Europees visserijfonds: Perspectief voor een duurzame visserij
Dit project is geselecteerd in het kader van het Nederlands Operationeel Programma ‘Perspectief voor een duurzame visserij’ dat wordt
medegefinancierd uit het EVF.
Physiological responses of
gametophytes and juvenile
sporophytes of Saccharina
latissima to environmental
changes
Report MSc Internship
Annelous Oerbekke
‘t Horntje, August 2014
1
Europees visserijfonds: Perspectief voor een duurzame visserij
Dit project is geselecteerd in het kader van het Nederlands Operationeel Programma ‘Perspectief voor een duurzame visserij’ dat wordt
medegefinancierd uit het EVF.
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
890927616020
2
Europees visserijfonds: Perspectief voor een duurzame visserij
Dit project is geselecteerd in het kader van het Nederlands Operationeel Programma ‘Perspectief voor een duurzame visserij’ dat wordt
medegefinancierd uit het EVF.
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
Europees visserijfonds: Perspectief voor een duurzame visserij
Dit project is geselecteerd in het kader van het Nederlands Operationeel Programma ‘Perspectief voor een duurzame visserij’ dat wordt
medegefinancierd uit het EVF.
Table of Content
1.
Introduction
1.1.
Background
1.2
Experimental organism: Saccharina latissima
5
5
5
2.
Research questions and hypotheses
8
3.
3.1
3.2
Material and Methods
General setup
Experimental setup
9
9
10
4
Results
4.1
Different methods of adding nutrients to gametophyte cultures
4.2
Effect of vitamins on the induction of gametophytes of different ages
4.3
Effect of kanamycin on the induction of gametes
4.4
Calibration line: Absorption – Amount of Gametophytes
13
13
18
25
28
5
Discussion
5.1
Different methods of adding nutrients to gametophyte cultures
5.2
Effect of vitamins on the induction of gametophytes of different ages
5.3
Effect of kanamycin on the induction of gametes
5.4
Calibration line: Absorption – Amount of Gametophytes
29
29
30
30
31
6
Conclusion
32
7
References
33
4
Europees visserijfonds: Perspectief voor een duurzame visserij
Dit project is geselecteerd in het kader van het Nederlands Operationeel Programma ‘Perspectief voor een duurzame visserij’ dat wordt
medegefinancierd uit het EVF.
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 1980. 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
Europees visserijfonds: Perspectief voor een duurzame visserij
Dit project is geselecteerd in het kader van het Nederlands Operationeel Programma ‘Perspectief voor een duurzame visserij’ dat wordt
medegefinancierd uit het EVF.
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
Europees visserijfonds: Perspectief voor een duurzame visserij
Dit project is geselecteerd in het kader van het Nederlands Operationeel Programma ‘Perspectief voor een duurzame visserij’ dat wordt
medegefinancierd uit het EVF.
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 12-17 °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
Europees visserijfonds: Perspectief voor een duurzame visserij
Dit project is geselecteerd in het kader van het Nederlands Operationeel Programma ‘Perspectief voor een duurzame visserij’ dat wordt
medegefinancierd uit het EVF.
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
Europees visserijfonds: Perspectief voor een duurzame visserij
Dit project is geselecteerd in het kader van het Nederlands Operationeel Programma ‘Perspectief voor een duurzame visserij’ dat wordt
medegefinancierd uit het EVF.
3. Material and Methods
3.1 General setup
3.1.1
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 10-12 °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.
3.1.2
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.
3.1.3
Medium
The main part of the medium consists of pasteurized seawater. Pasteurized seawater is stored at approximately 12 °C. P, K and GeO2
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. GeO2 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
2g
5 ml
GeO2
0.1 g **
0.5 ml
* Added to MilliQ
** Dissolved with 1ml/L 1 M NaOH
3.1.4
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 B12
2.5 mg
0.35 ml
Thiamine
50 mg
0.8 ml
Biotin
5 mg
0.8 ml
9
Europees visserijfonds: Perspectief voor een duurzame visserij
Dit project is geselecteerd in het kader van het Nederlands Operationeel Programma ‘Perspectief voor een duurzame visserij’ dat wordt
medegefinancierd uit het EVF.
* Added to MilliQ (West and McBride, 1999)
3.2 Experimental setup
3.2.1
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. 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 20-25 μmol m-2 s-1 in constant movement. The temperature will be 10-12 °C
and a light/dark cycle of 16:8. The titration plate will be incubated in red light (475 nm) with an intensity of 25-30 μmol m-2 s-1. The
temperature will be 10-12 °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 (Fm). Variable fluorescence (Fv) is estimated by subtracting F0 from Fm (Schreiber, 1997). Figure
3 shows schematically the process. A healthy culture gives a value of Fv/Fm around the 0.5-0.6.
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
Europees visserijfonds: Perspectief voor een duurzame visserij
Dit project is geselecteerd in het kader van het Nederlands Operationeel Programma ‘Perspectief voor een duurzame visserij’ dat wordt
medegefinancierd uit het EVF.
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 40-60 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
2014. 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
3.2.3
Standard medium without and with vitamins (gametophytes per ml)
Feb 2013
250,000 (100%)
187,500 (75%)
125,000 (50%)
62,500 (25%)
31,250 (12.5%)
Dec 2013
250,000 (100%)
187,500 (75%)
125,000 (50%)
62,500 (25%)
31,250 (12.5%)
Feb 2014
250,000 (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
Feb 2014
3.2.4
Standard medium and medium without kanamycin (gametophytes per ml)
250,000 (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
Europees visserijfonds: Perspectief voor een duurzame visserij
Dit project is geselecteerd in het kader van het Nederlands Operationeel Programma ‘Perspectief voor een duurzame visserij’ dat wordt
medegefinancierd uit het EVF.
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
Europees visserijfonds: Perspectief voor een duurzame visserij
Dit project is geselecteerd in het kader van het Nederlands Operationeel Programma ‘Perspectief voor een duurzame visserij’ dat wordt
medegefinancierd uit het EVF.
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.
4.1.1
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
Europees visserijfonds: Perspectief voor een duurzame visserij
Dit project is geselecteerd in het kader van het Nederlands Operationeel Programma ‘Perspectief voor een duurzame visserij’ dat wordt
medegefinancierd uit het EVF.
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
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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
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4.1.2
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 8.
Gametophytes per ml per treatment
20000
18000
Gametophytes/Ml
16000
14000
12000
10000
Medium
8000
Nuts
6000
Control
4000
2000
0
0
10
20
30
40
50
60
70
Day
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 Fv/Fm 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 Fv/Fm 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.
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Fv/Fm per treatment
0,700
0,600
Fv/Fm
0,500
0,400
Medium
0,300
Nuts
0,200
Control
0,100
0,000
0
10
20
30
40
50
60
70
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.
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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 2014. Another variable are the different starting densities and the last was the vitamins added to the medium.
4.2.1
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 2013. 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 2013. 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
2
268.069
0.000
1136.686
0.000
Density
4
13.385
0.000
76.942
0.000
Date * Density
8
8.204
0.000
31.348
0.000
Error (MS)
4197
0.838
0.158
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.
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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=0.0.886) 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.
Sporophyte Length
Sporophyte Width
Source
df
F
p
F
p
Vitamins
1
1.718
0.190
0.021
0.886
Density
4
3.721
0.005
20.522
0.000
Vitamins * Density
4
0.313
0.870
2.580
0.036
Error (MS)
586
0.232
Vitamins
1
22.559
0.000
23.158
0.000
Density
4
5.193
0.000
81.667
0.000
Vitamins * Density
4
1.574
0.179
2.209
0.066
Error (MS)
710
0.188
After induction
After 5 weeks
0.089
0.222
Table 7: Results of Tukey test over the different used densities for sporophyte length and width
12.5
25
Length
Width
25
ns
ns
50
ns
75
100
50
Length
Width
ns
ns
ns
ns
0.006
0.014
ns
0.000
0.046
75
Length
Width
0.005
ns
ns
0.000
ns
0.000
100
Length
Width
ns
0.001
Length
Width
12.5
19
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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
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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
Standard 12.5
Vitamine 25
Length
Width
ns
ns
Vitamine 50
Length
Width
ns
ns
Standard 25
Length
Width
ns
ns
Standard 50
Vitamine 75
Length
Standard 75
ns
Width
Vitamine 100
Length
Width
0.003
0.043
0.022
Standard 100
Table 9: Results of Tukey test over the different used densities for sporophyte length and width
12.5
Length
Width
25
0.026
0.000
50
0.000
75
0.003
100
0.000
25
50
Length
Width
0.000
ns
0.000
0.000
ns
0.000
ns
75
Length
Width
0.000
ns
0.034
0.000
ns
0.000
100
Length
Width
ns
ns
Length
Width
12.5
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).
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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.
Sporophyte Length
Sporophyte Width
Source
df
F
p
F
p
Vitamins
1
0.051
0.821
14.867
0.000
Density
4
57.407
0.000
64.141
0.000
Vitamins * Density
4
1.647
0.160
3.471
0.008
Error (MS)
848
0.156
Vitamins
1
22.859
0.000
67.090
0.000
After induction
0.043
Density
4
39.131
0.000
56.957
0.000
Vitamins * Density
4
16.039
0.000
14.180
0.000
Error (MS)
710
0.415
After 5 weeks
0.048
Table 11: Results of Tukey test over the different media treatments per densities.
Vitamine 12.5
Standard 12.5
Length
Width
ns
0.001
Vitamine 25
Vitamine 50
Length
Width
ns
ns
Standard 25
Length
Width
ns
0.000
Standard 50
Vitamine 75
Length
Width
ns
ns
Standard 75
Vitamine 100
Length
Width
ns
ns
Length
Width
Standard 100
Table 12: Results of Tukey test over the different used densities for sporophyte length and width
12.5
Length
Width
25
0.000
0.000
50
0.000
75
100
25
50
Length
Width
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.005
75
Length
Width
0.000
ns
ns
0.000
ns
ns
100
Length
Width
ns
ns
12.5
22
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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
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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
Standard 12.5
Length
Width
ns
ns
Standard 25
Vitamine 25
Length
Width
ns
ns
Standard 50
Vitamine 50
Length
Width
ns
ns
Standard 75
Vitamine 75
Length
Width
ns
0.022
Standard 100
Vitamine 100
Length
Width
0.003
0.043
Table 14: Results of Tukey test over the different used densities for sporophyte length and width.
12.5
25
Length
Width
25
0.026
0.000
50
0.000
75
100
50
Length
Width
0.000
ns
0.000
0.003
0.000
ns
0.000
0.000
ns
75
Length
Width
0.000
ns
0.034
0.000
ns
0.000
100
Length
Width
ns
ns
Length
Width
12.5
24
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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.
4.3.1
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
Source
df
Treatment
Density
Sporophyte Width
F
p
F
p
3
57.218
0.000
10.562
0.000
4
107.831
0.000
99.367
0.000
Treatment * Density
12
6.487
0.000
3.104
0.000
Error (MS)
1631
0.18
0.052
25
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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.
4.3.2
5 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
3
16.394
0.000
47.958
0.000
Density
4
241.453
0.000
186.038
0.000
Treatment * Density
12
49.936
0.000
22.887
0.000
Error (MS)
4240
0.297
0.051
26
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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.
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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 R2 = 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
R2, 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
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Figure 18: Calibration lines of S. latissima gametophytes stadium 8 after mixing the culture. A quadratic trend line with R2= 0,843.
5 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
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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 2014. 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
Europees visserijfonds: Perspectief voor een duurzame visserij
Dit project is geselecteerd in het kader van het Nederlands Operationeel Programma ‘Perspectief voor een duurzame visserij’ dat wordt
medegefinancierd uit het EVF.
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
Europees visserijfonds: Perspectief voor een duurzame visserij
Dit project is geselecteerd in het kader van het Nederlands Operationeel Programma ‘Perspectief voor een duurzame visserij’ dat wordt
medegefinancierd uit het EVF.
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 125,000.
32
Europees visserijfonds: Perspectief voor een duurzame visserij
Dit project is geselecteerd in het kader van het Nederlands Operationeel Programma ‘Perspectief voor een duurzame visserij’ dat wordt
medegefinancierd uit het EVF.
8 References
Alban, C., Job, D., Douce, R. (2000) Biotin metabolism in plants. Annual Review of Plant Physiology and Plant Molecular Biology.
Volume 51: 17-47
Anderson, G. J., Pedersen, M. G., Nielsen, S. L. (2013) Temperature acclimation and heat tolerance of photosynthesis in Norwegian
Saccharina latissima (Laminariales, Phaeophyceae). Journal of Phycology: 49, 689-700
Bartsch, I. & AL., E. (2008) The genus Laminaria senso lato: recent insights and developments. European Journal of Phycology, 43,
86.
Bolton. J. J. & Lüning, K. (1982) Optimal growth and maximal survival temperatures of Atlantic Laminaria species (Phaeophyte) in
culture. Marine Biology 66:89-94
Bourne, DG., Garren, M., Work, T. M., Rosenberg, E., Smith, G. W. & Harvell, C. D. (2009) Microbial disease and the coral holobiont.
Trends Microbiology 17: 554–562
Broch, O. J., Slagstad, D. (2012) Modelling seasonal growth and composition of the kelp Saccharina latissima. Journal of applied
phycology 24:759-776
Burton, T., Lyons, H., Lerat, Y., Stanley, M. & Rasmussen, M. B. (2009) A review of the potential of marine algae as a source of biofuel
in Ireland. Dublin: Sustainable Energy Ireland-SEI.
Croft, M. T., Lawrence, A. D., Raux-Deery, E., Warren, M. J. & Smith, A. G. (2005) Algae acquire vitamin B12 through a symbiotic
relationship with bacteria. Nature 438: 90-93
Duan, H., Ding, X., Song, J., Duan, Z., Zhou, Y., Zhou, C. (2009) Effects of kanamycin on growth and development of Arabidopsis
thaliana seedling, cotyledon and leaf. Pakistan Journal of Botany 41(4): 1611-1618
Egan, S., Harder, T., Burke, C., Steinberg, P., Kjelleberg, S., Thomas, T. (2012) The seaweed holobiont: understanding seaweedbacteria interactions. FEMS Microbiology Reviews 37: 462-476
Fang, Z.X., Ou, Y. L., Cui, J. J., Dai, J. X. (1978) Success in culturing gemetophytes of the gametophytes of Laminaria japonica (in
Chinese, with English abstract). Trans Sci 2:115-116
FAO, F. A. A. D. (2014) The State of World Fisheries and Aquaculture 2014 (SOFIA). E-ISBN 978-92-5-108276-8
Forbord, S., Skjermo, J., Arff, J., Handä, A., Reitan, K. I., Bjerregaard, R. & Lüning, K. (2012) Development of Saccharina latissima
(Phaeophyceae) kelp hatcheries with year-round production of zoospores and juvenile sporophytes on culture ropes for kelp
aquaculture. Journal of Applied Phycology, 1-7.
Fortes, M. D. & Lüning, K. (1980) Growth rates of North Sea macroalgae in relation to temperature, irradiance and photoperiod.
Helgolander Meeresun 34:15-29
Gerard, V. A. (1988) Ecotypic differentiation in fight-related traits of the kelp Laminaria saccharina. Marine Biology 97:25-36
Gerard, V. A. & Du Bois, K. R. (1988) Temperature ecotypes near the southern boundary of the kelp Laminaria saccharina. Marine
Biology 97:575-80
Goyer, A. (2010) Thiamine in plants: aspects of its metabolism and functions. Phytochemistry 71(14-15): 1615-24
Joint, I., Tait, K. & Wheeler, G. (2007) Cross-kingdom signalling: exploitation of bacterial quorum sensing molecules by the green
seaweed Ulva. Philosophicol Transactions of the Royal Society of London Series B: Biological Sciences 362: 1223–1233
Kaas, R. (1998) The seaweed resources of France. In: Critchley AT, Ohno M (eds) Seaweed resources of the world. JICA, Yokosuka,
Japan.
33
Europees visserijfonds: Perspectief voor een duurzame visserij
Dit project is geselecteerd in het kader van het Nederlands Operationeel Programma ‘Perspectief voor een duurzame visserij’ dat wordt
medegefinancierd uit het EVF.
Kain, J. M. (1964) Aspects of the biology of Laminaria hyperborean. III. Survival and growth of gametophytes. Journal of Marine
Biology Association. U.K. 44:415-33
Kain, J. M. (1979) A view of the genus Laminaria. Oceanography and Marine Biology: an annual review, 17.
Kain, J. M. & Dawes, C.P. (1987) Useful European seaweeds: past hopes and present cultivation. Twelfth International Seaweed
Symposium. Springer.
Kraan, S. (2013) Mass-cultivation of carbohydrate rich macroalgae, a possible solution for sustainable biofuel production. Mitigation
and Adaptation Strategies for Global Change, 1- 20.
Lüning, K. & Dring, M. J. (1972) Reproduction induced by blue light in female gametophytes of Laminaria saccharina. Planta, 104,
252-256.
Lüning, K. & Dring, M. J. (1975) Reproduction, growth and photosynthesis of gametophytes of Laminaria saccharina grown in blue
and red light. Marine Biology, 29, 195-200.
Lüning, K. & Neushul, M. (1978) Light and temperature demands for growth and reproduction of laminarian gametophytes in
southern and central California. Marine Biology, 45, 297-309.
Lüning, K. (1979) Growth strategies of three Laminaria species (Phaeophyceae) inhabiting different depth zones in the siblittoral
region of Helogoland (North Sea). Mar Ecol Prog Ser 1:195-207
Lüning, K. (1980) Critical levels of light and temperature regulating the gametogenesis of three laminaria species (Phaeophyceae).
Journal of phycology, 16, 1-15.
Lüning, K. (1990) Seaweeds: Their Environment, Biogeography and Ecophysiology. Publisher: John Wiley & Sons, Inc.
Machalek, K. M., Davison, I. R. & Falkowski, P. G. (1996) Thermal acclimation and photoacclimation of photosynthesis in the brown
alga Laminaria saccharina. Plant, Cell environment 19:1005-16
Nap, J.P., J. Bijvoet and W.J. Stiekema. 1992. The resistant of kanamycin in transgenic plants. Prophyta, 46(1): 48-51
Provasoli, L., Carlucci, A. F. (1974) Vitamins and growth regulators. Botanical monographs, volume 10. 741-789
Raven, P. H., Evert, R. F., & Eichhorn, S. E. (2005). Biology of plants. New York: W.H. Freeman and Co
Rosenberg, E., Koren, O., Reshef, L., Efrony, R. & Zilber-Rosenberg, I. (2007) The role of microorganisms in coral health, disease and
evolution. Nature Reviews Microbiology 5: 355–362
Sharma, C., Aggarwal, G., Srivastava, D. K. (2012) Effect of antibiotic kanamycin on cultured cotyledon and hypocotyl tissues of
Tomato (Solanum lycopersicon cv. Solan vajr) Intergrated Journal of Environmental Biotechnology 5(2): 77-82
Sjøtun K (1993) Seasonal Lamina Growth in two Age groups of Laminaria saccharina (L.) Lamour. in Western Norway. Botanica Marina
36:433–441
Sjøtun K, Gunnarsson K (1995) Seasonal growth pattern of an Icelandic Laminaria population (section Simplices, Lami- nariaceae,
Phaeophyta) containing solid- and hollow-stiped plants. European Journal Phycology 30:281–287
Schreiber, U., 1997. Chlorophyll Fluorescence and Photosynthetic Energy Conversion. Heinz Walz GmbH, Effleltrich. 73 pp.
Weinberger, F., Beltran, J., Correa, J. A. et al. (2007) Spore release in Acrochaetium sp. (Rhodophyta) is bacterally controlled.
Journal of Phycology 43: 235–241
Wu, C. Y., Pang, S. J., Jiang, B. Y. (1994) Establishment and application of vegetative gametophytes of Laminaria japonica. In: Wang,
S. J. (ed) Seaweed Biotechnology. Shanghai Scientific and Technical Publishers, Shanghai, pp 104-110 (in Chinese)
Wu, C., Li, D., Liu, H., Peng, G. & Liu, J. (2004) Mass culture of Undaria gametophyte clones and their use in sporeling culture. Asian
Pacific Phycology in the 21st Century: Prospects and Challenges, 153-156
34
Europees visserijfonds: Perspectief voor een duurzame visserij
Dit project is geselecteerd in het kader van het Nederlands Operationeel Programma ‘Perspectief voor een duurzame visserij’ dat wordt
medegefinancierd uit het EVF.
Xu, B., Zhang, Q. S., Qu, S. C., Cong, Y. Z., Tang, X. X. (2009) Introduction of a seedling production method using vegetative
gametophytes to the commercial farming of Laminaria in China. J Appl Phycol 21:171-178
Zhang, B. H., Liu, F., Liu, Z. H., Wang, H. M. & Yao, C. B. (2001) Effects of kanamycin on tissue culture and somatic embryogenesis in
cotton. Plant growth Regulation 33: 137-149
35
Europees visserijfonds: Perspectief voor een duurzame visserij
Dit project is geselecteerd in het kader van het Nederlands Operationeel Programma ‘Perspectief voor een duurzame visserij’ dat wordt
medegefinancierd uit het EVF.
Appendix
Appendix 1: P nutrients
Solution NPK-fertilizers 7+5+7 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 (P2O5)
7.0% in water soluble Potassium Oxide (K2O)
Micronutrients soluble in water






0.02 % Boron (B)
0.004 % Cuper (Cu)*
0.04 % Iron (Fe)**
0.02 % Manganese (Mn)*
0.002 % Molybdenum (Mo)
0.004 % Zinc (Zn)*
* Chelaatvormer EDTP
** Chelaatvormer DTPA
36
Europees visserijfonds: Perspectief voor een duurzame visserij
Dit project is geselecteerd in het kader van het Nederlands Operationeel Programma ‘Perspectief voor een duurzame visserij’ dat wordt
medegefinancierd uit het EVF.
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
NaNO3
75 g/L dH2O
1 ml
NaH2PO4 H2O
5 g/L dH2O
1 ml
Na2SiO3 9H2O
30 g/L dH2O
1 ml
First begin with 950 ml of dH2O, add the components and bring final volume to 1 liter with dH2O. Autoclave.
Component
Primary Stock Solution
Quantity
FeCl3 6H2O
-
3.15 g
Na2EDTA 2H2O
-
4.36 g
CuSo4 5H2O
9.8 g/L dH2O
1 ml
Na2MoO4 2H2O
6.3 g/L dH2O
1 ml
ZnSO4 7H2O
22.0 g/L dH2O
1 ml
CoCl2 6H2O
10.0 g/L dH2O
1 ml
MnCl2 4H2O
180.0 g/L dH2O
1 ml
For the primary stock solution, again start with 950 ml of dH2O, dissolve the thiamine, add 1ml of the primary stocks and bring final
volume to 1 litre with dH2O. Filter sterilize and store in refrigerator or freezer.
Component
Primary Stock Solution
Quantity
thiamine HCL (vit. B1)
-
200 mg
biotin (vit. H)
1.0 g/L dH2O
1 ml
cyanobalamin (vit. B12)
1.0 g/L dH2O
1 ml
Guillard, R.R.L. (1975) Culture of phytoplankton for feeding marine invertebrates. pp 26-60. 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. 8:229-239.
37
Europees visserijfonds: Perspectief voor een duurzame visserij
Dit project is geselecteerd in het kader van het Nederlands Operationeel Programma ‘Perspectief voor een duurzame visserij’ dat wordt
medegefinancierd uit het EVF.
Appendix 3: Stadium 4 S. latissima gametophytes
38