Physiological responses of gametophytes and juvenile sporophytes of Saccharina latissima to environmental changes

Transcription

1 Physiological responses of gametophytes and juvenile sporophytes of Saccharina latissima to environmental changes Report MSc Internship Annelous Oerbekke t Horntje, August

2 Physiological responses of gametophytes and juvenile sporophytes of Saccharina latissima to environmental changes MSc Internship Company: Hortimare Supervisor: Freek van den Heuvel University: Wageningen UR Department: Plant Physiology Supervisor: Harro Bouwmeester August 2014 Annelous Oerbekke

3 Abstract There is a growing interest for commercial cultivation of marine and freshwater algae. The marine algae (macro algae) are interesting for consumption by other aquaculture (for example fish or shellfish), human consumption, iodine extraction, algine extraction and agar extraction. One of the fastest growing species of macroalgae in European waters is sugar kelp Saccharina latissima (former Laminaria saccharina). S. latissima is dependent on a seasonal development, the growth and reproduction of gametophytes are influenced by different environmental factors like; light, temperature and day length. In this study the physical responses of gametophytes and sporophytes were monitored during changes in medium and density. The experiments were performed in Petri dishes, Erlenmeyer flasks and titration plates. First of all, the data showed a clear difference between sporophyte development in gametophyte cultures of different ages. The sporophytes grown after induction of gametophytes extracted in December 2013 where bigger than the ones grown from extractions in February. With these results we can conclude that the gametophytes extracted in December are stronger, probably due to the natural cycle of S. latissima. Another result was the clear effect of densities on the sporophyte development after induction. The higher the gametophyte density, the smaller the sporophytes became. Competition for space, light and/or nutrients may play a role in this effect. When adding vitamins to the medium, no clear positive effect was found. But when removing kanamycin during the induction phase, the sporophytes became larger in length and width. An explanation can be that kanamycin may have an effect on the protein syntheses. This study shows that a lot is known about gametophytes, but still a lot can be discovered to find the best method for inducing gametophytes. 3

4 Table of Content 1. Introduction Background Experimental organism: Saccharina latissima 5 2. Research questions and hypotheses 8 3. Material and Methods General setup Experimental setup 10 4 Results Different methods of adding nutrients to gametophyte cultures Effect of vitamins on the induction of gametophytes of different ages Effect of kanamycin on the induction of gametes Calibration line: Absorption Amount of Gametophytes 28 5 Discussion Different methods of adding nutrients to gametophyte cultures Effect of vitamins on the induction of gametophytes of different ages Effect of kanamycin on the induction of gametes Calibration line: Absorption Amount of Gametophytes 31 6 Conclusion 32 7 References 33 4

5 1. Introduction 1.1. Background There is a growing interest for commercial cultivation of marine and freshwater algae. According to the Food and Agriculture Organization (FAO) of the United Nations 37 marine and freshwater algae species are registered that are produced in aquaculture (FAO, 2014). In general there are three types of macro algae categorized by their type and pigment content. These are green, brown, and red algae (Burton 2009). Not all macro algae species can be used for aquaculture. Some grow to slow; others do not have interesting components. The Eacheuma seaweeds and Japanese kelps dominate the aquaculture production of macro algae at the moment (FAO, 2014). Eacheuma is a red seaweed that is interesting for carrageenan extraction. Other purposes are consumption by other aquaculture (for example fish or shellfish), human consumption, iodine extraction, algine extraction and agar extraction. The world production of seaweed has doubled between 2000 and 2012, especially in Asia. Estimation is that in 2012 approximately 9 million tonnes of farmed seaweeds were used for human consumption (FAO, 2014). In contrast with Asia, the European seaweed production mainly focussed on harvesting natural stocks (Burton, 2009). A couple of hurdles have to be taken before the aquaculture in Europe can develop further. A good location has to be found for seaweed farms along European shores. These shores can be exposed to unpredictable and frequent storms that make cultivation and harvesting difficult. On top of that seaweed beds need to consist of one single species at the same development stage (Kain and Dawes, 1987). In the late 1970s and early 1980s several seaweed farming systems designed in the U.S. were tested, but the offshore challenges were too great and the tests failed. In Europe, long-line systems were tested and showed that there is potential in the development of large-scale ocean cultivation of seaweeds. Todays challenges lie in further developing methodologies to grow, harvest, transport and process large quantities of macroalgae as cost-efficient as possible (Kraan, 2013). The seaweeds that are most interesting for cultivation in Europe is the genus Laminaria, also called kelp. Kelps are among the fastest growing seaweeds in the world. This genus is perfect for Europe, because it prefers temperate waters that stretch from northern Portugal to Northern Norway (Kraan, 2013). A difficulty that occurs in mass cultivation of kelps is the production of sporophytes on ropes, through zoospores that develop in female and male gametophytes (Bartsch et al. 2008). For cultivation it is important to accomplish a continuous production of zoospores or maintain cultures of gametophytes for year-round rope production (Xu, 2009). Laminaria gametophytes were successfully isolated and cultured in the 1970 s (Lüning and Neushul 1978; Fang et al. 1978). Lüning achieved vegetative growth and induction of gametophytes in cultures in In 1994 Wu et al. introduced a Laminaria seedling culture method using vegetative gametophytes instead of the extraction of zoospores from adult plants. With this knowledge different techniques of growing seedlings in hatcheries have been achieved (Kaas, 1998; Wu et al. 2004; Xu et al. 2009). In these hatcheries the perfect environment can be created for seedlings to grow. Small sporophytes are seeded on ropes and grown before transporting these to open sea. The growth depends on different parameters, among these the most important are: light, temperature, space and nutrients. Kelps are macro algae that can adapt to their environment by changes in morphology and/or growth. Less is known about the difficulties that come across when the seedlings are transferred from the hatcheries into unprotected environments like outside tanks and eventually the sea. During this research experiments will be conducted that try to give more insight in this process. Another goal will be to see if the Laminaria species can adapt to outside conditions during the formation of sporophytes and the weaning phase. 1.2 Experimental organism: Saccharina latissima In the northern hemisphere, especially in temperate to polar rocky coastal ecosystems, Laminaria is one of the most important macro alga order (Bartsch, 2008). Previous studies indicate that Laminariales species are well suited to environments characterized by low temperature, low light and periodic nutrient limitation (Bolton and Lüning, 1982; Gerard and DeBois, 1988; Lüning, 1990). One of the fastest growing species in European waters is sugar kelp Saccharina latissima (former Laminaria saccharina) (Forbord, 2012). S. latissima contains carbohydrates in high contents and is therefore interesting for large scale cultivation. S. latissima is dependent on a seasonal development with a maximum growth in the first half of the year followed by reduced growth during summer (Kain 1979; Lüning 1979; Bartsch et al. 2008). For aquaculture this life cycle is of big importance. The period of planting affects the preparation of ropes and gametophyte cultures. At the moment, planting and hatchery conditions are tuned to each other, but further research is being done on the subject of adapting indoor grown sporophyte seedlings to specific outdoor conditions. 5

6 Figure 1: Life cycle of Laminaria species. (Raven, 2005). The life cycle (figure 1) of Laminaria species consists of a diploid sporophyte phase and a haploid gametophyte phase (Kain, 1979). The adult plants form sporangia (sorus) from autumn to winter (Kain 1979; Lüning 1979; Bartsch et al. 2008). This sorus contains a large number of haploid zoospores. The zoospores will be released into the water and settle on substrates where they develop into male or female gametophytes. When moving into the reproductive phase, male gametophytes produce antheridia and female gametophytes oogonia (Kain, 1979). The antheridia develops sperm cells and the oogonia an egg cell. When fertilized, a zygote is formed which will develop into a sporophyte. This process of forming sperm and egg cells together with the formation of a zygote through fertilization is called induction. The growth and reproduction of gametophytes are influenced by different environmental factors like; light, temperature and day length (Kain, 1979; Lüning 1980). Temperature S. latissima can tolerate a broad range of temperatures, from New York and Portugal in the South all the way to Greenland in the North (Bartsch et al. 2008). The temperature optimum for S. latissima is between 10 and 15 C (Fortes and Lüning, 1980). In this range photosynthetic characteristics were almost identical, indicating thermal acclimation (Andersen, 2013). Higher temperatures are less suitable as sporophytes grown at 20 C suffered from tissue decline and the reduction of net photosynthetic 6

7 capacity (Andersen, 2013). The ability to adapt might indicate different ecotypes, which may also explain the wide distribution of S. latissima. Machalek et al. (1996) suggests that complex metabolic regulation optimizes the photosynthesis that made this acclimation over a wide range possible. The adult S. latissima plants have a wide temperature range but gametophytes require other conditions. Gametophyte cultures survive at 10 C according to Lüning (1969). Below 10 C growth is slow, above 10 C there is a broad optimum (just like the adult plants) and lethal limit is near 20 C (Lüning, 1980). For example, gametophytes of California Laminariales have a temperature optimum for vegetative growth in the range of C (Lüning and Neushul, 1978). The optimal temperature for the reproductive phase is 10 C in blue light, but 5 C in red light (Lüning and Dring, 1972). Light Kain (1964) found that light intensity affects the fertility and morphology of Laminaria gametophytes. The gametophyte cultures are best kept in red fluorescent light at an irradiance of 10 μmol/m/s (Lüning and Dring, 1972). The gametophytes will grow by branching, but will not go into reproductive stage. This reproductive stage is induced by blue light (Lüning and Dring, 1972; Lüning and Dring, 1975). After culturing in red light, blue light for a short time (6 hours or less) is necessary for inducing fertility. After 5-10 days gametophytes begin to form eggs after the blue light treatment, this suggests that blue light specifically affects the reproductive development of the gametophytes (Lüning and Dring, 1972; Lüning and Dring, 1975). The action spectrum for the induction of fertility has a main peak between 430 and 450 nm (Lüning and Dring, 1975). Ecotype differentiation in light-related traits has occurred between populations of S. latissima. Sporophytes from shallow, deep and turbid habitats exhibited differences in photosynthetic capacity and efficiency after acclimation to common garden conditions (Gerard, 1988). Nutrients Just as higher plants, macro algae need nitrogen, phosphorus and carbon. Phosphorus and nitrogen normally occur at low concentrations in seawater. This can cause nutrient limitation for macro algae (Lüning, 1990). There is a ratio, the Redfield stoichiometry, for carbon, nitrogen and phosphorus found throughout the deep oceans and organisms. This ratio is found to be C:N:P = 106:16:1. But throughout the year, the chemical composition of S. latissima varies considerably (Sjøtun and Gunnarsson, 1995). Kelps have a seasonal pattern of growth and storage of nutrients and polysaccharides (Broch, 2011). S. latissima stores nutrients in late winter and early spring. The nutrients will be used in the growth period through late spring into early summer. Growth is reduced in summer when the plant stores carbohydrates. During autumn the growth is still reduced and increases again during mid-winter. The stored carbohydrates are then used for growth (Sjøtun, 1993). Vitamins Many algae require vitamins because they are auxotrophic, in comparison with higher plants (Provasoli and Carlucci, 1974). Mainly Vitamin B12, Thiamine and Biotin are used in media for cultivation of micro algae. Algae require Vitamine B12 for growth and it is suggested that the source is a symbioses with bacteria (Croft, 2005). Thiamine plays a role as an enzymatic cofactor in metabolic pathways. It also functions other than an cofactor in abiotic an biotic stress (Goyer, 2010). Biotin is involved mainly in the transfer of CO2 during HCO3- dependent carboxylation reactions (Alban, 2000). 7

8 2. Research questions and hypotheses During this research the main focus of the experiments are the physiological responses of gametophytes and juvenile sporophytes of S. latissima to environmental changes. From the results practical consequences for conditions, treatments and weaning process can be concluded and advised. To reach this advice, different experiments will be conducted with gametophytes and with juvenile sporophytes. Both experiments will be conducted with gametophytes. The first experiment will focus on the addition of nutrients to cultured gametophytes. Research question: Is total medium refreshment necessary to maintain proper gametophyte cultures or will only addition of nutrients suffice? Hypotheses: There will be no difference between adding nutrients directly and refreshing the whole medium. The only time the whole medium had to be refreshed is when the propagation step is necessary. The second experiment will demonstrate if vitamins and antibiotics have an effect on the induction of gametophytes of different ages. Research questions: Do younger cultures show better induction results than older cultures? Will gametes overall induce better, when changing the medium by adding vitamins? Do different starting densities of gametophytes have an effect on the induction? Does kanamycin have an effect on induction of gametophytes? Hypotheses: Especially gametophytes in older cultures will induce better with vitamins. Induction in lower gametophyte densities will be better than in high densities. The induction of gametophytes is better in medium with kanamycin added than in medium without. 8

9 3. Material and Methods 3.1 General setup Gametophytes of S. latissima The collection of zoospores is an important step in the cultivation process. After extraction the zoospores have been cultured and will germinate into female and male gametophytes. The cultures are all kept in Erlenmeyer s with a volume of 400 ml under the same conditions. The temperature is approximately C and the cultures are in constant movement to keep the gametophytes from attaching to the surface of the Erlenmeyer. Red light (20-25 μmol m -2 s -1, 475 nm) with a 16:8 dark/light cycle prevents the gametophytes from moving into the reproductive phase. For the experiments gametes from Dutch origin of different ages (1 year, 6 months and 2 months) have been selected Sporophytes of S. latissima The induction of the gametophytes will be triggered by blue light. After refreshing the medium, the gametophytes will be incubated at 8-10 C in blue light (40-60 μmol m -2 s -1, 675 nm) for 2 weeks. A 12:12 dark/light cycle is used. After fertilization of the egg cells within the induced culture, the development of sporophytes starts. For the experiments sporophytes of Dutch background will be used Medium The main part of the medium consists of pasteurized seawater. Pasteurized seawater is stored at approximately 12 C. P, K and GeO 2 are added to a final concentration, which can be found in table 1. P is used as nutrient source. The used final concentration of P corresponds with a F/2 medium (appendix 2). Kanamycin is used for reducing the amount of bacteria in cultures. GeO 2 reduces growth of diatoms in the cultures (Protocol 8). Table 1: Medium preparation for stock and culture solution Stock solution (200 ml)* Added to culture medium (per 1 L) P 5 ml 5 ml K 2 g 5 ml GeO g ** 0.5 ml * Added to MilliQ ** Dissolved with 1ml/L 1 M NaOH Vitamins As seen in table 2, the medium that is used does not include any vitamins. Usually three vitamins, cyanocobalamin (vitamin B 12), thiamine and biotin, are added to algae media (Provasoli and Carlucci, 1974). The amount that will be used during different experiments can be found in table 2. Table 2: Vitamin preparation for stock and culture solution Stock solution (100 ml)* Added to culture medium (per 100 ml) Vitamin B mg 0.35 ml Thiamine 50 mg 0.8 ml Biotin 5 mg 0.8 ml 9

10 * Added to MilliQ (West and McBride, 1999) 3.2 Experimental setup Different methods of adding nutrients to gametophyte cultures As of right now the method for adding new nutrients to gametophyte cultures is refreshing the whole medium. For cultures that have a larger volume, like 8 L, it is more difficult to refresh the entire medium. This experiment will compare two ways of adding new nutrients. One treatment is refreshing the entire medium; the other treatment is only adding new nutrients (5 ml/1l of Pokon stock solution) after one month. The control stays without addition of nutrient or refreshment. The experiment is done in two separate versions, using an 18-well titration plate and Erlenmeyer flasks. This decision is made to have more control and measurement opportunities during the experiment. Experiment will be in triplicate. The Erlenmeyer flasks will be incubated in red light (475 nm) at an intensity of μmol m -2 s -1 in constant movement. The temperature will be C and a light/dark cycle of 16:8. The titration plate will be incubated in red light (475 nm) with an intensity of μmol m -2 s -1. The temperature will be C and a light/dark cycle of 16:8. In the titration plate 8 wells stay without any adding of nutrients or medium, in 8 wells only nutrients are added (pokon stock) and in the last 8 wells the old medium will be removed with a pipet and new medium will be added. A stadium 4-5 S. latissima culture will be used, with start absorption of approximately 0.4 in as well the Erlenmeyer flasks as the titration plate. Stadium 4-5 can be identified by the ball and dumble shape of the gametophytes. See appendix 1 for a picture. The measurements will be done by a liquid Pulse-amplitude modulator (PAM) and spectrophotometer, next to pictures. The pictures will be used when different measurements occur. The PAM measurement is to assess photosynthetic performance of the cultures. Figure 2: Chlorophyll fluorescence after Schreiber (1997). When chlorophyll a is exposed to light, it will absorb a part of the light s energy and use it, but it will also absorb and emit a part of this light s energy at a lower energy level. This is called fluorescence. Healthy marine algae will fluorescence when exposed to relatively high intensities of visible light. When a very weak amount of light is applied to the chlorophyll a by the PAM meter, they will weakly fluorescence. This is called Minimum Fluorescence (F0). When a brief pulse of intense light is applied to a dark-adapted sample, the fluorescence will be max (F m). Variable fluorescence (F v) is estimated by subtracting F0 from F m (Schreiber, 1997). Figure 3 shows schematically the process. A healthy culture gives a value of F v/f m around the The interesting wavelengths for the spectrophotometer are 475 nm and 675 nm. The 475 nm wavelength will give an indication of the carotene pigment, while 675 nm will give an indication of chloroplasts. Analyses of the titration plate will be done by eye. Colour and other differentiations in morphology will be noted and photographed. 10

11 3.2.2 Effect of vitamins on the induction of gametophytes of different ages In this second experiment, the effect of vitamins on the induction of gametophytes will be monitored. Induction will take place in an incubator with a temperature of 8-10 C and blue LED lights (intensity μmol m -2 s -1, 675 nm). One of the variables in this experiment is the different ages of the cultures. The cultures extraction dates are from February 2013, December 2013 and February Another variable are the different starting densities (see table 3 for complete set-up). All the cultures are of Dutch origin. Before starting the experiment, all cultures will be set on the same density of gametophytes (100%: 250,000 gametophytes per ml) using adsorption measurements and dilution of the cultures. For the experiment 5 different dilutions will be examined in Petri dishes with 8 ml content. These densities are approximately 250,000 gametophytes per ml (100%), 187,500 gametophytes per ml (75%), 125,000 gametophytes per ml (50%), 62,500 gametophytes per ml (25%) and 31,250 gametophytes per ml (12.5%). Petri dishes will be filled with the start culture according to table 3. Every week for 6 weeks, pictures will be taken through the microscope to analyse the development of the gametophytes. These pictures will be analysed with the Imagefocus program on amount of sporophytes, length and width of sporophytes (μm) and possible deviations. Table 3: Setup of the first experiment. Table gives the approximate number of gametophytes per ml in the Petri dishes. The Petri dishes contain 8 ml of culture. The experiment set up is the same for with and without vitamins. Culture Standard medium without and with vitamins (gametophytes per ml) Feb ,000 (100%) 187,500 (75%) 125,000 (50%) 62,500 (25%) 31,250 (12.5%) Dec ,000 (100%) 187,500 (75%) 125,000 (50%) 62,500 (25%) 31,250 (12.5%) Feb ,000 (100%) 187,500 (75%) 125,000 (50%) 62,500 (25%) 31,250 (12.5%) Effect of kanamycin on the induction of gametes Kanamycin is used in the medium to reduce the amount of bacteria. It is never tested if the kanamycin could have an effect on the induction of gametes. This experiment is based on experiment 1, also with vitamins and same dilution and the same conditions, but without kanamycin. The control will be the standard used medium. Only one recent culture (February 2014) will be used. The analyses will be the same as in the previous experiment. Every week (for 6 weeks) pictures will be taken through the microscope to analyse the development of the gametophytes. These pictures will be analysed with the Imagefocus program on amount of sporophytes, length of sporophytes and possible deviations. Table 4: Setup experiment without kanamycin in medium. Table gives the approximate number of gametophytes per ml in the Petri dishes. The dishes contain 8 ml of cultures. The experiment set up is the same for cultures with the standard medium and medium without kanamycin. Culture Standard medium and medium without kanamycin (gametophytes per ml) Feb ,000 (100%) 187,500 (75%) 125,000 (50%) 62,500 (25%) 31,250 (12.5%) Calibration line Absorption Amount of Gametophytes By using the spectrophotometer, absorption of a culture can be obtained. By taking absorption measurements of a gametophyte culture every week, it can be stated that when the absorption increases the amount of gametophytes will increase as well. But it is not clear to what amount. For this a calibration line will be developed that links the absorption to the amount of gametophytes. The calibration lines will be different per gametophyte stadium. In this case stadium 4 are small ball-shaped gametophytes and stadium 8 will be branched gametophytes. A clean culture of S. latissima will be selected and the gametophytes will be counted under the microscope using a Sedgewick Counting Chamber. This Sedgewick was specifically designed for the quantitative measurement of the exact number of particles in a set volume of liquid. The chamber is 50mm long by 20 mm wide and 1 mm deep and its base is a grid of 1000 x 1 mm squares. When put over the covering glass, 1 ml is caught. 11

12 When a homogenous distribution is made in de chamber, 15 squares of the grid will be counted to get an overall picture of the culture. Per absorption value, the gametophytes in the culture will be counted. By making a scattered figure, a trend line and formula can be obtained by SPSS. 12

13 4 Results 4.1 Different methods of adding nutrients to gametophyte cultures As of right now the method for adding new nutrients to gametophyte cultures is refreshing the whole medium. This experiment will compare two ways of adding new nutrients. The experiment is done in two separate versions, using a titration plate and Erlenmeyer flasks. One treatment is refreshing the entire medium; the other treatment is only adding new nutrients (5 ml/1l of Pokon stock solution) after one month. The control stays without addition of nutrient or refreshment Results Titration Plate The experiment was running for 8 weeks. The gametophytes started the experiment in stage 4-5, as can be derived from the pictures in figure 4. Figure 3: Start of the experiment (0-measurement). The culture had an absorption of 0.4. The 3 pictures have been made of different wells, but no treatment has been started yet. After the first 4 weeks, the gametophytes grow until stage 8. The gametophytes formed nice balls and there is a clear distinction seen between male gametophytes (thin rhizoids) and female gametophytes (thick rhizoids). No mutual differences were found yet, because no treatment was applied yet. Figure 4: 4-week monitoring moment. No treatment has been started yet. The first 3 pictures have been made under the microscope with 40x magnification. The other 3 pictures are 100x magnified. After 4 weeks (figure 5) the different treatments were applied. In 6 of the wells only Pokon stock was added, calculated to the normal used concentration. In 6 other wells, the whole medium was changed. And in the last 6 wells, nothing was added or changed (control). 13

14 After 8 weeks, clear differences can be seen between the three treatments (figure 6). The stadium 8 gametophytes in the control treatment seem smaller than the gametophytes in the nuts and medium treatment. When magnified 100x, the colour of the gametophytes is also different. The gametophytes in the nuts treatment seems to have more colour (brown) than the medium treatment, and especially more colour compared to the gametophytes in the control. The gametophytes also seem to have thicker rhizoids in the nuts treatment, than in the other two treatments. Figure 5: 8-week monitoring moment. Treatments are indicated above the pictures. The first row of pictures has been taken under the microscope with 40x magnification. The second row pictures are 100x magnified. The colour difference is also visible without a microscope. In figure 7, the whole titration plate is photographed. The gametophytes in the control treatment are much lighter than the ones in the nuts and medium treatment. What also is striking is that there is also a colour difference visible between the gametophytes in the nuts treatment and medium treatment. 14

15 Figure 6: Photograph of the titration plate after 8 weeks. The two columns on the left was the control treatment, the two columns in the middle was the nuts treatment and the two columns on the right was the medium treatment. 15

16 Gametophytes/Ml Europees visserijfonds: Perspectief voor een duurzame visserij Results Erlenmeyer Flasks Next to pictures, more tests have been performed with the cultures that were put in Erlenmeyer flasks. These tests were absorption measurements related to gametophytes amounts (figure 8) with the spectrophotometer and Pulse-amplitude modulator measurements (figure 9). Overall, all the cultures show an increase in biomass. The start absorption was 0.4 with corresponds to 65,000 gametophytes per ml, the end amount of gametophytes per ml lies between approximately 117,000 gametophytes per ml (absorption 0.7) and 218,000 gametophytes per ml (absorption 1.4). What is striking is that after the start of the different treatments all cultures show a drop in absorption, so in gametophytes. Also the control shows a drop, which is strange because nothing was done with these cultures. After the start of the treatments the cultures regained themselves, some faster than others. From the graph no clear difference between the treatments is found. When taken the data, after the first 4 weeks the average gametophyte amount for all treatments was between 155,000 and 163,000 (absorption between 0.94 and 1.0). After 8 weeks, the average gametophyte amount over all 3 Erlenmeyer flasks for the control was 160,000 gametes per ml (absorption of 0.98), for the medium treatment 155,880 (0.85) and for the nuts treatment 182,000 (1.13), seen in figure Gametophytes per ml per treatment Day Medium Nuts Control Figure 7: Absorption measurement each week per treatment. The data points are compared out of 3 measurements. The medium treatment is displayed with the blue line, the nuts treatment with the red line and the control treatment with the green line. The calibration line of figure 19 is used for transformation of the absorption into gametophyte amounts. The red line gives the start of the different treatments after 4 weeks. When the F v/f m is between 0.5 and 0.6 the culture can be seen as healthy. In the first couple of weeks almost all cultures where healthy (figure 8). When moving closer to the 4 week, all cultures had a negative trend. After addition of nutrients or medium refreshment, those cultures had a positive boost in contrast with the absorption values (figure 8). The F v/f m stayed the same for the control treatment, or showed a negative trend. One week after starting with the different treatments, all cultures showed a negative trend. 16

17 Fv/Fm Europees visserijfonds: Perspectief voor een duurzame visserij 0,700 0,600 0,500 Fv/Fm per treatment 0,400 0,300 0,200 Medium Nuts Control 0,100 0, Day Figure 8: PAM measurement each week per treatment. Fv/Fm gives the relative number of electrons produced per photon of light. The medium treatment is displayed with the blue line, the nuts treatment with the red line and the control treatment with the green line. The red line gives the start of the different treatments after 4 weeks. When looked more closely to the individual gametophytes of each treatment, they still seemed healthy (Figure 10). The gametophytes kept their colour and the cell wall was in place. Figure 9: Pictures of gametophytes that were cultured in the Erlenmeyer flasks. The pictures were made after 8 weeks at the end of the experiment. 17

18 4.2 Effect of vitamins on the induction of gametophytes of different ages The effect of vitamins on the induction of gametophytes was tested. The experiment was set up with 3 different variables. One of the variables was the different ages of the cultures. The extractions of the used cultures were done in February 2013, December 2013 and February Another variable are the different starting densities and the last was the vitamins added to the medium Date of extraction comparison The two variables are the age of the used gametophytes and the different densities. There is a significant difference found between sporophyte length and width of February 2013, December 2013 and February 2014 (both p<0.000). In figure 11 it is clearly visible that in general the sporophytes are larger over all densities when the gametophytes were extracted in December The different densities are also significantly different from each other (p<0.000) (table 5). As can be seen in figure 11, there is an overall clear negative trend over the densities visible. The negative trend is especially clear in the culture extracted in December The higher the culture density, how smaller the sporophytes were. For the gametophytes extracted in February 2013 it is remarkable that the sporophytes develop so much better in density 12.5 than in the other densities, as well for sporophyte length as width. This gap is not seen in the other cultures. The sporophytes in the culture from February 2014 seem to have the same development overall, no big differences in length or width. Table 5: Results of Uni-variate analyses for sporophyte length and width separately. Analyses were performed over all data. Data have been ln-transformed before analysis. Significant results are in bold. Sporophyte Length Sporophyte Width Source df F p F p Date Density Date * Density Error (MS) Figure 10: Mean (+/- 1 SE) sporophyte length and sporophyte width of different extraction dates, induced and grown in different densities. Note the different scales in the panels. 18

19 4.2.2 Effect of vitamins Below, the different extraction dates are split up and analysed for vitamin and density effects on the sporophytes. Data gained from the culture extracted in February 2013 were not sufficient enough for analyses. The culture was polluted and probably not viable anymore, concluded from the lack of induced gametophytes and formed sporophytes. So only the cultures extracted in December 2013 and February 2014 are analysed. December 2013 When all the data is pooled together, the length and width of the sporophytes were not significantly different between the two medium treatments (length, p=0.190; width, p= ) directly after induction. Mutually there was also no significance found (Table 7). As can be seen in figure 12a and 12b, it is not clear if vitamins in the medium will have a positive or a negative effect on the induction of sporophytes. The sporophyte length and width between different densities are significantly different (table 6), for sporophyte length (p=0.005) and for sporophyte width (p<0.001). These significances were found over all the data. When taken the densities separately and when compared the different densities mutually, only the densities 25 and 75 (p=0.014) were significant for sporophyte length. For sporophyte width, the densities 12.5 and 75 (p=0.006) and 25 and 75 (p=0.005) were significant. But overall, there is a negative density trend visible, especially for sporophytes width (Fig. 12b), with sporophytes length and width decreasing with increasing density. Table 6: Results of Uni-variate analyses for sporophyte length and width separately after 2 weeks of induction and after 5 weeks of growth after induction. Analyses were performed on data of all treatments (Standard and standard with vitamins) and densities (12.5, 25, 50, 75 and 100). Data have been lntransformed before analysis. Significant results are in bold. After induction After 5 weeks Sporophyte Length Sporophyte Width Source df F p F p Vitamins Density Vitamins * Density Error (MS) Vitamins Density Vitamins * Density Error (MS) Table 7: Results of Tukey test over the different used densities for sporophyte length and width Length Width Length Width Length Width Length Width Length Width ns ns 50 ns ns ns ns 75 ns ns ns 100 ns ns ns

20 Figure 11: Mean (+/- 1 SE) sporophyte length (a) and sporophyte width (b) after 2 weeks induction in different densities and two different media (standard medium and the standard medium with vitamins). Note the different scales in the panels. After 5 weeks of growth, the vitamin effect is changed in a significant difference between the treatment with standard medium and with added vitamins to the medium (p<0.000). Also here the significances found were from pooled data. When taken the vitamin effect per density separately for sporophyte length only at a density of 100 the treatment was significantly different (p=0.003). For sporophyte width at densities 75 (p=0.022) and 100 (p=0.043) there is a significant difference between the two media treatments (table 8). The densities after 5 weeks of growth are still significantly different from each other (p<0.000). The mutual significance is in contrast with table 6 for length of the sporophytes, because density 25 in combination with the other densities is not significant. This is also the case for densities 50 and 75. This is probably again caused by testing all data. For sporophyte width, only densities 75 and 100 are not significant from each other (table 9). However, the negative trend became more visible especially for the vitamin treatment (figure 13). 20

21 Figure 12: Mean (+/- 1 SE) sporophyte length (a) and sporophyte width (b) 5 weeks after induction in different densities and two different media (standard medium and the standard medium with vitamins). Note the different scales in the panels. Table 8: Results of Tukey test over the different media treatments per densities. Vitamine 12.5 Vitamine 25 Vitamine 50 Vitamine 75 Vitamine 100 Length Width Length Width Length Width Length Width Length Width Standard 12.5 ns ns Standard 25 ns ns Standard 50 ns ns Standard 75 ns Standard Table 9: Results of Tukey test over the different used densities for sporophyte length and width Length Width Length Width Length Width Length Width Length Width ns ns ns ns ns ns ns February 2014 The length of sporophytes is not significantly influenced by the two different medium treatments (p=0.821), but the sporophyte width is (p<0.000). This significance is when all data are pooled together (table 10). Between the medium treatments, for sporophyte length no difference between the media is found. For sporophyte width, the medium treatments of density 12.5 and 50 is significant different from each other (table 11). 21

22 When comparing all data for the density variable, both sporophyte length and width are significantly different after induction (p<0.000). For the length of the sporophyte, densities 12.5 and 25 compared with the other densities are significantly different form each other. This is the same for sporophyte width (table 12). Table 10: Results of Uni-variate analyses for sporophyte length and width separately after 2 weeks of induction and after 5 weeks of growth after induction. Analyses were performed on data of all treatments (Standard and standard with vitamins) and densities (12.5, 25, 50, 75 and 100). Data have been lntransformed before analysis. Significant results are in bold. After induction After 5 weeks Sporophyte Length Sporophyte Width Source df F p F p Vitamins Density Vitamins * Density Error (MS) Vitamins Density Vitamins * Density Error (MS) Table 11: Results of Tukey test over the different media treatments per densities. Vitamine 12.5 Vitamine 25 Vitamine 50 Vitamine 75 Vitamine 100 Length Width Length Width Length Width Length Width Length Width Standard 12.5 ns Standard 25 ns ns Standard 50 ns Standard 75 ns ns Standard 100 ns ns Table 12: Results of Tukey test over the different used densities for sporophyte length and width Length Width Length Width Length Width Length Width Length Width ns ns ns ns ns ns 22

23 Figure 13: Mean (+/- 1 SE) sporophyte length (a) and sporophyte width (b) after 2 weeks induction in different densities and two different media (standard medium and the standard medium with vitamins). Note the different scales in the panels. After 5 weeks of growth a significant difference was found between the medium treatments for sporophyte length when all data were pooled together (table 10). When the medium treatment data were compared with each other per density, only at a density of 100 there is a sporophyte length medium effect found. For sporophyte width a significant effect was found for densities 75 and 100 (table 13). The density significance is still present for both sporophyte length and width (p<0.000). In figure 15a the mean sporophyte length is visible, but it became a strange pattern. When taken the different densities separately, only density 12.5 compared with the other densities there are significant differences for sporophyte length. The negative trend is still visible for sporophyte width (figure 15b), only density 100 is not significantly different (table 14). 23

24 Figure 14: Mean (+/- 1 SE) sporophyte length (a) and sporophyte width (b) 5 weeks after induction in different densities and two different media (standard medium and the standard medium with vitamins). Note the different scales in the panels. Table 13: Results of Tukey test over the different media treatments per densities. Vitamine 12.5 Vitamine 25 Vitamine 50 Vitamine 75 Vitamine 100 Length Width Length Width Length Width Length Width Length Width Standard 12.5 ns ns Standard 25 ns ns Standard 50 ns ns Standard 75 ns Standard Table 14: Results of Tukey test over the different used densities for sporophyte length and width Length Width Length Width Length Width Length Width Length Width ns ns ns ns ns ns ns 24

25 4.3 Effect of kanamycin on the induction of gametes In this experiment the induction of gametophytes and growth of sporophytes was tested on different media. The used culture was extracted in December 2013 of Dutch origin. The gametophytes where induced in the standard used media, standard media with vitamins, standard media without kanamycin and the standard media without kanamycin but with vitamins After 2 weeks induction When comparing all densities, the length of sporophytes was significantly different between densities as well as for the width of sporophytes (Table 15). For sporophyte length only densities 75 and 100 are not significant (p=0.938), for sporophyte width densities 50 and 75 are not significant from each other (p=0.078) and densities 75 and 100 (p=0.178). Overall, there is a negative trend visible over the different densities for sporophyte length and width, with sporophytes length and width decreasing with increasing density (Figure 16). When focussing on the different treatments, the length of sporophytes was significantly different between treatments as well as for the width of sporophytes (Table 8). When comparing sporophyte length, the standard and vitamins treatment are not significant when comparing only the different treatments (p=0.999). For sporophyte width, the treatments no kanamycin and kanamycin are not significant (p=0.872). As can be seen in figure 16a, the sporophytes grown in the media without kanamycin are larger than the sporophytes grown in the media with kanamycin. This especially linked with lower densities. For sporophyte width the difference is not as clear. Table 15: Results of Uni-variate analyses for sporophyte length and width separately after 2 weeks of induction. Analyses were performed on data of all treatments (Standard, vitamins, no kanamycin and no kanamycin with vitamins) and densities (12.5, 25, 50, 75 and 100). Data have been ln-transformed before analysis. Significant results are in bold. Sporophyte Length Sporophyte Width Source df F p F p Treatment Density Treatment * Density Error (MS)

26 Figure 15: Mean (+/- 1 SE) sporophyte length (a) and sporophyte width (b) after 2 weeks induction in different densities and different media. Note the different scales in the panels weeks after induction The sporophytes grew for 5 weeks after induction and where measured again. There is still a clear significant difference between the treatments and the densities, for sporophyte length and sporophyte width (Table 9). The negative density trend is still visible even after 5 weeks of growth (Figure 17). For sporophyte length all densities are significantly different from each other (p<0.000). For sporophyte width only density 25 and 50 are not significant anymore (p=0.422). A change has occurred between the treatments for length and width of the sporophytes in comparison with the measurements directly after induction. The treatments standard and no kanamycin with vitamins are no longer significant (p=0.113) for length, as well as the vitamin and no kanamycin with vitamins (p=0.727). For sporophyte width only vitamins and no kanamycin are not significant (p=1.000). In figure 17 it looks like the higher densities are catching up with the lower densities with regards to sporophyte growth. Table 16: Results of Uni-variate analyses for sporophyte length and width separately 5 weeks after induction. Analyses were performed on data of all treatments (Standard, vitamins, no kanamycin and no kanamycin with vitamins) and densities (12.5, 25, 50, 75 and 100). Data have been ln-transformed before analysis. Significant results are in bold. Sporophyte Length Sporophyte Width Source df F p F p Treatment Density Treatment * Density Error (MS)

27 Figure 16: Mean (+/- 1 SE) sporophyte length (a) and sporophyte width (b) 5 weeks after induction in different densities and different media. Note the different scales in the panels. 27

28 4.4 Calibration line: Absorption Amount of Gametophytes By taking absorption measurements of a gametophyte culture for a certain period of time, it can be stated that when the absorption increases the amount of gametophytes will increase as well. But it is not clear to what amount these gametophytes will increase. For this a calibration line was developed that links the absorption to the amount of gametophytes. Figure 17: Calibration lines of S. latissima gametophytes stadium 4. The quadratic trend line with R 2 = 0,886. With SPSS a scatter plot is composed. In figure 18 and 19, a quadratic trend line was applied to the data. Both trend lines had a high R 2, so a good fit of the data. The formulas are given in each graph. In the graph the amount of gametophytes are plotted against absorption of the culture. 28

29 Figure 18: Calibration lines of S. latissima gametophytes stadium 8 after mixing the culture. A quadratic trend line with R 2 = 0, Discussion 5.1 Different methods of adding nutrients to gametophyte cultures Hypothesised was that there would be no difference between adding nutrients directly and refreshing the whole medium. From the experiment using Erlenmeyer flasks nothing could be concluded because the setup of the experiment had some errors, which became visible when analysing the results. But what was striking was that after adding nutrients or refreshing the medium, a dip was found in the absorption values. These values recovered themselves, but no clear explanation was found. From the titration plate experiment it can be concluded that there was a difference between adding nutrients directly and refreshing the whole medium. Although the gametophytes of both methods still looked healthy, the ones where nutrients were added directly had a brighter colour and thicker rhizoids. A possibility can be that with refreshing the whole medium the gametophytes lose their specific microbiome present in the medium. Many marine eukaryotes are in symbioses with bacterial partners and depend on them for growth, development and supply of nutrients. They can also protect from colonization and predation (Egan, 2012). This suggests that macroalgae and their microbiota form a holobiont the same as the coral holobiont (Rosenberg et al. 2007; Bourne et al., 2009). The epiphytic bacteria communities are likely to consist of both generalist and specialist populations and are dependent on the host algae as well as the surroundings (Egan, 2012). There is laboratory-based evidence that the health, performance and resilience of macroalgae are functionally regulated and assisted by epiphytic bacteria. In the red algae Acrochaetium sp., the bacterially generated N-acyl-homoserine-lactones (AHLs) regulated spore release (Weinberger, 2007). For the green algae Ulva the biofilms release these AHLs that attract zoospores. When zoospores detect AHLs, the swimming rate of the spores will reduce. The AHLs acts as a cue for the settlement, but is not directly involved as a signalling mechanism (Joint, 2007). The errors that were found in the Erlenmeyer flask part of the experiment where mainly focussed on the practical part. From the beginning of the experiment, the culture was already divided over the different Erlenmeyer flasks. Differences in growth in the first 4 weeks gave a different density at the start of the treatments. A better method would have been to keep the culture in one Erlenmeyer flask for 4 weeks and filled the other Erlenmeyer flask the day the treatments would start, this to have the same gametophyte density overall. Another remark was the used PAM method for measurements. The PAM is a good method to gain information about the photosynthetic efficiency of photosystem II in the gametophytes but in this case it gave data that resulted in 29

30 that the gametophyte cultures were unhealthy or even dead. But looking closely at the gametophytes under the microscope they still seemed healthy and viable. Maybe the gametophytes in the cultures became too big or the sample from the culture was not mixed properly before measurement. To homogenise the culture by mixing before using the PAM method would maybe work better. This homogenizing of the cultures may also be an idea before using the spectrophotometer. (This can be concluded from the calibration lines. There is a clear difference between the absorption of a gametophyte culture before and after mixing the cultures and a clear difference gametophyte amount before and after mixing. This because the mature gametophytes have been fractionated during mixing and each fragment becomes a gametophyte. A repetition of this experiment with these adaptations is recommended, as this would give a better picture of how it would work in bigger cultures. What also would be interesting to know is if the control gametophytes in the titration plate can recover themselves despite being nutrient starved for prolonged period of time. 5.2 Effect of vitamins on the induction of gametophytes of different ages From the vitamin experiment different conclusions can be drawn. It seems that age and period of extraction have a big influence on the induction and development of sporophytes. The gametophytes extracted in February 2013 were less viable than the gametophytes extracted in December 2013 and February But the youngest gametophytes did not induce the best, as was hypothesized. An explanation may be that the gametophytes extracted in December are stronger than the ones extracted in February. In a normal cycle, the mother plant releases the zoospores in autumn/winter, so December is a normal time for the zoospores to develop and be fertilized (Bartsch, 2008). It seems that there is a clear link between the density of gametophytes and development of sporophytes. The higher the gametophyte density, the smaller the sporophytes were. This means the hypothesis that induction at lower gametophyte densities will be better than in high densities can be confirmed. Different explanations can be given for these results. There can be competition for space, light and/or nutrients. As stated in the introduction these factors are all crucial for achieving good sporophyte growth. When the density is high, more gametophytes need space for growth and light for the development. For this development nutrients are needed as well. The main focus of this experiment was to see if vitamins would have an (positive) effect on the development of sporophytes. The hypothesis was that especially gametophytes in older cultures would induce better with vitamins in the medium. What can be concluded from the data was that vitamins do not have a positive effect on sporophyte development overall. It may even be a negative effect. The older culture used, February 2013, already was not viable enough so the hypothesis cannot be confirmed nor denied. An explanation can be that when the culture was set up in February 2013, no vitamins were added. Or the vitamins were added in the wrong concentrations or the vitamins lost their workable component. It also could be that the 3 vitamins chosen are not compatible (Provasoli and Carlucci, 1974). 5.3 Effect of kanamycin on the induction of gametes Another aspect was the effect of kanamycin on the induction/development of sporophytes. This was never tested before it became a regular method for reducing the amount of bacteria in the cultured gametophytes. The hypothesis was that the induction of gametophytes would be better in medium with kanamycin added than in medium without. The opposite occurred, the sporophytes in the medium without kanamycin became larger. Kanamycin is a bactericidal for a broad spectrum of both Gram-positive and Gram-negative bacteria. It is an aminoglycoside, acting as a selective agent that is used commonly in plant genetic engineering. Kanamycin is a good component to use because it kills plant wild cells (Nap, 1992). In literature, research has been done on the effect of kanamycin on development of different plants. A research conducted by Duan in 2009, the kanamycin effect on growth and development of Arabidopsis thaliana seedlings, cotyledon and leafs was tested. The cotyledons with kanamycin were very small and took on etiolation. When exposed longer to kanamycin, the cells in the epidermis tissue of these cotyledons were irregularly arranged, the intercellular space in the mesophyll tissue was large and the ability of cell division in the meristematic zone of the shoot tip weakened. These effects could be affected by restraining protein synthesis by the kanamycin. With cotton plants, cotyledon and hypocotyl explants and embryogenic calluses were highly sensitive to kanamycin as well (Zhang, 2001). The same effects were found by Sharma (2012) in tomato plants, especially with increasing kanamycin concentrations. The suggested statement by Duan (2009) that kanamycin can have an effect on protein synthesis can also be an explanation for the poor development of young sporophytes after induction. 30

31 5.4 Calibration line: Absorption Amount of Gametophytes Both linear and quadratic trend lines had a good fit with the data, but it is suggested that the quadratic trend line would be more realistic. When the densities become too high or the gametophytes branch, the gametophytes will capture light differently and this will give an incorrect absorption. So at some point, at high absorptions, the trend line weakens and will become steady. It has to be taken in account that different calibration lines have to be made because of observer bias. 31

32 6 Conclusion It is shown that still a lot can be learned before the best method for cultivation and induction of gametophytes can be found. The gametophytes extracted in autumn/beginning of winter develop in larger sporophytes than gametophytes extracted in winter. In a normal cycle, the mother plant releases the zoospores in autumn/winter, so December is a normal time for the zoospores to develop and be fertilized. The gametophytes extracted in autumn/beginning of winter seem to be stronger, but even in a high density their development is inhibited in comparison with low densities. Even in low densities older gametophyte cultures are not viable anymore, over time this viability seems to get lost. It is found that adding vitamins to the medium do not have a positive effect on the development of sporophytes after induction. It is possible that the vitamins are not compatible with each other. Adding kanamycin helps reducing bacteria in gametophyte cultures, but it inhibits the development of sporophytes after induction. This inhibiting effect of kanamycin may be explained by the link kanamycin has with protein synthesis during the induction. A lot of different aspects have to be taken in account while culturing gametophytes, next to temperature and light. 7 Recommendation Recommendations are stated on the experiments for the weaning process and will be: When adding nutrients to gametophyte cultures, it is possible to add stock solution of pokon instead of refreshing the entire medium; No usage of kanamycin during induction; Specific gametophyte density of the cultures can promote induction. The gametophyte amount has to lie between 50,000 and 125,

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36 Appendix Appendix 1: P nutrients Solution NPK-fertilizers with micronutrients 7.0 % Total Nitrogen (N) 2.4 % Nitrate Nitrogen 2.0 % Ammonium Nitrogen 2.6 % Urea Nitrogen 5.0 % in water soluble Phosphorus pentoxide (P 2O 5) 7.0% in water soluble Potassium Oxide (K 2O) Micronutrients soluble in water 0.02 % Boron (B) % Cuper (Cu)* 0.04 % Iron (Fe)** 0.02 % Manganese (Mn)* % Molybdenum (Mo) % Zinc (Zn)* * Chelaatvormer EDTP ** Chelaatvormer DTPA 36

37 Appendix 2: F/2 medium Start with 950 ml of filtered natural seawater and add the following components. After adding the different components, trace elements and vitamin solution bring the final volume to 1 litre with filtered natural seawater. Autoclave. Component Stock solution Quantity NaNO 3 75 g/l dh 2O 1 ml NaH 2PO 4 H 2O 5 g/l dh 2O 1 ml Na2SiO 3 9H 2O 30 g/l dh 2O 1 ml First begin with 950 ml of dh 2O, add the components and bring final volume to 1 liter with dh 2O. Autoclave. Component Primary Stock Solution Quantity FeCl 3 6H 2O g Na 2EDTA 2H 2O g CuSo 4 5H 2O 9.8 g/l dh 2O 1 ml Na 2MoO 4 2H 2O 6.3 g/l dh 2O 1 ml ZnSO 4 7H 2O 22.0 g/l dh 2O 1 ml CoCl 2 6H 2O 10.0 g/l dh 2O 1 ml MnCl 2 4H 2O g/l dh 2O 1 ml For the primary stock solution, again start with 950 ml of dh 2O, dissolve the thiamine, add 1ml of the primary stocks and bring final volume to 1 litre with dh 2O. Filter sterilize and store in refrigerator or freezer. Component Primary Stock Solution Quantity thiamine HCL (vit. B 1) mg biotin (vit. H) 1.0 g/l dh 2O 1 ml cyanobalamin (vit. B 12) 1.0 g/l dh 2O 1 ml Guillard, R.R.L. (1975) Culture of phytoplankton for feeding marine invertebrates. pp In Smith W.L. and Chanley M.H. (Eds.) Culture of Marine Invertebrate Animals. Plenum Press, New York, USA. Guillard, R.R.L. and Ryther, J.H. (1962). Studies of marine planktonic diatoms. I. Cyclotella nana Hustedt and Detonula confervacea Cleve. Canadian Journal of Microbiology. 8:

38 Appendix 3: Stadium 4 S. latissima gametophytes 38