I. Abstract
Some of the most interesting aspects of a kelp forest ecosystem are the herbivore
kelp interactions. Macrocystis pyrifera utilizes different methods to deter consumption by
herbivores. Both nutrient levels and defensive chemical concentrations may be allocated
differently to the different tissues of the kelp, showing defensive preference for those
tissues responsible for growth and reproduction. We examined this nutrient allocation as
well as differential distribution of polyphenolic concentrations in three different tissue
types (mature blades, apical meristems, sporophylls, and drift kelp) of Macrocystis pyrifera,
and those from drift kelp, and analyzed how these differences affected the growth of the
herbivore Strongylocentrotus purpuratus. Sea urchins were fed restricted diets of a single
type of algal tissue for 14 days, and their growth rates were compared. Urchins fed a diet of
mature blades experienced the strongest growth, as mature blades had low concentrations
of polyphenols and high nutritional value. Urchins fed diets of both sporophylls and apical
meristems experienced the least growth, as sporopyll and apical meristem tissue
palatability was low, due to either low nutrient levels (sporophylls) or high polyphenolic
concentrations (apical meristems). Urchins fed diets of drift kelp had strong growth rates,
because although drift kelp was low in nutrients, the tissue also contained low
polyphenolic concentrations. We concluded both nutrient allocation as well as differential
polyphenolic concentrations both played roles in palatability of kelp tissues and their effect
on herbivore growth rates.
II. Introduction
Kelp forests ecosystems provide a complex home to numerous organisms, including
invertebrates, herbivores, including echinoderms, gastropods, amphipods, and isopods
(Barrales & Christopher S. Lobban, 1975). It has been shown that herbivore-feeding
habits can exert a certain pressure on the evolution of kelps and the allocation of their
defenses, such as polyphenolic/phlorotannin concentrations (Hay & Fenical, 1988,
Wright, De Nys, Poore, & Steinberg, 2004). The relationship between these herbivores
and the giant kelp dominating the kelp forest ecosystems of the west coast of North
America, Macrocystis pyrifera, is vital for understanding kelp forest ecosystem dynamics
as a whole (Watanabe, 1984, Foster and Schiel, 1985, Wakefield, 1998).
Macrocystis pyrifera is comprised of many different types of tissues, including
meristems, reproductive tissues, and the non-reproductive tissue of the kelp blades
(Van Alstyne, McCarthy III, Hustead, & Kearns, 1999). The meristematic tissue of kelp is
responsible for the growth of the organism, while the reproductive tissue, or
sporophyll, is responsible for the production of the spores used in reproduction
(Barrales & Christopher S. Lobban, 1975). The non-reproductive tissue refers to the
blades of the organism, and is responsible for the majority of photosynthetic activity. It
has been shown that these different tissues may utilize different methods in the defense
of their parts, possibly both chemical defenses and differential nutrient allocations are
used (Bazzaz and Grace, 1997). Some of the specific anti-herbivore chemical defenses
include the production of polyphenols and/or phlorotannins on the surface of the kelp
tissues (Tugwell, 1989; Van Alstyne, McCarthy III, Hustead, & Kearns, 1999).
Phlorotannins are polymers of phloroglucinol and are ubiquitous in brown algae, and
can also serve as anti-biotic, anti-fungal, and anti-fouling agents (Ragan & Globmtiza
1986, Pavia et al. 1997, Wikstrom and Pavia 2004).
Multiple past studies have examined the variation in phlorotannin concentrations in
the different kelp tissues, but a more detailed understanding of the nutrient variations
that exist in these tissues is essential (Norderhaug, Fredriksen, & Nygaard, 2003). These
studies provide further explanations of the physiological evolution giant kelps have
undergone to protect their most important tissues for growth and development (Van
Alstyne, McCarthy III, Hustead, & Kearns, 1999).
The grazing pressure of herbivores is considered one of the most important factors
affecting the health of kelp beds (Dean et al. 1984, Schiel & Foster 1986). In the
northern pacific, grazing sea urchins have devastated many areas of kelp beds,
developing these once rich ecosystems into what are termed "urchin barens" (Estes &
Duggins 1995). Growth rates of the sea urchin Strongylocentrotus purpuratus, and other
herbivores, have been examined in past experiments (Ebert, 1968, Swan, 1961,
Watanabe, 1984)), but no research has been carried out to see how restricted diets on
the different kelp tissues of Macrocystis pyrifera affect growth rates of these sea urchins.
This research project specifically examines how kelp tissues differentially partitions
nutrients (as C:N ratios) and polyphenols in its different tissues. The apical meristem,
sporophylls, and mature blades will be the specific parts of the kelp that will be
analyzed, as well as drift kelp. The project will also address how restricted diets on
these specific kelp tissues affect growth rates of Strongylocentrotus purpuratus. The
data collected from these experiments will provide vital information on the macroalgae-
herbivore interactions present in the Monterey Bay region, and will aid in
understanding the complex ecosystem present in kelp forests around the world
(Hemmi & Jormalainen, 2002). There has been a lack of research looking at the growth
rates of Strongylocentrotus purpuratus and how different allocation of Macrocystis
pyrifera nutrients will affect them. This project will allow for these gaps in knowledge to
be filled.
I hypothesized the levels of nitrogen would be lowest and C:N ratios, as well as the
polyphenol levels, would be highest in the parts of the kelp responsible for growth and
reproduction (apical meristems and sporophylls) (Swan, 1961). Nitrogen levels may be
decreased in the meristems and sporophylls because of the evolutionary advantages
these tissues would experience for being less nutritious to herbivores (Diaz,
Güldenzoph, Molis, McQuaid, & Wahl, 2006). An increase in C:N ratios would
accompany an increase in carbon in the growing apical meristems and sporophylls,
which follow the source-sink phenomenon, which describes the importation of carbon-
rich materials to kelp tissues responsible for growth (C S Lobban, 1978; Schmitz, 1980).
An increase in carbon levels leads to an increase in C:N ratios. C:N ratios help signify
how nutrient rich certain plant material is, as nitrogen plays a major role in the nutrient
requirements of marine organisms (Hamilton, Lange, Boyd, & Peters, 2011). There have
been strong correlations between the amount of nitrogen with respective growth of
organisms (Davies, 1971, Lambers, 1992, (Howarth, Marino, Lane, & Cole, 1988).
Past experimental studies have shown that polyphenol levels are indeed higher for
those tissues that are most important for a kelp’s growth and reproduction, specifically
in the meristems, holdfasts, and sporophylls (Tugwell, 1989; Van Alstyne, McCarthy III,
Hustead, & Kearns, 1999). Based on these results, the polyphenol levels of both drift
kelp and mature blades should be significantly lower, as their role in the survival of the
kelp is not as vital (Lubchenco & Gaines, 1981).
I also hypothesized the nitrogen levels of the sporophylls and the meristems will be
very low, and the nitrogen levels in drift kelp and the mature blades will be significantly
higher, with lower C:N ratios as a result. Drift kelp refers to any parts of kelp that have
become dislodged from the substrate and drift near the ocean floor (Nosengo, 2011).
Because these tissues do not play as vital a role for the survival of the kelp, their high
nutrient levels do not play a disadvantage for the kelp’s endurance (Jackson, 1977).
The polyphenol levels of these parts will also be significantly lower, as the kelp does not
have incentives to as vigorously protect these tissues (Tugwell, 1989; Van Alstyne,
McCarthy III, Hustead, & Kearns, 1999; Watanabe, 1984). Past studies have also shown
that as kelp undergoes degradation at the hands of microbes, polyphenolic
concentrations have been shown to go decrease significantly (Van Alstyne, McCarthy III,
Hustead, & Kearns, 1999). This microbial breakdown of polyphenols may also decrease
the polyphenolic concentration of the drift kelp.
III. Methods
Field Sampling. Strongylocentrotus purpuratus individuals and tissues from
Macrocystis pyrifera were collected for laboratory experimentation along the California
coast in the Monterey Bay, specifically from the kelp forest adjacent to the Hopkins
Marine Station of Stanford University (36°36’ N, 121° 54’W). The kelp tissue samples
were collected between the depths of 0 and 10 meters. The collected kelp samples were
kept in a large tub with a continual flow of seawater, while the sea urchins were stored
in an aquarium also with a constant flow of seawater.
Growth Rate Studies. The quality of nutrients of different tissues of kelp as a bio¬
assay for herbivore growth was determined by restricting the diets of
Strongylocentrotus purpuratus to those specific tissues in the laboratory, over a period
of two weeks.. In the growth rate trials, four experimental groups of each tissue type
(mature blades, apical meristems, sporophylls, and drift kelp) were used, as well as one
control group, receiving no kelp tissue. For each tissue group, four different arenas
were created to separate the sea urchins and prevent competition. Each arena with a
single urchin was initially provided with 15 grams of kelp tissue (wet weight), after
starving for 72 hours to remove confounding effects of their previous diets.
Before the experiment, each sea urchin’s diameter was measured with vernier
calipers to the nearest 0.1 mm. The urchins were placed between the jaws of the
calipers and were rotated until the greatest diameter was determined.
Each arena was subjected to natural light-dark cycles, and provided with a continual
supply of running seawater (n-4). Food was replaced in the arenas ad libitum, when
little food remained. All arenas were maintained with similar amounts of food.
At the end of the experiment, the diameters of the sea urchins were measured in the
same manner as the initial diameters, with the maximum diameter being recorded, to
the nearest 0.1 mm. The growth was then determined, subtracting the initial diameters
from the final diameters.
Nutrient and Polyphenol Determination. In order to determine both the nutrient
and polyphenol levels in the different tissues of the kelp, 10 g of kelp (wet weight) were
dried at 120° C for 48 hours. The dried kelp samples were then removed, and placed in
canisters of a Wiley ball mill for 30 seconds ensuring grinding into fine powder. This
kelp tissue powder was then used in the determination of nutrient allocation (n-5) as
well as the polyphenol concentrations (n-4).
To determine the nitrogen and carbon levels in the different kelp tissues, the
powder of the dried tissues was processed in a Carlo Erba CN Elemental Analyzer. 4-7
mg of powder was measured by microscale and placed in tin capsules for carbon and
nitrogen content, and these tins, after being placed in the analyzer, were run. A
standard curve was developed before the procedure with acetanilide, and applied to the
experimental samples for standardization.
For determination of the polyphenol levels, -75 mg of the dried tissue sample was
used according to a modified Folin-Ciocalteu method, according to Hendrik Poorter &
Yvonne de Jong-Van Berkel, from Utrecht University (Van Alstyne, 1995). In this revised
method, the dry plant material was first extracted with 5 ml of 80% ethanol in a
centrifugation tube. The tubes were placed in a heating block for 30 minutes at 30° C,
and then centrifuged at 4500 rpm for 10 minutes. After the supernatant had been
removed, the pellet was extracted for a second time with 2.5 ml of 80% ethanol, and
again placed in a heating block for 30 minutes at 30° C. After a second centrifugation at
4500 rpm for 10 minutes, the supernatant was removed and added to the supernatant
from the first extraction. To remove chlorophyll, the pellet was extracted a third time
with 5 ml chloroform and 2.5 ml deionized water. The chloroform is removed after
centrifugation for 10 minutes at 4500 rpm. 0.1 ml of the ethanol-water fraction is mixed
well with 5 ml of reagent A (75 ml Folin-Ciocalteu reagent made up to 750 ml with DI
water). After 0.5-8 minutes, 3.5 ml of reagent B is added (57.5 g sodium carbonate
dissolved in 500 ml of DI water). The tubes were then incubated at 40° C for an hour.
After incubation, 1 ml of the mixture was added to a cuvet, and the absorption at 765
nm was measured with a spectrophotometer. A standard solution of 0.05 g of p-
Coumaric acid was dissolved in 2 ml 96% ethanol and filled up to 50 ml of DI water. The
standard was diluted to a range of 0, 0.1, 0.2, 0.4, 0.6, 0.8 and 1 mg/ml. After
determination of the standard curve, the measured absorption allowed for the
calculation of polyphenolic concentration of the tissues.
Statistical Analyses. Single-factor ANOVA testing was used to test and analyze all
data, including polyphenolic levels, nutrient levels (nitrogen levels, carbon levels, and
C:N ratios), and the effect of tissue identity on growth rates. Significant (Pæ0.05) tissue
nutrients and growth rate results were used to indicate whether certain tissues were
beneficial for the growth of sea urchins and also what defensive mechanisms kelp
organisms may employ.
IV. Results
The growth rates of Strongylocentrotus purpuratus differed significantly (p-0.0006)
when feeding on restricted diets of the different tissues of kelp. Figure 1 shows the
averages of the growth rates of the sea urchins feeding on different diets of the kelp
tissues. Sea urchins fed the mature blades had the highest average growth at 0.136 mm
per 2 weeks, while those fed apical meristems grew the least, on average 0.0422 mm;
those fed sporophylls 0.0558 mm, and those fed drift kelp grew 0.114 mm. A control
group of sea urchins fed nothing grew on average only 0.015 mm.
The results from the nutrient allocation in the different parts of the kelp are
significant, in terms of % nitrogen levels, % carbon levels, and C:N ratios (P= 0.0001,
0.0008, and 0.0012 respectively). Figure 2 displays the % nitrogen levels from the
different parts of kelp. The average for the mature blades was 2.838%, apical
meristems 2.318%, sporophylls had the lowest value of 1.446%, and drift kelp had
1.911% nitrogen.
Figure 3 displays the % carbon levels from the different kelp tissues involved in the
experiment. The average % carbon level for mature blades was 24.182%, apical
meristems with the lowest average of 20.433%, sporophylls with 28.051%, and drift
kelp with the highest average of 29.231%.
Based off these % carbon and nitrogen values, C:N ratios could be calculated. Figure
4 shows these C:N results for their respective tissues of kelp. Mature blades had an
average C:N value of 8.573, the lowest value of the experimental variables. Apical
meristems had the next lowest value, of 8.857, sporophylls had the highest ratio of
19.560, and drift kelp had a C:N value of 15.399.
The polyphenol values for the different kelp tissues were also significant
(P=0.0022). Figure 5 shows the respective values of the polyphenolic concentrations
measured from the different tissues. Apical meristems had the significantly highest
value of 0.202 mg/ml, while drift kelp had the lowest value of 0.00585 mg/ml, mature
blades with 0.0273 mg/ml, and sporophylls with a concentration of 0.0121 mg/ml.
Tables 1-5 display the results from the single factor ANÖVA analysis, with the test
degrees of freedom, error degrees of freedom (df), number of replicates (n), P values,
and F values displayed.
V. Discussion
The significance in the difference of growth rates between the sea urchins feeding
on different kelp tissues may further assist in the knowledge of preferential feeding
patterns of these organisms in a kelp forest ecosystem. Many experiments have been
performed to attempt to understand food choice in marine invertebrates (A. G. B. Poore
& P. D. Steinberg, 1999; Wakefield & Murray, 1998; Watanabe, 1984), but little work
has been done linking possible nutrient variations and defensive mechanisms employed
by algae to explain how growth rates of those invertebrates may be different with their
different feeding patterns. The data in these studies leads to the belief that sea urchins
are a good stand in for other herbivores that may benefit from consumption of tissues
that are high in nitrogen levels, low C:N ratios, and low in polyphenol levels.
The growth rates of those sea urchins that have fed on mature blades and drift kelp
is much higher than those growth rates of the urchins feeding on both apical meristems
and sporophylls. For mature blades, their high nutrient levels and low polyphenolic
levels make them very palatable for urchins, thereby providing the ability for strong
growth. Drift kelp, meanwhile, has a somewhat intermediate value of nutrients, but
their significantly low polyphenolic concentrations allow their tissues to be extremely
palatable for the urchins (Tugwell, 1989; Van Alstyne, McCarthy III, Hustead, & Kearns,
1999). With their low polyphenolic values, the urchins may be able to consume enough
drift kelp to satisfy their nutrient needs, and allow for another strong growth rate.
The lower growth rates of urchins fed both sporophylls and apical meristems seem
to follow the hypotheses that organisms have evolved to allocate their defenses in such
a way as to enhance survival (Rhoades, 1979; Tugwell, 1989). While sporophylls have
extremely low values of nitrogen, and the highest of C:N values, apical meristems had
the second highest percentage of nitrogen, and the second lowest value of C:N, which
could possibly lead to increased grazing by herbivores, if no other protective
mechanism were present. So in order to protect these extremely important tissues for
growth, kelp has evolved to allocate very high concentrations of polyphenols to these
tissues. Because of this high concentration, apical meristem tissues may not be
palatable to urchins, and therefore the urchins fed restricted diets of meristematic
tissue will be unable to consume this tissue, and will therefore be unable to obtain
enough nutrients to support growth. For those urchins on diets of sporophylls,
however, they are not deterred to consume the tissue of the sporophylls, but the lack of
nutrients does not support strong growth.
The high levels of nutrients in the mature blades can be explained by the notion that
because of the relative lack of their necessity to growth and reproduction, as the
meristems and sporophylls are. As a result, the plant may not need to protect these
tissues in any manner, and have evolved to decrease the amount of costly chemical
defenses to these tissues. The high nitrogen levels are explained by the source-sink
theory, which says that mature blades produce an excess of nitrogen levels, and because
of their lack of growth, are sources for nitrogen for the rest of the kelp organism
(Schmitz, 1980; C S Lobban, 1978).
The high relative nutrient levels examined in apical meristems is intriguing. The
high nitrogen levels could signify the large amount of nitrogen transported to the
meristems to support growth (Schmitz, 1980), while the low carbon levels could
represent very high turnover due to the rapid expansion and elongation consuming the
carbon for that growth of the kelp organism.
It has been shown that sporophylls receive significantly less translocated products,
such as nitrogen containing amino acids, than other parts of the kelp, supporting the
finding of the low % nitrogen of the sporophylls (CS Lobban, 1978). The same study
describes sporophylls as growing the slowest of the kelp tissues, which translates to
having the highest carbon levels, as herbivores that have strong growth rates do not
consume as much carbon (C S Lobban, 1978). Because of the high C:N values, and
therefore low nutrient enrichment, these tissues have evolved to need lesser defenses,
as they may not be targeted by certain herbivores. While other experiments have found
reproductive tissue to have high concentrations of defensive chemicals, the low nutrient
levels themselves may be enough to deter herbivores from consuming these important
tissues of the kelp (Tugwell, 1989).
The low concentration of defensive chemicals, like polyphenols, in the drift kelp
samples is supported by past experiments, which have shown that bacterial
degradation of drift kelp decreases the amount of polyphenols produced by the kelp
tissues (Norderhaug et al., 2003; Van Alstyne, McCarthy III, Hustead, & Kearns, 1999).
It is also possible that this bacterial degradation led to a decrease in nitrogen levels in
the drift kelp, influencing the C:N value. But because of such low levels of polyphenols,
sea urchins feeding on this drift kelp did not experience nitrogen limitation, and
therefore experienced strong growth (Norderhaug et al., 2003).
This study has shown that kelps differentially allocate both their defensive
chemicals and nutrients in their different tissues. Nutrient values and the concentration
of polyphenols can help determine the importance of certain tissues, in the event kelp
may preferentially defend those tissues most important for growth and reproduction by
decreasing their palatability either through a decrease in the nutrient levels or by an
increase in the polyphenolic concentrations. Sea urchins fed restricted diets on these
different kelp tissues displayed growth rates positively correlated with nutrient levels
and negatively correlated with polyphenolic concentrations. Those fed mature
Macrocystis blades and drift kelp, which are high in nutrients and low in polyphenols,
grew the most, while those fed meristematic tissue, also high in nutrients but
significantly higher in polyphenolic concentrations, grew at a much slower rate. Those
urchins fed sporophylls also grew at a very slower rate, as these tissues have very low
polyphenol values, but also have very low nutrient values. These conclusions help to
clarify herbivore-kelp relationships, and can further develop the complexity present in
giant kelp forest ecosystems. It will be important to further examine how herbivores
may preferentially choose one kelp tissue over another, whether that choice is cased by
differences in nutrient levels or defensive chemicals.
VI. Acknowledgements: I would like to thank all the participants in BIOHOPK
175H, including lan Markham, Sharon Beltracchi, and Acata Felton. He would also like
to thank Sarah Lee and Vanessa Michelou, his mentors for their extremely generous
help throughout this research. I also need to extend a heartfelt thank you to the entire
student body and faculty of Hopkins Marine Station, where everyone’s love of the
marine world drove my interest. Thank you all!
Tables
ANOVA Results for Sea Urchin Growth
MS
P-value
F-value
0.0012
149.7717
142.535
Test
Error
16
Table 1: Sea Urchin Growth. Results from single factor ANOVA analysis. Displays degrees
of freedom (df) for both test and error, number of replicates (n), P-Value, F-Value, and
Mean Squares value.
ANOVA Results for % Nitrogen for Different Kelp Tissues
P-value
F-value
MS
Test
0.0001
19.4947
0.006
Error
16
Table 2:% Nitrogen for Different Kelp Tissues. Results from single factor ANOVA
analysis. Displays degrees of freedom (df) for both test and error, number of replicates (n),
P-Value, F-Value, and Mean Squares value.
ANOVA Results for % Carbon for Different Kelp Tissues
MS
P-value
F-value
0.212
0.0008
Test
9.3683
Error
16
Table 3:% Carbon for Different Kelp Tissues. Results from single factor ANOVA
analysis. Displays degrees of freedom (df) for both test and error, number of replicates (n),
P-Value, F-Value, and Mean Squares value.
ANOVA Results for C:N Ratios for Different Kelp Tissues
MS
P-value
F-value
0.0006
0.010
Test
10.0457
13
Error
Table 4: C:N Ratios for Different Kelp Tissues. Results from single factor ANOVA
analysis. Displays degrees of freedom (df) for both test and error, number of replicates (n),
P-Value, F-Value, and Mean Squares value.
ANOVA Results for Polyphenol Concentrations of Different Kelp Tissues
F-value
P-value
MS
39.6659
Test
0.0022
0.018
Error
Table 5: Polyphenol Concentrations for Different Kelp Tissues. Results from single
factor ANOVA analysis. Displays degrees of freedom (df) for both test and error, number of
replicates (n), P-Value, F-Value, and Mean Squares value.
Figures
0.18
0.14
30.12
0.1
0.08
2 006
0.04
0.02
Apical
Sporophylls Drift Kelp Control
Mature Blades
Meristems
Figure 1: Sea Urchin Growth. Average growth of sea urchins over 2-week period, fed
restricted diets of different kelp tissues (+ 1 SD). P-0.0006. Standard Error Bars shown
(n=4)
3.5
2.5
1
0.5
Mature Blades Apical Meristems
Sporophylls
Drift Kelp
Figure 2:% Nitrogen Levels of Different Kelp Tissues. Average % nitrogen levels from
the different experimental kelp tissues (+ 1 SD). P= 0.0001. Standard Error Bars shown
(n=5)
25
20
15
Mature Blades Apical Meristems
Sporophylls
Drift Kelp
Figure 3:% Carbon Levels of Different Kelp Tissues. Average % carbon levels from the
different experimental kelp tissues (+ 1 SD). P= 0.0008. Standard Error Bars shown (n=5)
25
10
Sporophylls
Drift Kelp
Mature Blades Apical Meristems
Figure 4: C.N of Different Kelp Tissues. Average C.N ratios for the different experimental
kelp tissues, taken as the ratio between the %C and %N levels (+ 1 SD). P= 0.0012.
Standard Error Bars shown (n=5)
0.25
0.15
01
0.05
I
Sporophylls
Mature Blades Apical Meristems
Drift Kelp
Figure 5: Polyphenol Concentrations for Different Kelp Tissues. Polyphenolic
concentrations for the different kelp tissues (+ 1 SD).
P= 0.0022. Standard Error Bars shown (n-4)
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