The Importance of Water Flow for Culture of Dysidea Avara Sponges
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The importance of water flow for culture of Dysidea avara sponges DM-thesis final_03-05-08.indd 1 3/4/08 9:31:42 PM Promotoren: prof. dr. ir. R. H. Wijffels, hoogleraar in de Bioprocestechnologie, Wageningen Universiteit. prof. dr. ir. J. L. van Leeuwen, hoogleraar in de Experimentele Zoölogie, Wageningen Universiteit. Overige leden promotiecommissie: prof. dr. J. A. J. Verreth, Wageningen Universiteit. prof. dr. M. J. Uriz, Centro de Estudios Avanzados de Blanes, Spanje. prof. dr. S. A. Pomponi, Harbor Branch Oceanographic Institution, Verenigde Staten. prof. dr. P. Aerts, Universiteit Antwerpen, België. Dit onderzoek is uitgevoerd binnen de onderzoekschool VLAG. DM-thesis final_03-05-08.indd 2 3/4/08 9:31:42 PM The importance of water flow for culture of Dysidea avara sponges Dominick Mendola Proefschrift ter verkrijging van de graad van doctor op gezag van de rector magnificus van Wageningen Universiteit, prof. dr. M. J. Kropff in het openbaar te verdedigen op woensdag 9 april 2008 des namiddags te vier uur in de Aula DM-thesis final_03-05-08.indd 3 3/4/08 9:31:42 PM Dominick Mendola The importance of water flow for culture ofDysidea avara sponges Ph.D. Thesis Wageningen University, The Netherlands, 2008 – with Dutch and English Summaries ISBN: 978-90-8504-908-1 DM-thesis final_03-05-08.indd 4 3/4/08 9:31:43 PM Contents Chapter 1 Introduction . 1 Chapter 2 Aquaculture of three phyla of marine invertebrates to yield Bioactive metabolites: process development and economics . .9 Chapter 3 Environmental flow regimes for Dysidea avara sponges . 39 Chapter 4 Morphology induced flow patterns by Dysidea avara sponges in a flow tank . .55 Chapter 5 Oscular outflow rates for Dysidea avara sponges . 75 Chapter 6 Re-plumbing in a Mediterranean sponge . 95 Chapter 7 General Discussion and Conclusions . 107 Summary . 119 Samenvatting . 123 Acknowledgements . 128 Training and Supervision Activities . 129 Biography . .130 DM-thesis final_03-05-08.indd 5 3/4/08 9:31:44 PM DM-thesis final_03-05-08.indd 6 3/4/08 9:31:44 PM Chapter Introduction to the 1 Thesis DM-thesis final_03-05-08.indd 1 3/4/08 9:31:45 PM INTRODUCTION TO THE THESIS The title of this thesis, “The importance of water flow for the culture of Dysidea avara sponges” begs two key questions: • why does man want to culture this sponge, and • why is water flow important in the culture process? Dysidea avara is a violet-colored encrusting sponge native to the Mediterranean Sea. It is the natural source of terpenoid compounds avarol and avarone which have shown activity against some of man’s most pernicious ailments, including inflammatory diseases, viral and bacterial infections, and some tumoral cancers. Demand for these compounds as natural products is projected to increase. Aquaculture has been proposed to replace natural collections as a more sustainable method of supply of sponge biomass for natural product extraction. Sponge science fulfills the role of informing sponge aquaculturists on scientific findings related to the culture requisites of these valuable sponges. For any aquaculture process, in situ (in the sea) or ex situ (in tanks), water flow past the cultured organisms is of paramount importance for sustaining organism health and vitality. Too much flow and the organisms could be physically harmed, fail to catch their food, or stop growing (and energy resources would be wasted for moving water too quickly through the system). Too little flow and the organisms could be harmed for lack of sufficient exchange of metabolic gases, deteriorating water quality, or access to food. In the case of ex situ culture in controlled environment tank systems, too little flow could also harm the metabolic balance of the microorganisms in the bio-filters. Within the aims of a funded EU CRAFT project, Wageningen University was charged with the investigation of water flow requirements for sponge culture — including Dysidea avara. This thesis reports on the findings of those sponge-flow interactions. Aim and goal of this research The principal aim of this research was to understand the optimal water flow requirements for the marine sponge Dysidea avara, so that flow parameters could be set for ex situ culture in tanks, or in situ culture in the sea. The ultimate goal was to help insure the success of large- scale aquaculture of the species for obtaining biomass for extraction of its valuable terpenoid natural product avarol. Research overview The first phase of the research was directed towards understanding natural flow regimes surrounding individual Dysidea avara sponges that were members of a mixed sponge fauna found within a rocky cove on the N. E. coast of Spain. Using SCUBA and employing a 3-D Doppler acoustic velocimeter we recorded bulk current flow in the open water of the cove, and also close to aggregations of sponges on rocky ledges and inside small caves. A heated 2 CHAPTER 1 DM-thesis final_03-05-08.indd 2 3/4/08 9:31:45 PM Introduction thermistor flow sensor was used to record fine-scale flow speed in very close proximity to individual sponges, and outflow velocity from their oscula. Flow velocities and flow regimes were characterized over a year-long period from September, 2005 through October, 2006. In the laboratory, a special-built flow tank was employed together with laser-illuminated particle tracking velocimetry and high-speed video photography, to visualize and record flow passing over the complex surface morphology of specimens. Oscular outflow velocity and outflow rate were calculated over a range of background flow velocities modelled after nature. The flow tank was also taken to Spain, to compare oscular outflow of freshly-collected wild specimens to that of the tank-reared specimens. From outflow velocities and oscula dimensions, volume outflows were calculated. For specimens that showed over-sized ostia inflow rates were also calculated. Three-D computational fluid dynamic modeling (CFD) was begun to aid in better understanding fine scale flow around the mm-sized cone-shaped conules which characteristically cover the surface of the sponge. Some preliminary results were obtained which will form the base for future CFD work that will continue past the completion of the thesis project. Before introducing the Thesis Chapters, I will first give a brief overview of the biology of sponges for the lay reader, and a brief history of flow research in marine biology. What are sponges…? Sponges are sessile (i.e., living attached to a substratum) marine and freshwater invertebrate animals with only a cell-level body plan (e.g., they possess no true tissues). An outer epithelial layer of cells, the pinacoderm is separated from the internal cell layer called the choanoderm by the mesohyle, a matrix of amoeboid cells, skeletal elements, and sometimes dispersed or filamentous collagen (called spongin, as in Dysidea avara). The entire sponge body comprises little more than an open hydraulic network of branching inhalant and exhalant canals with flagella-powered cellular pumps in-between. In demosponges (95% of all living species, including D. avara) ambient water enters the inhalant canals through a myriad of 5-50 Figure 1. A schematic representation a single μm-sized inlet ostia spread across the dermal osculum sponge with cut-away showing (in surfaces, and flows through the inhalant canals order of flow through the aquiferous system): to the food-filtering choanocytes. It exits via the the ostia, inhalant canals, choanocyte chambers, exhalant canals, and finally the exhalant canals which lead to one or more mm osculum. (Drawing not to scale; after original or larger-size exit holes called oscula or oscules. drawing by R. Osinga.) CHAPTER 1 3 DM-thesis final_03-05-08.indd 3 3/4/08 9:31:47 PM The system is efficiently designed to pump large amounts of ambient water for filtering-out micrometer-sized food particles such as bacteria, micro-algae and small organic aggregates. The specialized flagellated pumping cells (choanocytes) are arranged 50-200 each into 25-65 μm-dia. spherical or cylindrically-shaped mini-pumping chambers (Bergquist; 1978, Simpson, 1984). It has been estimated that a cubic millimeter of sponge cell mass contains as many as 10 million choanocyte chambers, and with all of its flagella beating simultaneous and continuously, a one kg sponge could pump as much as 23,000 liters of water in a single day (calculated from data of Reiswig, 1974). Sponges are among the earliest evolved multi-cellular organisms, the Metazoans; with fossil ancestors dating from the pre-cambrian period, some 580 million years in the past (Chia-Wei et al. 1998). Science has so far described approximately 15,000 sponge species, placing all known and fossil species into one of three taxonomic classes within the phylum Porifera. Each taxonomic class is differentiated mainly on the bases of the chemical and fine-scale structure of its inorganic skeletal elements. The Demospongiae class contains 95% of all living sponge species. Members generally have siliceous spicules dispersed in an organic matrix of collagen (some do not, including Dysidea avara). It is the dried skeleton of members of the Demospongiae which is the familiar “sponge” for human uses. The Hexactinellida contains the exclusively marine and mostly deep-sea glass sponge with hexactine-formed siliceous skeletal elements. The third class, Calcarea, are also exclusively marine and generally considered the most primitive of living sponges, with all members making a calcareous skeleton from dissolved calcium carbonate. Most sponges also contain aggregations of various autotrophic and/or heterotrophic sponge-specific symbiotic bacteria which live commensally within the cells of the mesohyl. The symbionts provide nutritional components biosynthesized from dissolved nutrients, and sometimes allelochemicals, exploited by the sponge for defence against would-be predators or competitors vying for precious colonization space.