due to the adaptation of lecithotrophic propagules at a prolonged developmental period in cold environments 301. Further studies on congeneric species of other latitudes should be conducted to confirm this pattern. Finally, the embryogenesis described in the haplosclerid species of this study was not unexpected, since studies on other haplosclerid sponges have either reported that brooding embryos are scattered in the mesohyl as observed here in H. pilosus 126,136,302,303, or that embryos accumulate in brooding chambers as in H. penicillata 112,302. By concentrating resources and nurse cells in particular areas of the tissue, brooding chambers may provide energetic advantages and potentially higher reproduction and survival rates to this species. In haplosclerids, asynchronous development, as observed in this study, has been previously reported for the Antarctic haploscerid Haliclona bilamellata 289. Similarly, in haplosclerids, the heterosynthetic formation of yolk by nurse cells and the phagocytosis of intact nurse cells by the oocytes seem to be common characteristics 96,98,112,136,290. Surprisingly, the congeneric Mediterranean species Hal- iclona fulva, had oviparous and gonochoristic traits 108, following very different reproductive strategy than most of haplosclerid species studied so far. The female gametic and embryonic ultrastructural features of these six species were described in detail for the first time in this study, identifying slight differences in the reproductive strategies even of species from the same order. These differences might be special adaptations to allow the coexistence of these species and the formation of high aggregations at the same habitat.

41 Key results and conclusions from Chapter I:

• Reproductive activity is observed in six different sponge species from the Antarctic Peninsula (Dendrilla antarctica, Phorbas areolatus, Kirkpat- rickia variolosa, Isodictya kerguelenensis, Hemigellius pilosus, and Hal- iclona penicillata) during the Austral summer (January-February of different years). • All studied species are brooders with embryos within their tissues. Devel- opment is synchronous in some species, but asynchronous in other species. • In all species, nurse cells are present providing nutrients to the oocytes and embryos, indicating heterosynthetic formation of yolk. • The late embryo of I. kerguelenensis is among the largest (1 mm) ever reported in sponges. • In most species, embryos are scattered in the mesohyl or close to the canals, but interestingly in H. penicillata they are located in brooding chambers.

42 Chapter II: Exploring the reproductive biology of the genus Geodia from boreo-arctic North- Atlantic deep-sea sponge grounds (Paper II)

Results In this study, the reproductive activity and strategy of five species of the genus Geodia (order Tetractinellida) were analysed with histological observations: G. barretti 304, G. macandrewii 304 , G. hentscheli 305 , G. phlegraei 306, and G. atlantica 307. Studied specimens were collected between the beginning of April and the end of September in several locations and different years (Kosterfjord, Swedish coast; Korsfjord and Hordaland coast, Norwegian coast; Rosemary Bank Seamount, Scotland; Sula reef, Norwegian Sea; Schulz Bank, Greenland Sea; Tromsøflaket, Barents Sea).

Histological observations of gametogenesis in Geodia spp. All Geodia species studied here were gonochoric, as oocytes and spermatic cysts were always detected in separate individuals, and oviparous, since no embryos or larvae were observed in the tissue. Gametogenesis was mostly synchronous at individual and population level for most species, mainly with the exception of G. atlantica that showed asynchronous development at population level in Korsfjord (Fig. 1). The cytological features during the progress of gametogenesis were very similar in all studied species (Figs. 2, 3).

43 Figure 1. Average diameter of the oocytes detected per individual and per species population, collected from different locations and months.

At the beginning of oogenesis, PV oocytes had an ameboid shape and a size of 17−20 μm max. diameter (Fig. 2A). They were found almost without any yolk or bacterial symbionts within their ooplasm (Fig. 2B−C), even though bacteria were abundant in the mesohyl of the sponge around the oocytes (Fig. 2B). Although PV oocytes were spread in the mesohyl, in a later developmental stage, Vi oocytes migrated close to the canals, ready to be released into the water column (Fig. 2D). Vi oocytes were larger (>50–60 μm, 150 μm max. diameter), had a rounder shape, and were surrounded by a thick layer of collagen (Fig. 2D−E). They contained a high amount of yolk, mainly of lipid content and scarce protein platelets, and in most of the species these oocytes contained bacterial symbionts acquired by vertical transmission (Fig. 2E−H). Bacteria were phagocytosed by oocyte pseudopods, and stored in single vesicles without any visible signs of digestion (Fig. 2F). Nurse cells were not observed around or within the oocytes, indicating that the yolk within the oocyte was produced via homosynthesis (by the oocyte itself), mostly by the endoplasmic reticulum. Mitochondrial clouds were observed close to the nucleus, with nuage granules present among them (Fig. 2H).

45 Figure 2. Cytological features of oocytes from different studied species. A. PV oocytes (oc) in the mesohyl of G. hentscheli (~12 μm). B. TEM image of PV oocyte (oc) of G. hentscheli with a well-developed nucleus (n) and nucleolus (nu), an ooplasm with many vesicles (v) and around the oocyte, a thin layer of collagen (co) and a large number of bacteria are observed in the mesohyl of the sponge. C. Close-up of the ooplasm of the oocyte from 2B. The empty vesicles (v) are visible, and few lipid droplets (li) have formed. Only very few bacteria (b) can be observed within the ooplasm. D. Vi oocytes (oc) of G. atlantica (~70 μm) accumulated around the canals (ca). Insert. Vi oocytes (oc) of G. barretti (~50 μm) in clusters already released in a canal (ca). E. TEM image of Vi oocyte (oc) of G. barretti with nucleus (n) with chromatin compactions, ooplasm filled with lipids (li) and some proteinic (p) yolk. A thick layer of collagen (co) surrounds the oocyte. F. TEM image of Vi oocyte of G. macandrewii filled with lipid yolk (li), and bacterial symbionts (b) in vesicles. Note the extended pseudopodia (ps). A thick layer of collagen surrounds the oocyte (co). G. TEM image of ooplasm from Vi oocyte of G. hentscheli from Schulz Bank, August with lipid droplets (li) and bacterial symbionts (b). H. Close-up of Fig.1F of G. maca- drewii. The endoplasmic reticulum (ER) can be seen in the periphery of the nucleus, and dense accumulations of mitochondria and associated nuage (ng) granules are present in the ooplasm and close to the nucleus.

Similar to PV oocytes, at the beginning of their development, SP_I spermatic cysts were scattered throughout the mesohyl of male individuals. They had an average size of 20–40 μm in all species, containing only a few cells in early developmental stages, namely spermatogonia with an average size of 5 μm (Fig. 3A). At a more mature stage, SP_II spermatic cysts were observed close to the canals, measuring on average 60−70 μm and containing a large number of cells in different developmental stages (asynchronous development within each spermatic cyst) (Fig. 3B−C). In SP_I cysts, early spermatogonia were in the outer part of the spermatic cyst, while more mature spermatocytes were found in the inner part (Fig. 3C). From primary spermatocytes to mature spermatids, there was a decrease in cell size (from 5 to 2 μm) and an increase in chromatin compaction (Fig. 3C). Primary and secondary spermatocytes were clearly dividing as indicated by the structures of synaptonemal complexes and cytoplasmic bridges (Fig. 3D). Some amoeboid-like cells were observed in the periphery and within the spermatic cyst (Fig. 3D). In the spermatogonium stage, the axoneme structure was internalized (Fig. 3E−F), while in further stages it seemed to be absorbed while the basal body appeared, indicating a synthesis of a de novo flagellum after the spermatid stage. No follicle was observed around the spermatic cyst. Remarkably, a few bacteria were present within the spermatogonia (Fig. 3F), but no bacteria were observed in spermatic cells in further developmental stage.

46 Figure 3. A. Developmental stage I (SP_I) spermatic cyst (sc) in the mesohyl of G. hentscheli. B. Developmental stage II (SP_II) spermatic cysts (sc) of G. phlegraei accumulated around the canals (c). C. TEM image of SP_II spermatic cyst from G. phlegraei with asynchronous development: spermatogonia (s) in the outer part of the cyst, and primary (ps) to secondary spermatocytes (ss), spermatids (sd) and mature spermatids/sperm (sp) with acrosome (ar) in the inner part of the spermatic cyst. D. primary spermatocytes during division, with well- formed synaptonemal complexes (syn) in the nucleus and cytoplasmic bridge (cb), before completed cytokinesis. Insert. Primary spermatocyte with two nuclei and chromatin compaction just after nuclear division. E. Spermatogonium with nucleated (nu) nucleus (n) and internalized axonemal structure of the flagellum (f). F. Spermatogonium/ primary spermatocyte with a distinct nucleus (n), little chromatin compaction, vesicles (v) and few bacteria (b). The axonemal structure of the flagellum (f) is in the interior of the cell.

47 Seasonality of gametogenesis Reproductive activity of all five Geodia spp. was detected between May and September. However, since no samples were available from other months of the year, conclusions on the annual reproductive activity of Geodia spp. are not definite. Both female and male specimens were found in all the species. During late spring-early summer (May-June), most of the females had PV oocytes and males had SP_I spermatic cysts, maturing along the reproductive season (Fig. 1, Fig. 2 in Paper II). However, some species (G. barretti, G. atlantica) also contained mature oocytes during this same period (Fig. 1, Fig. 2 in Paper II), suggesting two reproductive periods for these sponges. Finally, one specimen of G. macandrewii from Svalbard contained previtellogenic oocytes in September. From the above, and given the average time of oocyte maturation, the spawning period of Geodia spp. was estimated to span from May-June to November, depending on the studied area. In regard to the size of oocytes and spermatic cysts, G. hentscheli had the smallest oocytes (30.4 ± 16.9 μm) and spermatic cysts (44 ± 3.4 μm), while G. atlantica had the largest female gametes (95.4 ± 6.8 μm) and spermatic cysts (154 ± 56.8 μm). The density of oocytes was ~ 7.2 ± 3.6 oocytes/mm2 of sponge tissue (Fig. 4). The number of oocytes produced during oogenesis in each species (calculated from individuals with the most mature oocytes in each species using the adjusted Elvin’s formula as discussed further in Paper II), fluctuated from ~4 million oocytes for G. hentscheli to ~116 million oocytes for G. macandrewii (Fig. 5). In most species the number of produced oocytes was comparable to their volume of the sponge (~ 10 times), with the exception of G. macandrewii in which the number of oocytes produced was disproportionately large (~30 times) (Fig. 5). Finally, at population level, 60 to 90% of the individuals were reproductive in most species. However, variations within the same species were found, depending on the sampling month (further analysed in Paper II). The number of females was either larger or equal to that of males in all Geodia spp. except for G. phlegraei, in which males were three times more abundant than females.

48 Figure 4. Average number of oocytes /mm2 calculated in specimens with premature and mature oocytes from of each species.

Figure 5. Diagram depicting the theoretical average volume of each species on the left y axis and the calculated number of the released oocytes of each species on the right y axis.

49 Discussion The Geodia spp. examined in this study showed similar reproductive strategies and cytological features to other species of the genus or even the suborder Astrophorina 143,308–313, suggesting the presence of phylogenetic constraints behind their reproductive strategy. In particular, gonochorism and oviparity was described for all Geodia species in this and other studies, with oocytes being derived from archaeocytes, and spermatic cysts from choanocyte chambers. The spermatogonia resorbed the choanocyte flagellum (axoneme structure was observed within the cells) in order to multiply, and spermatocytes in the stage of spermatids generate a de novo sperm flagellum. Some other common characteristics describing the oocytes of Geodia spp. were the homosynthesis of yolk by the oocyte, the high lipid yolk, lack of nurse cells, vertical transmission of the symbionts, and an external collagen layer surrounding the female gametes but not the male gametes. All the Geodia spp. studied here, had an overall high reproductive effort: the density of oocytes and the number of individuals/population engaged in reproduction were similar or higher to those observed in oviparous sponges from temperate and tropic habitats 133,314,315. This pattern, could explain the dense aggregations of these sponges in these areas. The positive correlation of reproductive output to body size has been considered as the generalized para- digm 156, and has been previously observed in sponges 316,317. The bacterial symbionts within the oocytes were not digested, so they were not directly used for nutritional needs at that stage. However, the vertical transmission of symbiotic bacteria, might act as a potential nutritional boost, either via their direct digestion or via uptake of their metabolic by–products by the sponge during later developmental stages or settlement. Although basic reproductive patterns were common in all the studied Geo- dia spp. here, variations in their reproductive traits were found, such as a slight shift in the timing of gametogenesis, in the size and density of the gametes generated, in the amount of yolk produced or in the proportion of individuals engaged in reproduction. These slightly different adaptations may permit the different species to coexist in the same habitat, and to efficiently exploit the few resources that are offered in these oligotrophic environments 142. Fluctu- ations in reproductive timing were detected even within the same species from different locations or year of collection, and these could be also explained by differences in environmental factors, and more certainly by variations in the availability of food sources 318,319. This phenomenon has been previously observed in populations of the deep-sea coral Lophelia pertusa 320, which demonstrated variability in reproductive timing, duration of reproductive cycle and several other reproductive traits due to differences in nutrient availability among locations 321,322.

50 Deep-sea habitats are oligotrophic and they generally face stable environmental conditions all year round. The reproductive cycle of the organisms living in these habitats is determined by impactful seasonal changes, such as the enrichment of the seafloor with nutrients after a primary production event in surface waters 323,324. Reproduction often occurs during summer-autumn, following the spring nutrient bloom at the ocean’s surface 324,325. The abundance of nutritional resources has a prominent role in shaping various aspects of the reproduction of benthic species, accelerating for example the initiation of gametogenesis, vitellogenesis and the timing of spawning 326–329. In the Geo- dia spp. of the present and other studies 143, it is plausible that gametogenesis, and particularly the stage of vitellogenesis was synchronized with the nutrient blooms at the different locations. This is the most detailed study to date describing the reproduction of Geodia spp. from deep-sea sponge grounds, providing essential information for the development of conservation strategies in these areas.

Key results and conclusions from Chapter II: • The Geodia spp. studied here are all gonochoristic and oviparous, with synchronous development of the gametes at the individual level. • All species share similar cytological features regarding the formation of their gametes. • The main cytological observations of oogenesis in Geodia spp. are: homosynthesis of lipid origin yolk, vertical transmission of bacteria within the oocytes, a thick collagenous layer around the oocytes, the lack of follicle, and the lack of nurse cells. • The main cytological features of spermatogenesis in Geodia spp. are: spermatic cysts derived from choanocyte chambers, absorption of choanocyte flagellum and formation of a de novo flagellum in later stages, asynchronous development within the spermatic cyst, and absence of symbionts within the spermatic cysts. • The gametogenesis is observed from May to late September, with one or two reproductive periods depending on the species and location. • The gametogenesis is likely driven by nutrient blooms in each studied location. Differences in food supplies in different locations may cause variation in time of gametogenesis, even within the same species. • All studied species have a high reproductive effort. • Across species, some reproductive traits differ slightly, possibly allowing the various species to coexist in the same oligotrophic habitats.

51 Chapter III: The molecular machinery of gametogenesis in Geodia demosponges (Porifera): evolutionary origins of a conserved toolkit across animals (Paper III)

Results In this chapter, RNAseq analysis was used to compare the gene expression levels between individuals of five sponge species of the genus Geodia that were morphologically characterized as female, male and nonreproductive at the time of collection. An additional proteomic analysis was performed in a subset of the female and male individuals in order to detect the regulation of gametogenesis at the protein level, and to compare the proteomic data with the transcriptomic data for any concurrent results. Based on the morphological data of these five Geodia species (described in Papers II and III), individuals of each species were grouped based on the maturation stage of their oocytes and spermatic cysts. The different levels of oocyte maturation considered here were PV, Vi_I, Vi_II and Vi_III (largest size and highest amount of yolk) and of spermatic cysts were SP_I (containing only spermatogonia and primary spermatocytes) and SP_II (containing secondary spermatocytes, spermatids, and spermatozoa). For each studied species, one transcriptome assembly including all the individuals of the species was generated, and used as reference for the further DGE analysis. In summary, the percentage of reads aligned against each transcriptome was between 80.6 and 90.7% and the completeness of each assembly was between 69 and 85%. The GC content in the different assemblies ranged from 51.77% to 55.38% and the N50 from 485 to 646 bp likely due to a large proportion of bacterial short sequences obtained (the detailed statistics of these assemblies are presented in Table S1 in Paper III). Using DGE analysis, the overexpressed genes during oogenesis (pairwise comparisons vs NR and vs males) and spermatogenesis (pairwise comparisons vs NR and vs females) in Geodia species were studied together with their enriched GO categories. The function of 19% to 69% of these differentially upregulated genes could be identified through blastx using the Swissprot metazoan database (Table S2 in Paper III). DGE analysis for the oogenesis was

52 based on the species G. phlegraei (PV), G. barretti (Vi_I), and G. macandrewii (Vi_II), and for spermatogenesis on the species G. hentscheli (SP_I) and G. phlegraei (SP_I and SP_II), covering almost all the developmental stages along gametogenesis, and trying in parallel to extract as much information as possible (e.g., using species with highest number of replicates; highest functional information obtained). G. atlantica (Vi_III and SP_II stage), although analysed, was not used in the DGE analysis, as it returned too few DE genes and so too little information regarding gametogenesis could be extracted. For the proteomic analysis, female individuals of G. macandrewii (Vi_II) and G. atlantica (Vi_III) as well as male individuals of G. atlantica (SP_II) and G. phlegraei (SP_I) were used. The list of all the upregulated genes in each comparison and species, together with the annotation (function) of each gene, can be found in Table S3 in Paper III, while the GO enriched categories that were related to gametogenesis and extensively discussed in Paper III are summarized in Table S8 in Paper III. Some of the results described below, regarding genes regulating both oogenesis and spermatogenesis, were supported by the proteomic analysis and are discussed in detail in Paper III. As expected, the patterns of expressed genes in the transcriptomic analysis and those of detected peptides in the proteomic analysis were not always congruent (Table S5 in Paper III) as in the proteomic analysis no biological replicates were used but instead a single proteomic profile of each specimen was observed. Furthermore, many genes related to gametogenesis have a post-transcriptional regulation, and the mRNAs of others are silenced in order to be expressed at a later stage, namely during embryogenesis (see below). Briefly, less than 1% of the expressed genes in the RSEM database of the studied specimens was recovered as translated protein in their proteome (Fig. S1 in Paper III). Nevertheless, many GO enriched categories were common as genes transcribed in stages Vi_I and Vi_II often had a protein regulation in Vi_II and Vi_III stages respectively (Tables S6−S9 and Fig. S1 in Paper III).

DGE analysis

Germ cell formation and initiation of gametogenesis In both female and male individuals, especially those with gametes in early gametogenic stages, GO enriched categories and upregulated genes related to germ cell formation and germ cell identity (germ cell granules, nuage) were found (Fig. 1; Table S8 in Paper III). Among the upregulated genes, common genes involved in primordial germ cell proliferation such as sushi domain- containing 2 (susd2) and tudor domain-containing 1 (tdrd1) were found in both females and males (Fig. 1A). Furthermore, genes related to nuage, such as musashi homolog 2 (msi2), piwi-ago3, nanos 1, and pumilio (pum3) were also upregulated during gametogenesis, especially in males during the early

53 stages of spermatogenesis (Fig. 1A). In addition, three transcription factors of the Sox family belonging to the groups B, E/F, and C were upregulated during the early stages of gametogenesis, (Fig. 1A) (the phylogeny of the Sox genes is discussed in detail in Paper III). Most genes triggering the retinoic acid pathway, which activates downstream molecular signals responsible for the transition of future gametes from the germ line fate to the meiotic cycle, were overexpressed, at least in the majority of the male individuals of G. hentscheli and G. phlegraei (Fig. 1B), as further discussed in Paper III.

Figure 1. A. Heatmaps with expression level of germline related genes across individuals and studied species. B. Schematic representation of the RA pathway (modified by Griswold, 2016), indicating the different elements of this pathway, sig- nalled by a red arrow when their coding genes were overexpressed in male or female specimens. A heatmap depicting the expression level of those genes accom- panies the scheme. The scale for relative expression values increases from blue to red. The asterisks indicate the upregulated genes; pink found in females, green in males, and purple in both.

54 Oogenesis During oogenesis in Geodia spp., GO enriched categories and overexpressed genes related to meiotic cell cycle and cycle arrest, cell differentiation, maturation, and vitellogenesis were identified (Fig. 2A; Tables S3, S8 in Paper III). Genes related to cell cycle arrest such as structural maintenance of chromosomes protein (smc) and meiosis-specific nuclear structural protein 1 (mns1), were upregulated in females of early (PV and Vi_I) oogenetic stages (Fig. 2A). Genes regulating transcription and growth factors which play a role in cell growth and differentiation (such as taf, fgfr, and eps) were also upregulated. As expected, in females with gametes in all the developmental stages, GO categories of structural morphogenesis, extracellular matrix (ECM), collagen production and vitellogenesis were enriched (Table S8, Paper III). Fur- thermore, tolloid (tll1/tll2), fibulin (fbln5/fbln7), and fibronectin (fn1), as also genes coding for both fibrillar and nonfibrillar collagen ̶ which are main components of the extracellular matrix and regulate its assembly and reorganization ̶ were overexpressed in females with vitellogenic oocytes (Fig. 2A; Table S3 in Paper III) (the phylogeny of collagen genes is discussed in detail in Paper III). Upregulated genes related to yolk formation (vitellogenesis) were also identified in female Geodia individuals with mature (Vi_II) oocytes. Among the most important overexpressed genes were the putative vitellogenin receptor (yl), oxysterol binding protein (obp9), and long-chain fatty acid lig- ases (acsbg1) genes (Fig. 2A).

55 Figure 2. Heatmaps with the expression level of selected genes across individuals and species, related to A. oogenesis B. spermatogenesis. The genes are separated into key categories according to their function. The scale for relative expression values increases from blue to red. The asterisks indicate the differentially expressed genes in female and male specimens respectively.

Spermatogenesis GO enriched categories and overexpressed genes related to all the different stages of spermatogenesis from mitotic and meiotic cycle, to chromatin condensation, flagellum formation, spermatid development and sperm capacitation were identified in male individuals. First of all, genes regulating the processes that occur along the different phases of the meiotic cycle were upregulated in male individuals, enabling to trace the molecular regulation of the whole meiotic cycle, until completion. Genes from the breaking of the double strands, to the formation of the synaptonemal complex, and those supporting the DNA recombination and the overall meiotic process were overexpressed, mainly in males with SP_II spermatic cysts (Fig. 2B; Tables S3, S8 in Paper III). Few examples of such genes were the histone-lysine N-methyltransferase PRDM9 (prdm9); fanconi anemia group J homolog (brip1); meiotic recombination DMC1 LIM15 (dmc1), telomere repeats-binding bouquet formation 1- 2 (terb1-2), testis-expressed and cyclin-A1 (ccna1) genes (Fig. 2B). Regarding the chromatin condensation process during spermatogenesis, genes coding for specialized histones such as the h2ax and histone–arginine methyltransferase gene (carm1) were overexpressed in males. All genes related to the construction of the flagellum were also overexpressed in males (Fig. 2B), including

56 genes related to the formation of the skeleton of the flagellum or the so-called axoneme (e.g. tubulin, dynein, tektin and hydin genes) as well as the formation of the outer structures of the sperm flagellum (e.g. outer dense fibre and fi- brous sheath genes) (Fig. 2B). Finally, the potential mechanism for sperm capacitation was also upregulated in males, as related GO enriched categories (Table S8 in Paper III) and genes coding for ion channels (cation channels of sperm relative genes, sodium and potassium channel genes, and cAMP- and cGMP-related channels) and other genes regulating the cascade of sperm capacitation were overexpressed (Fig. 3, Table S3 in Paper III) in the late stages of spermatogenesis in G. phlegraei.

57 Figure 3. Illustration of Ca+2 driven regulation cascades related to sperm motility and capacitation before fertilisation in marine invertebrates and mammals. The illustration is a modified version according to Morisawa and Yoshida (2005), Ickowicz et al. (2012), and Rahman et al. (2017). The different elements of this pathway are sig- nalled by a red arrow when their coding genes were found overexpressed in male individuals of G. phlegraei. The illustration is accompanied by the expression level of the relevant genes in all the G. phlegraei male specimens and presented in a heatmap. The expression level increases from blue to red.

Discussion In this study, genes regulating all stages of oogenesis and spermatogenesis in other animals were overexpressed in female and male individuals of Geodia spp. respectively. First of all, it seems that there is a well-established germline in Geodia spp. at the beginning of gametogenesis, as molecular signals regulating the germ cell specification similar to those in other animals 330–334 were present and overexpressed in individuals undergoing gametogenesis. Apart from the histological observation of potential nuage in oocytes of G. macandrewii (results from Paper II), overexpressed genes of the Germline Multip- otency Program (GMP) (e.g. nanos, pumilio, musashi, piwi) in Geodia spp. indicate that a similar molecular mechanism regulates the animal germ cell development and maintenance in Porifera and other Metazoa 32,335–343. Further- more, differentiation of the gametic cells in Geodia spp. might also be triggered by the activation of the retinoic acid pathway as happens in vertebrates and some other invertebrates 344. During oogenesis, signals of meiotic regulation as well as meiotic arrest and maturation were observed across the female samples. However, those signals involved different genes than those known in other animals. For example,

58 while mos genes have been previously identified from placozoans to mammals 345 and they regulate the meiotic cycle arrest from cnidarians to bilaterians, they were absent in Geodia spp. Nevertheless, the downstream ERK1/ERK2 MAPK pathway, activated by mos in other organisms, including Cnidaria 346, was enriched in Geodia spp. and thus it is possible that mos genes are not necessary for downstream activation of this pathway, which probably partici- pates in meiotic cell cycle regulation in sponges as well. Although morphological features of cell cycle arrest were not observed in histological sections (results from Papers II, III), it is suggested here that oocytes of Geodia spp. might be arrested at prophase I (before vitellogenesis starts), and meiosis might be potentially resumed prior to oocyte release or after fertilization, to avoid parthenogenetic cleavage. The further meiotic progressions might be regulated by cyclins-B, as happens in sea urchins 347, as several genes coding for cyclins-B were only upregulated in Vi_II females (Table S3.D in Paper III). The next stages of oogenesis such as maturation, growth and vitellogenesis seem to be regulated by very similar molecular signals. Oocyte growth is structurally supported by the ECM and its components 348–351, which also play essential role in nutrient accumulation and communication with the surroundings of the oocyte. GO categories related to ECM reorganization and genes involved in this process were largely enriched in the RNA-seq data. This enrichment was in accordance with the histological analysis which showed oocytes changing size and shape during their vitellogenesis, with a parallel increase of the collagenous layer around the oocyte (results from Papers II, III). The yolk formed in the oocytes during the vitellogenic stages consisted mainly of lipids (results from Papers II, III), and many genes and GO categories related to lipid formation and lipid transport were upregulated in females. Fi- nally, the vertical transmission of symbiotic bacteria to the oocytes, observed cytologically, was also followed by upregulation of genes involved in recog- nition of symbiotic organisms during oocyte development. In oviparous species, oocytes must contain the essential molecular machinery for successful fertilization, zygotic gene activation and early embryo development within the water column. In fish, the majority of mRNAs detected during the post-vitellogenic oocyte stage are translated after fertilization and they control the first steps of embryonic development 353. Several upregulated categories and genes in female Geodia spp. might be related to embryogenesis, as several genes related to mammalian oocyte competence and early zygotic development were upregulated in this study. For instance, the genes embryonic polyadenylate-binding protein B (epabp-b) and YTH domain-containing family protein 2 (ythdf2) (Table S3 in Paper III) play an important role in gametogenesis and mainly in oocyte maturation and early morphogenesis 354,355 355, while the gene nuclear migration protein nudC (nudc) is important for cytokinesis and mitosis during embryonic cell division 356–358, playing a

59 role in nuclear positioning 359. Genes related to epigenetic regulation of embryogenesis were also upregulated (e.g. histone-lysine N-methyltransferase setd2) 353. Continuous and quick changes happen during all these processes which are controlled by transcription factors and signalling pathways. GO categories of Wnt and Notch signalling pathways that play a role in embryonic development and morphogenesis in sponges as well 86,87,89,91,360, were enriched in female Geodia spp. Finally, the spliceosome (which is responsible for mRNA editing, silencing or stability) and the proteasome machinery (which entails necessary proteins of the signalling pathways that have a short-half time such as MAP kinases and cyclins) are activated during oogenesis/embryogenesis, and tightly related to egg quality 361, and several of their regulatory genes were overexpressed in female specimens of Geodia spp. The expression patterns found here in Geodia spp. indicate that sponges might employ similar molecular control for a successful oogenesis and subsequent embryo development. According to both histological observations and the gene expression patterns (results from Papers II, III), the process of spermatogenesis in Geodia spp. follows the same course (from early spermatogonia to mature spermatozoa) as in the rest of metazoans. Spermatogenesis in sponges involves meiotic processes as in all Metazoa, and this is indicated by the upregulation, among others, of the eukaryotic-conserved set of meiotic genes 151,362. In Geodia spp., spermatogonia are derived from choanocytes, which lose their flagellum to transform into gametes. They then undergo several mitoses and two meiotic divisions, and at the end of the spermatogenesis they make a flagellum de novo (results from Papers II, III). This is the most common strategy in sponges, even though some species retain the flagellum during the process 97,98. The main element of the flagellum is the axoneme, and the entire molecular toolkit regulating the axoneme formation was activated in male individuals of the studied Geodia spp. This indicates that molecular mechanism of axoneme formation it is also present in sponges and is similar to the molecular machinery behind the axonemal structure of the mammalian sperm 363. Histological observations of spermatic cells of Geodia spp. together with the transcriptomic data (results from Papers II, III) indicated that chromatin condensation also happens in Geodia spp. and is controlled by very conserved molecular mechanisms. Finally, the activation of sperm capacitation in Geodia spp. might be regulated by a similar mechanism to that in other marine invertebrates and even mammals. After sperm release and attraction to the oocyte by female chemoattractants, activation of ion channels in the sperm membrane causes its hyperpolarization, in turn activating downstream cascades that regulate the flagellum motility 364–367. In Geodia spp., this mechanism was activated prior to the sperm release into the water column, which could explain the high speed observed directly upon early release of sperm in other Geodia species 368. This is the most comprehensive molecular study on the gametogenesis of sponges

60 so far and has profound implications for the understanding of the evolution of sexual reproduction in animals.

Key results and conclusions from Chapter III: • The presence of a germline is observed in Geodia spp., as morphological features and genes regulating germ cell lines were upregulated in Geodia spp. during the initial stages of gametogenesis. • The retinoic acid pathway in both females and males possibly regulates the entrance of the germline into the meiotic cycle and initiates gamete differentiation in Geodia spp. • The progress of oogenesis in Geodia spp. involves a cell cycle arrest where maturation, growth, and yolk formation within the oocyte occur, as indicated by overexpression of essential genes regulating these main stages of oogenesis in other animals. • The progress of spermatogenesis consists of several mitoses and two subsequent meiotic divisions, chromatin condensation, de novo flagellum formation, and sperm capacitation, with the involvement of essential genes known to regulate all meiotic machinery, axoneme formation, and sperm capacitation in other Metazoa.

61 Chapter IV: Oogenesis and lipid metabolism in the deep-sea sponge Phakellia ventilabrum: a histological, lipidomic and transcriptomic approach (Manuscript IV)

Results In this chapter the seasonality and reproductive strategy of the boreal deep-sea sponge Phakellia ventilabrum 320 was described with histological observations. Furthermore, the lipid profile of this species was investigated with UPLC-MS and the signal intensity of lipid groups was compared between females with PV oocytes, females with Vi oocytes and nonreproductive individuals in order to understand the lipid patterns during oogenesis in this species. Individuals were grouped in each reproductive stage according to the size of the oocyte and the amount of yolk accumulated. Further RNA-seq comparative analysis was performed between one Vi_I and two NR individuals, and between one Vi_II and two NR individuals, in order to compare expression levels of genes related to lipid metabolism and to find potential correlations with the lipid profile.

Seasonality and cytological features of oogenesis The histological analyses revealed that P. ventilabrum is an oviparous species. Reproductive activity was detected both in Kosterfjord (Swedish coast) in March 2019 and in Korsfjord (Norwegian coast) in September 2016. Female specimens with developing oocytes were observed among the specimens collected, but no individual with male gametes was identified. NR specimens were also detected, meaning that not all the individuals of a population were engaged in reproduction in the same season, or that these individuals may be females or males that are still not actively producing gametes or that have already released their gametes. The oogenesis was not synchronous at the population level, as there were some specimens with PV oocytes and others with mature Vi_II oocytes. Asynchronous development was observed in the same individual as well in some specimens. While oocytes at PV stage measured ~20 μm, Vi_I oocytes were about 40 μm (Fig. 1A), and they reached the size of ~80 μm in the latest developmental

62 stage (Vi_II) recorded in the present study (Fig. 1B). In this latter stage, oocytes migrated to the canals (Fig. 1B), in order to be released. This species was categorized as an LMA sponge, with only sparse bacteria detected within the mesohyl, and no vertical transmission of bacterial symbionts to the oocytes was observed. The yolk accumulated within the oocytes during the vitellogenic stages was mainly characterized by platelets with heterogenous composition (mix of lipids and protein) (Fig. 1C). However, homogeneous droplets of lipid and protein origin were also found, especially in the later stages (Fig. 1D). In total, 30% of the total oocyte area was occupied by yolk. Nurse cells providing the oocyte with nutrients were detected in close proximity to the oocytes (Fig. 1C).

Figure 1. Cytological observations of female gametes of P. ventilabrum. A. Previtellogenic oocytes (~20 μm) distributed in the mesohyl of a female sponge. Insert: a close-up of a previtellogenic oocyte with a well-formed nucleus. B. Oocytes of vitellogenic stage_II (60-80 μm), accumulated around the canal (ca). C. Ultrastructure of a vitellogenic stage_I oocyte with a well-formed nucleus (n), ooplasm filled with vesicles (v) and some large heterogeneous (y) and other homogenous lipid (li) droplets. A nurse cell (nc) with protein (p) and lipid (li) yolk in close proximity. D. Ultrastructure of vitellogenic stage II oocyte: ooplasm full of homogenous lipid (li) and proteic (p) yolk and heterogenous droplets (y). Fibers (f) are observed around the oocyte and a bacterium (b) in the mesohyl nearby the oocyte.

63 Lipidomic analysis Lipids were extracted from all thirteen sponge samples from both locations and different reproductive groups (NR, PV, and Vi), whereas oxylipin extraction was conducted only from samples collected from Korsfjord (Norwegian coast) in September. In total 691 different lipids from several lipid groups were isolated with UPLC-MS. These lipids included among others: 113 FFA with 14-29 carbons and 0-5 unsaturated bonds, 157 PC with 30-46 carbons and up to 5 unsaturated bonds, 65 LPC with 14-29 carbons and 0-2 unsaturations, 19 PG with 30-40 carbons and up to 2 unsaturated bonds, 6 LPG with 15-17 carbons and only one saturation, 24 PE with 32-52 carbons and up to 3 unsaturations, 8 LPE with 16-25 carbons and 0-1 saturated bonds, 211 TG with 42-72 carbons and up to 6 unsaturated bonds, and 26 sphingolipids/ glycosphingolipids of 40-44 carbons and 0-2 unsaturated bonds (Table S2 in Manuscript IV). Furthermore 62 oxylipins were identified and quantified, belonging to the groups: AA, LA, ALA, EPA, DHA, and others such as PGD2, PGE2, PGF2α, HETE, HETrE, DiHETE, DiHETrE, 12-HHTrE, KETE, EpETrE, and LXA4 which are derived from AA; HODE, KODE derived from LA; PGE3, PGF3α, HEPE, DiHETE, EpETE, and LXA5 derived from EPA; and HDoHE and EpDPE which are derived from DHA (Table S2 in Manuscript IV). Overall, significant differences were identified in the signal intensity of most lipid groups when comparing samples between the different locations, with most of the lipids showing a statistically higher average signal in Koster- fjord compared to Korsfjord, e.g. PUFA-FFAs, MFA-PCs, PGs, PEs, LPEs, (Fig. 2), while TGs on average had a statistically higher signal in Korsfjord (Fig. 2). Apart from the variance in the average signal of each lipid group per location/month, fluctuations in signal between the two locations/months were tested for each identified lipid separately within each lipid group, finding statistical differences in several lipids within each lipid group (SF1 in Manu- script IV).

Figure 2. Boxplots depicting the average signal [au/mg] extracted from all the lipids of each lipid group in the two different sampling locations/months. Fluctuations in signal [au/mg] were calculated separately for SFA, MFA, and PUFA lipids within each lipid group.

No significant variation in the signal of lipids within the studied lipid groups, was found when comparing individuals with PV oocytes, Vi oocytes or with no oocytes at all (NR) with ANOVA analysis, due to the high intragroup variation and the low number of replicates per reproductive category. However, fluctuations in lipid signals among the NR, PV, and Vi categories were still observed (SF2 in Manuscript IV). Of the total number of lipids identified in each lipid group, approximately 50 % of the FFA (either SFA, MFA or PUFA) had a tendency of decreasing their signal from NR to Vi, while less than a 15 % of the FFA showed an increasing signal. A high percentage of the total identified PE, PG, sphingolipids, and oxylipins had a decreased signal in reproductive specimens as well. On the contrary, 40 % of the SFA-TG and MFA-TG had an increased signal from NR to Vi stage (Fig. 3). The main TGs with the tendency of signal increase from NR to Vi category are indicated in Figure 4.

65 Figure 3. Barplot presenting the sum of SFA-, MFA-, and PUFA- lipids within each lipid group with a trend of total signal decrease or increase in female, compared to nonreproductive individuals within each lipid group.

Regarding the trends of oxylipin signal during oogenesis, the signal of AA seemed stable between NR and individuals with female gametes, while some derivatives of AA (5(6)-EpETrE, 12-HHTrE, 14,15-DiHETrE) tended to have an increasing signal in Vi stage. A different pattern was observed for LA, the signal of which showed a strong increase in Vi stage, but lipids derived from LA did not have any correlation with oogenesis (SF2 in Manuscript IV).

66 Figure 4. dotplot depicting TGs with a tendency of signal increase [au/mg] towards the females with Vi oocytes.

67 Transcriptomic analysis The constructed transcriptome assembly had 78.2% completeness according to BUSCO, with an overall alignment rate of 96.5%, an N50 equal to 1,240 and a GC content of 45%. DGE analysis was conducted separately for Vi_I and Vi_II individuals vs the NR specimens, as possibly different processes of vitellogenesis could occur in the different vitellogenic stages. That way, more information could be extracted, even though no replicates were taken into account for the reproductive stages. In the pairwise comparison Vi_I vs NR, 3,490 genes were upregulated. Likewise, 4,774 genes were upregulated for the female individual in the Vi_II vs NR comparison. Blast hits occurred for about 67% and 50% of the upregulated genes in Vi_I and Vi_II stages respectively. GO enriched categories related to oogenesis were found in both vitellogenic stages (female germline, extracellular matrix reorganisation, and vitellogenesis) and among those, several categories were related to lipid metabolism (Fig. 5 in Manuscript IV). More specifically, upregulated genes and KEGG pathways derived from these genes were related to PL metabolism, unsaturated FA and TG biosynthesis, FA elongation, FA degradation and beta- oxidation (Table S6, S9 and Fig. S1 in Manuscript IV). All the genes, regulating the TG biosynthetic and the FA beta-oxidation pathways were upregulated in females (especially in Vi_I when the vitellogenic process starts) when compared to NR individuals (Fig. 5).

68 Figure 5. TG biosynthetic pathway in the endoplasmic reticulum (ER) and FA beta- oxidation pathway in mitochondria, accompanied by heatmaps with gene expression levels of the genes regulating these pathways. Relative expression level increases from blue to red. DE genes were overexpressed in females (either Vi_I or Vi_II) compared to nonreproductive specimens. The red arrows close to the genes in the pathways also indicate gene overexpression in female individuals of the Vi_I and/or Vi_II stages.

Discussion Specimens of P. ventilabrum with Vi_II oocytes were found in both locations and sampling months, indicating either a variation in the reproductive cycle among locations, or two reproductive cycles within a year, as in other deep- sea sponges 143 and results from Geodia spp. (Paper II). However, due to the restricted sampling time, no definite conclusions can be drawn for P. ventilabrum. In any case, the reproductive cycle of this species seems to be synchro- nised with the seasonality of nutrient blooms in the sampling areas (as seen also in Geodia spp. in Paper II), as there is a spring bloom in March-April followed by another bloom in August-September 369–371. Nutrients from the surrounding environment would provide sufficient energy to the adult to trigger oocyte development and they would also accumulate within the oocytes. As P. ventilabrum is an oviparous species with either a lecithotrophic larval stage or direct development, vitellogenesis is crucial as the same nutrients will supply energy to the propagules during their further development in the water column 352. Among the identified lipids in the comparative analysis, most of the TGs (~40%) showed an increasing signal from nonreproductive specimens to specimens with vitellogenic oocytes. On the other hand, the majority of lipids identified in the rest of the lipid groups mostly decreased in signal towards the female with mature oocytes. Therefore, TGs are suggested as the main component of stored lipids in the yolk of P. ventilabrum, with potential contribu- tion of other lipid groups but to a lesser extent, or with higher selectivity as a lower percentage of those increased in signal during oogenesis. This also co- incides with the transcriptomic data, in which all the genes of the TG biosynthetic pathway 372 were upregulated, apart from the gene coding for the penul- timate enzyme of this pathway (dgat) which only showed a tendency for increased expression. TGs constitute one of the main forms of energy storage in marine organisms 373–375, including deep-sea sponges 206, and it is used as lipid storage as well as in the ovaries and eggs of marine reptiles and crustaceans 115,376–378. Lipids when oxidized yield twice the energy per unit dry mass than carbohydrates or proteins and TGs are the most compact form of energy storage compared to other lipid groups as they are stored in the most anhydrous stage 379.

69 On the other hand, the SFA-, MFA- and PUFA-FFA groups contained a high percentage (one of the highest) of identified lipids with decreasing signal during oogenesis. Furthermore, the DGE analysis revealed overexpression of the molecular toolkit regulating all the different stages of the FFA degradation pathway in specimens under oogenesis, from its activation and transportation to the mitochondria to its beta-oxidation, until the generation of acetyl-CoA, the initial component of the citric acid cycle (Fig. 5) 380. FFAs are used as a backbone for the synthesis of other lipids and therefore, a decrease in the signal of FFAs could possibly indicate their incorporation in the formation of other lipids such as TGs. Furthermore, adult sponges have high energetic requirements during their gamete development, and lipid oxidation is the main energy provider for their physiological processes, including reproduction 381,382. Thus, FFA beta-oxidation could also be the main energy source for the adults of P. ventilabrum during gametogenesis. However, another possibility is that the NR specimens of this study had just spawned prior to collection, and thus they had already suffered from reproductive stress and starvation. In that case, it is possible that NR specimens were catabolising lipids from their energy stock, including TGs, to cover their metabolic demands. This may have led to the observation of more TGs with decreasing signal and in parallel, more FFAs with increasing signal. Similar patterns have been previously observed in fishes and corals 116,383,384. Changes in lipid signals (Fig. 2) were observed between locations in most lipid groups, with a tendency for an increase in the signal detected in most PLs in Kosterfjord in March during the intense spring bloom 120,385,386. This was expected, given that P. ventilabrum depends on the nutrients of the surrounding waters to cover its energetic demands and to sustain its physiological processes, as has been previously observed in the deep-sea coral Lophelia pertusa 387. As a LMA species, P. ventilabrum might be especially dependent on its heterotrophic diet and have relatively high pumping rates in comparison to other sponges in order to cover their nutritional needs 388. The current analysis cannot determine whether the observed lipids are incorporated directly from the environment or synthesized via available carbon precursors absorbed by the sponge. Sponges are known to possess the necessary enzymatic machinery to catalyse such processes of elongation and unsaturation 389,390. From the above, it can be derived that sponges potentially store energy for the survival of the future propagule mainly in the form of TGs during oocyte formation, and that they accumulate or produce other lipids such as PLs (PC, PG, PE) mainly for using them to other physiological processes (e.g. defence, membrane reconstruction, etc). However, further studies are necessary to confirm this hypothesis. This study unveils the rich lipid content of P. ventilabrum and describes the lipid metabolic processes associated to female gametogenesis in sponges for the first time.

70 Key results and conclusions from Chapter IV: • Phakellia ventilabrum is an oviparous species, and oogenesis is observed both in March on the Swedish coast and in September on the Norwegian coast. No individuals with male gametes were detected. • The yolk in the oocytes has a mainly heterogenous composition, but some homogenous lipid and protein droplets are also present. • The lipidomic analysis identified 691 lipids in P. ventilabrum with very long-chain carbons and a high number of unsaturated bonds (up to 6) belonging among others to: FFA, PC, LPC, PG, LPG, PE, LPE, TG. • The oxylipin analysis extracted 65 oxylipins belonging to the AA, LA, ALA, EPA, DHA groups and their derivatives. • Lipids of P. ventilabrum from all the different lipid groups have a significantly different signal intensity between locations regardless of the reproductive status. • Regarding the reproductive factor, about half of the identified TGs show a tendency of signal increase and about half of the identified FFAs show a tendency of signal decrease from NR to individuals with Vi oocytes. • Lipid data are in accordance with transcriptomic data which reveal overexpression of genes regulating the PL and TG biosynthetic pathway and the FFA beta-oxidation pathway. • TGs could be the main lipid group used for storage in oocytes while FFA catabolism might be necessary for energy provision to the adult during energetically demanding processes, such as oogenesis.

71 General Discussion and future directions

Reproductive strategies in extreme environments The work of this thesis is focused on assessing the reproductive patterns of sponge species found in shallow cold waters of Antarctica and deep cold waters of the boreo-arctic area of the North Atlantic Ocean. These habitats, even though geographically remote, are both characterized as “extreme” environments with unfavorable abiotic conditions for the marine benthos, compared to tropical and temperate habitats. However, a high biodiversity and abundance of sponge species thrive in both environments 47,210,391. Why species are abundant in an ecosystem might be the result of biological, ecological, and physico-chemical factors, and here I explore the biology of these communities by studying their reproductive strategies using a combination of morphological, molecular and chemical techniques. All the Antarctic and deep-sea sponge species studied in this work had a reproductive activity during the summer months of each area, with one or two annual reproductive cycles around this period, possibly synchronized with abiotic changes and seasonal nutrient blooms 128,142,392,393. However, species from the different habitats had completely different reproductive modes, but species from the same habitat shared many common reproductive features, despite belonging to different genera and orders. In particular, the dendroceratid (Dendrilla antarctica), poecilosclerid (Phorbas areolatus, Kirkpatrickia variolosa, Isodictya kerguelenensis) and haplosclerid (Hemigellius pilosus, Haliclona penicillata) Antarctic species were all most likely (successive) hermaphrodites and viviparous, brooding their embryos within their maternal tissues (Chapter I). On the other hand, the deep-sea North Atlantic species (five Geodia spp. and Phakellia ventilabrum) were all gonochoristic, with distinct male and female individuals, and oviparous, releasing their gametes to the water column for external fertilization (Chapters II, IV). Furthermore, most of the oocytes and embryos of the Antarctic species had a well-developed follicle layer around them (Chapter I), a typical characteristic of viviparous species, while the female gametes of the boreo-arctic species had only a collagenous layer supporting them (Chapters II, IV). The vitellogenic oocytes of the Ant- arctic species were much smaller, as the main investment occurred in the embryonic stage (Chapter I), while the oocytes of the oviparous boreo-arctic species were much larger, since all the nutrients for the future propagules were accumulated during that stage (Chapters II, IV).

72 The different reproductive modes found in the studied species are probably a combination of strict consequences of phylogenetic constraints and fine tuning to specific environmental conditions. Actually, the fact that viviparous species dominate the shallow waters of Antarctica and oviparous species are mainly abundant in the North Atlantic deep-sea sponge grounds indicate that each reproductive mode might be successfully adapted to provide the most successful survival and perpetuation of generations in each habitat. In the shallow regions of Deception Island (South Shetland Islands), where samples were collected, intense annual fluctuations of temperature, salinity, light and nutrients occur. In detail, two phytoplankton blooms (one in spring and another one in summer) occur, with chlorophyll-a levels reaching the maximum record in western Antarctic Peninsula during the summer months 394. The sea-surface temperature also increases by 3 degrees and the daylight can last ~20h per day 394. In addition, the annual primary production in this area is relatively high compared to other Antarctic areas, with no particular nutrient limitation to be observed 395. Because of the nutrient abundance in the surrounding during the summer months, sponges can sustain their brooded offspring with external food supplies. In parallel, brooding protects the propagules from preda- tion and other abiotic factors (e.g. UV light), thus enhancing the fertilization rates. In deep-sea areas where conditions are mostly stable with only a very short period of nutrient availability 369,370,396, sponges might not have the necessary energetic supplies to develop their oocytes and the embryos, and thus oviparity, which is characterized by shorter periods of gamete formation (~2 months) with high numbers of gametes produced, is a much more advantageous strategy in these environmental conditions. In this sense, oviparity and broadcast spawning could be analogous to an “r-strategy”, in which parents do not invest lots in their offspring and in turn produce massive amounts of gametes to ensure fertilisation 397. Viviparous sponges are all spermcast spawners (i.e., release of sperm but retaining of oocytes within the tissue and then brooding of embryos), a strategy with higher fertilization rates than broadcast spawning, because even low concentrations of sperm in dilute suspension can be filtered by the sponge and fertilize the eggs through an extended period of time 398–400. The asynchronous oocyte and embryo development observed within the individuals of Antarctic sponges studied here, can also support this (Chapter I). On the contrary, in oviparous species, a high density of gametes and their synchronous release must occur for a successful fertilization. Indeed, female and male specimens were undergoing gametogenesis during similar periods of time in Geodia spp., suggesting a synchronous spawning event, and the density of oocytes that were produced (~7 oocytes/ mm2) was comparable and even higher to that in oviparous temperate species (~4-7 oocytes / mm2 ) 133. Although in the vast area of the deep sea, oviparity still sounds like an im- probable way of ensuring fertilisation, in other marine invertebrates, several

73 tactics seem to accompany oviparity for a better success. For instance, maintenance of the female gametes on the surface of the sponge, accumulation of oocytes in clumps, increased size of the eggs or regulation of viscosity of the spawned gametes have shown to increase the fertilization success as the period in which eggs can be encountered by sperm is increased 401,402. Some of these tactics were also observed in the oviparous species of this thesis: the accumulation of several female gametes in clusters enveloped by mucous material before their release and an increased surface of oocytes (larger size) in these species (Chapter II) compared to their shallow-water counterparts. The embryonic development and subsequent dispersal of gametes/larvae must be taken into account in order to understand the effectiveness of the reproductive mode adopted by each species in each habitat. In fact, all Den- droceratida, Poecilosclerida and Haplosclerida (species studied in Chapter I) have a larval stage (parenchymella) with ciliated cells for active swimming 110. Even though the larval pelagic duration in sponges is usually very short (<2 weeks), sponge larvae swim relatively fast 403–405, therefore probably increasing their dispersal capabilities. Indeed, high genetic connectivity was found for populations of Dendrilla antartica around the Antarctic Peninsula and the South Shetlands 406. In turn, the development of most Tetractinellida and all Bubarida (Chapter II and Chapter IV respectively) is completely unknown as no larva has ever been collected. A single unique type of larva has been described for the tetractinellid family Thoosidae, which actually pos- sesses the longest-drifting duration in sponges, the hoplitomella 110. On the other hand, one of the very few reports on development of tetractinellid sponges has been performed on the family Tetillidae, which completely lack a larval stage, and in turn the oocytes are fertilized in the canals, then released and the embryo settles immediately to develop into the juvenile sponge 124. Since the larva of Geodia or Phakellia spp. has never been observed despite active research in sponge reproduction for many years 123, the possibility of direct development, and therefore only gamete dispersal, appears. In either case of developmental stage (larva or direct development), it is expected that oocytes are maintained on the surface of the adult or in close proximity for increased possibilities of fertilization through the pumping of adult sponges, however further evidence is needed. The physical properties of the gametes such as the buoyancy, the dispersion rate and the viscosity, correlated with other factors such as the water flow should be tested in order to understand how this oviparity can be successful in these habitats and maintain the high abundance of these populations. Finally, slight variations in the reproductive strategy (e.g. spawning period, size and number of gametes, amount and type of yolk) were also found among the studied species within each habitat, even among species of the same order or the same genus. These variations, though, are more likely due to specific adaptations to the environmental conditions, which make these species able to coexist in the same habitat. This fine tuning of reproductive dynamics has

74 been proposed before as a mechanism to allow co-existence in temperate sponges 130,133,291. Sponges have high energetic requirements during gamete/embryonic development, so avoiding temporal overlapping of gamete/embryo formation, or exploiting a different nutrient regime could be a strategy to avoid intense competition for nutrients among genera and species, especially in habitats with limited food supplies 142. The different timing of gamete or larval release has also been proposed as a strategy to avoid spatial competition in corals 407 and as sponges have lecithotrophic larvae with a limited dispersal capacity, this strategy is also necessary in habitats with high sponge densities. In this thesis, strategies such as asynchronous gamete/embryonic development within an individual (in: K. variolosa, H. pilosus, H. penicillata, P. ventilabrum), population (in: Geodia spp., P. ventilabrum) or species (in: Antarctic species, Geodia spp.) were observed, as well variability in the yolk content and/or yolk formation strategy between genera (comparing Phakellia with Ge- odia spp.), indicating that sponges have evolved different mechanisms to cope with limiting factors of their surroundings.

Yolk, its role in survival and dispersal of the propagule Vitellogenesis is a crucial process during which the oocyte or embryo/larva is provided with the necessary nutrients until the juvenile sponge is able to feed. In sponges, the type and amount of yolk is most likely a character under phylogenetic constrains, although both can also be adjusted to specific conditions of the habitat, allowing different species from the same habitat to exploit limiting nutrients in different ways. In this sense, different biological characteristics of the genera might favour the formation of one or another type of yolk. For instance, sponges use the bacterial lipids as precursors to build their own lipids 68,203,408,409. Recently, it has been shown that one of the significant inputs of the symbiotic bacterial community in cold water, deep-sea sponges, including Geodia spp., is to provide lipid building blocks to sponges, contributing to construction of their host’s very long chain lipids 204,205. So, the high microbial abundance in Geodia spp. might be the explanation for the high lipid content observed in their oocytes (Chapter II), and even further, the vertically transmitted symbionts within the oocytes might also have a direct role during the homosynthetic activity of lipid formation (Chapter II). On the other hand, the LMA P. ventilabrum had less homogeneous lipid droplets (~7% compared to ~15% in Geodia spp.) and the nurse cells packed and transferred yolk (heterosynthesis) mainly of heterogeneous form (Chapter IV), as observed for the Antarctic species (Chapter I). On the other hand, incorporation of heterosynthetic nutrients in the gametes is a much faster process than synthesizing the nutrients by the gamete itself 184, so the durations of gamete formation and maturation are shorter, giving an ecological advantage for faster gamete release and faster colonization of the ecological niches, or dispersal for longer

75 times compared to species in which gametogenesis takes longer. Unfortu- nately, we did not have samples from consecutive months during the reproductive period of P. ventilabrum in order to estimate the duration of oogenesis. For the successful survival and dispersal of the sponge propagule, the content and quality of yolk accumulated during oogenesis and embryogenesis are crucial elements. Lipids are fast fuels, provide twice the energy than proteins and also serve for buoyancy 121,122. Although both proteins and lipids are catab- olized during embryogenesis in the shrimp Macrobrachium borellii, lipids are consumed earlier than proteins, and considered as the main fuel during this period 115. All the different yolk characteristics described above can determine the duration of dispersal of the studied species. In the present thesis, gametes of Geodia spp. and P. ventilabrum contain different types of yolk. One possibility is that highly lipidic Geodia propagules drift further than those of P. ventilabrum, but after settlement in the substrate, P. ventilabrum may have higher probabilities for survival due to their larger proteinic energy stock. In turn, in the juvenile stage, Geodia spp. may be sustained with the help of their large stock of bacterial symbionts that are transferred to the oocyte. The other possibility is that as lipids are fast fuels, gametes of Geodia spp. drift for shorter periods of time until lipid depletion, compared to those of P. ventilabrum, which could drift for longer periods making use of the protein yolk for energy and the lipid stock for buoyancy. To better understand the ecological behaviour of these species and the adaptation of their reproductive strategies to their habitats, further studies are needed, combined with precise nutrient availability information of the area, oceanographic models, and connectivity studies.

Lipid content and chemical ecology in Phakellia ventilabrum In P. ventilabrum particularly, the analysis of reproductive strategy was accompanied by the comparative lipidomic analysis (with UPLC-MS) between female and nonreproductive specimens in order to understand which lipids are engaged to yolk formation during vitellogenesis (Chapter IV). The fact that a large number of TGs, and much fewer PLs showed a tendency of signal increase, together with a slight increase in the total average signal of TGs in females with more mature oocytes, led to the hypothesis that TGs may be the main component of the lipidic yolk in this species while PLs may participate to yolk formation in a lesser extent, or in a more specific way, or used in other physiological processes. Among the lipids, TGs are the most “efficient” source of energy, having a very compact structure 379. As thus, although cytologically this species seems to provide the propagule with fewer lipid yolk droplets compared to Geodia spp., this lipid yolk is of high energetic value. It

76 was also found an intense FFA beta-oxidation in females, indicating that females catabolize other lipids to cover their energetic demands during gametogenesis. Due to the fact that the lipidomic analysis was performed on the whole individual and not particularly to the gametes, no clear conclusion can be extracted regarding the lipid origin of the yolk. However, these results are also supported by studies conducted in terrestrial and marine animals with lecithotrophic development, e.g. reptiles and fish, which mention that TGs are used as main component of the vitellus 373,410,411. The strong correlation of the lipid signals with the sampling location in this study, reflects the dependency of this species on its diet, so with this analysis further information regarding the food web interactions of this species can also be extracted. In this thesis, it is the first time to our knowledge, that such a broad lipidomic analysis has been conducted in sponges. Although the fatty acids of P. ventilabrum have been previously characterized by GC-MS, identifying only 21 FAs 72, here a total of 691 different lipids were identified, opening new insights for better understanding the chemical ecology of this species and further comparative analyses with other genera. Furthermore, it is the first attempt to investigate the lipid metabolism of P. ventilabrum during oogenesis, an energetically costly process.

Molecular studies on the reproductive toolkit of sponges from the deep sea In this work, reproduction was not only described and discussed with histological and chemical approaches, but also with molecular analyses, studying the mechanisms that regulate the female and male gamete formation in all five species of Geodia (Chapter III) and very briefly in P. ventilabrum (Chapter IV). Even though previous studies focused on understanding the evolutionary footprint of the molecular markers behind the embryogenesis and morphogenesis in sponges, and found several mechanisms with similar functions as those of more recent metazoans, e.g. Wnt, Notch/Delta, TGF-b and NF-kappaB pathways 88,89,360,412–414, no studies on the comprehensive molecular regulation of the gametogenesis had been conducted to date. Previous studies have examined the molecular mechanisms of several aspects of sexual reproduction, e.g. germ cell line, meiotic cycle, vitellogenesis, oogenesis, in other early metazoans such as Cnidaria and Placozoa, finding that most processes are under similar molecular control in these phyla and bilaterians 186,342,346,415–417. The significance of identifying similar mechanisms related to gametogenesis in Porifera and bilaterians, is that these mechanisms were already present in the first diverging phylum of multicellular animals, and therefore potentially appeared in the ancestor of all Metazoa (e.g. the Urmetazoa). Here, genes related

77 to germ cell formation, and genes regulating the oogenesis and spermatogenesis were found overexpressed in female and male individuals during gametogenesis, showing that the molecular toolkit regulating the basic processes of gametogenesis is well conserved from Porifera to mammals. Even signalling pathways that are activated in specialized accessory structures of gametic organs and regulate stages of gametogenesis in mammals (e.g. retinoic acid pathway) are activated in similar stages and potentially with similar roles in sponges. Thus, the main changes in the evolution of gametogenesis could potentially be the increase in the complexity of cellular components and organ development, as well as the assignment of each structure to a specific role during the progress of gametogenesis. Similar enriched categories were found in all Geodia species (Chapter III) and P. ventilabrum (Chapter IV) during oogenesis, hypothesizing that molecular mechanisms regulating gametogenesis are rather conserved at least among Demospongiae. This comes in accordance with previous findings, identifying germ cell markers and few other reproductive genes in all sponge classes 32,87,188. It is expected that gametogenesis is therefore regulated by similar molecular patterns in all the other sponge classes, but further studies need to be conducted to prove this assumption. Although germ cell molecular markers were activated in both sexes, with some markers more involved in female and others in male gametogenesis, it was not possible to identify which markers are behind the specification of the gamete sex. The molecular markers that regulate and trigger sex determination in Porifera still remain unknown. The fem1 gene, with a clear role in sex determination 418, was activated in Suberites domuncula 419 during increased temperatures in areas where gametes were formed 420. Additionally, fem1 gene was previously detected in all four sponge classes and another sex-determination gene, the dmrt 421, only in homoscleromorph sponges 87. Only the fem1 gene was detected in the transcriptomes of Geodia and Phakellia spp. but it was not upregulated during gametogenesis. So, if this gene plays a role in sex determination, it might be overexpressed before the onset of gametogenesis. In several marine invertebrates, the initial triggering factor is a change in the abiotic conditions, which determines the sex and activates gametogenesis 422,423. As abiotic conditions seem to trigger gametogenesis in Porifera 97, it is expected that nutrient blooms in the case of Geodia and Phakellia spp. and nutrients, light and/or temperature shifts in Antarctic species are the factors that activate relevant molecular cascades which determine the sex and trigger gametogenesis.

Future studies The present work is a foundational step into our understanding of sponge reproduction, covering the morphological and molecular bases of reproductive processes and unraveling ecological and evolutionary implications. However,

78 further studies would help understand the reproductive strategies of these and other keystone species in sponge grounds, which will allow to paint a more accurate picture of the potential of these communities to survive perturbations (human or else) and maintain their populations. The logistic constraints to sample deep-sea habitats are such that even the great efforts performed by the SponGES consortium with multiple campaigns over the course of four years were still not enough to completely assess the reproductive dynamics of the most common species. To further understand the ecological implications of the sponge reproductive patterns in these vulnerable habitats, more key sponge species from these areas are necessary to have an accurate view of the panoply of reproductive strategies that these sponges have developed to adapt to these extreme habitats. In addition, when possible, I would include samples from a wider range of locations and depths, covering all seasons to assess their complete reproductive cycle and understand the adaptability of different species to the different abiotic conditions. These studies would allow to assess the level of vulnerability of these Antarc- tic and deep-sea habitats. Furthermore, I would tag a certain number of specimens in shallower areas where sampling is more accessible, to monitor their reproductive activity, fecundity rates, and metabolic activity for several years, and thus, assess the population dynamics and the energetic demands required regarding reproductive activity. For instance, in some shallow-water oviparous species, it is known that only a portion of the population is reproducing each year because it is a highly demanding process that sometimes entails starvation and death after spawning. Monitoring of the temperate shallow-water oviparous Geodia cydonium, though, has shown that all tagged specimens reproduced all the three years of the study. So, it would be interesting to compare such observations with the reproductive patterns of the deep-sea Geodia populations studied here. Of course, such comprehensive approach is relatively easier to be applied on Antarctic shallow-water species, but quite challenging for the deep-sea sponges. However, the progress in ROV technology makes more and more accessible the manipulation and experimentation of the sponges in vivo. In order to understand the links between the reproductive strategies of these species, their energetic demands, the food availability in the surroundings, and the source of yolk nutrients they provide their propagules with, I would expand the lipidomic analysis pioneered in this study with additional techniques and further approaches. A natural next step in the characterization of the lipid profile of the sponges with UPLC-MS would be to quantify the concentrations of lipids of interest such as the TGs in nonreproductive and reproductive specimens with GC-MS, in order to further understand the actual levels of these lipids and their role. Furthermore, additional information could be retrieved with a spatial identification of lipids, combining histological sections with spectrometry. Several techniques have been recently developed, in which di-

79 rect visualization, identification and quantification of the different lipid classes can be obtained simultaneously (e.g. MALDI-MSI). Using this approach, the role of the different types of lipids in oogenesis in sponges (e.g. detecting the type of lipids used for the yolk) could be properly defined. Similarly, the conclusions regarding the activated molecular toolkit during gametogenesis, by using a comparative transcriptomic approach, could be expanded with newly developed techniques such as spatial transcriptomics. With such a technique, RNA sequencing is conducted directly on histological sections, revealing spatial patterns of gene expression in different areas of the sponge tissue. In this way, it would be possible to identify the role of the different cells in oogenesis (e.g. germ cell identity or true role of accessory cells on the regulation of gametogenesis). Up to now, these spatial lipidomic and transcriptomic techniques are in preliminary stages and need optimization for application on non–model organisms such as sponges. The main difficulty with sponges resides in that they do not have organs and gametes are widely spread in the mesohyl, as well as the presence of highly silicified skeletal spicules, rendering these techniques quite challenging to try. Furthermore, the genome of these species would be a fundamental resource in the future, comple- menting the transcriptomic studies, in order to verify that the genes that were not found in the present study, are actually present or absent in sponges and whether or not the reproductive complement shows any synteny across Meta- zoa. In addition, functional studies are the necessary next step, to verify the function of the reproductive toolkits. However, gene manipulation in sponges is still far from being a reality, although many efforts are currently being car- ried out by several laboratories.

80 Acknowledgments

I wholeheartedly thank my supervisor Dr. Ana Riesgo, for giving me the opportunity to participate in the SponGES Project. It was a once in a lifetime experience, that allowed me to broaden my knowledge around the subject of sponges, to travel to distant places, to meet numerous highly esteemed scien- tists and to cultivate true friendships with nice spongers from all over the world. Ana, I am very thankful for introducing me to the fascinating world of sponge reproduction and electron microscopy, sitting close to me in the dark room and teaching me all the secrets of the cells. I really appreciate your gen- erosity in sharing with me all your expertise and insisting on doing the most colorful graphs in the world. You had the role of supervisor but also of a mum who was paying my lunch, going out for shopping and treated me as another member of your family. Most importantly I really appreciate your great patience, and tolerating my rather frequent meltdowns. I would also like to thank Dr. Paco Cárdenas, for giving me the chance to be affiliated with Uppsala university and thus making it possible for me to receive my PhD degree from the pharmacognosy department, but also for being a very good supervisor. Paco, I am very thankful for your willingness to transmit your knowledge and for your support and guidance throughout my PhD studies, for explaining me with patience every little detail on the chemical structures of sponges and on the Geodia sterasteres. I feel privileged for the opportunity you gave me to participate in the workshop in Srilanka and having my first teaching experience and sponge hunting but also for accumulating beautiful memories. I would also like to thank Prof. Anders Backlud, for his assistance and sustenance in obtaining my degree. I would also like to thank Prof. Ulf Göransson for his support during my PhD, as head of the Pharmacognosy group. Moreover, I would like to express my gratefulness to the late Prof. Hans Tore Rapp, the coordinator of the SponGES Project, who unfortunately passed away and although it is not possible for him to admire his achievements and impact on the final outcome of the entire Project, it would not be possible for me to complete my PhD without his proactive involvement. I would also like to thank Dr. Joana Xavier for all her efforts in administering the Project and rendering it feasible. Without the assistance of external collaborators, my PhD would not be complete, therefore I wanted to thank Dr. David Balgoma for introducing me

81 into the world of lipidomics, for elucidating me in their chemical background as well as running the LC-MS analyses, and Maria Conejero, Dr. Hannah Armer and Dr. Alex Ball for their tutoring on the electron microscopy sectioning. Additionally, I wanted to sincerely thank Dr. Javier Cristobo, Dr. Pilar Ríos and Dr. Paco Sanchez for providing me with the opportunity to participate in the scientific expedition in the Cantabric Sea and for being very help- ful, especially with the construction of special sponge larvae collectors. I wish to extend special thanks to Dr. Javier Cristobo for taking care of me during the Blue Symposium, and introducing me to all the sponge scientific community. Furthermore, I would like to recognize the support received from Prof. Shirley A. Pomponi by hosting me in her lab and sharing her knowledge in sponge cell separation. I would further like to show my gratitude to Prof. Det- mer Sipkema, who was always available to help on board with the sample collection. I am deeply thankful and honored that I was part of the Riesgo lab, which I viewed as my London family. I thank all the people who passed from the Riesgo Lab (Alex, Connie, Anna, Olivia, Jenny and many more) for all the fun and the weirdest discussions in the common room. I have captured in my memory all the unique moments we shared together. Especially, Nadia: thank you for your great support and positive spirit during multiple RNA extractions in the “dungeon”, for inhaling so much HF for my samples, for teaching me how to dance mapale and for showing me the Colombian take on life. Sergi: thank you for your support in and out of the lab, for keeping your house doors always open for me and for counting on me in many of your works. Nathan: thank you for teaching me the weirdest “kiwi” phrases, for being so annoying with me like an older brother, and for being readily available whenever I needed your expertise either in bioinformatics (and actually in everything regarding to work) or in drinking beer (cool bananas!). Patri: you will always be my fili mou, thank you for your support and the loughs you offered me as also for your best company in the remote conferences and cruises during these years, and together with Carlos, thank you for just being crazy, and always there for me, available to fix sponges on board and open for any discussion or party! Belen and Angi: “compis” thank you for all the moments we spent together, for the musical and theater plays you brought me to, for the original “chilian” moments and for being able to share our everyday dramas. I have no doubts that the “dinosnore” nights at the museum would be much less interesting without you by my side. Ele: although I did not really enjoy the fact that you left me poorer than I was before we met with your “two pounds”, I would change for nothing the moments we spent together in and out of the lab. Bruna: it was such a pleasure sitting next to you and the patience you demonstrated with me was astonishing, to say the least. Cristina: thank you for helping me with R and biostatistics and for re-introducing me to the world of roller skating. Aida: thank you for assisting me with the proteomics and for always

82 offering your help with a smile. Vanina: your visit definitely gave an extra positive spirit and lots of audiovisual material of crazy moments in Riesgo lab. Besides the Riesgo lab family, I also wish to express my thankfulness to all the people I met at Uppsala University, who made me feel like home during my short stay there. Karin: my PhD sister: thank you for the support and advice on matters pertaining to work, explaining to me how the UU system functions, for offering me your sofa so many times, for all the special experiences (remember our diving success in Sri Lanka) we shared together from the very beginning of our PhD life and for continuously transmitting your positive energy. Astrid: thank you for always welcoming me to your office (together with Karin) for a beer and for introducing me to the virtual reality world. With- out you, I would not be able to submit a “normal” kappa, so thank you for being present during the eleventh-hour. Raquel: thank you for helping me with the Phakellia subsampling and Luke, Erik, Camila, and so many other people from UU, thank you for the nice moments we spent together. Asimenia and Kathrin: thank you for making the overnight ROV transects much more intriguing and for helping me with sampling, but also for the beautiful moments during the GAMs. Emilio and Fernanda: thank you for open- handedly offering your help and your valuable advice on sponge reproduction and the amazing caipirinhas; I will never forget our “swimathon”! Michele and Antonia: thank you so much for your editorial support and the last-minute corrections. I would also like to express my gratitude to my non-biologist friends, who were always supportive, even thought they could not understand why I was putting all this effort in studying sponges. Last but not least, I would not be able to complete my PhD without the support of my family who taught me to be passionate with my work, always encouraged me to keep moving forward without any hesitation and always supported me. Special thanks to Alexandros for standing ceaselessly by my side and always supporting me, especially during the last period of writing.

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Acta Universitatis Upsaliensis Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy 288 Editor: The Dean of the Faculty of Pharmacy

A doctoral dissertation from the Faculty of Pharmacy, Uppsala University, is usually a summary of a number of papers. A few copies of the complete dissertation are kept at major Swedish research libraries, while the summary alone is distributed internationally through the series Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy. (Prior to January, 2005, the series was published under the title “Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy”.)

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