effects of quantitative and qualitative variation in ... · and the echinoid ~tron~locentr~t~s...

J. Exp. Mar. Biof. Ewi., 1988, Vol. 120, pp. 221-237 Elsevier

221

JEM 01111

Effects of quantitative and qualitative variation in phenolic compounds on feeding in three species of marine

invertebrate herbivores

Peter D. Steinberg School of Bio~~a~ Sciences, U~.~r~ of Sydney, Sydney, New South Wales, A&r&a

(Received 10 August 1987; revision received 18 April 1988; accepted 13 May 1988)

Abstract: The deterrent effects of brown algal phenolic compounds and the terrestrial polyphenolic tannic acid on feeding by three species of invertebrate herbivores from central California, including the gastropods Tegula funebralis (Adams) and Tegula brunnea (Phillipi) and the echinoid Strongylocentrotus purpuratus (Stimpson) were examined. Algal phenolics used were the monomeric phenolic phloroglucinol, and polyphloroglucinols from Fucus vesiculosus (Linnaeus), Halidrys siiiquosa (Linnaeus) Lyngbye, and Etienia arboreu Areschoug. All of the polyphenolics deterred feeding by all three herbivores at concentrations of 5 mg . ml - ‘ in agar disks. Concentrations of 2 mg . ml- ’ also generally deterred feeding by the gastropods (these levels were not tested against S. purpuratus). Relative amounts of deterrence by diflerent compounds were similar, especially for the gastropods. Phloroglucinol deterred feeding by the echinoids, but not by T. jknebral~. Responses of the echinoids were otherwise similar to the gastropods, but more variable. I also demonstrated deterrence of S. p~uTa~ by tannic acid using the “tanned” kelp technique of Steinberg (1985). Reactivity of the different phenolic compounds in the Fohn-Denis procedure, a common colorime- tric assay used to estimate levels of phenolics in plant tissue, was similar. This suggests that measuring phenolic levels in brown algae by this technique will not be greatly confounded by the occurrence of different kinds of phenolic molecules in different brown algae. This result, in combination with the similarity of the deterrent effects of the compounds used in this study, increases the validity of previous studies in the northeastern Pacific Ocean which correlate algal phenolic levels and diets or feeding preferences of invertebrate herbivores. For plants and herbivores in this region, this assay is a reasonable measure of a biologically meaningful phenomenon - levels of phenolic deterrents in the algae.

Key words: Brown alga; Feeding deterrence; Herbivory; Potyphenol

The importance of secondary metabolites in terrestrial plants as chemical defenses against herbivores (and other natural enemies) is now well known (Rosenthal & Janzen, 1979; Denno 8c McClure, 1983; Hedin, 1983). Benthic marine macroalgae also contain

a wide diversity of secondary metabolites (Faulkner, 1984, 1986). Although these compounds in algae have also been proposed to function as defenses against herbivores (Ogden & Lobel, 1978; Norris & Fenical, 1982), until recently there was little known about their ecological effects. However, at least some algal secondary metabolites do

Correspondence address: P. D. Steinberg, School of Biological Sciences, MaCleay Building A12, Uni- versity of Sydney, Sydney, New South Wales 2006, Australia.

~22-~81/88/$03.50 0 1988 Elsevier Science Publishers B.V. (Biomedic~ Division)

7-I- -‘.._ P. Il. STEINBERG

have deleterious effects against marine herbivores, and thus can function as defenses (Geiselman & McConnell, 198 1; Targett & McConnell, 1982; Targett et ~1.. 1986; Paul & Fenical, 1983, 1986; Steinberg, 1984, 1985; Hay et ul., 1987a,b). These compounds have other ecological effects as well (Bakus et al., 1986).

To date, the most well-studied chemical defenses in temperate algae are the polyphenolic compounds found in brown algae (Phaeophyta). These compounds consist of phloroglucinol (1,3,5_trihydroxybenzene) and its polymers (e.g.. Glombitza, 1977; Ragan & Glombitza, 1986). Polyphloroglucinols are thought to be ubiquitous within the brown algae (Ragan, 1976; Ragan & Glombitza, 1986), and can comprise up to lS-ZO~/, of the dry weight of an algal thallus (Geiselman, 1980; Ragan & Glombitza, 1986; Steinberg, 1985, 1986, in prep.). The chemistry and biology of brown algal polyphenoli~s are reviewed by Ragan & Glombitza ( 1986).

There is both indirect (correlative) and direct (experimental) evidence for the deterrent effects of brown algal polyphenolics against marine herbivores. Indirect evidence stems from the observation that feeding preferences of gastropod and echinoid herbivores are often strongly negatively correlated with quantitative variation in polyphenolic levels among brown algae (Anderson & Velimirov, 1982; Steinberg, 1984, 1985; Johnson & Mann, 1986).

Direct experimental evidence for the deterrent effects of algal polyphenolics against marine herbivores comes from the work of Geiselman & McConnell (1981). They showed that addition of purified polyphenoli~s from the fucoid algae Ascup~yl~~~ ~o~o~~~ (Linnaeus) Le Jolis and FUCUS ve~~c~~~~~~ (Linnaeus) to otherwise palatable agar discs significantly deterred feeding by the gastropod Littorina littorea (Linnaeus). The terrestrial plant tannin tannic (polygallic) acid had similar deterrent effects (Geiselman & McConnell, I981), as was also shown by Steinberg (1985). The amount of deterrence exhibited by different molecular weight fractions in their experiments varied, suggesting that different sizes of algal polyphenolics differed in their effects on L. littorea (Geiselman & McConnell, 1981). However, the predominant effect was simply a quantitative (dosage dependent) one; as the concentration of phenolics in the agar increased, feeding activity by L. littorea decreased.

In this paper, I investigate the deterrent effects of a number of kinds of phenolic ~~~lnpounds against three species of marine herbivores from the i~o~heastern Pacific Ocean, including thegastropods ~e~l~f~nebra~is (Adams) and Teg& hrunnea (Phillipi), and the echinoid ~tron~locentr~t~s purpuratus (Stimpson). The purposes of this study are three-fold: (1) to experimentally test the deterrent effects of phenolic compounds strongly implied by such studies as Steinberg (1984, 1985), Johnson & Mann (1986), and Anderson & Velimirov (1982); (2) to examine the effects that quantitative (dosage) vs. qualitative (structural) variation in phenolics has on feeding by marine herbivores; and (3) to attempt to correlate the deterrent effects of purified compounds with the calorimetric techniques commonly used to estimate phenolic levels in brown algae (e.g., Geiselman, 1980; Anderson & Velimirov, 1982; Steinberg, 1984, 1985; Johnson & Mann, 1986).

ALGAL PHENOLIC FEEDING DETERRENTS 223

MATERIALS AND METHODS

HERBIVORES

The herbivores used in this study are all common species along the coast of California, and were collected in central California, near Santa Cruz. T. funebrulis occurs in the intertidal, from + 1.5 to - 0.2 m (Morris et al., 1980). T. brunnea (Morris et al., 1980) overlaps with T. funebrulis in the low intertidal, and extends into the sublittoral, where it is common in giant kelp Mucrocystis pyriferu (Linnaeus) C. Agardh communities

(Riedman et al., 1981; Watanabe, 1984b). S. purpuratus is one of the two most common species of echinoids on rocky coasts in California and occurs both intertidally and subtidally (Morris et al., 1980). All three species are generalist herbivores and readily consume brown algae in the field and in the laboratory (Best, 1964; Leighton, 1966; Vadas, 1977; Duggins, 1983; Steinberg, 1984, 1985; Watanabe, 1984a; pers. obs.). T. funebralis and T. brunnea used here were between 25 and 30 mm in maximum shell diameter. S. purpurutus were between 50 and 70 mm maximum test diameter.

COMPOUNDS TESTED

Five kinds of phenolic compounds were tested for their deterrent effects against the herbivores (Table I), although not all compounds were tested against each herbivore. Four of these compounds are brown algal metabolites (phloroglucinol, and polyphenolics from the fucoids F. vesiculosus, Halidy siliquosa (Linnaeus) Lyngbye, and the kelp Eiseniu arborea Areschoug). Compounds from two separate extractions of F. vesiculosus were used in the experiments, designated as Fucus 1 and 2 (in Results). Details

TABLE I

Phenolic compounds used in feeding experiments.

Compound tested Characteristics Source Reference

F. vesiculosus polyphenolics High molecular weight (> 10000)

M. A. Ragan, eastern Canada

H. siliquosa polyphenolics

E. arborea polyphenolics

Mix of polymers, mostly more than eight phloroglucinol units in size

Mix of polymers, mostly less than six phloroglucinol units

K. W. Glombitza, Roscoff, France

K. W. Glombitza, Vancouver Island, Canada

Phloroglucinol

Tannic (polygallic) acid

Dihydrate monomer Sigma Chemicals (1,3,5-trihydroxybenzene)

Terrestrial polyphenol Fisher Scientific

Ragan (1985), Ragan & Craigie (1976), Ragan & Glombit- za (1986)

Glombitza et al. (1980), Ragan & Glombitza (1986)

Glombitza & Gerst- berger (1985), Ra- gan & Glombitza (1986)

Ragan & Glombitza (1986)

Swain (1979)

224 P. D. STEINBERG

of extraction techniques and structures for the algal polyphenolics are in Ragan & Craigie (1976), Glombitza et al. (1980), Glombitza & Gerstberger (1985) Ragan (1985) and Ragan & Glombitza (1986). The fifth compound, tannic (polygallic) acid, is a polyphenolic found in a variety of terrestrial plants.

Although the algal polyphenolics were extracted from algae collected from different locations (Table I) than where the herbivores are found, all three species of herbivores probably contact E. arborea and Halidrys spp. in the field as living plants or drift, and the distribution of T. funebralis in the intertidal overlaps with congeners of F. vesiculosus. Moreover, the differences in structure (either in molecular weight or in the type and arrangement of bonds between phloroglucinol units) among the four types of algal phenolics used here are substantial. These compounds are probably a fair representation of the variety of polyphenolics that these herbivores contact in the field in California.

EXPERIMENTAL

Two techniques were used to study the effects of phenolic compounds on the herbivores. In all experiments except one, deterrence was examined by feeding herbivores agar disks containing various levels of phenolic compounds, similar to the methods of Geiselman & McConnell (198 l), Targett & McConnell (1982), and Targett et al. (1986). Agar disks were made by heating an aliquot of kelp extract, adding agar, boiling, and letting cool to 50 ’ C. Kelp extracts were prepared from M. pyrzzra, a preferred food of all three herbivores (Leighton, 1966; Watanabe, 1984a; Steinberg, 1985), by homo- genizing the kelp blades in seawater in a ratio of 1 : 3 w/w kelp : seawater, and then decanting the liquid portion. The phenolic compounds were dissolved in an aliquot of kelp extract at room temperature, added to the (cooled) aliquot containing agar, and shaken vigorously. This mixture was immediately poured into 60-mm plastic Petri dishes and allowed to harden. Standard concentrations of phenolics in the final mixture were 0,2, or 5 mg . ml _ ‘, with the exception of phloroglucinol, for which concentrations as high as 22 mg . ml - ’ were used. Since polyphenolics bind to agar in solution, apparently preventing hardening, the concentration of agar in the disks was varied between 2.5 and 3.4% ofthe final mixture (w : w) to insure that disks containing different levels of polyphenolics were similar in texture. In an experiment in which I compared feeding by T. jiunebralis on disks which varied only in their percent composition of agar, I found that T. funebralis did not select among disks containing varying levels of agar.

The second method used was the “tanned kelp” technique used by Steinberg (1985) to experimentally demonstrate the deterrent effects of tannic acid against T. ,funebrulis. In this method, blades of A4. pyrifera are soaked in solutions of tannic acid to allow tannins to adsorb onto or into the kelp. Elevated tannin levels persist sufficiently long (in running seawater) for feeding experiments to be conducted (Steinberg, 1985). This technique has the advantages of using a natural food of the herbivores in the experiments [but does not seem to work with algal (F. vesiculosus) polyphenolics]. I used this technique here to measure the effect of tannic acid against S. purpuratus.


Feeding experiments were conducted in 22-1 aquaria (snails) or 25-l plastic bins (echinoids) in running seawater at the Joseph Long Marine Laboratory, University of California, Santa Cruz, at ambient water temperature and natural photoperiod. All grazers were maintained on a variety of algae in the laboratory for at least 6 wk prior to the experiments, and were starved for 36-48 h prior to each experiment. For experiments with the gastropods, 15 snails (occasionally 12) were placed in each tank with two or four agar disks (removed from the Petri dish molds). For S. purpurutus, five animals were placed in each bin with three to six disks. Between nine and 18 bins (replicates) were used in each experiment. The duration of the gastropod-feeding experiments was 48 h with the exception of the second T. funebmlis/Fucus tannin no choice (see below) experiment (28 h) and the T. funebrulis/phloroglucinol experiment (6 h). Experiments with S. purpurutus lasted between 7 and 42 h. Experiments using phloroglucinol were of shorter duration because phloroglucinol leaches out of the agar disks. In one instance (with compounds from F. vesiculosus), an experiment was repeated to test for changes in the responses of the herbivores over time.

Deterrence was determined by measuring the amount consumed of the different types of agar disks in an experiment. Weighing the agar after it had cooled prior to adding it to the aquaria, and then again after the experiment, following a standard blotting procedure, proved a reliable method. In all experiments at least one control tank was run to measure the change in weight of the disks in the absence of herbivores. This change was never > 3.5% of the initial weight of the disks and it was generally much less (= 1%). The amount of agar consumed by the herbivores was corrected accordingly. Consumption data are presented in Figs. l-4 as the amount consumed relative to the consumption of disks lacking added phenolics (0 mg * ml- ‘) in each experiment. This facilitates comparison of deterrent effects among different compounds and/or herbivores.

In some instances, two kinds of feeding experiments were done for a given type of phenolic. In most experiments only one kind of disk was present in each tank and no choice between phenolic concentrations was offered to the herbivores. In other “choice” experiments, however, herbivores in one tank could choose between agar disks containing different levels of phenolics. Herbivores may or may not be presented with a choice of foods in nature, and the deterrent effects of a compound may well be affected by the presence or absence of other foods nearby. However, since in this study I am primarily interested in deterrence, as opposed to preferences, most experiments were of the no-choice sort.

PHENOLIC ASSAYS

Phenolic assays were done calorimetrically using the Folin-Denis technique for measuring total phenols (Swain 8z Hillis, 1959; Ragan & Jensen, 1977; Anderson & Velimirov, 1982; Steinberg, 1985). Absorbance was measured at 725 nm. Compounds (and agar disks containing phloroglucinol) were dissolved in 70% aqueous methanol

276 P.D.STEINBERG

for the assay. Concentrations of between 0 and 15 pg. ml ’ of the pure compounds were

used to develop standard curves of phenolic concentration vs. absorbance (Table IV).

STATISTICAL ANALYSES

The significance of the deterrent effects of the compounds in no-choice experiments

was determined using single-factor model I ANOVAs for comparisons of three or more

treatment groups, and t tests when there were only two (as for S. purpuratus). Means

of different groups were compared using the S-N-K procedure (Sokal & Rohlf, 1969).

Results of choice experiments in which only two levels of phenolics were present were

analyzed with paired t tests. In one instance, a choice experiment was done using three

levels of compounds [ Tegulu funebrulis tannic acid choice experiment (Table I, Fig. 3)].

Since consumption of disks in this experiment were not independent, simple ANOVAs

are inappropriate. Instead n/3 replicates were chosen randomly from different aquaria

for each phenolic concentration (treatment), and a single-factor ANOVA done on this

reduced data set. All data were analysed as amount consumed (as opposed to amount

consumed relative to consumption of 0 mg . ml - ’ disks, as in Figs. l-4).

Variances of treatment groups were often heteroscedastic and proportional to the

means, in which case the data were transformed by In(x) prior to analyses. If transfor-

mation did not achieve homoscedasticity, results of the ANOVAs are presented along

with the results of Bartlett’s test for homogeneity of variances so that heteroscedasticity

can be estimated. All probabilities for pairwise tests given are one-tailed, testing the

hypothesis that a greater concentration of phenolics results in greater feeding deterrence.

RESULTS

TEGULA FUNEBRALIS

All polyphenolics used in the feeding experiments significantly deterred feeding by

T. ,funebraZis at a concentration of 5 mg * ml _ ’ in the agar disks (Fig. 1, Table II). Except

for compounds from H. siliquosa, and tannic acid in a no-choice experiment, they were

also significantly deterrent at a concentration of 2 mg * ml _ ’ (Fig. 1, Table II). In every

experiment the amount consumed of disks containing 2 mg * ml - ’ polyphenolics was

intermediate between the amount of 0 and 5 mg . ml - ’ disks consumed. The relative amount of feeding deterrence exhibited by the 5 different kinds of

polyphenolics were similar, especially at higher concentrations. Concentrations of

5 mg . ml _ ’ reduced feeding by 51-69”/6, relative to disks lacking added polyphenolics.

Polyphenolics from E. arborea and tannic acid caused the greatest deterrence, and those

from the first F. vesiculosus extraction (Fucus l), the least (Fig. 1). Concentrations of

2 mg . ml- ’ reduced feeding by 22-51x. The most deterrent compounds at this con-

centration were tannins from Fucus 1 in a choice experiment, and the least were these

same compounds in a no-choice experiment.


Polyphenolic Concentrations

0 0 mg/mi added

m 2 mg/ml added

a 5 mg/ml added

m 5 mg/ml Fucus 1 tanninsodded

Tannic Acid (N=4)

Fucus 1 Fucus 2 (N= 5) (N=3)

No Choice Expts.

tialidrys (N-4)

Eisenia : Tanmc Acid Fucus 1 (N=4) : (N=6) (N= 6)

Choice Expts.

Type of Phenolic

Fig. 1. Results of no-choice and choice feeding deterrence experiments with T.fitnebralh Data are expressed as percent eaten, relative to mean consumption of 0 mg.ml-i disks in each experiment. N = number of replicates for each treatment (phenolic concentration) in each experiment (differences in numbers of replicates for certain treatments are shown in parentheses above approp~ate history). Data

are X + SE. ND = not done.

Responses of T. funebralis to polyphenolics in its food were consistent over time as judged by the two experiments using polyphenolics extracted from F. vesiculosus, done in March 1984 (with Focus I pheno~cs) and June 1986 (with MUCUS 1 and 2 compounds). The two batches of polyphenols used were extracted at different times, but by the same procedure (Methods; M.A. Ragan, pers. comm). At a concentration of 5 mg * ml- ‘, Fucus 1 polyphenolics decreased feeding by T. jiunebralis by 5 1% in the first experiment, and by 53% in the second experiment (Fig. 1). In the second experiment, Fucus 2 polyphenolics decreased feeding by 60% (relative to cons~ption of 0 mg * ml - ’ disks), which was not si~~c~tly different from the deterrent effects of Focus 1 polyphenolics (Table II).

The feeding responses of T. funebralis dflered depending on whether a range of phenolic concentrations were offered or not. Both choice and no-choice experiments were conducted for tannic acid and Focus 1 phenolics. In each case, feeding deterrence was less in the no-choice experiments (Fig. 1). This ditference was substantial for Fucus 1 polyphenolics, but marginal for tannic acid. In no choice experiments with Fucus 1 compounds, a concentration of 2 mg * ml- ’ deterred feeding by 22x, relative to 0 mg + ml- 1 disks (P < 0.05, S-N-K test, Table II). In the choice experiment, feeding deterrence by 2 mg * ml- ’ disks was z 50% (paired t test, P = 0.001, Table II).

Phloro~ucinol, the monomer of which algal ~lyph~o~cs are comprised, did not deter feeding by T. @zebra&s (Fig. 2, Table II). Because phloroglucinol leaches out of the agar disks when they are placed in seawater, this experiment was run for a shorter

22x P. D. STEINBERG

Statistical analyses of feeding experiments. No choice experiments in which three concentrations ol phenolics or more were used (e.g., 0, 2, 5 mg ml _ ‘) were analysed by single-factor model 1 ANOVA. followed by S-N-K multiple range test (Sokal & Rohlf, 1969). Those concentrations (0, 2, or 5) sharing underline are not significantly different at 0.05 level. Experiments using only two concentrations ofphenolics were analysed with paired (choice experiments) or unpaired (no-choice) t tests. Probabilities given fort tests are one-tailed (see Methods). In several instances, data were transformed by In (x) prior to analysis in order

to ensure homogeneity of variances; these instances are marked by “$“. Exceptions to these general procedures are noted below or in Methods.

-. % funebrah (Figs. 1,2) No-choice experiments

Tannic acids” F. vesiculosus 1s

F. vesiculosus 2 H. siliquosa E. arborea

Phloroglucinol expt. Choice experiments

Tannic acid’

F. vesiculosis 1 T. brunneu (Fig. 3)

Tannic acid (no-choice)s Tannic acid (choice) F. vesiculosus (no-choice) E. arborea’” (no-choice)

S. purpurarus (Fig. 4) Tannic acid

Choice 1 Choice 2 Choice I and 2

No choice I No choice 2 No choice I and 2s

F, vesiculosus (choice) Phloroglucinol (choice)

-

F = 8.4

F = 27.29 F = 22.81 F = 9.28 F = 52.66 F = 22.96

F’ = 46.8

I = 4.80

F = 32.53 I = 2.88 F = 33.70 I = 3.86

t = 3.35 t= 5.11

t = 5.88 t = 5.14 I = 1.70 I = 3.92 t = 1.80 I = 4.80

df = 2,8 df= 2,ll

df = 3,8 df = 2,9 df = 2.9 df = 2.13

df = 2.3

df= 5

df = 2,Y df= 5 df = 2.X df= 5

df= 5 df=6 df= I2

df= 7 df= 8 df= 17

df= 6 df= 5

P < 0.01 P 0.001 < P 0.001 < P <O.Ol P<O.OOl

P c 0.001

P < 0.01 P = 0.001

ND

P<O.OOl P < 0.025 P < 0.001 P <: 0.01

0 2 5

(I 3 5

P = 0.02 P < 0.001 P < 0.001 P < 0.01

NS

P < 0.01

P = 0.06 P < 0.001

* 5 mg ml ’ Fucus 1 tannins. “ Bartlett’s test for homogeneity of variances; P i 0.025. h Ph = 22 mg ml _ ’ phloroglucinol: TA = 4 mg ml -- ’ tannic acid. ’ Analysis by ANOVA on random selection of replicates from each treatment (Methods)

” F,,,;,, test for homogeneity of variances; P < 0.01.

period of time (6 h) than the previous ones. Tannic acid was included as a treatment

in this no-choice experiment to insure that 6 h was sufficient time to detect any deterrent

effects. In 6 h, the concentration of phloroglucinol in the disks decreased from

22 mg . ml ’ to 4.3 + 0.38 mg * ml- ’ (X & SD, n = 3), as measured by the Folin-Denis

procedure. These levels are comparable to or higher than the levels of polyphenolics

used in the previous experiments. Disks containing 4 mg . ml _ ’ of tannic acid signili-

cantly deterred feeding in this experiment (Fig. 2, Table II), and at levels comparable

to those observed in earlier experiments.


Phenolic Concentrations

0 0 mg/ml added to agar

m 22 mg/ml phloroglucinol added

m 4 mg/ml tannic acid added

Fig. 2. Results of no-choice experiment with ~~~ebru~, p~oro~ucinol, and taxmic acid. Concentration ofphloroglucinol shown is at beginning of experiment. Data are 3 + SE, and expressed as percent eaten (see

Fig. 1). N = 6.

TEGULA BRUNNEA

Results for T. brumea (Fig. 3) were very similar to to those for T. funebralis. All three polyphenolics used significantly deterred feeding at concentrations of 5 mg * ml - ’

(Fig. 3, Table II). The degree of deterrence at this concentration, 45-65x, was within a similar range as that observed for T. f~~ebrali~. Pol~h~o~cs from E. arborea and tarmic acid were again more deterrent than polyphenolics from F. v~sicu~~~~. The principal difference in the responses of the two species of snails was that T. brunnea was not deterred by polyphenolics from F. vesiculosus at a concentration of 2 mg * ml- ‘.


0 0 mg/ml added

m 2 mg/ml added

m 5 mg/ml added

- Tannic Acid Tannic Acid $ -2

(N-4) (N=6) (N = 4)

Type of Phenolic

Fig. 3. Results of feeding deterrence experiments with T. bnmzeu. All data are from no-choice experiments except for tannic acid* (choice). Data (given as X + SE) as in Fig. 1.

Feeding by the echinoid S. purpurahes was also deterred by the addition of poiy- phenolics to its food, although its responses differed somewhat from those of the gastropods. In particular, variation within and between experiments was higher, there were greater differences between choice and no-choice experiments, and phloroglucinol did deter feeding by the urchins (Fig. 4).

.z 125 z E 100 z S 75

0

f

50

25

5 a 0


f 0 mg/ml added

m 6 mg/ml added

m 20 mg/mi phloroglucinol added

1 (N-5) 2(N=5) : l(N=6) Tannic Acid : Tannic Acid

L Phloro.

NoChoice Expts. : Choice Expts

Type of Phenolic

Fig. 1. Results of feeding deterrence experiments with S. purpurutus. Concentration of phloroglucinol shown is at start of experiment. Data (Z + SE) as in Fig. 1

Due to a shortage of algal compounds, most experiments used only tannic acid. Tannic acid was highly and significantly deterrent in the two choice experiments, decreasing feeding by 81 and 737; at a concentration of 5 mg . ml _ ’ (Fig. 4, Table II).

The effects of tannic acid in no-choice experiments were variable. In one experiment, 5 mg . ml of tannic acid in the disks decreased feeding by 67% (P < 0.01, Table II). In the second experiment, feeding was only decreased by 34%, a nonsignificant result (Table II). When the two experiments were lumped together and analysed, deterrence was significant (Table II).

Both polyphenolics from F. vesiculosus, and phloroglucinol, deterred feeding by S. purpuratus (Fig. 4, Table II), but the effect was marginally non-significant for Fucus 2 polyphenolics (P = 0.06), despite a 50% decrease in food consumed. (However, the analysis used here, a paired t test with df = 6, is a fairly weak test.) Deterrence by phloroglucinol was highly significant, comparable to that of tannic acid in choice experiments (Fig. 4).


The deterrent effects of tannic acid on S. pupuratus was also investigated using the “tanned kelp” technique (Steinberg, 1985). Blades of M. pyrifera soaked for 18 h in a 3.5 % solution of tannic acid in seawater were highly unpalatable to the urchins, relative to kelp blades soaked in seawater (Table III). Levels of phenolics in the tanned kelp decreased from z 2 1% (by dry weight) at the beginning of the experiment to a mean of 3.7% at its finish (Table III). Initial levels of phenolics in the tanned kelp are high relative to values in naturally phenolic-rich species of algae (Steinberg, 1985), but previous experiments indicate that most of the leaching occurs in the first 12 h or so (Steinberg, 1985, pers. obs.), with concentrations rapidly decreasing to 8 % or less. This is followed by a much slower leaching (pers. obs.).

TABLE III

Feeding rate over 36 h of S. putpuratus on “tanned” and “untanned” M. pyrifera blades. Difference in amount consumed of tanned vs. untanned blades is highly significant (t test, df = 5, P < 0.001). Data are

? f 1 SE.

Concentration of Amount tannic acid soak eaten (g, n = 6)

Total phenolic levels in blades (percent dry weight)

Before feeding After feeding

3.5% 3.63 f 0.72 21.50 * 1.02 3.68 f 0.18 0 (seawater) 16.18 + 1.26 0.79 * 0.14 not done

COLORIMETRY OF POLYPHENOLICS

Slopes of the regression lines for plots of phenolic concentration vs. absorbance in the Folin-Denis assay are similar (Table IV), varying at most by 28% (tannic acid vs. compounds from F. vesicukms). Comparing algal phenolics only, then the slopes differ by a maximum of 24%. Thus different phenolic compounds react similarly in the Folin-Denis assay.

TABLE IV

Equations for regression lines of relationship between phenolic concentration and absorbance at 725 nm in Folin-Denis assay. Equations are of the form, Absorbance = m (pg. ml - ’ of phenolics) + Absorbance (as y intercept). N = sample sizes. Range of concentrations spanned, O-15 pg. ml- ‘. All slopes are signiti-

cantly different from 0 at P < 0.001 (t test). Calculated r* values all > 0.99.

Type or source of phenolic compound

Regression N

Tannic acid Phloroglucinol E. arborea H. siliquosa F. vesiculosus

y = 0.081x + 0.019 8 y = 0.077x + 0.012 14 y = 0.071x + 0.065 6 y = 0.068x + 0.059 6 y = 0.059x + 0.048 5

231 P. D. STEINBERG

Small differences in reactivity to the assay among the different algal compounds are roughly related to the size of the molecules, with the monomer phloroglucinol and the smaller polymers from E. arborea more reactive (i.e., have higher slopes) than the larger polymers from H. siiiquosa and F. vesiculosus. This is to be expected if the compounds contain ether bonds between phloroglucinol units [as do polyphenolics from all three species (Glombitza et al., 1980; Glombitza & Gerstberger, 1985; Ragan & Glombitza, 1986)], as these bonds reduce the number of hydroxyl groups available to react to the assay reagents. Additional data on the reactivity of algal polyphenolics to the Folin-Denis assay are found in Ragan & Glombitza (1986, Table XIV).

DISCUSSION

The levels of phenolics or polyphenolics produced by different brown algae (or tissues within a single alga) are inversely correlated with these algae’s palatability to a wide variety of invertebrate herbivores (Anderson & Velimirov, 1982; Steinberg, 1984, 1985; Johnson & Mann, 1986). In this paper, I experimentally demonstrate that brown algal polyphenolics, and the terrestrial polyphenolic polygallic (tannic) acid, all consistently deter feeding by three species of marine herbivores. These results support the conclusions of the above authors regarding the deterrent effects of algal polyphenolics, and in particular strengthen the conclusions of Steinberg (1985) who argued that brown algal phenolics were generally effective deterrents against marine invertebrate herbivores in the northeastern Pacific Ocean. The results in this paper also parallel those of Geiselman & McConnell (198 l), who showed that polyphenolics from the brown algae A. nodosum and F. vesiculosus deterred feeding by the herbivorous gastropod L. littorea.

An important result of the work described here is that the different polyphenolics (excluding phloroglucinol) caused similar levels of feeding deterrence. With the exception of several experiments with tannic acid and S. purpuratus, concentrations of 5 mg . ml - ’ of polyphenolics in the agar disks resulted in feeding deterrence of 45-69% (relative to 0 mg * ml - ’ disks). This consistency in response is seen most strongly for the two species of Tegula; responses of the echinoid S. purpuratus were more varied.

The similarity in the deterrent effects of these compounds is somewhat surprising since different species of herbivores were used, there are substantial structural differences among the compounds (Table I), and different experimental designs were used. These results suggest that the herbivores, especially the gastropods, are responding mainly to some general property of polyphenolics, rather than to specific characteristics of different polyphenolics [as was once proposed for the effects of terrestrial plant tannins on insect herbivores (Feeny, 1976)]. The response of the herbivores in this study to polyphenolics differs from that of some marine herbivores to other secondary metabolites, where small differences in structure can elicit very large differences in the response of the herbivores (Targett & McConnell, 1982). These results also differ somewhat from those of Geiselman & McConnell (198 l), who found that different sizes (molecular weights) of polyphenolics difIered in their effects on feeding rates of L. littoretr.


Although most of the effects of polyphenolics appear to be due to quantitative (concentration) effects, qualitative differences among the polyphenolics may be important to a lesser degree. Polyphenolics from E. arborea, and tannic acid, generally exhibited greater deterrence in no-choice experiments against all three herbivores than did polyphenolics from F. vesiculosus. This may be a function of the size of the molecules. Both Eisenia polyphenolics and tannic acid are relatively small polymers compared to the compounds from Fucus. The size of various tannins from terrestrial plants has been suggested to be related to their deterrent effects against natural enemies

(Zucker, 1983). The principal effect of qualitative differences among the compounds was the lack of

deterrence exhibited by the monomer phloroglucinol against T. funebrulis, although this compound did deter feeding by S. purpurati,s. Phloroglucinol also deterred feeding by the gastropod L. littorea (Geisehnan & McConnell, 1981). However, since Geiselman & McConnell (198 1) make no mention of the possibility of phloroglucinol leaching out of their agar disks, their conclusions must be viewed with caution. Phloroglucinol does differ from polyphenolics in that, unlike the polyphenolics, it does not bind to proteins or agar. But these differences do not easily explain why this compound would deter some herbivores but not others.

Another difference among the responses of the three species of herbivores was the greater variability in the responses of S. purpurcztus. Variation within treatments of an experiment was usually high for the urchins, as was variation among repeated choice experiments with tannic acid (Fig. 4). These results suggest that, compared to gastropods, echinoids are better able to tolerate high levels of phenolics in their food if more palatable plants are rare or unavailable.

The levels of feeding deterrence resulting from the various concentrations of phenolics in this study are consistent with previous work correlating consumption of algae and their phenolic levels (Steinberg, 1985). In the present study, 5 mg * ml - * of polyphenolics generally resulted in 50-60x deterrence of feeding by T.funebrulis. This corresponds to about half the concentration of phenolics found in species of algae that were unpalatable to T. funebralis in the study by Steinberg (1985), indicating that phenolics can account for much of the differences in consumption rates of palatable vs. nonpalatable algae exhibited by T. funebralis.

This conclusion is tempered somewhat by the difficulty in directly equating concentrations of phenolics in agar disks with concentrations in algae. Within a.lgae, phenolics are encapsulated in vesicles termed physodes (Ragan, 1976) which are part of a very heterogeneous structure (the thallus). In agar the compounds are embedded in a homo- geneous matrix. The accessibility (to an herbivore) of polyphenolics in agar disks and in algae are certainly different, with the effective concentration (e.g., what an herbivore senses when feeding) in the agar disks probably much higher.

Although qualitatively similar, my results differ quantitatively from previous research on the effects of algal phenolics on the gastropod L. Zittorea (Geiselman & McConnell, 1981). Although phenolics consistently deterred feeding by L. littorea, the concentra-

233 P. D. STEINBERG

tions at which deterrence was detected was generally much lower than in this study. Many of the fractions investigated in their study exhibited deterrence at concentrations in agar of 0.01 y0 fresh weight (fi: 0.1 mg . ml - I), and all fractions were highly deterrent at concentrations of O. l-0.2% fresh weight. Tannic acid significantly inhibited feeding at concentrations as low as 0.001% (fresh weight). Moreover, qualitatively different compounds (either different molecular weight fractions or different solvent partitions) varied significantly in their deterrent effects, although these differences primarily occurred at concentrations below those found in phenolic-rich plants.

Other secondary metabolites, particularly terpenes, may be more highly deterrent than the polyphenolics studied in this paper. Targett et al. (1986) showed that

1 mg * ml - ’ of < 4.9 diacetoxyudoteal and 1.7 mg . ml ~ ’ of caulerpenyne both signiti- cantly inhibited feeding by parrotishes. Other terpenoid metabolites from tropical green algae are toxic to, and/or highly deterrent to juvenile conchs and damselfishes at < 1 .O 7; dry weight. (Paul & Fenical, 1986). Several terpenoids from tropical algae deter invertebrate and vertebrate herbivores at concentrations of < 1.5% dry weight (Hay et al., 1987a,b). Geranylacetone, a terpene extracted from the Australian fucoid alga Cystophora monilijknis (Van Altena, 1988), reduces feeding by the echinoid Trtpneustes gratilla and the gastropod Turbo (Subninella) undulata by > 60% at a concentration in agar discs of 2.0 mg* ml- ’ (Steinberg & Van Altena, in prep.). Terpenes in terrestrial systems are often more deterrent than polyphenolics at equal concentrations (Feeny, 1976; Rhoades & Cates, 1976).

My results also shed light on the common practice of correlating the results of calorimetric assays of phenolic levels in algae with their palatability to herbivores (Anderson & Velimirov, 1982; Steinberg, 1984, 1985; Johnson & Mann, 1986; and numerous terrestrial studies, for examples, see review by Swain, 1977; also Zucker, 1982; Tahvanainen et al., 1985; Coley, 1986). The reactivity of structurally different compounds to the Folin-Denis assay in this study were very similar (differing by 28 y0 or less). In a similar comparison, Ragan & Glombitza (1986, Table 14) found that for polyphenolics with known structures (and phloroglucinol), reactivity of different compounds in the Folin-Denis test differed by less than a factor of two for 12 of 13 compounds. Thus in most instances this assay is not strongly influenced by the presence of different sorts of polyphloroglucinols in different algae. Moreover, all the compounds used in this study, with the exception of phloroglucinol, had similar deterrent effects against the herbivores (especially the gastropods). These results taken together suggest that for the herbivores in this study, and perhaps more generally for invertebrate herbivores in the northeastern Pacific Ocean (Steinberg, 1985), the Folin-Denis procedure (and perhaps other calorimetric assays) really does estimate a biologically meaningful phenomenon, e.g., levels of deterrent compounds. Algae which differ in levels of phenolics as measured by the Fohn-Denis technique probably do differ in the amounts of phenolic deterrents they contain. High levels of phloroglucinol would complicate this interpretation, since it is both the most reactive compound in the Folin-Denis assay and varies in its deterrent effects against different herbivores.

ALGALPHENOLICFEEDINGDETERRENTS 235

However, high concentrations (> 0.1% dry weight), of free phloroglucinol appear to be rare in brown algae (Ragan & Glombitza, 1986).

ACKNOWLEDGEMENTS

Part of this work is from my Ph.D. thesis done at the University of California, Santa Cruz. M. A. Ragan and Professor K. W. Glombitza generously supplied algal poly- penolics. I thank my advisors, D. C. Potts, and L. R. Fox, for their help and guidance. I am very grateful to the Institute of Marine Studies at Santa Cmz, and its director, B. Doyle, for support. I also thank F. Winter, N. Andrew, R. Evans, J. Hansen, J. Jeffries, and C. Johnson. During the writing of this paper, I was supported by a Queen Elizabeth II Research Fellowship from the National Research Council of Australia, and the School of Biological Sciences, University of Sydney.

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effects of quantitative and qualitative variation in ... · and the echinoid ~tron~locentr~t~s...

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