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Marine Biological Laboratory

Ecosystems Center

Semester in Environmental Science

Effects of nutrients on palatability and concentration of anti-herbivory compounds in macroalgae

Independent research paper SES 2012

Michael Marty-Rivera

Universidad de Puerto Rico Recinto Río Piedras

Avenida Juan Ponce De León, San Juan, PR 00931

Advisor: Dr. Joe Vallino

Marine Biological Laboratory

7 MBL Street, Woods Hole, MA 02543

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Abstract:

Macroalgae are a key component in aquatic ecosystems providing a food source for many organisms.

Many marine macroalgae have developed defensive mechanisms, such as anti-herbivory compounds. In

this study I will test for differences in herbivory on macroalgae grown under different nutrient

conditions by two amphipods, Jassa falcata and Gammarus oceanicus. Algal anti-herbivory compounds

and palatability was assayed by constructing synthetic algae leaf grids made up with agar and

dried/ground algae (Fucus vesiculosus and Gracilaria spp.) and feeding these to amphipods given the

selection of algae coming from two different nutrient availability conditions. A bioassay showed

Amphipods preference for Fucus vesiculosus coming from Sage Lot Pond, a low nutrient site, with a

consumption of 46% over the Child’s river (high nutrient site) variety. Phenolic compounds found in

Fucus vesiculosus did not appear to inhibit amphipod grazing. No preference was observed for Gracilaria

spp. and this can serve as an indication of no chemical defenses against Jassa falcata and Gammarus

oceanicus.

Keywords: Macroalgae, Amphipod, herbivory, nutrients, Anti-herbivory compounds, bioassay.

Introduction:

Macroalgae form part of the trophic web and are an important primary producer in aquatic ecosystems,

therefore a key part of the ecosystem energy and nutrient cycle. Recent research has shown that

macroalgae can serve as a bioremediation agent via nutrient removal from eutrophic waters (Marinho-

Soriano 2009 et al.; Marinho-Soriano et al. 2011). There are also some commercial products derived

from algae such as carrageenan, agar, fertilizers (Chynoweth 2001). Algae biomass has also been found

to be a good candidate for biofuel production since the 1970’s (Roesijadi 2010).

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Macroalgae have several needs for its optimal growth such as light, temperature, salinity, available

space and nutrients (Pederson et al. 1996) and have developed mechanisms to prevent herbivory, this

gives them an opportunity to thrive in ecosystems with many herbivores and be exploited for

anthropogenic benefits. The types of algae defenses can be separated as structural, chemical, seasonal

(Hay and Fenical 1988). This study will focus on the chemical defenses of macroalgae based on its

growing environment. Two sites in the Waquoit Bay area (Figure 1), Child’s River (CR) an estuary which

receives a nutrient loading of 407 kg N * Ha-1 * year-1, and Sage lot Pond (SLP) which receives only 7.6 kg

N * Ha-1 * year-1 (Hauxwell et al. 2003), were used to compare the effects of the brown algae Fucus

Vesiculosus, the red algae Gracilaria spp. and the green algae Ulva lactuca anti-herbivory compounds

against common herbivores in the area, the amphipods Gammarus oceanicus and Jassa falcata.

Macroalgae grown in a nutrient enriched area will have a higher growth rate than those grown in an

oligotrophic environment (Hemmi 2004) and ideally algae growth inhibited by a lack of nutrients will

have more resources to allocate into chemical defenses (Hemmi 2004). With those assumptions I

hypothesize that algae from the Child’s River site will be more susceptible to herbivory due to having a

greater nutrient availability for growth, therefore not needing to invest energy in defensive chemicals.

Methods:

I collected macroalgae from the coastlines of both study sites, cleaned with seawater to remove visible

epiphytes, oven-dried at 60°C and ground to a fine powder. I prepared an agar mixture using 1L filtered

seawater and 20g of Agar reagent and autoclaved for 3 minutes. Once the agar cooled down, I poured

50mL into a beaker along with 5g of ground species of algae and mixed thoroughly using a glass stir rod

and once homogeneous it was poured over a sheet of wax paper with four plastic grid squares in its

center. A new sheet of wax paper was placed on top of the mixture and a cinderblock was placed on top

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to weigh down on the agar to force it into the plastic grids (Figure 2). With these, an incubation tank was

set up using algae square grids (AG) of two different species and 3 amphipods (either Jassa falcata or

Gammarus oceanicus) per AG. This was done for both Gracilaria spp. and Fucus Vesiculosus algae. In

order to test differences in amphipod selectivity of algae from the same site I set up another tank

experiment with two replicates of Gracilaria spp. and Fucus Vesiculosus and Ulva lactuca AG from

Child’s River and 6 Jassa falcata or Gammarus oceanicus per AG.

I performed an adaptation of Folin-Ciocalteau’s for determination of phenolic compounds, where tannic

acid was used instead of gallic acid as a standard. I extracted dried and ground algae with 40% Ethanol,

sonicated for 2 minutes and shaked for 2 hours. Then I filtered the extracted material using a 47mm

swinnex filter and analyzed in a Shimadzu UV-Vis spectrometer at a wavelength of 790nm.

I did the Elemental analysis using a Europa ANCA-SL elemental analyzer - gas chromatograph

preparation system attached to a continuous-flow Europa 20-20 gas source stable isotope ratio mass

spectrometer following standard operation procedures for plant material of the Ecosystems Center -

Marine Biological Laboratory Stable Isotope Laboratory. I analyzed Nitrate and Ammonia in water

samples from both CR and SLP following Soloranzo (1969) and Wood et al. (1967) procedures,

respectively.

Results:

I performed several tests to look for differences between sites, algae and herbivory. Figure 3 shows δ15N

values of Fucus vesiculosus and Gracilaria spp. coming from both study sites where higher value can be

seen for both species at CR ( 6.74 and 7.38 0/00 vs Air respectively ). Figure 4 shows carbon and nitrogen

molar ratio (C:N) for Fucus vesiculosus and Gracilaria spp. where CR algae have a lower C:N than their

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SLP counterpart, a lower value meaning less carbon per every nitrogen. Nitrate and ammonia were also

measured for SLP and CR (Table 1). Again CR has a higher value for both of these nutrients. With this

ongoing trend of data I assumed both sites to be considerably different and performed a series of

bioassays. The first assay is shown in Figure 5 where both amphipods consumed 40 to 50% more from

the CR Fucus spp. than the SLP variety. The Gracilaria spp. assay showed a small difference between

algae consumed of about 76 to 92% in favor of the CR algae (Figure 6). The third assay comparing algae

from CR showed both amphipods showing preference to the Ulva spp. over the other algae. In the assay

using Gammarus oceanicus the Gracilaria spp. was also heavily grazed on (Figure 7).

Discussion:

Isotope values from CR can serve as an indication of an anthropogenic input of nutrients to the system

because human generated wastes generally have a higher δ15N value (Valiela 2000). And the C:N ratio

can help confirm this idea since the algae from CR have a lower ratio, meaning there are less carbon per

mole of nitrogen. Inorganic nutrients analyzed give another solid confirmation of differences in nutrients

available in both sites, showing a greater amount at CR.

My results indicate that there is a preference for Fucus vesiculosus from the SLP site, contrary to my

expectations; the phenolic compounds, shown in figure 8, produced by the Fucus spp. appear to have no

detrimental effect against either Jassa falcata or Gammarus oceanicus and may even be a beneficial

compound for such amphipods; as other herbivores can benefit from chemical compounds produced by

plants. However another way of analyzing my data is that amphipods might be consuming less algae

from CR because of its higher nutritional content and do not need to feed that often.

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No apparent preference was observed for either amphipod in the Gracilaria spp. bioassay as they both

consumed a similar amount of algae from both sites. During this experiment I observed amphipod’s

behavior and noticed how both species of amphipod were hasty towards hiding underneath AG, this

type of behavior leads me to think that amphipods will prioritize hiding over feeding preferences.

Results from CR bioassay show that amphipod’s were consuming algae and had a higher preference for

the Ulva spp. over both Gracilaria spp. and Fucus spp. which can be due to being as less palatable than

Ulva spp.; this was expected as Ulva spp. is generally consumed by many herbivores. The assay also

showed that amphipods have different feeding rates as each bioassay had either Gammarus or Jassa

outcompeting one another.

Acknowledgments: I thank my advisor Joe Vallino for all of his help and advice throughout this project. I

also thank the course TA’s Rich McHorney, Carrie Harris, and Alice Carter, Ivan Valiela and Scott Lindell

for advice on project ideas, Jimmy Nelson for advice on bioassay preparation and Ken Foreman for his

advice on chemical compounds tests. This project was funded by the Semester in Environmental Science

Program 2010 with lab space and equipment provided by the Marine Biological Laboratory.

References:

Chynoweth, D.P., C.E. Turick, J.M. Owens, D.E. Jerger, Peck M.W. 1993.Biochemical methane potential of

biomass and waste feedstocks. Biomass and Bioenergy. 5:95-111.

Geiselman, J.A. 1980. Ecology of Chemical Defenses of Algae Against the Herbivorous Snail, Littorina Littorea, In the New England Rocky Intertidal Community. PhD thesis, Massachusetts Institute of Technology/Woods Hole Oceanographic Institution, Massachusetts, USA.

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Hauxwell, J., J. Cebrian, I. Valiela. 2003. Eelgrass Zostera marinaloss in temperate estuaries: relationship to land-derived nitrogen loads and effect of light limitation imposed by algae. Marine Ecology Process Series. 247:59-73

Hay, M.E., and W. Fenical. 1988. Marine Plant-herbivore Interactions: The Ecology of Chemical Defense.

Ann. Rev. Ecol. Syst. 19:111-45 Hay, M.E., J.J. Stachowicz, E. Cruz-Rivera, S. Bullard, M.S. Deal, and N. Lindquist. 1998. Bioassays with

marine and freshwater macrwrganisms. Pages 39-141 in K.F. Haynes and J.G. Millar (eds.) Methods in Chemical Ecology. Volume 2, Bioassay Methods, Chapman and Hall, New York.

Hemmi, A., T. Honkanen, V. Jormalainen. 2004. Inducible resistance to herbivory in Fucus vesiculosus – duration, spreading and variation with nutrient availability. Marine Ecology Progress Series. 273: 109-120.

Marinho-Soriano, E., R.A Panucci,. M.A.A. Carneiro, D.C. Pereira.2009. Evaluation of Gracilaria Caudata J. Agardh for bioremediation of nutrients from shrimp farming wastewater. Bioresource Technology. 100:6192-6198.

Marinho-Soriano, E., C.A.A. Azevedo, T.G. Trigueiro, D.C. Pereira, M.A.A. Carneiro, M.R. Camara. 2011. Bioremediation of aquaculture wastewater using macroalgae and artemia. International Biodeterioration & Biodegradation. 65:253-257.

Pedersen, M.F. and J. Borum.1996. Nutrient control of algal growth in estuarine waters. Nutrient limitation and the importance of nitrogen requirements and nitrogen storage among phytoplankton and species of macroalgae. Marine Ecology Progress Series. 142:261-272.

Roesijadi, G., S.B. Jones, L.J. Snowden-Swan, Y. Zhu. 2010. Macroalgae as a Biomass Feedstock: A Preliminary Analysis. Pacific Northwest National Laboratory. PNNL-19944.

Soloranzo, L. 1969. Determination of ammonia in natural waters by the phenolhypochlorite method.

Limnol. Oceanogr. 14:799.

Valiela I., M. Geist, J. McClelland and G. Tomasky. 2000. Nitrogen loading from watershed to estuaries:

Verification of the Waquoit Bay Nitrogen Loading Model. Biogeochemistry. 49:277-293.

Wood, E.D., F.A.G. Armstrong, and F.A. Richards. 1967. Determination of nitrate in seawater by

cadmium-copper reduction to nitrite. J.Mar. Biol. Assoc. U.K. 47:23

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Tables:

Table 1. Dissolved Inorganic Nutrient analysis for CR and SLP sites.

Sage Lot Pond Child's River

NH4 0.063 0.048

NO3 0.060 3.440

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Figures:

Figure 1. Study site. Point A is Child’s River area, point B is Sage lot Pond.

41°33’ N , 70°31’ W

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Figure 2. Algae square grid used for the bioassay.

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Figure 3. δ15N values for Fucus vesiculosus and Gracilaria spp. from SLP and CR.

4.82

6.74

4.63

7.83

0

1

2

3

4

5

6

7

8

9

Fucus SageLot Pond

Fucus Child'sRiver

GracilariaSage Lot Pond

GracilariaChild's River

Isotopic composition

d15N (o/oo vs. AIR)

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Figure 4. Carbon and nitrogen ratio of Fucus vesiculosus and Gracilaria spp.

21.88

15.59

11.94

8.63

0

5

10

15

20

25

FucusSage Lot

Pond

FucusChild'sRiver

GracilariaSage Lot

Pond

GracilariaChild'sRiver

Carbon & nitrogen composition

Mole C:N

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Figure 5. Bioassay for the algae Fucus vesiculosus from SLP and CR. Average of four replicates.

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Figure 6. Bioassay for the algae Gracilaria spp. from SLP and CR. Average of four replicates.

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Figure 7. Bioassay for Child’s River macroalgae. Two replicates per algae.

-10

0

10

20

30

40

50

60

Jassa falcata Gammarus oceanicus

% o

f al

gae

co

nsu

me

dChild's River Assay

Ulva

Gracilaria

Fucus

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Figure 8. Phenolic compounds concentration in Fucus vesiculosus, Gracilaria spp. and Ulva lactuca from

SLP sites.

0

0.5

1

1.5

2

2.5

3

FucusSage Lot

Pond

FucusChildsRiver

GracilariaSage Lot

Pond

GracilariaChildsRiver

UlvaChildsRiver

Co

nce

ntr

atio

n (

mg/

L)

Phenolic compounds