Abstract: Wounding of the meristem region in the subtidal kelp, Laminariacomplanata,resulted in increases of phenolics by 120% after the first 24 hours and over 190% after three days. This shows the chemical defense mechanism induced by herbivory or natural wounding of the plant, and has, in past studies, shown to repel grazers and may be conducive to the healing of the plant.
 
 
 

Secondary compounds in plants, both terrestrial and marine (Steinberg, 1988), are widely known to deter grazers. Phaeophyta are known to contain secondary compounds composed mainly of phloroglucinol and its polymers (Ragan and Glombitza, 1986). These polyphenolic substances alter the ingestion of tissue by such grazers as gastropods, isopods, and echinoids by aggregation of proteins, but may be precluded by other factors such as surfactants and gut alkalinity produced by the grazers (Tugwell and Branch, 1992). In addition to being an herbivore deterrent, polyphenolic compounds may also aid in the healing ability of the plant by means of protein coagulation.

 Although much attention has been given to the study of the effects of polyphenols on grazers, little is known about how grazers or wounding in general affect the levels of phenolics in these alga or how synchronous the production of these secondary phenolic compounds is. In a study on Alaria marginata, Steinberg (1984) noted that resources for the production of such defensive products will be conserved for the parts of plants or the times when the plants need the protection, just as some alga (e.g. Fucus distichus) will allocate products for growth and reproduction on the basis of need (Van Alstyne, 1990). Thus, an allocation of energy and resources to the production of polyphenols may not only lead to the protection of the alga, but may also hinder the growth and reproduction, causing over the long term.

variation in the population of the alga (Van Alstyne, 1988). If the alga can control the output and allocation of the phenolic defenses with respect to grazing and wounds, then the plant will have a much higher survivability rate.

 In this study, I show that the brown alga, Laminaria complanata, is capable of producing phenolic compounds as a result of being grazed upon, represented here by wounding.
 
 
 
 

PLANTS

 Laminariacomplinata is a large benthic kelp found subtidally in regions of rocky substrates and usually low wave energies. It has been found in the Northeast Pacific Ocean around Friday Harbor (4836' N., 12321' W.), Washington and James Bank (4832' N., 12300' W.), British Columbia (Druehl, 1966). As well as being locally abundant, this alga has one of the highest levels of polyphenolic compounds for the vicinity (Duggins, personal observations). Only large sporophytes (blade length > 1.0 m) without obvious damage and confined within a 10 m diameter area were used.

STUDY SITE

 The collection of samples and meristem wounding was conducted subtidally at a spot known as Cantilever, approximately 15 m off the Friday Harbor Laboratories property and 8 to 12 m deep at low, low tide. The field study area was a rocky substrate which was subjected to tidal currents of up to three knots, available for herbivore grazing, and prone to other environmental stresses.

WOUNDING

 Thirty plants were chosen within the specific study area and individually tagged with numbers 1 through 30. At day 0, each of these plants was wounded in the meristem region with 5 disks (1.0 cm diameter ) being removed and radiating in an arc about the center of the meristem to assure even variability of core thickness (Fig. 1). Ten of these plants were chosen at random and were again wounded, this time with three disk punches, two to the sides of the meristem region and inside the original arc and one in the center of the blade above the original arc (Fig. 1). Ten more plants were chosen at random without replacement and on day 3 were wounded as were the day 1 plants. All disks were saved for later analysis.

PHENOLIC ASSAYS

 On day 0, each of the five tissue disks from each plant were cut approximately in half. Five half disks for each plant were wet weighed, dried at 60C for 24 hours, and weighed again to get a wet/dry weight ratio. The other five half disks were frozen at -80C for 48 hours and used for the phenolic assay. The actual wet weights for these five half disks was assumed to be equal to the mean of the wet weights for the other five half disks. For days 1 and 3, the three collected disks were cut in half, and three half disks (total weight 0.10 g) were frozen at -80C for 48 hours and used for the phenolic assay.

 The half disks were ground in an 80% aqueous methanol solution and left to extract in the dark at 4.0C. The Folin-Denis method (Steinberg, 1985) was used to assess the total phenolic content with absorbencies read at 765 nm. Standard curves of phenolic content vs. absorbance were made for regressions at each sampling date using standards with phloroglucinol concentrations between 0 and 0.25 mg/ml.
 

The estimated mean percentage of dry weight phenolic content of the day 0 (unwounded) plants was 2.25 0.51 %. For the day 1 wounded plants and the day 3 wounded plants, the percent phenols of dry weight means were 2.50 0.10 % and 4.14 0.47%, respectively (Fig. 2). Using a paired t-test, the day 1 group was significantly higher in polyphenols than the day 0 group and day 3 group was significantly higher than the day 1 group in polyphenols.  Although 3 out of the 10 day 1 plants showed decreases in phenolic content after the first 24 hours after wounding (Fig. 3), there were phenolic increases in all plants from day 1 to day 3 (Fig. 4).
 

Trends in the phenolic contents of the data series are all uniform with the exception of the data from day 0. This could be due to the fact that an average estimate was used for all sample weights in this series. The trend may have been more uniform if true sample wet weights were used. The overall trend in the data suggests that the polyphenolic content within a plant increases after wounding and furthermore, does so to a greater extent the first 24 hours. During the first 24 hours, the phenolic content is low, but this may be due to the inability of the plant to manufacture these compounds within a short time. This may also allow for the plant to produce just as much phenolic material as needed and to not overproduce and expend excess amounts of energy and storage products, for if this is a response to grazing, then it allows some time for the grazer to move on, and if the plant receives no further damage, it can preserve the energy and resources needed for other interests, such as growth and reproduction. After the first period of time, a more definite increase in the phenolic levels is seen, reflecting a dedicated shift to either healing a wound or severely detracting grazers.

A more extensive project should be undertaken to determine the apex of the phenolic compound production, both temporally and quantitatively. Other factors such as translocation of the phenolics and merely the distribution of phenolics should also be studied. In addition, it is not known as to what specific phenolic compounds increased . Just a raise in the levels of phoroglucinol could have been enough to raise the phenolic level, or all present phenolics could have been raised. More study is needed in this area to determine what specific phenolics are raised or even created. Also in need of study is the plants' means of detecting a grazer or wound. According to Van Alstyne (1988), these signal should not be waterborne. There has been little or no study on the algal cues for inducing chemical responses, for the kelps are unlike vascular plants in that they have either a non direct or primitive transport system. Lastly, the effects that the phenolic compounds has on a community needs to be known. This includes predator-prey interactions as well as plant vs. plant competition.

I would like to thank Meagan Dithier, David Duggins, and Terry Klinger for their support, advice, and assistance throughout this project.

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Ragan, M. A., and K. Glombitza, 1986. Phlorotannins, brown algal polyphenols. Pages 129-241 in J. A. Hellebustand J. S. Craigie, editors. Handbook of phycological methods. Volume II. Cambridge University Press, Cambridge, England.

Steinberg, P. D. 1984. Algal chemical defenses against herbivores: allocation of phenolic compounds in the kelp Alaria marginata. Science 223:405-407.

-----. 1985. Feeding preferences of Tegulafunebralisand chemical defenses of marine brown algae. Ecological Monographs 55:339-349.

-----. 1988. Effects of quantitative and qualitative variation in phenolic compounds on feeding in three species of marine invertebrate herbivores. Journal of Experimental Marine Biology and Ecology 120:221-237.

Tugwell, S., and Branch, G. M. 1992. Effects on herbivore gut surfactants on kelp polyphenol defenses. Ecology 73:205-215.

Van Alstyne, K. L. 1988. Herbivore grazing increases polyphenolic defenses in the intertidal brown alga Fucus distichus. Ecology 69:655-663.

-----. 1990. Effects of wounding by the herbivorous snails Littorinasitkana and L. scutulata (Mollusca) on growth and reproduction of the intertidal alga Fucusdistichous (Phaeophyta). Journal of Phycology 26:412-416.