The effects of macrobenthic deposit-feeding on the degradation of chloropigments in sandy sediments

J. Exp. Mar. Biol. Ecol., 1988, Vol. 122, pp. 243-255 Elsevier

243

JEM 01150

The effects of macrobenthic deposit-feeding on the degradation of chloropigments in sandy sediments Thomas S. Bianchi’, Rodger Dawson’ and Pichan Sawangwong2 ‘Chesapeake 3~~o~.cai Laboratory, University of Mawland, ~olorno~, Maryfund. U.S.A. ; 2~epa~me~t of Aquatic Sciences, Faculty ofSciences, ~~a~a~nwirot University, Bangsaen, Chonb~~, Thailand

Received 24 March 1988; revision received 11July 1988; accepted 23 July 1988 Abstract:

Microcosms with sediments containing either the bivalve Macoma balthica (surface depositfeeder), the polychaete ~ejtosco~o~Zos~a~~ (subs~Face deposit-feeder), or both animals were m~nt~ned in a flow-through seawater system. Two different plant-derived food sources (Ulva rotundata or Z&era marina) were added (weekly) to the microcosms. The conversion of chlorophyll a to phaeophorbide a was significantly higher in microcosms containing macrofauna than in the controls with no macrofauna. Microbenthos in the controls were only capable of converting chlorophylls a and b to phaeoph~ins. The highest production of phaeophorbide a was in isolated deposit-feeder treatments with U. rotxndata as the food source. Even though the total macrofaunal biomass was greater in the treatments with the two depositfeeders together, more phaeophorbide was produced in the isolated treatments. Because M. balthica has a different feeding mode than L. jiagiks, feeding interference between surface and subsurface deposit-feeding activities may have occurred in the combined treatments, thus producing less phaeophorbides. Phaeophorbides may represent good feeding markers for macrobenthic deposit-feeding processes. The amount of ingested chlorophyll that is converted to phaeopigments is dependent upon the quality of plant source materials and the interactions between coexisting species. Key words: Chloropi~ent;

Deposit-feeder; High-perfo~ance

liquid chromato~aphy

INTRODUCTION

Although previous studies have described the distribution and composition of plant pigments in marine and lacustrine surface sediments (Gorham, 1959, 1960; Daley, f973; Daley & Brown, 1973; Watts &al., 1977; Repeta & Gagosian, 1982, 1987), mech~ism of their degradative tr~sformation in sediments are poorly understood. The degradation products of chlorophylls and carotenoids, resulting from benthic heterotrophy, represent the predominant form of pigments in sediments (Daley, 1973; Repeta & Gagosian, 1987). Benthic and water column metabolic processes are irn~o~~t in controlling the concentrations of pigment degradation products in sediments (Daley, 1973; Repeta& Gagosian, 1987). However, studies examining pigment transformations have emphasized the importance of macrozoopl~kton grazing activities, and have largely ignored the macrobenthos. The activities of macrobenthos, particularly depositCorrespondence address: T. S. Bianchi, Institute of Ecosystem Studies, New York Botanical Garden, Box AB, Millbrook, NY 12545, U.S.A. 0022-0981~88~$03,500 1988 Elsevier Science Publishers B.V. (Biomedical Division)

T. S. BIANCHI

244

ETAL.

feeders, can substantially alter the chemical properties of sediments (Darwin, 1881; Dapples, 1942; Rhoads, 1974; Schink & Guinasso, 1977; Aller, 1978, 1982; Aller & Yingst, 1985; Rice et al., 1981; Rice, 1986). Moreover, the feeding activities of macrobenthic deposit-feeders can drastically alter the abundance, metabolic activity and composition of the microbial community (Hargrave, 1970; Yingst & Rhoads, 1980; Morrison & White, 1980; Bianchi & Levinton, 198 1; Levinton & Bianchi, 198 1; Bianchi 1988), which ultimately affects the biogeochemistry of the sediment.

BREAKDOWN

OF CHLOROPHYLLS

IN PELAGIC

AND

BENTHIC

SYSTEMS

Herbivorous grazing activity si~i~c~tly affects the fo~ation of chlorophyll degradation products (phaeopi~ents) in pelagic systems. The 3 dominant phaeopigments found in marine and freshwater systems are phaeophytin, phaeophorbide, and chlorophyllide. These tetrapyrrole derivatives are formed during bacterial, viral, or autolytic cell lysis, and by grazing activities. Early work demonstrated that copepod fecal pellets contain high concentrations of chlorophyll degradation products (Cut-tie, 1962). In fact, the intensity of grazing activity in the water column can be estimated from phaeopigment concentrations (Welschmeyer & Lorenzen, 1985). For example, chlorophyll is degraded to phaeophytin (Mg atom removed from tetrapyrrole) as a result of grazing by salps (Hallegraeff, 1981) and waterfleas (Daley, 1973). Phaeophorbide formation has been observed (Mg atom and the phytol chain removed) after grazing by copepods (Shum~ & Lorenzen, 1975; Bathmann & Liebezeit, 1986). On the other hand, elevated concentrations of chlorophyllide (phytol chain removed) in the water column are usually found in the presence of chlorophyllase activity which is associated with cell senescence (Jeffrey, 1974) but may also be an artefact of sample manipulation causing enzyme induction (Jeffrey & Hallegraeff, 1987). To date, studies that have investigated the effects of macrobenthic grazing activity on phaeopigment formation have focused on filter-feeders (Ansell, 1974; Mann, 1977; Babinchak & Ukeles, 1979; Gelder & Robinson, 1980; Hawkins et ai., 1986). However, in a recent study, the concentration of phaeopigments in sandy sediments was used to determine the effects of grazing by a deposit-flog polychaete on benthic diatom production (Bianchi, 1987; Bianchi & Rice, 1988). The variety of potential sedimentary food resources for deposit-feeders (bacteria, microalgae, protozoa, fungi, meiofauna, and non-living organic matter) (Lopez & Levinton, 1987), makes it particularly di.Bicult to determine the source pigment materials. Moreover, in a situation where depositfeeders are ingesting sediments consisting primarily of non-living plant material (i.e., diatom and vascular plant detritus), it is important to distinguish between phaeopigments formed by remineralization and gut passage. To investigate the importance of chlorophyll digestion by macrobenthic deposit-feeders on pigment chemistry in surface sediments, it is necessary to use analyses that can accurately quantify the individual phaeopigments. In most of the studies cited above, ~hlorophyIls and phaeopi~ents were determined

DEPOSIT-FEEDING

AND DEGRADATION

OF CHLOROPIGMENTS

245

by spectrophotometric and spectrofluorometric analyses. However, mixed samples containing chlorophylls and their derivatives cannot be distinguished with these methods because their absorbance and fluorescence bands overlap (Yentsch & Menzel, 1963; Holm-Hansen et al., 1965; Lorenzen, 1967). In recent years, more advanced techniques using high-performance liquid chromatography (HPLC) have allowed for separation and quantification of chlorophylls and phaeopigments (Brown et al., 1980; Gieskes & Kraay, 1983a,b; Mantoura & Llewellyn, 1983; Wright & Shearer, 1984; Klein et al., 1986; Daemen, 1986). In this study we investigate the effects of 2 deposit-feeders on the degradation of chlorophylls from 2 sources of plant detritus. We have chosen 2 deposit-feeders that have distinctly different feeding modes, Leitoscoloplosfrugilis (Orbiniidae : Polychaeta), a subsurface deposit-feeder, and the surface deposit-feeder Mucoma bulthicu (Tellinidae : Bivalvia). Using laboratory microcosms we will examine how the feeding activities of these 2 deposit-feeders affect the transformation of chlorophylls from 2 plant sources, the green macroalga Ulvu rotundutu and the marine angiosperm Zosteru marina. This is the first study of its kind, that uses HPLC, to investigate the effects of macrobenthos on the degradation of plant pigments in marine sediments. Our longrange objective is to be able to use pigment markers, that are identified using laboratory microcosm experiments, to understand how different benthic metabolic processes and source plant materials affect the chemistry of pigment transformations in natural sediments.

MATERIALS EXPERIMENTAL

ANIMALS,

The experimental

animals,

SEDIMENTS,

AND

METHODS

AND FOOD RESOURCES

L. frugilis and M. bulthicu were collected from the intertidal sediments of Solomons Island, Maryland, using a 500 pm sieve. Solomons Island is located at the mouth of the Patuxent River and Mid-Chesapeake Bay. Sediment was collected from the field, wet-sieved (< 1 mm), dried at 60 ‘C and allowed to stand at room temperature before being used in the laboratory microcosms. This experimental sediment was also subsampled for determination of pigment composition (blank) and then subtracted from the final pigment concentrations. The 2 plant sources added to the experimental microcosms were each collected from different field locations, Ulvu rotundutu was collected from the Chesapeake Bay, Maryland and Z. marina from Great South Bay, New York. After both plant materials were frozen and lyophilized, each was ground to pass through a 125 pm mesh sieve and stored frozen. Before using these detrital sources in the microcosms, subsamples were taken of each analysis of molar pigment composition and organic carbon and nitrogen content.

T. S. BIANCHI ETAL.

246 LABORATORY

Sixteen

MICROCOSMS

cylindrical

polypropylene

microcosms

(8 cm diameter

x 6 cm depth)

were

Then, 2 M. balthica (representing filled with 5 cm of experimental sediment. 400 clams * m - ‘) or 8 L. frugilis (representing 1600 worms * m- “) were added to each of two replicate microaquaria. These experimental densities are within the range of field densities

at Solomons.

In a third treatment,

2 A4. balthica and 8 L. frugilis were added

together to each of 2 replicate microaquaria. Two replicate control microaquaria (having no animals) were also included. To each ofthese 8 microaquaria, 100 mg of dried ground U. rotundutu (< 125 pm) were added weekly for 8 weeks. Similarly, to another 8 microaquaria having the same replicate treatments described above we added 100 mg of Z. marina. All 16 microaquaria were submerged and randomly arranged in a flowthrough seawater system with ambient water from the area where the animals were collected. The incoming seawater was filtered (2 ,um) before entering the aquarium to preclude any contamination from planktonic pigment sources. All microcosms were kept in the dark throughout the entire 8 weeks to minimize photo-oxidative degradation of pigments. This experiment ran from the end of October through mid December 1987 for 60 days where the ambient water temperature and salinity ranged from 17-13 ‘C and 12-16%. At the end of the experiment 2 syringe mini-cores (5.0 cm deep, 2.0 cm inner diameter) were taken from each microaquarium and sectioned at 1 cm depth intervals for determination of chloropigments. Any animals found in each of the sectioned intervals were removed prior to pigment analysis. The reported pigment concentrations represent the sum of pigments in the depth interval from O-5 cm. Pigments were extracted from sediments using 100% acetone. Jeffrey & Hallegraeff (1987) recommend that 100% acetone be used for extraction to reduce the artifactial production of chlorophyllide. Pigment extracts were then ultrasonicated for 5 min and allowed to stand overnight in the dark at 4 “C prior to centrifugation at 3000 rpm for 3 min in 15 ml polypropylene centrifuge tubes. The tubes were capped and stored deep frozen 2 days prior to pigment analysis. Pigments were determined by ion pairing, reversed-phase HPLC (Mantoura & Llewellyn, 1983). The equipment employed consisted of a gradient pumping system (Constametric III, LDC Milton-Roy) controlled by an MP3000 controller and dual channel acquisition system. Dual channel detection was achieved with an LDC UV monitor set to 440 nm for absorbance and a Perkin Elmer 650-105 fluorescence detector, excitation 440 nm and emission 670 nm. An A-1000 auto-injector equipped with a Rheodyne model 7126 valve was connected via a precolumn to a water radial compression system with a 8 mm x 10 cm C18, 5 pm cartridge. A typical gradient program from 100% A (80: 10: 10, methanol; water; tetrabutyl ammonium acetate) to 100% B (70:30, methanol; acetone) in 10 min with a hold for 17 min gave sufficient resolution of all pigments of interest. Standard chlorophyll and degradation products were prepared as described in Mantoura & Llewellyn (1983).

DEPOSIT-FEEDING AND DEGRADATION OF CHLOROPIGMENTS

241

RESULTS PLANT SOURCES The pigment composition of the 2 plant materials are shown in (Fig. 1). The concentrations of both chlorophyll a and b was substantially higher in U. rotund&u (Table I). The molar C: N ratio was also significantly (t-test, P < 0.05) lower in U. rotund&u. MICROCOSM PIGMENTS The concentration of chlorophyll a was significantly (one-way ANOVA,

P < 0.05) higher in the control and mixed macrofaunal treatments than in the isolated L. frugilis

8 111 !&Q

rotundata

Zostera

marjna

7

34

56

1

2

I

I

10

5

15 TIME (min)

$0

25

, 10

5

I 15

2’0

2h

TIME (min)

Fig. 1. Absorbance chromatograms of pigments in Ulvu rotundata and Zosteru marina, analyzed by ionpairing reversed-phase HPLC: 1, chlorophyllide a; 2, phaeophorbide a; 3, fucoxanthin; 4, neoxanthin; 5, violaxanthin; 6, zeaxanthin; 7, lutein; 8, lutein epoxide; 9, chlorophyll b; 10, chlorophyll b’; 11, chlorophyll a allomer; 12, chlorophyll a; 13, chlorophyll a’; 14, phaeophytin b; 15, phaeophytin b’; 16, b-carotene; 17, phaeophytin a; 18, phaeophytin a’. TABLE I

Concentration

*N=2,

+l

of chloropigments (nmol g dry plant material- ‘) and the molar organic ratio in plant materials added to the laboratory microcosms. Plant material

Chl a

Chl b

b:a

C/N

Ulva rotundata Zostera marina

73.28 33.41

20.11 5.94

0.27 0.18

17.6 f 0.5* 38.3 k 3.4*

SD.

carbon/nitrogen

248

T. S. BIANCHI

ETAL.

or M. bafthica treatments, with U. rotundata as the plant source (Fig. 2). The concentration of chlorophyll b was also significantly (one-way ANOVA, P < 0.05) higher in the control and mixed macrofaunal treatments than in the isolated treatments with U. rotundata (Fig. 3). The highest concentrations of chlorophyll a (2.66 + 0.78 nmol *g dry sed. - ‘) and b (1.20 & 0.27 nmol . g dry sed. - ‘) were in the U. rotundata controls (least significant difference (LSD) test, P < 0.05) (Figs. 2 and 3). There were also significant differences (one-way ANOVA, P < 0.05) in the amount of phaeophorbide a and phaeophytin b produced in the microcosms, for both plant sources (Figs. 2 and 3). The concentrations of other chloropigments were not significantly different among treat-

.50

_m .40 ‘d B

..\... ,.,... j9.. .,j. :.,;.:: :; j; 6 cn .30

:.:.: ,.> ... :.:.:.: ,::;:

H 2

.20

:i;i’i; ... .. .A. ..’ .. .., .:.... ... .

.,O

:_i:l.

x +l

8

~

Lt&!Ls

.. . :+:I :: .y: .... .... :.,.:.,.

COlltrOl

M. balthica

L. fraoilis

Fig. 2. Concentrations of chlorophyll a and phaeophorbide a (nmol . g dry sed. - ‘) in sediments microcosms containing different macrofaunal composition and source pigment materials,

from

Fig. 3. Concentrations of chlorophyll b and phaeophytin b (nmol ‘g dry sed. - ‘) in sediments from microcosms containing different macrofaunal composition and source pigment materials.

DEPOSIT-FEEDING

ments

for both

plant

P < 0.01) phaeophorbide in the control

or mixed

AND DEGRADATION

sources

(Table II).

a was produced

Significantly

more

(one-way

in the 2 isolated macrofaunal

macrofaunal

249

OF CHLOROPIGMENTS

treatments,

for both

plant

ANOVA,

treatments sources

than

(Fig. 2).

However, the concentration of phaeophorbide a was significantly higher (two-way ANOVA, P < 0.05). in the U. rotundata than in the Z. marina treatments (Fig. 2). Moreover, there appears to be almost no phaeophorbide a in the control treatments for both plant sources. On the other hand, the concentration of phaeophytin b shows no significant difference among the 4 treatments with Z. marina as the plant source (Fig. 3). In the U. rotundata treatments there was significantly (LSD test, P < 0.05) more phaeophytin b produced in the isolated macrofaunal treatments than in the mixed (Fig. 3). The concentration of phaeophytin b is significantly higher (two-way ANOVA, P < 0.05) in the isolated macrofaunal treatments with U. rotunduta as the plant source. Unlike the concentrations of phaeophorbide a, phaeophytin b is substantially higher in the control treatments for both plant sources. Fig. 4 shows a sample chromatogram of

c

0

,

,

4

,

,

8

,

,

,

12

,

,

18

,

,

20

,

24

Time (mid

Fig. 4. Chromatogram of chloropigments in sediments from a microcosm containing both Macoma balthica and Leitoscoloplosfragdis.

a h4. balthica treatment conversion

of chlorophyll

with U. rotundata as the plant a (with the allomer included)

source where there is a high to phaeophorbide

a.

The phaeophorbide a : chlorophyll a ratio is significantly higher (one-way ANOVA, P < 0.05) in the isolated treatments of macrofauna than in the control or mixed macrofaunal treatments for both plant sources (Fig. 5). Moreover, the ratio is significantly (LSD test, P < 0.05) higher in the isolated macrofaunal treatments with U. rotundata as the plant source. The phaeophytin b : chlorophyll b ratio is not significantly different among treatments with Z. marina as the plant source (Fig. 6). However, there are significantly higher (LSD test, P < 0.05) phaeophytin b : chlorophyll b ratios in the isolated macrofaunal treatments than in the control and the mixed macrofaunal treatments with U. rotundatu as the plant source.

of chloropigments and macrofaunal

Control M. baithica L frag& M. balthica and L. fragiiix Zostera marina Control M. balthica L fragilis M. balthica and L. fragilis

Treatment

Concentration plant material

+ k k +

+ 0.27 k 0.23 & 0.22 + 0.09

2.66 0.93 0.75 1.25

1.20 0.50 0.50 0.57

0.78 0.25 0.25 0.20

Chl a

composition.

TABLE

II

0.14 0.18 0.16 0.23

0.35 0.21 0.17 0.26 f 0.05 & 0.05 & 0.07 + 0.05

& 0.02 f 0.04 * 0.07 + 0.04

Chl b

0.09 0.11 0.14 0.08

0.07 0.05 0.04 0.05 * + f 2

k f f 2 0.01 0.02 0.10 0.03

0.02 0.02 0.01 0.02

Chl c

0.10 0.09 0.11 0.09

0.08 0.14 0.17 0.13 * f f *

* + + * 0.01 0.02 0.04 0.04

0.03 0.02 0.05 0.02

Chlorophyllide

a

0.02 0.27 0.27 0.14

0.05 1.12 0.83 0.46

0.02 0.24 0.25 0.06 & 0.01 & 0.10 f 0.09 f 0.10

* f + f

a

0.16 0.18 0.12 0.17

0.24 0.30 0.27 0.18

k f f +

k f + +

0.04 0.04 0.03 0.02

0.10 0.11 0.08 0.07

Phaeophytin

a

0.19 0.22 0.12 0.18

0.29 0.35 0.44 0.20

f f + +

f f f +

0.05 0.06 0.04 0.02

0.05 0.04 0.03 0.04

Phaeophytin

b

(O-5 cm depth) (N = 2, k 1 SD) containing different that were found in the initial experimental sediment.

Phaeophorbide

‘) in sediment collected from laboratory microcosms All concentrations have been corrected for any pigments

(nmol g dry sed.

z

;;1

z

!+ v m F

DEPOSIT-FEEDING

AND DEGRADATION

OF CHLOROPIGMENTS

251

1.60

Control

L. fragilis

M. balthiia

&

balthica -ziiTL. fragilis

Fig. 5. Phaeophorbide u/chlorophyll a ratios (nmol g dry sed. - ‘) in sediments from microcosms containing different macrofaunal composition and source pigment materials.

mlJivarotundata

q Zostera

marina

. : ., :.:: ::. ll.--lk L_i! .’ .:

._

:._ : ::. ::. (_‘. <:: .:.

:.:._ ;:_ ::. ::. ::_ ::_ ::. ::. ;:.

Control

.‘_‘. :: ::

M. balthiia

L. fraailis

@ balthica %a--L. fraoilis

Fig. 6. Phaeophytin b/chlorophyll b ratios (nmol g dry sed. _ ‘) in sediments from microcosms containing different macrofaunal composition and source pigment materials.

DISCUSSION

The high concentrations

of phaeophorbide

a observed

in the experimental

micro-

cosms appears to be directly related to the deposit-feeding activities of M. balthica and L. frugdis (Fig. 2). Phaeophorbide a represents z 40-60% of the total phaeopigments produced in any of the microcosms with macrofauna present (Table II). On the other hand, phaeophorbide a represented only 3-4% of the total phaeopigments in the control treatments where no macrofauna were present (Table II). Moreover, there are higher

252

T. S. BIANCHI ETAL.

concentrations of chlorophyll a in the control treatments (Fig. 2). Other studies also show that chlorophyll is converted to phaeophorbide during herbivorous digestion (Shuman & Lorenzen, 1975; Billett et al., 1983; Bricelj, 1984; Hawkins et al., 1986; Bianchi, 1987). Concentrations of phaeophorbide a may have been lower in the mixed macrofaunal treatments than the isolated h4. balthica and L. frugilis treatments because of feeding interference. If we assume that phaeophorbide a accumulation in the microcosms represents the amount of feeding activity that occurred, both deposit-feeders fed less in the presence of each other. Earlier studies have shown this type of interference response between benthic macrofaunal species with different feeding modes (in this case subsurface and surface deposit-feeders), to be quite common (Rhoads, 1963; Levinton, 1972, 1977). Similar concentrations of phaeophytins a and b in all the treatments suggests that microbial activity may primarily be responsible for this degradative pathway (Fig. 3 and Table II). Phaeophytins a and b represent =40-60% of the total phaeopigments produced in the control treatments, where no macrofauna were present (Table II). In the treatments with macrofauna present, phaeophytins represented only = 20-25 % of the total phaeopigments (Table II). The higher concentrations of phaeophytin b in the isolated M. balthica and L. frugiiis treatments may be due to combined effects of macrofaunal grazing and microbial processes (Fig. 3). The xanthophylls fucoxanthin and peridinin were found in most of the treatments, indicating the presence of diatoms and dinoflagellates (Wright & Jeffrey, 1987; Kleppel & Pieper, 1984). Degradation of photosynthetic pigments derived from these two sources may have contributed to the accumulation of phaeopigments in microcosms. However, in the case of dinoflagellates, the source of peridinin may have been from inactive cysts in the sediments (D. Brownlee, personal communication). Because U. rotund&a is a more labile plant source material (Rice & Tenore, 1981), significantly more of the chlorophyll in U. rotundata was converted to phaeophorbide a and phaeophytin b than in Z. marina (in the isolated L. fragilis and At. balthica treatments (Figs. 5 and 6). Differences in the decompositional rates of “aging” macroalgae (U. rotundata) and vascular plants (Z. marina) are mainly determined by the chemical components of the source material (Harrison & Mann, 1975; Tenore & Rice, 1980; Rice & Tenore, 1981). Vascular plants that contain more ligneous material and secondary metabolites, such as phenolic acids (Harrison, 1982), provide a less suitable resource for deposit-feeders and the microbial community. The molar organic carbon: nitrogen ratio of the U. rotundata used in this study (17.6 k 0.5) was significantly lower than 2. marina (38.3 + 3.4). Nitrogenous components of macroalgae are more likely to contribute to population growth of deposit-feeders than marine angiosperms (Tenore, 1977, 1982; Findlay & Tenore, 1982). The feeding activities of deposit-feeding macrofauna can significantly affect the transformation of chloropigments in marine sediments. Differences in the rate at which macrofauna can produce phaeopigments will primarily be dependent upon the quality of the food resource and the species composition of the benthic community. For

DEPOSIT-FEUDING

AND DEGRADATION

OF CHLOROPIGMENTS

253

example, there will be less phaeopi~ents produced by deposit-feeding macrofauna in a benthic community where there is feeding interference occurring (i.e., due to different feeding behavior). Seasonal variability in the quality of the plant food resources will also change the abundance of phaeopigments produced. By using laboratory microcosm experiments it may be possible to obtain characteristic pigment markers (for different macrofauna and plant materials) that can be used to determine the seasonal importance of macrofauna feeding activities on sediment pigment chemistry in the field. ACKNOWLEDGEMENTS

We thank R. F. C. Mantoura, S. Findlay, and A. Marsh for suggested revisions on earlier stages of this manuscript. Special thanks are due to J.M. Bianchi for graphic illustrations. T. S. Bianchi received postdoctoral support from an NSF grant (OCE 8442759) to D. L. Rice.

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