Chemical ecology of red algal bromophenols. I. Temporal, interpopulational and within-thallus measurements of lanosol levels in Rhodomela larix (Turner) C. Agardh

J. Exp. Mar. Biol. Ecol., 1982, Vol. 58,

pp. 285-293

285

Elsevier Biomedical Press

CHEMICAL ECOLOGY OF RED ALGAL BROMOPHENOLS. I. TEMPORAL, INTERPOPULATIONAL MEASUREMENTS R~~~~~ELA

AND WITHIN-THALLUS

OF LANOSOL LEVELS IN LARIX

(Turner) C. Agardh

D. W. PHILLIPS and G. H. N. TOWERS’ Department of Botany, University of British Columbia, Vancouver, B.C., Canada

Ahstreet: High-performance liquid chromatography was used for the separation and quantitative determination of 2,3-dibromo-4,5-dihydroxybenzyl alcohol (lanosol) from Rhodomeia larix (Turner) C. Agardh. Temporal variation in lanosol content ranged from 1.2 to 3.87: on a dry-weight basis. Populational comparisons showed slight differences in lanosol concentrations among algae from separate low-intertidal sites and considerably higher levels of the compound in tide-pool forms of this species. The highest levels in a single plant were found in the youngest regions (growing tips) of the thallus and the lowest levels in the holdfast. Seasonal stress is considered responsible for the changing lanosol levels, implying no correlation of changes with reproductive state. The ecological impli~tions of bromophenol production by red algae are introduced and their potential uses as antibiotic, antiepiphyte. or antiherbivore agents are discussed.

The recent increased interest in marine natural products chemistry has led to the discovery of a number of unusual carbon-halogen compounds from marine algae. Red algae (Rhodophyta) contain a diverse array of these compounds representing at least four main chemical classes: low molecular weight alkenes, phenols, terpenoids, and fatty acids. The halophenois and in particular the bromophenols have been extensively investigated in a large number of red algal species from a variety of locations (Fenical, 1975). Fenical (1975) has suggested that red algal halometabolites, rather than being involved in primary metabolic pathways, function in an exocrine system providing the alga containing them with a selective environmental advantage. The nature of this advantage, if it exists, is unclear; however, excretion of dissolved organic matter (DOM) by algae is well documented (Hellebust, 1974; Wangersky, 1978; Ragan & Jensen, 1979). Such materials may be only waste products of metabolism, but they also may be actively produced as antibiotic substances (Langlois, 1975; Ragan & Jensen, 1978). The study of variations in the temporal abundance of bromophenols in red algae ’ Author to whom correspondence

should be addressed.

OO22-0981/82/0000-0000/$02.75 8 1982 Elsevier Biomedical Press

%drywt

P. morrowii P. urceolata

P. lanosa

h

;

m

I

1.0 0.07 0.08 0.005 0.02 0.03 0.02-0.06* (0.003) 0.003

0.5, 0.005 0.8 0.006 0.005 l-5, 2-3

0.1 (0.024)

h h i j e f g k k

Polysiphonia brodiaei

0%2.0*

F

0. dentata

(0.003) (0.002) (0.003) (0.003) (0.003) (0.01) (0.004) (0.00s) (2.0)

(% wet wt)

d e! f

C

a b a b

Compounds

Odo~thal~ corymbifera

H. pinastroides

Hatopytis incurvus

Alga

Craigie 62 Gruenig, 1967 Glombitza & Stoffelen, 1972 Craigie & Gruenig, 1967

Hodgkin et al., 1966; Ragan & Craigie, 1978 Glombitza & Stoffelen, 1972 Saito & Ando, 1955 Knrata et al., 1976

Craigie & Gruenig, 1967 Craigie & Gruenig, 1967; Glombitza & Stoffelen, 1972 Pedersen et al.. 1974; Lundgren et al.. 1979; Lundgren et al., 1979

Kurata ef al., 1973

Combaut et al., 1978

Chantraine et al., 1973

Reference

summary of data avaiiable on quantitative aspects of bromophenols in red algae: all estimates are based on the abundance of the naturally occurring phenol, not on derivatives which may have been prepared in isolation procedures; the compounds listed are as follows: a, 3,5-dibromo-4-hydroxyphenylacetic acid; b, 3,5~dibromo-4-hydrox~henylpyruvic acid; c, perdesmethylcyclotribromoveratrylene; d, 2,3-dibromo-4,5-dihydroxybenzylmethyl ether; e, 2,3,2’,3’-tetrabromo-4,5,4’,5’-tetrahydroxydiphenylmethane; f, 2,3-dibromo-4,5-dihydroxybenzyl alcohol; g, 2,3-dibromo-5-hydroxybenzyl-1’,4disulfate (potassium); h, 3,5-dibromo-4-hydroxybenzyl alcohol; i, 2,3-dibromo-4,5_dihydroxybenzaldehyde; j, 2,3-dibromo-4,5-dihydroxybenzyl ethyl ether; k, 3-bromo_4,5dihydroxybenzaldehyde; 1, 3,3’-dibromo-4,4’,5,5’-tetrahydroxybibenzyl; m, 3-bromo-4,5dihydroxybenzyl methyl ether; n, 3,5dibromo-4hydroxybenzyl methyl ether; o, 2,4-dibromo-1,3,5trihydroxybenzene; p, 5,6,3’,5’-tetrabromo-3,4,2’,4’,6’-pentahydroxydiphenylmethane; q, bis(2,3,6-tribromo-4,5-dihydroxybenzyl) ether; *, estimated abundance; !, tentative identification.

A

TABLE I

QUANTIFICATION

OF LANOSOL IN RHODOMELA

LARIX

287

288

D.W.PHILLIPSAND G.H.N.TOWERS

has received little attention. Our present knowledge consists only of quantitative data derived from algal extractions which, unless very carefully done, must invariably lead to underestimation of compounds in the plants. Table I summarizes the available literature in this area and illustrates the great variability in the data even for multiple extracts of a single species. As a result, the physiological and ecological significance of bromophenols in red algae is unknown. Bromophenols have been shown to be effective antibiotics (Silva & Bittner, 1979) but no information is available on quantitative aspects of their occurrence, which might be helpful in assessing their importance to the plant. Ragan & Jensen (1978) have shown that a knowledge of seasonal variation in concentrations of polyphenols in brown algae can provide useful information regarding their significance. For this reason we chose to examine the red alga Rhodomelu lurix (Turner) C. Agardh for changes in the levels of 2,3-dibromo-4,5dihydroxybenzyl alcohol (lanosol), the major bromophenol (when considered as the free phenol) in this species (Weinstein et al., 1975), over a I-yr period. Also examined were differences in bromophenol content among three distinct populations and within a single plant. R. Zurix is a common alga in British Columbia and is known to contain high levels of some bromophenols (Table I). An extensive quantitative chemical examination of this alga should lead to a clearer understanding of the raison d’etre of bromophenols in red algae.

MATERIAL AND METHODS COLLECTIONOFALGALSAMPLES

Unless otherwise indicated, all algal samples were collected in the lower intertidal to upper subtidal zone off Bath Island, British Columbia, Canada, during the highest tide of the month. The plants for each sample were randomly selected over a large area, cleaned of epiphytes and returned to the laboratory in plastic bags over ice. Algae were then used immediately or deep frozen ( -20 “C). Other samples came either from tidepools on Bath Island or from the low intertidal zone near Barnfield, British Columbia. EXTRACTIONPROCEDURES

All extractions were done in triplicate; dry weights were taken in duplicate, the samples having been dried for 1 wk at 90 “C. One g of each algal sample was ground in a Virtis microhomogenizer in boiling 80% methanol. Following one hour’s reflux on a steam bath, particulate matter was filtered off and the methanol removed in vacua. The residual water layer was acidified to pH 2 with 1.0 N HCl, heated to 60 “C for 15 min (to hydrolyze ester sulphates) and then continuously extracted with ethyl acetate (4 h). The ethyl acetate was removed and the residue taken up in 5 ml methanol for subsequent chromatographic analysis.

QUANTIFICATION

OF LANOSOL

IN RHODOMELA

LARIX

289

Collections from the other two locations (see above) were treated in the same way for relative comparison of phenol content among populations. For measurement of lanosol concentration in different parts of the plant, 1 g each of growing tips, branches with many laterals, branches with few laterals and holdfasts were excised and extracted as before. CHROMATOGRAP~C

METHODS

Quantitative determination of lanosol was performed using a Varian Model 5000 Liquid Chromatography (HPLC) linked to a Variscan 634-S Spectrophotometer set at 292 nm, the maximum absorption of lanosol (Weinstein ef af., 1975). The chromatograph was fitted with a 10 yl calibrated loop. Injections of this volume were always used to minimize potential error in this part of the quantification procedure. A column of Micropak MCH-10 (Varian) was used with mixture of acetonitrile (Fisher Scientific Co.) and water (4 : 6) containing 10 mM each of NaH,PO, buffer (pH 3.2) and tetramethylammonium chloride (Aldrich Chemical Co.) as eluant. The flow rate was 1 ml/min. Lanosol was identified by comparison with an authentic standard kindly provided by Dr. J. S. Craigie, Atlantic Regional Laboratory, Halifax, N.S. Calibration curves for lanosol in methanol were made by measuring peak areas by the width at half height method and converting to mg/ml of lanosol and subsequently to mg/g dry weight as quantities in the alga. The life history data for algae in these samples were provided by Ulla Visscher (pers. comm.). RESULTS

AND

DISCUSSION

HPLC is an effective and rapid method for the separation, identification and quantification of red algal phenols (Phillips & Towers, 1981). Fig. 1 shows a typical HPLC separation of the bromophenols and other UV absorbing materials from R. larix. Lanosol was chosen as an indicator of the level of total bromophenols in the plant. The peak corresponding to lanosol is well separated and can easily and accurately be quantified by this method. The yearly range of lanosol levels in R. lark is 1.2 to 3.8% on a dry-weight basis (Fig. 2). The levels in winter are essentially three times those of the summer months. Ragan & Jensen (1978) found similar maxima and minima in the brown algae which they examined for polyphenols. The results of the interpopulational comparisons are shown in Table II. The wide range of lanosol levels among the three populations examined here is typical of the variation observed for this species (Table I). The tidepool form had higher lanosol levels in the summer months than did either the subtidal Bath Island or Bamfield populations. The latter two forms are morphologically similar (large

1

T

0.02

AU

_L

1

c+JfJI

I, 2

I

0

, I I 6 4 TI?IE (min)

IIll 8

10

Fig. 1. A typical HPLC separation of Rhodomela lark bromophenols and other UV absorbing materials used for the quantitative determination of lanosol (indicated by the arrow): the chromatographic conditions are listed in the text.

+-0

50 -

-r----veg-

~“~g-------t--$

40 -

30 -

20 -

10 -

O-

I

I

I

I

I

I

I

I

I

I

I

I

J

F

M

A

El

J

J

A

S

0

N

D

TIME

(months)

Fig. 2. Seasonal variation of lanosol content in Rhodomela lark (-) and changes in the dry to wet weight ratio over the period of 1 yr (- - - - -) : the deviation from the mean for all samples is indicated by vertical bars and the reproductive state of the plants by horizontal bars; @, tetrasporic plants; 9, carposporic plants; veg, vegetative plants. TABLE II

Quantitative comparison of lanosol concentrations in three distinct populations (collected in August 1978).

of Rhodomela lark

Population

Lanosol (mgig dry wt)

Reproductive condition

Bamtield (low intertidal) Bath Is. (low inter- to high subtidal) Tidepool (Bath Is.)

11.8 kO.30 13.9 f 0.17 21.7 f 0.55

Tetrasporic Vegetative and tetrasporic Vegetative

QUANTIFICATION

OF LANOSOL IN RHODOMELA

LARIX

291

plants with terete branching) but quite distinct when compared to the tidepool form (small thin plants with irregular branching patterns); however, all three are still considered to be the same species (Phillips & Towers, in press). Ragan & Jensen (1978) have pointed out that the higher levels of polyphenols in brown algae do not occur during the period of maximum potential epiphytization (spring and summer) but during the fall and winter months when colonization by the smaller marine algae is at a minimum. For Rho~melu the same pattern was observed. Both tidepool and subtidal plants are typically epiphytized in the early spring increasing to a maximum epiphyte cover by early to midsummer. Thereafter the diversity of Rhodomela epiflora decreases until in winter none is observed. Data for intrathallial variation in lanosol content (Fig. 3) show maximal levels LANOSOL bSlg dry wt.)

41.6

21.0

REGION OF LATERALS

9.0

HOLDFAST

Fig. 3. Lanosol content in different parts of the Rhodomelu Zurix thallus (collected from tidepools on Bath Is., B.C. in September, 1979).

of the compound in the youngest and most rapidly growing portion of the algal thallus. Rhodomelu is almost invariably epiphytized in the older regions of the thallus as are many other species of marine algae (Ballantine, 1979). This could be a result of the lower concentration of lanosol in these portions, as contrasted to the growing tips. Leaching out or exudation of the compounds during a period of less active growth (summer) would lead to lower levels of the compounds for the entire plant. This could explain the rapid drop in lanosol content as summer proceeds. Higher levels of insolation and higher water temperatures, or a combination of factors might affect the levels or rate of change in the levels of lanosol in the plants. Further study correlating the period of most active growth with changes in lanosol levels should provide a better understanding of the role of lanosol in epiphyte control. Lanosol has been shown to be an effective antibacterial and antifungal agent (Silva & Bittner, 1979). Again, as with brown algal phenols (Ragan & Jensen, 1978),

D. W. PHILLIPS

292

AND

G. H. N. TOWERS

the ecological signifmnce of bromophenols to red algae may be in the control of pathogens rather than epiphytes. The control of herbivory by these compounds is also not excluded. The authors have shown (Phillips, 1980) that lanosol and, at much higher concentrations, its dipotassium sulphate salt are effective as repellants against tidepool snails. Similarly, Geiselman (1980) has demonstrated strong antiherbivore activity for halometabolites isolated from North Atlantic red algae. This effect could extend to predators other than snails as well. The possibility that these compounds are waste products of me~bolism (Peders&n et al., 1979) seems highly unlikely. Biochemical evolutionary trends do not generally lead to the formation of by-products more complex and potentially more toxic than the starting materials from which they are synthesized (Swain, 1977). Neither should bromophenols be considered storage products since their minimum accumulations occur during the period when environmental conditions would favor increased photosynthesis and, subsequently, increased food storage. The scope of possible functions for bromophenols is thus considerably narrowed. A more thorough assessment of the effectiveness of lanosol as an antibiotic, antiepiphyte, or antiherbivore agent is required before the significance of bromophenols in the ecology of red algae is understood.

ACKNOWLEDGEMENTS

We wish to thank Dr. R. E. DeWreede for assistance during the many collecting trips, Dr. J. S. Craigie for supplying the lanosol standard and the National Science and Engineering Research Council of Canada for financial support.

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on macrophytc

hosts offshore

from

Vol. 22, pp. 107-l 11.

J.-M., G. COMBAUT & J. TESTL 1973. Brominated phenols of a red alga, Haiopyti.r incurws: carboxylic acids. Phyroch~misrr~, Vol. 12. pp. 1793.-1796. CH~VOL~T-MAC~~EUR. A.-M., A. CAVF, P. POTIER.J. Tbsrl . A. CIIIARONI & C. Rlc~t. 1976. Composes bromes de RyIiphiea rincrorio (Rhodophyceae). Phy~ochemisrr.r. Vol. IS, pp. 767 -771. CIIAYI‘RAINE.

COMBAUT.

G., J.-M.

CIIANTRAINE,

J. TESTY & K.-W.

GI.OMBITZ.A, 1978.. Phenols bromes des algues

nouveau derive ohtenu au tours de I‘extraction de Hnlopyric rouges: cyclotribromoveratrylene. pinustroides. Phytochemisrry. Vol. 17. pp. 1791.. 1792. CKAIWE. J. S. & D. E. GRUENIG. 1967. Bromophenols from red algae. &fence. Vol. 157. pp. IO%- 1059. F~.SIC‘AL,

W..

1975. Halogenation

in the Rhodophyta:

a review. J. Phycol.. Vol.

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GtisELMAN. J. A., 1980. Ecology of chemical defenses of algae against the herbivorous snail, Lirrorinu litrorea, in the New England rocky intertidal community. Ph.D. thesis. ‘Massachusetts Institute of

(;I

Technology/Woods Hole Oceanographic Institution, 209 pp., OMHITZA. K.-W. & H. STOFFELI‘X. 1972. 2.3-Dibromo-5-hydroxybenzyl-1,4-disulfat

(Dikaliumsalz)

aus Rhodomelaceen. Plunru Med.. Vol. 22, pp. 391-395. H~LLEBUST. J. A.. 1974. Extracellular products. In. AlRalphy.sio/o,q>,md hiorhrmrsfr~~. edited by W. D. P. Stewart.

Univ.

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Press, Berkeley.

pp. 838-863.

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LARIX

293

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