Growth and metabolism of sponges

Sponges (phylum Porifera) are multi cellular filter-feeding invertebrate animals living attached to a substratum in mostly marine but also in freshwater habitats. The interest in sponges has increased rapidly since the discovery of potential new pharmaceutical compounds produced by many sponges. An enormous amount of different chemical structures have been found. Thus far no sustainable production technique has been developed for these marine natural products, because not sufficient knowledge is present about the needs of sponges for both growth and bioactive compound production. The aim of this thesis was to get a better understanding of the growth and metabolism of sponges and of their nutritional needs. Aquaculture is thus far the best method to produce these compounds, although also this technique is not fully developed. To gain more insight in the nutritional needs for growth, we studied the growth rate of Haliclona oculata in its natural environment, Oosterschelde, the Netherlands, and monitored environmental parameters in parallel (Chapter 2). A stereo photogrammetry approach was used for measuring growth rates. Stereo pictures were taken and used to measure volumetric changes monthly during 1 year. The volumetric growth rate of Haliclona oculata showed a seasonal trend with the highest average specific growth rate measured in May: 0.012±0.004 day−1. In our study a strong positive correlation (p<0.01) was found for growth rate with temperature, algal biomass (measured as chlorophyll a), and carbon and nitrogen content in suspended particulate matter. Thus growth rate seems to be dependent on these factors. No correlation was found with dissolved organic carbon, suggesting that Haliclona oculata is more dependent on particulate organic carbon. To obtain more knowledge about the carbon requirements for growth by sponges, respiration rate and clearance rate were measured in situ in Haliclona oculata and compared to the earlier measured growth rate (Chapter 3). The net growth efficiency, being the ratio of carbon incorporated in biomass and the total carbon used by the sponge for respiration and growth, was found to be 0.10 ± 0.013. Thus, about 10% of the total used carbon was fixed in biomass and over 90% was used for generating energy for growth, maintenance, reproduction and pumping. H. oculata had 2.5 μmol C available for every μmol O2 consumed. A value of 0.75 for the respiratory quotient (RQ in μmol CO2 μmol O2 -1) is the average value reported in literature for different marine invertebrates. Thus, carbon was available in excess to meet the respiratory demand. We found that only 34% of the particulate carbon pumped through the sponge was used for both respiration and growth. Oxygen was not the limiting factor for growth, since only 3.3% of the oxygen pumped through the sponge body was used. Our results indicate that both oxygen and carbon availability are not limiting. The low growth efficiency agrees with the low growth rates found for many sponges. In order to produce drugs by culturing sponges their growth must be improved. To improve growth, basic knowledge about how food sources are used by the sponge is needed. To find the exact relation between food retained and food converted to sponge biomass we need to be able to distinguish between feed components and sponge biomass, which means we need biomarkers for the feed and for the sponge. The fatty acid (FA) composition of organisms is specific and can therefore be used as biomarkers. We identified and compared fatty acid profiles of five different sponges in three habitats with those in the suspended particulate matter (SPM) in the surrounding water (Chapter 4). Haliclona oculata and Haliclona xena from the Oosterschelde, Haliclona xena and Halichondria panicea from Lake Veere, both in The Netherlands and Dysidea avara and Aplysina aerophoba from the Mediterranean were studied. In the SPM we found comparable FAs to the FAs of sponges up to chain lengths of 28 C-atoms. Different species of sponges showed similarities, but also very different FA profiles, while they were collected from the same habitat at the same moment. The biomarkers for diatoms and dinoflagellates were abundantly found in all sponges except A. aerophoba as this sponge relies mostly on bacterial food sources based on the many bacterial FAs found in this sponge. In all species, except A. aerophoba, C26:3(5,9,19) and C26:2(5,9) were very abundantly present. These FAs were also abundant in the SPM, while it was stated in literature that these compounds are very typical for sponges. Several FA biomarkers were found for the different sponges. Fatty acid composition is dependent on different factors like food availability and temperature and thus the composition will change in the different seasons. We have studied fatty acid composition and stable isotope 13C natural abundance of suspended particulate matter (SPM) from seawater and sponges in different seasons in the same locations as in chapter 4 (Chapter 5). 13C natural abundance can be used to find the origin of compounds, as the 13C values of compounds are similar to the values from their original producers. The FA concentration variation in sponges was related to changes in fatty acid concentration in SPM. 13C natural abundance in sponge specific FAs showed very limited seasonal variation at all sites. Algal FAs in sponges were mainly acquired from the SPM through active filtration in all seasons. Sponge specific FAs had similar 13C ratios as algal FAs in May at the two Dutch sites, suggesting that sponges were mainly growing during spring and probably summer. During autumn and winter, they were still actively filtering, but the food collected during this period had little effect on sponge 13C values suggesting limited growth. The bacterial sponge A. aerophoba relies mostly on the symbiotic bacteria. In all sponges we found that the ω7 longer chain FAs, C24:1(17) and C26:3(5,9,19) could be traced back to be of bacterial origin. Using a 13C pulse-chase approach metabolic rate can be studied inside organisms. The carbon metabolism of two marine sponges, Haliclona oculata from the Oosterschelde (The Netherlands) and Dysidea avara from the Mediterranean (Spain), has been studied (Chapter 6). The sponges were fed 13C labelled diatom (Skeletonema costatum) for 8 hours in a closed system during which they took up between 75 and 85 % of the diatoms added. At different times whole sponges were sampled for total 13C enrichment, fatty acid composition and 13C enrichment in these fatty acids. During the first day the level of 13C label inside the sponges stayed the same after which the 13C label was metabolized and excreted. Algal biomarkers present in the sponges were highly labeled after feeding and their labeling levels decreased from the second day until no label was left 10 days after enrichment. The sponge specific long chain C26 fatty acids incorporated 13C label already during the first day and the amount of 13C label inside these FAs kept increasing until 3 weeks after labeling. Thus, the algae fed to the sponges were taken up by the sponges within 8 hrs and first conversion started during the first day. Conversion of label occurred at least until at least 3 weeks after feeding. In different studies it was shown that sponges grow slow, but are able to regenerate damaged tissue fast. Moreover, it has been found that damaged tissue coincides with higher secondary metabolite production. Therefore, we were interested in carbon metabolic rate changes after damaging sponge tissue. We have examined the change of carbon metabolic rate of fatty acid synthesis due to mechanical damage of sponge tissue in Haliclona oculata and Dysidea avara (Chapter 7). Metabolic studies were performed by feeding sponges with 13C labeled biomass of diatom, Pheaodactylum tricornutum, either after or before damaging and tracing back the 13C content in the damaged and healthy tissue. Filtration and respiration rate in both sponges responded quickly to damage. For the finger-sponge H. oculata the rate of respiration was reduced immediately after damage. 6 Hours after damage the filtration rate increased to a level that was higher than the starting value, while the respiration rate returned to the initial value before damage. For the encrusting sponge D. avara the filtration rate also decreased directly after damage, but in this case it did not return to the value before damage after one day. Respiration was not measured for D. avara. The 13C data revealed that H. oculata has a higher metabolic rate in the tips where growth occurs compared to the rest of the tissue and that the metabolic rate is increased after damage of the tissue. For D. avara no differences were found between damaged and non damaged tissue. Thus far it is still not fully understood why, when, where and how bioactive metabolites are produced in sponges. For the near future sea-based sponge culture seems to be the best production method. However, for controlled production in a defined system it is better to develop in vitro production methods. This could be in vitro sponge culture or sponge cell culture, culture methods for symbionts or transfer production routes into another host. We still have insufficient information about the background of metabolite production in sponges. Before culture methods are developed we should focus on factors that induce metabolite production, which could be done in the natural habitat by studying the relation between stress factors (such as predation) and the production of bioactive metabolites. Next, the biosynthetic pathway of metabolite production should be unraveled, as well as the genes involved. The location of production within the sponge should be identified in order to choose between sponge cell culture and symbiont culture. Alternatively the biosynthetic pathways could be introduced into hosts that can be easily cultured in bioreactors. Chapter 8 discusses the current state of sponge metabolite production and the steps that need to be taken to develop commercial production techniques. The different possible production techniques are also discussed.